**Section 2**

### **Environmental and Nutritional Issues**

106 Public Health – Methodology, Environmental and Systems Issues

[146] Domenici, E., et al. Plasma protein biomarkers for depression and schizophrenia by multi analyte profiling of case-control collections. PLoS One 5, e9166 (2010). [147] Levin, Y., et al. Global proteomic profiling reveals altered proteomic signature in

[148] Melas, P.A., et al. Epigenetic aberrations in leukocytes of patients with schizophrenia:

[149] Kurian, S.M., et al. Identification of blood biomarkers for psychosis using convergent

[150] Kuzman, M.R., Medved, V., Terzic, J. & Krainc, D. Genome-wide expression analysis

[151] Dempster, E.L., et al. Disease-associated epigenetic changes in monozygotic twins

[152] Rutten, B.P. & Mill, J. Epigenetic mediation of environmental influences in major

[153] Heijmans, B.T. & Mill, J. Commentary: The seven plagues of epigenetic epidemiology.

[154] Kumarasinghe, N., Tooney, P. & Schall, U. Finding the needle in the haystack: A

[155] de Jong, S., et al. Expression QTL analysis of top loci from GWAS meta-analysis highlights additional schizophrenia candidate genes. Eur J Hum Genet (2012). [156] Gibbs, J.R., et al. Abundant quantitative trait loci exist for DNA methylation and gene

[157] Richards, A.L., et al. Schizophrenia susceptibility alleles are enriched for alleles that affect gene expression in adult human brain. Mol Psychiatry 17, 193-201(2011).

association of global DNA methylation with antipsychotic drug treatment and

of peripheral blood identifies candidate biomarkers for schizophrenia. J Psychiatr

discordant for schizophrenia and bipolar disorder. Hum Mol Genet 20, 4786-4796

review of microarray gene expression research into schizophrenia. Aust N Z J

[144] Perks, B. New regulations urged for UK health research. Nat Med 17, 142 (2011). [145] Rawlins, M., Barnett, D. & Stevens, A. Pharmacoeconomics: NICE's approach to

[143] Rawlins, M. A new era for UK clinical research? Lancet 377, 190-192.

decision-making. Br J Clin Pharmacol 70, 346-349 (2010).

schizophrenia serum. Mol Psychiatry 15, 1088-1100 (2009).

functional genomics. Mol Psychiatry 16, 37-58 (2011).

psychotic disorders. Schizophr Bull 35, 1045-1056 (2009).

expression in human brain. PLoS Genet 6, e1000952 (2010).

disease onset. FASEB J (2012).

Res 43, 1073-1077 (2009).

Int J Epidemiol 41, 74-78 (2012).

Psychiatry (2012).

(2011).

**6** 

*Canada* 

**Iron Deficiency Anemia:** 

Christopher V. Charles *University of Guelph,* 

**A Public Health Problem of Global Proportions** 

Iron deficiency anemia (IDA) is the most common micronutrient disorder in the world, negatively affecting the health and socio-economic wellbeing of millions of men, women, and children (Baltussen et al., 2004). According to the World Health Organization (WHO), IDA constitutes a significant public health problem requiring immediate attention from governments, researchers and healthcare practitioners (McLean et al., 2008). Iron deficiency (ID) is inherently associated with poverty, and is thus particularly prevalent in the developing world where the problem is often exacerbated by limited access to appropriate

Iron deficiency and IDA result from a long term negative iron balance, culminating in decreased or exhausted iron stores (Allen, 2000; Clark 2008; Ramakrishnan & Yip, 2002). Iron, a component of every living cell, is intrinsically involved in numerous biochemical reactions in the body and is associated with oxygen transport and storage, energy

Although the etiology of IDA is multifaceted, it generally results when iron demands are not met by iron absorption for any number of reasons. Individuals with IDA may have inadequate intake of iron due to poor quantity and/or quality of diet, impaired absorption or transport of iron, or chronic blood loss due to secondary disease (McLean et

Consequences of IDA are devastating: inhibited growth, impaired cognitive development, poor mental and motor performance, reduced work capacity, and an overall decreased quality of life (Macdougall et al., 1975; Newhouse et al., 1989; Preziosi et al., 1997; Soewondo

Prevention and control is typically achieved through iron fortification of food staples like flour, rice, and pasta, and/or through administration of iron supplements most often in iron pill or, more recently sprinkle form (Baltussen et al., 2004; Faqih et al., 2006; Mumtaz et al. 2000; Ramakrishnan & Yip, 2002). Although iron supplements are widely available and fortified foods constitute a major component of the diet in the developed world, access

production, DNA synthesis, and electron transport (Crichton et al., 2002; Theil, 2004).

healthcare and treatment (DeMaeyer & Adiels-Tegman, 1985).

et al., 1989; Walter et al., 1989; Zhu & Haas, 1997).

is limited in the developing world and cost if often prohibitive.

**1. Introduction** 

al., 2008).

### **Iron Deficiency Anemia: A Public Health Problem of Global Proportions**

Christopher V. Charles *University of Guelph, Canada* 

#### **1. Introduction**

Iron deficiency anemia (IDA) is the most common micronutrient disorder in the world, negatively affecting the health and socio-economic wellbeing of millions of men, women, and children (Baltussen et al., 2004). According to the World Health Organization (WHO), IDA constitutes a significant public health problem requiring immediate attention from governments, researchers and healthcare practitioners (McLean et al., 2008). Iron deficiency (ID) is inherently associated with poverty, and is thus particularly prevalent in the developing world where the problem is often exacerbated by limited access to appropriate healthcare and treatment (DeMaeyer & Adiels-Tegman, 1985).

Iron deficiency and IDA result from a long term negative iron balance, culminating in decreased or exhausted iron stores (Allen, 2000; Clark 2008; Ramakrishnan & Yip, 2002). Iron, a component of every living cell, is intrinsically involved in numerous biochemical reactions in the body and is associated with oxygen transport and storage, energy production, DNA synthesis, and electron transport (Crichton et al., 2002; Theil, 2004).

Although the etiology of IDA is multifaceted, it generally results when iron demands are not met by iron absorption for any number of reasons. Individuals with IDA may have inadequate intake of iron due to poor quantity and/or quality of diet, impaired absorption or transport of iron, or chronic blood loss due to secondary disease (McLean et al., 2008).

Consequences of IDA are devastating: inhibited growth, impaired cognitive development, poor mental and motor performance, reduced work capacity, and an overall decreased quality of life (Macdougall et al., 1975; Newhouse et al., 1989; Preziosi et al., 1997; Soewondo et al., 1989; Walter et al., 1989; Zhu & Haas, 1997).

Prevention and control is typically achieved through iron fortification of food staples like flour, rice, and pasta, and/or through administration of iron supplements most often in iron pill or, more recently sprinkle form (Baltussen et al., 2004; Faqih et al., 2006; Mumtaz et al. 2000; Ramakrishnan & Yip, 2002). Although iron supplements are widely available and fortified foods constitute a major component of the diet in the developed world, access is limited in the developing world and cost if often prohibitive.

Iron Deficiency Anemia: A Public Health Problem of Global Proportions 111

iron stores to compensate for iron loss through menstruation, pregnancy, and lactation

In food, two basic forms of iron exist: non-heme (inorganic) and heme (organic) (Bothwell, 1995; Charlton & Bothwell 1983). In an average diet, non-heme iron accounts for approximately 90% of total dietary iron content, while heme iron constitutes the remaining

Heme iron is highly bioavailable, and present in meat, fish, and poultry. In contrast, nonheme iron is not as readily bioavailable absorption is greatly influenced by diet composition (Harvey et al., 2000). Enhancers, such as ascorbic acid, and inhibitors, such as phytates and polyphenols, significantly affect inorganic iron absorption (Baynes & Bothwell, 1990, Tseng et al., 1997). Although total iron content in a meal is an important consideration, it is crucial to appreciate that the overall composition of the meal is of far greater significance for iron

Dietary iron digested from food and/or supplements is absorbed by the mature villus enterocytes of the duodenum and proximal jejunum (McKie et al., 2001). Non-heme and heme iron are absorbed via different pathways, though the understanding of heme iron

Non-heme iron in ferrous form is transported across the apical membrane of enterocytes by a non-specific divalent metal transporter (DMT1) (Aisen et al., 1999, Hentze et al., 2004). Because much of the iron that enters the gastrointestinal tract is in the oxidized or ferric form, a duodenal ferric reductase (Dcytb) in the apical membrane of enterocytes reduces

In contrast, heme iron molecules bind to an apical membrane protein and are absorbed intact. With the discovery of heme carrier protein 1 (HCP1), understanding has improved (Shayeghi et al., 2005). HCP1 is a polypeptide belonging to a superfamily of transporter proteins, and is predicted to have nine transmembrane domains by which heme iron is taken up. Though the mechanism is unclear, research has shown that by altering gene expression in animal models, heme absorption can be enhanced or limited by

Duodenal basolateral iron export into blood is mediated by the transmembrane protein ferroportin 1 (FPN1) (Zoller et al., 2001). The exact mechanism by which FPN1 functions is unclear, though it is thought to be facilitated by the ferroxidase activity of a membrane

After moving into the plasma, iron binds to transferrin and is transported by the blood to sites of use and storage (Bailey et al., 1988). Cellular iron uptake is mediated by transferrin receptor 1 (TfR)-mediated endocytosis (Fleming & Bacon, 2005). Once inside the cell, iron has two possible fates: incorporation into iron proteins (usually as heme) or storage as

overexpressing or silencing HCP1 genes, respectively (Shayeghi et al., 2005).

bound oxidase called hephaestin (Fleming & Bacon, 2005; Han & Kim 2007).

ferritin for later use during times of iron deficiency (Bleackley et al., 2009).

nutrition than the amount of total iron provided (McLean et al., 2008).

(Bothwell, 1995).

**3.2 Dietary iron sources** 

10% (Bothwell et al., 1979).

**3.3 Dietary iron absorption** 

absorption is somewhat more limited.

dietary iron prior to uptake (Latunde-Dada et al., 2002).

National, regional and global efforts to combat the problem of iron deficiency and IDA have garnered momentum in recent years, but the prevalence does not appear to be decreasing and the disorder remains a severe global public health problem. The current review will provide a general summary of the problem, touching upon the physiological aspects related to iron and hemoglobin, the etiology and epidemiology of IDA, and current prevention and control measures.

#### **2. Defining iron nutritional status**

Iron deficiency is defined as a condition in which there are no mobilizable iron stores, resulting from a long-term negative iron balance and leading to a compromised supply of iron to the tissues (Beutler et al., 2003). Iron status can be considered as a continuum: the ideal stage is normal iron status with varying amounts of stored iron within defined ranges; this is followed by iron deficiency, characterized by the absence of measurable iron stores; next, iron-deficient erythropoiesis shows evidence of a restricted iron supply in the absence of anemia; finally, the most significant negative consequence of ID is anemia, usually microcytic, hypochromic in nature (McLaren et al., 1983).

Anemia in general is characterized by a decrease in number of red blood cells or less than the normal quantity of hemoglobin. The condition is determined by the expected normal range of hemoglobin in a population, and is defined as existing in an individual whose hemoglobin concentration (Hb) has fallen below a threshold lying at two standard deviations below the median for a healthy population of the same demographic characteristics, including age, sex and pregnancy status (McLean et al., 2008). Anemic conditions can result from a myriad of causes that can be isolated, but more often than not co-exist. These causes include hemolysis with malaria and other infectious diseases, enzyme deficiencies, a variety of hemoglobinopathies, and other micronutrient deficiencies (McLean et al., 2008). That said, the most significant contributor to the onset of anemia worldwide is iron deficiency, and thus the terms ID, IDA, and anemia are often falsely used interchangeably. IDA represents the most severe form of iron deficiency, and has corresponding alterations in hematological laboratory values and observable signs and symptoms. Currently, the World Health Organization accepts that generally a little less than 50% of all anemias can be attributed to iron deficiency (McLean et al., 2008).

#### **3. Biochemical and physiological importance of iron in the blood**

#### **3.1 Human iron metabolism:**

Iron is important in the formation of a number of essential compounds in the body, including but not limited to hemoglobin, myoglobin, and other metalloproteins (Lynch, 1997). Most well-nourished adults in industrialized countries contain approximately 3 to 5 grams of iron, of which about 65% is in the form of hemoglobin (Bothwell, 1995). The remaining iron in the body is in the form of myoglobin, other heme compounds that promote intracellular oxidization, or is stored as ferritin in the reticuloendothelial system and cells of liver hepatocytes, bone marrow, and spleen (Frazer & Anderson 2005). Typically men have more stored iron than women, as women are often required to use iron stores to compensate for iron loss through menstruation, pregnancy, and lactation (Bothwell, 1995).

#### **3.2 Dietary iron sources**

110 Public Health – Methodology, Environmental and Systems Issues

National, regional and global efforts to combat the problem of iron deficiency and IDA have garnered momentum in recent years, but the prevalence does not appear to be decreasing and the disorder remains a severe global public health problem. The current review will provide a general summary of the problem, touching upon the physiological aspects related to iron and hemoglobin, the etiology and epidemiology of IDA, and current prevention and

Iron deficiency is defined as a condition in which there are no mobilizable iron stores, resulting from a long-term negative iron balance and leading to a compromised supply of iron to the tissues (Beutler et al., 2003). Iron status can be considered as a continuum: the ideal stage is normal iron status with varying amounts of stored iron within defined ranges; this is followed by iron deficiency, characterized by the absence of measurable iron stores; next, iron-deficient erythropoiesis shows evidence of a restricted iron supply in the absence of anemia; finally, the most significant negative consequence of ID is anemia, usually

Anemia in general is characterized by a decrease in number of red blood cells or less than the normal quantity of hemoglobin. The condition is determined by the expected normal range of hemoglobin in a population, and is defined as existing in an individual whose hemoglobin concentration (Hb) has fallen below a threshold lying at two standard deviations below the median for a healthy population of the same demographic characteristics, including age, sex and pregnancy status (McLean et al., 2008). Anemic conditions can result from a myriad of causes that can be isolated, but more often than not co-exist. These causes include hemolysis with malaria and other infectious diseases, enzyme deficiencies, a variety of hemoglobinopathies, and other micronutrient deficiencies (McLean et al., 2008). That said, the most significant contributor to the onset of anemia worldwide is iron deficiency, and thus the terms ID, IDA, and anemia are often falsely used interchangeably. IDA represents the most severe form of iron deficiency, and has corresponding alterations in hematological laboratory values and observable signs and symptoms. Currently, the World Health Organization accepts that generally a little less than 50% of all anemias can be attributed to iron deficiency (McLean

**3. Biochemical and physiological importance of iron in the blood** 

Iron is important in the formation of a number of essential compounds in the body, including but not limited to hemoglobin, myoglobin, and other metalloproteins (Lynch, 1997). Most well-nourished adults in industrialized countries contain approximately 3 to 5 grams of iron, of which about 65% is in the form of hemoglobin (Bothwell, 1995). The remaining iron in the body is in the form of myoglobin, other heme compounds that promote intracellular oxidization, or is stored as ferritin in the reticuloendothelial system and cells of liver hepatocytes, bone marrow, and spleen (Frazer & Anderson 2005). Typically men have more stored iron than women, as women are often required to use

control measures.

et al., 2008).

**3.1 Human iron metabolism:** 

**2. Defining iron nutritional status** 

microcytic, hypochromic in nature (McLaren et al., 1983).

In food, two basic forms of iron exist: non-heme (inorganic) and heme (organic) (Bothwell, 1995; Charlton & Bothwell 1983). In an average diet, non-heme iron accounts for approximately 90% of total dietary iron content, while heme iron constitutes the remaining 10% (Bothwell et al., 1979).

Heme iron is highly bioavailable, and present in meat, fish, and poultry. In contrast, nonheme iron is not as readily bioavailable absorption is greatly influenced by diet composition (Harvey et al., 2000). Enhancers, such as ascorbic acid, and inhibitors, such as phytates and polyphenols, significantly affect inorganic iron absorption (Baynes & Bothwell, 1990, Tseng et al., 1997). Although total iron content in a meal is an important consideration, it is crucial to appreciate that the overall composition of the meal is of far greater significance for iron nutrition than the amount of total iron provided (McLean et al., 2008).

#### **3.3 Dietary iron absorption**

Dietary iron digested from food and/or supplements is absorbed by the mature villus enterocytes of the duodenum and proximal jejunum (McKie et al., 2001). Non-heme and heme iron are absorbed via different pathways, though the understanding of heme iron absorption is somewhat more limited.

Non-heme iron in ferrous form is transported across the apical membrane of enterocytes by a non-specific divalent metal transporter (DMT1) (Aisen et al., 1999, Hentze et al., 2004). Because much of the iron that enters the gastrointestinal tract is in the oxidized or ferric form, a duodenal ferric reductase (Dcytb) in the apical membrane of enterocytes reduces dietary iron prior to uptake (Latunde-Dada et al., 2002).

In contrast, heme iron molecules bind to an apical membrane protein and are absorbed intact. With the discovery of heme carrier protein 1 (HCP1), understanding has improved (Shayeghi et al., 2005). HCP1 is a polypeptide belonging to a superfamily of transporter proteins, and is predicted to have nine transmembrane domains by which heme iron is taken up. Though the mechanism is unclear, research has shown that by altering gene expression in animal models, heme absorption can be enhanced or limited by overexpressing or silencing HCP1 genes, respectively (Shayeghi et al., 2005).

Duodenal basolateral iron export into blood is mediated by the transmembrane protein ferroportin 1 (FPN1) (Zoller et al., 2001). The exact mechanism by which FPN1 functions is unclear, though it is thought to be facilitated by the ferroxidase activity of a membrane bound oxidase called hephaestin (Fleming & Bacon, 2005; Han & Kim 2007).

After moving into the plasma, iron binds to transferrin and is transported by the blood to sites of use and storage (Bailey et al., 1988). Cellular iron uptake is mediated by transferrin receptor 1 (TfR)-mediated endocytosis (Fleming & Bacon, 2005). Once inside the cell, iron has two possible fates: incorporation into iron proteins (usually as heme) or storage as ferritin for later use during times of iron deficiency (Bleackley et al., 2009).

Iron Deficiency Anemia: A Public Health Problem of Global Proportions 113

Aerobic metabolism is critically dependent on maintaining normal concentrations of Hb, and the protein's ability to combine with oxygen in a reversible manner is essential for normal physiological functioning (White & Beaven, 1954). Oxygen binds with Hb in the lungs during respiration and is later released in peripheral tissue capillaries in the form of molecular oxygen where the gaseous tension of the molecule is much lower than in the lungs (Campbell, 1927). This is a cooperative process as the binding affinity of hemoglobin

In addition to hemoglobin's ability to bind oxygen, the protein can also bind with carbon dioxide and carbon monoxide, though not in a cooperative manner (Christiansen et al., 1914; Hill, 1913). In the presence of carbon monoxide, hemoglobin's ability to bind with oxygen is hampered as both gases compete for the same binding site with a much greater binding affinity for carbon monoxide than oxygen (Douglas et al., 1912). As a result, small amounts of carbon monoxide can dramatically reduce the oxygen transport in the body and carbon monoxide poisoning can ensue (Hill, 1913). On the other hand, hemoglobin's ability to bind carbon dioxide is a necessary process to allow for removal of carbon dioxide and byproducts from the system. Because carbon dioxide occupies a different binding site on the hemoglobin molecule, this type of ligand binding is allosteric in nature (Christiansen et al.,

The primary factor regulating the production of hemoglobin is tissue oxygenation. The peptide hormone erythropoietin (EPO), responding to a feed-back mechanism measuring blood oxygenation, is synthesized in times of decreased tissue oxygenation within 24 hours of the stimulus (Faura et al., 1969). EPO release triggers erythrocyte production in the bone marrow in an effort to achieve homeostasis of tissue oxygenation (Fandrey, 2004). As erythrocyte production increases, transferrin from plasma directly from diet and/or from iron stores enters the erythroblasts of bone marrow and is delivered to the mitochondria

Over the past three decades a large number of studies on the relationship between iron status and cognitive development have been conducted, often with varying results (Lozoff & Georgieff 2006). Iron and other micronutrient deficiencies often occur in the context of poverty and among individuals and families who are influenced by multiple stressors that may interfere with health and well-being, further confounding the issue. While an association between IDA and impaired cognitive development has been reported, research that takes into consideration the multi-diseased state common among individuals with IDA

Experiments employing animal models have demonstrated a key role for iron in brain development and function (Beard et al., 2006; Dallman et al., 1975; Felt & Lozoff, 1996;

where heme synthesis occurs, thus inducing the formation of hemoglobin.

**5. Functional consequences of iron deficiency and anemia** 

for oxygen is increased by the oxygen saturation of the molecule (Perutz, 1980).

**4.2 Reversible oxygenation of hemoglobin:** 

**4.3 Physiological control of hemoglobin levels:** 

1914; Roughton, 1970).

**5.1 Cognitive development** 

is needed (Lozoff & Georgieff, 2006).

#### **3.4 Regulation of iron homeostasis**

Since the discovery of the hormone hepcidin in 2000, the understanding of how iron homeostasis is achieved has shifted (Krause et al., 2000; Park et al. 2001). Hepcidin, a peptide hormone that is produced and predominately expressed in the liver, appears to be the master regulator of iron homeostasis in humans and other mammals (Ganz, 2003).

When iron levels are high, several regulatory molecules including hemochromatosis gene product, hemojuvelin and transferrin receptor 2, increase hepatic hepcidin expression, stimulating downstream molecular pathways. With up-regulation of hepcidin expression, iron levels are effectively regulated by binding to FPN1 which is found on the surface of iron storage cells. When iron levels are high, hepcidin causes internalization and degradation of FPN1, leading to decreased iron release from iron storage cells and a reduction in intestinal iron uptake (Dunn et al., 2007). In addition, hepcidin may also play a role in negatively regulating divalent metal transporter-1 (DMT1) and duodenal cytochrome-b (Dcytb) which are involved in intestinal iron absorption; currently, the mechanism and extent of control is unknown (Viatte et al., 2005).

In situations where iron requirements are increased, during periods of increased erythropoietic activity, anemia and hypoxia, the down-regulation of hepcidin expression is observed, though again the mechanism is not clear (Dunn et al., 2007; Pak et al., 2006; Vokurka et al., 2006)

#### **4. Hemoglobin**

#### **4.1 Formation of hemoglobin**

Hemoglobin is an allosteric protein with primary function of binding and transporting of oxygen in the blood to tissues in order to meet metabolic demands (Baldwin & Chothia, 1979). Synthesis of Hb involves a series of complex steps occurring in the erythrocytes, with production continuing through the early phases of the development and maturation of red blood cells (London et al., 1964). The coordinated production of heme, the group that mediates reversible oxygenation, and globin, which is responsible for protection of the heme group during transport, is required during synthesis (Schwartz et al., 1961).

Fully functional hemoglobin molecules consist of four globular protein subunits, each made of a protein chain that is tightly associated with a non-protein heme group (Perutz 1969, 1976; Perutz et al., 1960). The first step in the synthesis of Hb is the binding of succinyl-CoA (formed during the Krebs cycle) with glycine to form a pyrrole molecule. Next, four pyrroles combine to form protoporphyrin IX, which subsequently binds with iron to form the heme molecule. Each heme molecule then combines with a ribosomalderived long polypeptide chain called a globin, forming a globular subunit of hemoglobin called a hemoglobin chain. Lastly, four Hb chains are loosely bound to produce a whole hemoglobin molecule. The most common form of Hb in adult humans, hemoglobin A, is a combination of two alpha and two beta chains arranged as a set of alpha-helix structural segments connected in a globin fold arrangement (Forget, 1979).

#### **4.2 Reversible oxygenation of hemoglobin:**

112 Public Health – Methodology, Environmental and Systems Issues

Since the discovery of the hormone hepcidin in 2000, the understanding of how iron homeostasis is achieved has shifted (Krause et al., 2000; Park et al. 2001). Hepcidin, a peptide hormone that is produced and predominately expressed in the liver, appears to be the master regulator of iron homeostasis in humans and other mammals (Ganz,

When iron levels are high, several regulatory molecules including hemochromatosis gene product, hemojuvelin and transferrin receptor 2, increase hepatic hepcidin expression, stimulating downstream molecular pathways. With up-regulation of hepcidin expression, iron levels are effectively regulated by binding to FPN1 which is found on the surface of iron storage cells. When iron levels are high, hepcidin causes internalization and degradation of FPN1, leading to decreased iron release from iron storage cells and a reduction in intestinal iron uptake (Dunn et al., 2007). In addition, hepcidin may also play a role in negatively regulating divalent metal transporter-1 (DMT1) and duodenal cytochrome-b (Dcytb) which are involved in intestinal iron absorption; currently, the

In situations where iron requirements are increased, during periods of increased erythropoietic activity, anemia and hypoxia, the down-regulation of hepcidin expression is observed, though again the mechanism is not clear (Dunn et al., 2007; Pak et al., 2006;

Hemoglobin is an allosteric protein with primary function of binding and transporting of oxygen in the blood to tissues in order to meet metabolic demands (Baldwin & Chothia, 1979). Synthesis of Hb involves a series of complex steps occurring in the erythrocytes, with production continuing through the early phases of the development and maturation of red blood cells (London et al., 1964). The coordinated production of heme, the group that mediates reversible oxygenation, and globin, which is responsible for protection of the heme group during transport, is required during synthesis (Schwartz et

Fully functional hemoglobin molecules consist of four globular protein subunits, each made of a protein chain that is tightly associated with a non-protein heme group (Perutz 1969, 1976; Perutz et al., 1960). The first step in the synthesis of Hb is the binding of succinyl-CoA (formed during the Krebs cycle) with glycine to form a pyrrole molecule. Next, four pyrroles combine to form protoporphyrin IX, which subsequently binds with iron to form the heme molecule. Each heme molecule then combines with a ribosomalderived long polypeptide chain called a globin, forming a globular subunit of hemoglobin called a hemoglobin chain. Lastly, four Hb chains are loosely bound to produce a whole hemoglobin molecule. The most common form of Hb in adult humans, hemoglobin A, is a combination of two alpha and two beta chains arranged as a set of alpha-helix structural

mechanism and extent of control is unknown (Viatte et al., 2005).

segments connected in a globin fold arrangement (Forget, 1979).

**3.4 Regulation of iron homeostasis** 

2003).

Vokurka et al., 2006)

**4.1 Formation of hemoglobin** 

**4. Hemoglobin** 

al., 1961).

Aerobic metabolism is critically dependent on maintaining normal concentrations of Hb, and the protein's ability to combine with oxygen in a reversible manner is essential for normal physiological functioning (White & Beaven, 1954). Oxygen binds with Hb in the lungs during respiration and is later released in peripheral tissue capillaries in the form of molecular oxygen where the gaseous tension of the molecule is much lower than in the lungs (Campbell, 1927). This is a cooperative process as the binding affinity of hemoglobin for oxygen is increased by the oxygen saturation of the molecule (Perutz, 1980).

In addition to hemoglobin's ability to bind oxygen, the protein can also bind with carbon dioxide and carbon monoxide, though not in a cooperative manner (Christiansen et al., 1914; Hill, 1913). In the presence of carbon monoxide, hemoglobin's ability to bind with oxygen is hampered as both gases compete for the same binding site with a much greater binding affinity for carbon monoxide than oxygen (Douglas et al., 1912). As a result, small amounts of carbon monoxide can dramatically reduce the oxygen transport in the body and carbon monoxide poisoning can ensue (Hill, 1913). On the other hand, hemoglobin's ability to bind carbon dioxide is a necessary process to allow for removal of carbon dioxide and byproducts from the system. Because carbon dioxide occupies a different binding site on the hemoglobin molecule, this type of ligand binding is allosteric in nature (Christiansen et al., 1914; Roughton, 1970).

#### **4.3 Physiological control of hemoglobin levels:**

The primary factor regulating the production of hemoglobin is tissue oxygenation. The peptide hormone erythropoietin (EPO), responding to a feed-back mechanism measuring blood oxygenation, is synthesized in times of decreased tissue oxygenation within 24 hours of the stimulus (Faura et al., 1969). EPO release triggers erythrocyte production in the bone marrow in an effort to achieve homeostasis of tissue oxygenation (Fandrey, 2004). As erythrocyte production increases, transferrin from plasma directly from diet and/or from iron stores enters the erythroblasts of bone marrow and is delivered to the mitochondria where heme synthesis occurs, thus inducing the formation of hemoglobin.

#### **5. Functional consequences of iron deficiency and anemia**

#### **5.1 Cognitive development**

Over the past three decades a large number of studies on the relationship between iron status and cognitive development have been conducted, often with varying results (Lozoff & Georgieff 2006). Iron and other micronutrient deficiencies often occur in the context of poverty and among individuals and families who are influenced by multiple stressors that may interfere with health and well-being, further confounding the issue. While an association between IDA and impaired cognitive development has been reported, research that takes into consideration the multi-diseased state common among individuals with IDA is needed (Lozoff & Georgieff, 2006).

Experiments employing animal models have demonstrated a key role for iron in brain development and function (Beard et al., 2006; Dallman et al., 1975; Felt & Lozoff, 1996;

Iron Deficiency Anemia: A Public Health Problem of Global Proportions 115

1977) and Indonesian tea pickers (Basta et al., 1979) note significant gains in productivity following treatment of those individuals with significant IDA. One investigation conducted in China revealed that a rise of 10 g/L in Hb level was associated with an improvement in production efficiency of 14% in response to iron supplementation to treat IDA (Li et al.,

A meta-analysis of 29 studies demonstrated a strong causal effect of severe and moderate IDA on aerobic work capacity in animals and humans (Haas & Brownlie, 2001). The presumed mechanism for this effect is reduced oxygen transport and reduced cellular oxidative capacity due to tissue iron deficiency (Haas & Brownlie, 2001; Davies et al., 1984). In laboratory and field trials, iron deficiency and IDA at all levels of severity also appears to affect energetic efficiency (Zhu & Haas 1997; Li et al., 1994) and endurance capacity (Edgerton et al., 1972; Rowland et al., 1988). Conversely, iron supplementation has been shown to improve endurance and aerobic work capacity in iron-depleted humans (Hinton et

Two meta-analyses drawing upon the same published studies reported on an association between ID and maternal mortality. In a 2001 paper, Brabin et al. suggested that there is an association between a higher risk of maternal mortality with severe anemia (Brabin et al., 2001). Stoltzfus and colleagues, using a methodologically-different analysis, corroborated these findings, suggesting that the risk of maternal mortality increased with decreasing hemoglobin levels, though not in a linear manner. Causal evidence for the role that mild or moderate anemia may play in maternal mortality is lacking (Stoltzfus et al.,

In spite of these findings, a causal link between iron deficiency and mortality related to pregnancy and childbirth (ie. maternal mortality) remains unclear due to methodological concerns. To date there have been no large scale, placebo-controlled, prospective interventions to test the effect of iron supplementation on maternal mortality as large sample sizes would be required and it is considered unethical to withhold treatment from pregnant, anemic women. In addition, research in this field often does not take into consideration other possible causes of anemia and maternal mortality, such as concurrent micronutrient deficiencies, infectious disease, and other related conditions (Allen, 2000; Rush, 2000). For this reason, better observational data that controls for confounders are

A negative correlation between maternal IDA with length of gestation is well established (Allen, 2001). There are currently two widely accepted biological mechanisms that explain this phenomenon (Allen, 2001). One theory suggests that anemia (leading to hypoxia) and iron deficiency (which increases serum nor-epinephrine concentrations) induces maternal and fetal stress, ultimately leading to stimulation of the production of corticotropin-releasing hormone (CRH) (Allen, 2001; Dallman, 1987; Emanuel et al., 1994). Elevated CRH is a major risk factor for preterm labour, pregnancy-induced hypertension, eclampsia, premature rupture of the membranes, maternal infection (leading to yet more

al., 2000; Brownlie et al., 2004; Brownlie et al., 2002).

**5.4 Maternal mortality** 

required (Stoltzfus et al., 2005).

**5.5 Preterm delivery and growth** 

1994).

2005).

Jorgenson et al., 2005; Nelson et al., 2002). Iron-containing enzymes and hemoproteins are necessary in many important development processes such as myelination, dendritogenesis, synaptogenesis, and neurotransmission. Iron deficiency disrupts these processes in a regionally specific manner depending on brain development at the time of deficiency. This disruption may lead to a variety of neurodevelopmental effects that usually do not respond to iron replenishment (Lozoff & Georgieff, 2006).

In humans, the majority of research has focused on developmental and behavioural effects of ID on infancy during 6-24 months of age. Delayed psychomotor development, cognitive performance, and social/emotional functioning have been observed in numerous studies (Grantham-McGregor & Ani, 2001; Lozoff& Georgieff, 2006).

A number of observational studies have found that children who suffered from IDA early in life continued to demonstrate lower academic performance during their school-age years. In Costa Rican children born at term and free of health problems other than moderate iron deficiency, persistence of motor differences, more grade repetition, anxiety, depression, and other social problems have been observed (Lozoff et al., 2000). When compared with children that were not anemic during infancy, these children achieved lower scores on intelligence and other cognitive performance tests upon entry into school, despite controlling for socioeconomic factors that may have acted as confounders (Lozoff et al., 1991). A recent meta-analysis estimated the long term effects on IQ to be 1.73 points lower for each 10 g/L decrease in hemoglobin during infancy (Stoltzfus et al., 2005).

The detrimental effects of iron deficiency have been ameliorated with iron supplementation. Randomized controlled trials of iron supplementation consistently show improvement in motor (Moffatt et al., 1994), social-emotional (Williams et al., 1999), and language outcomes (Stoltzfus et al. 2001).

#### **5.2 Resistance to infection**

The role that iron deficiency plays in decreased immune response has been reported in both animal and human studies (Dallman, 1987). Leukocytes (neutrophils, in particular) appear to have a reduced capacity to ingest and neutralize microorganisms (Chandra, 1973; Macdougall et al. 1975; Srikantia et al. 1976), while mitogen-stimulated lymphocytes exhibit a decreased ability to replicate (Neckers & Cossman, 1983). Additionally, depressed T-cell responses have been widely documented, with the depression proportional to the severity of iron deficiency (Chandra, 1973; Srikantia et al., 1976, Bagchi et al., 1980; Prema et al., 1982). Treatment regimens such as iron supplementation and food fortification programs have been reported to reduce morbidity from infectious disease, further implicating a role for iron in immune response (Walter et al., 1997).

#### **5.3 Working capacity**

Anemia has long been known to impair work performance, endurance, and productivity (Walker 1998). Studies in developing countries in South America (Walker, 1998; Desai et al., 1984), East Africa (Davies, 1973; Davies &Haaren, 1973), and Sri Lanka (Gardner et al., 1977) report a linear relationship between ID and work capacity. Iron supplementation studies carried out on Indonesian rubber tappers (Basta et al., 1979), and Sri Lankan (Gardner et al., 1977) and Indonesian tea pickers (Basta et al., 1979) note significant gains in productivity following treatment of those individuals with significant IDA. One investigation conducted in China revealed that a rise of 10 g/L in Hb level was associated with an improvement in production efficiency of 14% in response to iron supplementation to treat IDA (Li et al., 1994).

A meta-analysis of 29 studies demonstrated a strong causal effect of severe and moderate IDA on aerobic work capacity in animals and humans (Haas & Brownlie, 2001). The presumed mechanism for this effect is reduced oxygen transport and reduced cellular oxidative capacity due to tissue iron deficiency (Haas & Brownlie, 2001; Davies et al., 1984). In laboratory and field trials, iron deficiency and IDA at all levels of severity also appears to affect energetic efficiency (Zhu & Haas 1997; Li et al., 1994) and endurance capacity (Edgerton et al., 1972; Rowland et al., 1988). Conversely, iron supplementation has been shown to improve endurance and aerobic work capacity in iron-depleted humans (Hinton et al., 2000; Brownlie et al., 2004; Brownlie et al., 2002).

#### **5.4 Maternal mortality**

114 Public Health – Methodology, Environmental and Systems Issues

Jorgenson et al., 2005; Nelson et al., 2002). Iron-containing enzymes and hemoproteins are necessary in many important development processes such as myelination, dendritogenesis, synaptogenesis, and neurotransmission. Iron deficiency disrupts these processes in a regionally specific manner depending on brain development at the time of deficiency. This disruption may lead to a variety of neurodevelopmental effects that usually do not respond

In humans, the majority of research has focused on developmental and behavioural effects of ID on infancy during 6-24 months of age. Delayed psychomotor development, cognitive performance, and social/emotional functioning have been observed in numerous studies

A number of observational studies have found that children who suffered from IDA early in life continued to demonstrate lower academic performance during their school-age years. In Costa Rican children born at term and free of health problems other than moderate iron deficiency, persistence of motor differences, more grade repetition, anxiety, depression, and other social problems have been observed (Lozoff et al., 2000). When compared with children that were not anemic during infancy, these children achieved lower scores on intelligence and other cognitive performance tests upon entry into school, despite controlling for socioeconomic factors that may have acted as confounders (Lozoff et al., 1991). A recent meta-analysis estimated the long term effects on IQ to be 1.73 points lower

The detrimental effects of iron deficiency have been ameliorated with iron supplementation. Randomized controlled trials of iron supplementation consistently show improvement in motor (Moffatt et al., 1994), social-emotional (Williams et al., 1999), and language outcomes

The role that iron deficiency plays in decreased immune response has been reported in both animal and human studies (Dallman, 1987). Leukocytes (neutrophils, in particular) appear to have a reduced capacity to ingest and neutralize microorganisms (Chandra, 1973; Macdougall et al. 1975; Srikantia et al. 1976), while mitogen-stimulated lymphocytes exhibit a decreased ability to replicate (Neckers & Cossman, 1983). Additionally, depressed T-cell responses have been widely documented, with the depression proportional to the severity of iron deficiency (Chandra, 1973; Srikantia et al., 1976, Bagchi et al., 1980; Prema et al., 1982). Treatment regimens such as iron supplementation and food fortification programs have been reported to reduce morbidity from infectious disease, further implicating a role

Anemia has long been known to impair work performance, endurance, and productivity (Walker 1998). Studies in developing countries in South America (Walker, 1998; Desai et al., 1984), East Africa (Davies, 1973; Davies &Haaren, 1973), and Sri Lanka (Gardner et al., 1977) report a linear relationship between ID and work capacity. Iron supplementation studies carried out on Indonesian rubber tappers (Basta et al., 1979), and Sri Lankan (Gardner et al.,

for each 10 g/L decrease in hemoglobin during infancy (Stoltzfus et al., 2005).

to iron replenishment (Lozoff & Georgieff, 2006).

(Stoltzfus et al. 2001).

**5.3 Working capacity** 

**5.2 Resistance to infection** 

for iron in immune response (Walter et al., 1997).

(Grantham-McGregor & Ani, 2001; Lozoff& Georgieff, 2006).

Two meta-analyses drawing upon the same published studies reported on an association between ID and maternal mortality. In a 2001 paper, Brabin et al. suggested that there is an association between a higher risk of maternal mortality with severe anemia (Brabin et al., 2001). Stoltzfus and colleagues, using a methodologically-different analysis, corroborated these findings, suggesting that the risk of maternal mortality increased with decreasing hemoglobin levels, though not in a linear manner. Causal evidence for the role that mild or moderate anemia may play in maternal mortality is lacking (Stoltzfus et al., 2005).

In spite of these findings, a causal link between iron deficiency and mortality related to pregnancy and childbirth (ie. maternal mortality) remains unclear due to methodological concerns. To date there have been no large scale, placebo-controlled, prospective interventions to test the effect of iron supplementation on maternal mortality as large sample sizes would be required and it is considered unethical to withhold treatment from pregnant, anemic women. In addition, research in this field often does not take into consideration other possible causes of anemia and maternal mortality, such as concurrent micronutrient deficiencies, infectious disease, and other related conditions (Allen, 2000; Rush, 2000). For this reason, better observational data that controls for confounders are required (Stoltzfus et al., 2005).

#### **5.5 Preterm delivery and growth**

A negative correlation between maternal IDA with length of gestation is well established (Allen, 2001). There are currently two widely accepted biological mechanisms that explain this phenomenon (Allen, 2001). One theory suggests that anemia (leading to hypoxia) and iron deficiency (which increases serum nor-epinephrine concentrations) induces maternal and fetal stress, ultimately leading to stimulation of the production of corticotropin-releasing hormone (CRH) (Allen, 2001; Dallman, 1987; Emanuel et al., 1994). Elevated CRH is a major risk factor for preterm labour, pregnancy-induced hypertension, eclampsia, premature rupture of the membranes, maternal infection (leading to yet more

Iron Deficiency Anemia: A Public Health Problem of Global Proportions 117

Worldwide, the prevalence of anemia is highest in non-industrialized nations where prevalence is three to four times higher than developed countries (Table 2). Africa, Eastern Europe and the Western Pacific have a large burden of disease, with over 1 billion people in these three regions estimated to be anemic (McLean et al., 2008). That said, anemia in South-East Asia is more prevalent than any other region in the world, with nearly 800 million affected. While the prevalence of IDA among women and children in the developed world is lower when compared to the developing world, a high prevalence is still reported in high-risk

**Preschool-aged Children Pregnant Women Non-pregnant Women** 

**Number affected (millions)** 

17.2 (15.9-18.5)

> 3.9 (2.8-5.0)

18.1 (16.4-19.7)

> 2.6 (2.0-3.3)

> 7.1 (6.1-8.0)

> 7.6 (7.1-8.1)

56.4 (53.8-59.1) **Prevalence (95% CI)** 

47.5 (43.4-51.6)

17.8 (12.9-22.7)

45.7 (41.9-49.4)

19.0 (14.7-23.3)

32.4 (29.2-35.6)

21.5 (20.8-22.2)

30.2 (28.7-31.6)

**Number affected (millions)** 

69.9 (63.9-75.9)

39.0 (28.3-49.7)

182.0 (166.9-197.1)

40.8 (31.5-50.1)

39.8 (35.8-43.8)

97.0 (94.0-100.0)

468.4 (446.2-490.6)

**Prevalence (95% CI)** 

57.1 (52.8-61.3)

24.1 (17.3-30.8)

48.2 (43.9-52.5)

25.1 (18.6-31.6)

44.2 (38.2-50.3)

30.7 (28.8-32.7)

41.8 (39.9-43.8)

Table 2. Anemia prevalence and number of individuals affected in pre-school aged children,

**Age or gender group Hemoglobin (g/L)** 

**6 – 59 months** 110 **5 – 11 years** 115 **12 – 14 years** 120 **Non-pregnant women (>15 years)** 120 **Pregnant women** 110 **Males (>15 years)** 130 Table 1. Hemoglobin levels below which anemia is present in a population.

groups, including preschool-aged children and pregnant women.

**Number affected (millions)** 

83.5 (79.4-87.6)

23.1 (21.1-25.1)

115.3 (107.3-123.2)

> 11.1 (7.9-14.4)

0.8 (0.4-1.1)

27.4 (25.9-28.9)

293.1 (282.8-303.5)

pregnant women, and non-pregnant women by WHO region.

Source: McLean et al., 2008.

**Prevalence (95% CI)** 

67.6 (64.3-71.0)

29.3 (26.8-31.9)

65.5 (61.0-70.0)

21.7 (15.4-28.0)

46.7 (42.2-51.2)

23.1 (21.9-24.4)

47.4 (45.7-49.1)

Source: (McLean et al., 2008).

WHO Region

Africa

Americas

South-East Asia

Europe

Eastern Mediterranean

Western Pacific

Global

CRH synthesis), and increased fetal cortisol production (inhibiting longitudinal growth of the fetus) (Allen, 2001; Falkenberg et al., 1999; Lin et al., 1998; Linton et al., 1990, McLean et al., 1995). A second theory suggests that iron deficiency may increase oxidative damage to erythrocytes and the fetal-placental unit (Cester et al., 1994; Poranen et al., 1996).

Maternal iron deficiency with and without anemia is also strongly associated with low birth weight and impeded growth (Stoltzfus et al., 2005). While full-term infants are normally born with sufficient iron stores, infants have high iron requirements and the diets offered to infants in the developing world are frequently inadequate in terms of satisfying the iron requirements for growth. Although iron in breast milk is highly bioavailable, maternal iron reserves are depleted after 4-6 months of feeding, thus infants commonly develop iron deficiency and IDA if the diet is not altered to include a readily absorbable source of iron (Friel et al., 1990).

Iron supplementation of infants appears to ameliorate the problem of impaired growth. A number of studies conducted in Indonesia (Soewondo et al., 1989), Kenya (Latham et al. 1990), Bangladesh (Briend et al., 1990), and the United Kingdom (Aukett et al., 1986) provide evidence that iron supplementation of iron deficient children leads to improved growth.

#### **5.6 Heavy-metal absorption**

An important consequence of iron deficiency is an enhanced ability for heavy-metal uptake, leading to heavy-metal poisoning. Iron deficiency is strongly associated with an increased absorption capacity that is not specific to iron, resulting in the uptake of divalent heavymetals like lead, cadmium, mercury and arsenic from the environment (Peraza et al., 1998). Heavy metal poisoning is a particular concern in children, as impaired cognitive development and irreversible physical and mental disability can result (Byers, 1959; Cebrian et al., 1983). For this reason, prevention of iron deficiency is important, predominantly in areas where exposure to heavy metals is common.

#### **6. Prevalence and epidemiology**

#### **6.1 Prevalence of iron deficiency and iron deficiency anemia**

Globally, nearly two billion people are affected by anemia (McLean et al., 2008). The majority of those affected live in developing countries where the problem is exacerbated by limited access to inadequate resources and appropriate treatment (Baltussen et al., 2004). IDA is unique in that it is the only nutrient deficiency which is significantly prevalent in virtually all industrialized nations as well. Currently there are no figures specifically for IDA, but it is widely accepted that approximately 50% of all cases of anemia are caused by iron deficiency (McLean et al., 2008; DeMaeyer & Adiels-Tegman, 1985). While the extent to which anemia is a problem in women and children has been widely documented, data on the prevalence of anemia in adolescents, men, and the elderly are scarce.

The level of hemoglobin concentration in the blood is used as an indicator to estimate the prevalence of anemia. Hemoglobin values that indicate the threshold for anemia have been published by the WHO and are widely accepted (Table 1) (McLean et al., 2008).

CRH synthesis), and increased fetal cortisol production (inhibiting longitudinal growth of the fetus) (Allen, 2001; Falkenberg et al., 1999; Lin et al., 1998; Linton et al., 1990, McLean et al., 1995). A second theory suggests that iron deficiency may increase oxidative damage to erythrocytes and the fetal-placental unit (Cester et al., 1994; Poranen et al.,

Maternal iron deficiency with and without anemia is also strongly associated with low birth weight and impeded growth (Stoltzfus et al., 2005). While full-term infants are normally born with sufficient iron stores, infants have high iron requirements and the diets offered to infants in the developing world are frequently inadequate in terms of satisfying the iron requirements for growth. Although iron in breast milk is highly bioavailable, maternal iron reserves are depleted after 4-6 months of feeding, thus infants commonly develop iron deficiency and IDA if the diet is not altered to include a readily absorbable source of iron

Iron supplementation of infants appears to ameliorate the problem of impaired growth. A number of studies conducted in Indonesia (Soewondo et al., 1989), Kenya (Latham et al. 1990), Bangladesh (Briend et al., 1990), and the United Kingdom (Aukett et al., 1986) provide evidence that iron supplementation of iron deficient children leads to improved growth.

An important consequence of iron deficiency is an enhanced ability for heavy-metal uptake, leading to heavy-metal poisoning. Iron deficiency is strongly associated with an increased absorption capacity that is not specific to iron, resulting in the uptake of divalent heavymetals like lead, cadmium, mercury and arsenic from the environment (Peraza et al., 1998). Heavy metal poisoning is a particular concern in children, as impaired cognitive development and irreversible physical and mental disability can result (Byers, 1959; Cebrian et al., 1983). For this reason, prevention of iron deficiency is important, predominantly in

Globally, nearly two billion people are affected by anemia (McLean et al., 2008). The majority of those affected live in developing countries where the problem is exacerbated by limited access to inadequate resources and appropriate treatment (Baltussen et al., 2004). IDA is unique in that it is the only nutrient deficiency which is significantly prevalent in virtually all industrialized nations as well. Currently there are no figures specifically for IDA, but it is widely accepted that approximately 50% of all cases of anemia are caused by iron deficiency (McLean et al., 2008; DeMaeyer & Adiels-Tegman, 1985). While the extent to which anemia is a problem in women and children has been widely documented, data on

The level of hemoglobin concentration in the blood is used as an indicator to estimate the prevalence of anemia. Hemoglobin values that indicate the threshold for anemia have been

1996).

(Friel et al., 1990).

**5.6 Heavy-metal absorption** 

areas where exposure to heavy metals is common.

**6.1 Prevalence of iron deficiency and iron deficiency anemia** 

the prevalence of anemia in adolescents, men, and the elderly are scarce.

published by the WHO and are widely accepted (Table 1) (McLean et al., 2008).

**6. Prevalence and epidemiology** 


Table 1. Hemoglobin levels below which anemia is present in a population. Source: McLean et al., 2008.

Worldwide, the prevalence of anemia is highest in non-industrialized nations where prevalence is three to four times higher than developed countries (Table 2). Africa, Eastern Europe and the Western Pacific have a large burden of disease, with over 1 billion people in these three regions estimated to be anemic (McLean et al., 2008). That said, anemia in South-East Asia is more prevalent than any other region in the world, with nearly 800 million affected. While the prevalence of IDA among women and children in the developed world is lower when compared to the developing world, a high prevalence is still reported in high-risk groups, including preschool-aged children and pregnant women.


Table 2. Anemia prevalence and number of individuals affected in pre-school aged children, pregnant women, and non-pregnant women by WHO region. Source: (McLean et al., 2008).

Iron Deficiency Anemia: A Public Health Problem of Global Proportions 119

Interventions to control iron deficiency and associated anemia are available, affordable, and sustainable (McLean et al., 2008, ACC/SCN, 2000). Food-based approaches are the most desirable and sustainable method of preventing IDA, with dietary improvement representing the most cost-effective and sustainable option. Advancements in the fortification of food staples and compliment have also shown promise. In addition, supplementation using multivitamins and vitamin complexes containing high levels of iron are accessible, though

Efforts towards promoting the availability of, and access to iron-rich foods are a key prevention technique. Foods containing high levels of iron include: meat and organs from cattle, fish, and poultry, as well as non-animal foods such as legumes and green leafy

As overall meal composition is as important as total iron content of a meal, it is important to promote the consumption of foods that enhances iron absorption and while limiting the consumption of foods that act as inhibitors (Layrisse et al. 1969; Layrisse & Martinez-Torres, 1968; Hallberg et al., 1986; Hallberg et al. 1991; Hallberg et al., 1989). Foods that enhance absorption of iron typically contain high levels of vitamins A, vitamin C, and folic acid; this includes various fruits, vegetables, and tubers. Conversely, phytates, found in cereal grains, tannins and other polyphenols found primarily in tea and coffee, and calcium from milk and milk products should be avoided where possible to limit the

Typically, diets of individuals in the developing world do not provide adequate iron. In a typical South-East Asian diet consisting of rice, vegetables and spices, iron absorption was reported to be inadequate (Hallberg et al. 1974; Hallberg et al. 1977). Even with the addition of fruit, meat and fish to these simple meals, iron absorption remained lower than the estimated requirement. These findings would suggest that individuals consuming such a diet could only maintain their iron balance in a state of iron deficiency, and would therefore greatly benefit from ID and IDA treatment and prevention programs

Iron fortification involves the addition of iron to an appropriate food vehicle that is distributed widely to the general population. Fortified flour and other cereals have historically been the most commonly used (Baltussen et al., 2004; Ramakrishnan & Yip 2002). Research into self-fortification through plant breeding is also gaining momentum and in the future may have a great impact on improving nutritional status (Lucca et al.,

(Bhargava et al., 2001; Thankachan et al., 2007).

often at a higher cost than other preventive and treatment methods.

**7. Prevention and control** 

**7.1 Dietary improvement** 

vegetables (WHO, 2001).

inhibition of iron absorption.

(Hallberg et al., 1974).

**7.2 Iron fortification** 

2001).

malnutrition, poor education regarding health and hygiene, and greater presence of concomitant disease when compared to populations of higher socioeconomic status

Women and children are hardest hit by this nutritional disorder due to increased iron requirements during periods of growth as well as during menstruation and pregnancy. Nearly 40% of preschool children and women (aged 15-59 years) and more than 50% of all pregnant women in developing countries estimated to be anemic (McLean et al., 2008).

#### **6.2 Etiology of iron deficiency and iron deficiency anemia**

The prevalence of ID and IDA varies greatly from population to population according to a variety of host and environmental factors. The etiology of anemia is multifaceted and often several factors are at play in an anemic individual. Nutritional anemia as a result of iron deficiency is the most common cause of anemia worldwide, with approximately 50% of all cases attributed to a lack of iron in the diet. A number of host and environmental factors are associated with iron deficiency, and in more severe forms contribute to IDA as well. These include:


malnutrition, poor education regarding health and hygiene, and greater presence of concomitant disease when compared to populations of higher socioeconomic status (Bhargava et al., 2001; Thankachan et al., 2007).

### **7. Prevention and control**

118 Public Health – Methodology, Environmental and Systems Issues

Women and children are hardest hit by this nutritional disorder due to increased iron requirements during periods of growth as well as during menstruation and pregnancy. Nearly 40% of preschool children and women (aged 15-59 years) and more than 50% of all pregnant women in developing countries estimated to be anemic (McLean et al., 2008).

The prevalence of ID and IDA varies greatly from population to population according to a variety of host and environmental factors. The etiology of anemia is multifaceted and often several factors are at play in an anemic individual. Nutritional anemia as a result of iron deficiency is the most common cause of anemia worldwide, with approximately 50% of all cases attributed to a lack of iron in the diet. A number of host and environmental factors are associated with iron deficiency, and in more severe forms contribute to IDA as well. These

1. *Inadequate dietary iron intake*: Diets low in iron or diets low in adequate amounts of bioavailable iron are a major cause of IDA, particularly in non-industrialized countries. Typically, high levels of IDA are also observed in old age when dietary quality and quantity

2. *Menstruation and pregnancy*: Blood losses associated with menstruation and pregnancy are common causes of ID and IDA. Typically non-menstruating women lose about 1 mg of iron per day, while menstruating women lose an additional 10 mg of iron per day during menses. Pregnancy is associated with an iron loss of approximately 1000 mg in a

3. *Infectious disease:* In the developing world common infections which may be both chronic and recurrent are associated with blood loss leading to iron deficiency, and ultimately to IDA. Hemolytic malaria and parasitic infections such as hookworm, trichuriasis, amoebiasis, and schistosomiasis are particularly common diseases that contribute to the depletion of iron stores and often result in IDA (Oppenheimer, 2001). 4. *Interactions with medication*: Several pharmacological agents can interfere with iron uptake and/or transport leading to iron loss or defective absorption. These include H2 blockers, proton pump inhibitors, aspirin or non-steroidal anti-inflammatory drug use

5. *Gastrointestinal conditions*: Both acute and chronic gastrointestinal illness is associated with IDA and is an important consideration in clinical diagnosis of the condition. Duodenal or gastric ulcers, carcinoma, polyps, irritable bowel disease, erosive gastritis, celiac disease, altered hepatic function for any number of reasons, and/or compromised

6. *Periods of growth*: Iron deficiency and IDA are particularly prevalent during peak periods of growth. Though full-term infants are normally born with adequate iron stores, if complementary foods containing iron are not introduced to the diet after six months of age then an infant is at risk of developing ID, and ultimately IDA. Iron requirements on a body weight basis are proportional to growth velocity, thus iron deficiency and IDA are common in preschool years and during puberty (McLean et al.,

7. *Socioeconomic status*: Iron deficiency and IDA are most common among groups of low socioeconomic status for a number of reasons, including but not limited to:

**6.2 Etiology of iron deficiency and iron deficiency anemia** 

deteriorates (Clark, 2008; Fiatarone-Singh et al., 2000).

55kg woman (Bothwell, 1995).

(Rockey & Cello, 1993).

protein status may lead to IDA (Clark, 2008).

2008; Tolentino & Friedman 2007; Turner et al., 2003).

include:

Interventions to control iron deficiency and associated anemia are available, affordable, and sustainable (McLean et al., 2008, ACC/SCN, 2000). Food-based approaches are the most desirable and sustainable method of preventing IDA, with dietary improvement representing the most cost-effective and sustainable option. Advancements in the fortification of food staples and compliment have also shown promise. In addition, supplementation using multivitamins and vitamin complexes containing high levels of iron are accessible, though often at a higher cost than other preventive and treatment methods.

#### **7.1 Dietary improvement**

Efforts towards promoting the availability of, and access to iron-rich foods are a key prevention technique. Foods containing high levels of iron include: meat and organs from cattle, fish, and poultry, as well as non-animal foods such as legumes and green leafy vegetables (WHO, 2001).

As overall meal composition is as important as total iron content of a meal, it is important to promote the consumption of foods that enhances iron absorption and while limiting the consumption of foods that act as inhibitors (Layrisse et al. 1969; Layrisse & Martinez-Torres, 1968; Hallberg et al., 1986; Hallberg et al. 1991; Hallberg et al., 1989). Foods that enhance absorption of iron typically contain high levels of vitamins A, vitamin C, and folic acid; this includes various fruits, vegetables, and tubers. Conversely, phytates, found in cereal grains, tannins and other polyphenols found primarily in tea and coffee, and calcium from milk and milk products should be avoided where possible to limit the inhibition of iron absorption.

Typically, diets of individuals in the developing world do not provide adequate iron. In a typical South-East Asian diet consisting of rice, vegetables and spices, iron absorption was reported to be inadequate (Hallberg et al. 1974; Hallberg et al. 1977). Even with the addition of fruit, meat and fish to these simple meals, iron absorption remained lower than the estimated requirement. These findings would suggest that individuals consuming such a diet could only maintain their iron balance in a state of iron deficiency, and would therefore greatly benefit from ID and IDA treatment and prevention programs (Hallberg et al., 1974).

#### **7.2 Iron fortification**

Iron fortification involves the addition of iron to an appropriate food vehicle that is distributed widely to the general population. Fortified flour and other cereals have historically been the most commonly used (Baltussen et al., 2004; Ramakrishnan & Yip 2002). Research into self-fortification through plant breeding is also gaining momentum and in the future may have a great impact on improving nutritional status (Lucca et al., 2001).

Iron Deficiency Anemia: A Public Health Problem of Global Proportions 121

1997; Faqih et al., 2006, Mumtaz et al. 2000; Menendez et al. 1997; Suharno et al. 1993; Schultink et al. 1995; Berger et al. 1997; Viteri, 1997). It is becoming increasingly clear that a main target group for iron supplementation in the developing world should be all women of reproductive age, regardless of pregnancy status at the time, thereby ensuring adequate iron reserves for both the mother and fetus during pregnancy and lactation (Yip & Ramakrishnan, 2002). Of concern is the relative cost of iron supplements in developing nations, coupled with issues surrounding delivery to infants and children. Other problems with iron supplementation include: undesirable side effects (including gastrointestinal irritation, black stools, and constipation); poor adherence to treatment guidelines; awareness and motivation of the target group to take supplements, often due to inadequate health and nutrition education; quality and packaging of iron supplements; and risk of iron overload if supplementation guidelines are not followed correctly (WHO,

Research conducted in the latter half of the 20th century has reported on the use of iron pots for cooking as an innovative way to reduce IDA, with the first study conducted in 1986 (Martinez & Vannucchi, 1986). Wistar rats fed a basal diet low in iron though cooked in an iron pot demonstrated comparable hemoglobin, hematocrit, protoporphyrin, serum iron, and transferrin saturation levels to those rats fed a complete diet, thus implicating the iron

Since this time several studies have examined this supplementation technique in humans with similar findings. Experiments conducted on Ethiopian children aged 2-5 years and pre-term infants (between months 4 and 12) from Brazil reported that cooking food in iron pots led to lower rates of anemia than children whose food was cooked in non-iron pots (Adish et al. 1999; Borigato, Martinez 1998). Significantly improved hematologic values between iron pot and non-iron pot groups were noted, including increased hemoglobin, hematocrit, mean corpuscular volume, free erythrocyte protoporphyrin, and serum ferritin. In addition, the Ethiopian study indicated moderate height and weight gains in children assigned to treatment groups (Adish et al. 1999). A more recent study conducted in Malawi verifies this research, noting a reduction in iron deficiency among children and increased hemoglobin levels in adults living under malarial endemic conditions (Geerligs

Research into the beneficial aspects of contaminant iron and adventitious iron sources, should be conducted. This supplementation technique has the possibility of providing a low-cost and sustainable way of improving dietary iron content, and may be particularly

Anemia is a global public health problem with serious consequences for human and socioeconomic health and development. Despite a concerted effort to improve treatment and prevention of iron deficiency and anemia in recent years, the problem does not appear

effective in the developing world where resources are limited.

2001).

**7.4 The use of adventitious iron sources** 

pot as an adventitious source of iron.

et al. 2003a, 2003b).

**8. Conclusion** 

to be going away.

#### **7.2.1 Fortification in the developed world**

Fortification has played an important role in the reduction of ID in the developed world since the latter half of the 20th century (Ramakrishnan & Yip 2002; Rees et al., 1985). The addition of elemental iron powder to flour and other cereals has since been commonplace, with levels of enrichment ranging from approximately 30 to 60 μg/g (Baltussen et al., 2004). In Canada, for example, it has been mandatory to enrich all white flours, enriched pastas, enriched precooked rice and certain substitute foods since 1976 (Guggenheim, 1995).

As the demand for processed foods has increased over the past 50 years, vitamins and minerals have slowly been added to an increasing array of foods. Ready-to-eat cereals in particular play an important role in daily iron intake in the Western world. Research in the last decade suggests that approximately 40% of total iron intake in women of reproductive age from the U.S. (Ramakrishnan& Yip, 2002), and approximately 78% of total iron intake in German children aged 2 to 13 years (Guggenheim, 1995) can be attributed to ready-to-eat cereals.

The fortification of infant formulas and foods provides particularly convincing evidence for the benefits of food fortification (Walter et al., 1993). Following the introduction of fortification guidelines in the U.S. in the late 1960's a clear reduction in IDA among infants and young children was noted (Yip et al., 1987).

#### **7.2.2 Fortification in the developing world**

In developing countries a much lower consumption of food from animal sources is observed and typically the overall nutritional value of the diet is lower when compared to developed nations (Yip & Ramakrishnan, 2002). In addition, both a high relative cost and a decreased availability of fortified products like cereal flours, ready-to-eat cereals, and infant formula leads to an overall decreased use of industry-prepared food that would otherwise benefit the population (Yip & Ramakrishnan, 2002). Thus, the relative absence of fortified food products from diets in developing nations could at least partly explain the high prevalence of iron deficiency and IDA and why current fortification practices have not ameliorated the situation. Research into fortifiable products that are culturally acceptable and desirable in developing nations should be conducted.

#### **7.3 Iron supplementation**

Iron supplementation is the most common and cost-effective strategy used to control ID and IDA in the developing world and is used as both a preventive measure and a treatment option (Baltussen et al., 2004). World Health Organization guidelines suggest that iron supplementation should include administration of 60 mg of iron daily with a dose of 400 μg of folic acid for women of reproductive age, 30 mg of iron and 250 μg of folic acid for schoolaged children, and approximately 2 mg/kg body weight per day for preschool-aged children (McLean et al., 2008). Weekly iron supplementation also exists, though is considered to be a less effective treatment option and requires additional research and evaluation (ACC/SCN, 2001).

The majority of supplementation studies to date have examined a variety of treatments in women of reproductive age, as infants, preschool and school-aged children (Preziosi et al., 1997; Faqih et al., 2006, Mumtaz et al. 2000; Menendez et al. 1997; Suharno et al. 1993; Schultink et al. 1995; Berger et al. 1997; Viteri, 1997). It is becoming increasingly clear that a main target group for iron supplementation in the developing world should be all women of reproductive age, regardless of pregnancy status at the time, thereby ensuring adequate iron reserves for both the mother and fetus during pregnancy and lactation (Yip & Ramakrishnan, 2002). Of concern is the relative cost of iron supplements in developing nations, coupled with issues surrounding delivery to infants and children. Other problems with iron supplementation include: undesirable side effects (including gastrointestinal irritation, black stools, and constipation); poor adherence to treatment guidelines; awareness and motivation of the target group to take supplements, often due to inadequate health and nutrition education; quality and packaging of iron supplements; and risk of iron overload if supplementation guidelines are not followed correctly (WHO, 2001).

#### **7.4 The use of adventitious iron sources**

120 Public Health – Methodology, Environmental and Systems Issues

Fortification has played an important role in the reduction of ID in the developed world since the latter half of the 20th century (Ramakrishnan & Yip 2002; Rees et al., 1985). The addition of elemental iron powder to flour and other cereals has since been commonplace, with levels of enrichment ranging from approximately 30 to 60 μg/g (Baltussen et al., 2004). In Canada, for example, it has been mandatory to enrich all white flours, enriched pastas,

As the demand for processed foods has increased over the past 50 years, vitamins and minerals have slowly been added to an increasing array of foods. Ready-to-eat cereals in particular play an important role in daily iron intake in the Western world. Research in the last decade suggests that approximately 40% of total iron intake in women of reproductive age from the U.S. (Ramakrishnan& Yip, 2002), and approximately 78% of total iron intake in German children aged 2 to 13 years (Guggenheim, 1995) can be attributed to ready-to-eat

The fortification of infant formulas and foods provides particularly convincing evidence for the benefits of food fortification (Walter et al., 1993). Following the introduction of fortification guidelines in the U.S. in the late 1960's a clear reduction in IDA among infants

In developing countries a much lower consumption of food from animal sources is observed and typically the overall nutritional value of the diet is lower when compared to developed nations (Yip & Ramakrishnan, 2002). In addition, both a high relative cost and a decreased availability of fortified products like cereal flours, ready-to-eat cereals, and infant formula leads to an overall decreased use of industry-prepared food that would otherwise benefit the population (Yip & Ramakrishnan, 2002). Thus, the relative absence of fortified food products from diets in developing nations could at least partly explain the high prevalence of iron deficiency and IDA and why current fortification practices have not ameliorated the situation. Research into fortifiable products that are culturally acceptable and desirable in

Iron supplementation is the most common and cost-effective strategy used to control ID and IDA in the developing world and is used as both a preventive measure and a treatment option (Baltussen et al., 2004). World Health Organization guidelines suggest that iron supplementation should include administration of 60 mg of iron daily with a dose of 400 μg of folic acid for women of reproductive age, 30 mg of iron and 250 μg of folic acid for schoolaged children, and approximately 2 mg/kg body weight per day for preschool-aged children (McLean et al., 2008). Weekly iron supplementation also exists, though is considered to be a less effective treatment option and requires additional research and

The majority of supplementation studies to date have examined a variety of treatments in women of reproductive age, as infants, preschool and school-aged children (Preziosi et al.,

enriched precooked rice and certain substitute foods since 1976 (Guggenheim, 1995).

**7.2.1 Fortification in the developed world** 

and young children was noted (Yip et al., 1987).

**7.2.2 Fortification in the developing world** 

developing nations should be conducted.

**7.3 Iron supplementation** 

evaluation (ACC/SCN, 2001).

cereals.

Research conducted in the latter half of the 20th century has reported on the use of iron pots for cooking as an innovative way to reduce IDA, with the first study conducted in 1986 (Martinez & Vannucchi, 1986). Wistar rats fed a basal diet low in iron though cooked in an iron pot demonstrated comparable hemoglobin, hematocrit, protoporphyrin, serum iron, and transferrin saturation levels to those rats fed a complete diet, thus implicating the iron pot as an adventitious source of iron.

Since this time several studies have examined this supplementation technique in humans with similar findings. Experiments conducted on Ethiopian children aged 2-5 years and pre-term infants (between months 4 and 12) from Brazil reported that cooking food in iron pots led to lower rates of anemia than children whose food was cooked in non-iron pots (Adish et al. 1999; Borigato, Martinez 1998). Significantly improved hematologic values between iron pot and non-iron pot groups were noted, including increased hemoglobin, hematocrit, mean corpuscular volume, free erythrocyte protoporphyrin, and serum ferritin. In addition, the Ethiopian study indicated moderate height and weight gains in children assigned to treatment groups (Adish et al. 1999). A more recent study conducted in Malawi verifies this research, noting a reduction in iron deficiency among children and increased hemoglobin levels in adults living under malarial endemic conditions (Geerligs et al. 2003a, 2003b).

Research into the beneficial aspects of contaminant iron and adventitious iron sources, should be conducted. This supplementation technique has the possibility of providing a low-cost and sustainable way of improving dietary iron content, and may be particularly effective in the developing world where resources are limited.

#### **8. Conclusion**

Anemia is a global public health problem with serious consequences for human and socioeconomic health and development. Despite a concerted effort to improve treatment and prevention of iron deficiency and anemia in recent years, the problem does not appear to be going away.

Iron Deficiency Anemia: A Public Health Problem of Global Proportions 123

Aukett, M.A., Parks, Y.A., Scott, P.H. & Wharton, B.A. (1986). Treatment with iron

Bailey, S., Evans, R.W., Garratt, R.C., Gorinsky, B., Hasnain, S., Horsburgh, C., Jhoti,

Baldwin, J. & Chothia, C. (1979). Haemoglobin: the structural changes related to ligand binding and its allosteric mechanism. *Journal of Molecular Biology* 129: 175-220. Baltussen, R., Knai, C. & Sharan, M. (2004). Iron Fortification and Iron Supplementation

Basta, S., Karyadi, D. & Scrimshaw, N. (1979). Iron deficiency anemia and the productivity of adult males in Indonesia. *American Journal of Clinical Nutrition* 32: 916-925. Baynes, R.D. & Bothwell, T.H. (1990). Iron Deficiency. *Annual Review of Nutrition* 10: 133-148. Beard, J.L., Felt, B., Schallert, T., Burhans, M., Connor, J.R. & Georgieff, M.K. (2006).

Berger, J., Aguayo, V.M., Tellez, W., Lujan, C., Traissac, P. & San Miguel, J.L. (1997).

Beutler, E., Hoffbrand, A.V. & Cook, J.D. (2003). Iron deficiency and overload. *Hematology/the Education Program of the American Society of Hematology. pp* 40-61. Bhargava, A., Bouis, H.E. & Scrimshaw, N.S. ( 2001). Dietary Intakes and

Bleackley, M.R., Wong, A.Y.K., Hudson, D.M., Wu, C.H. & MacGillivray, R.T.A. (2009).

Borigato, E.V.M. & Martinez, F.E. (1998). Iron Nutritional Status Is Improved in

Bothwell, T.H. (1995). Overview and mechanisms of iron regulation. *Nutrition Reviews* 53:

Bothwell TH, Charlton RW, Cook JD, Finch CA. (1995). *Iron metabolism in man.* 

Brabin, B.J., Hakimi, M. & Pelletier, D. (2001). An Analysis of Anemia and Pregnancy-

Briend, A., Hoque, B.A. & Aziz, K.M. (1990). Iron in tubewell water and linear growth

Brownlie, T.,IV, Utermohlen, V., Hinton, P.S., Giordano, C. & Haas, J.D. (2002). Marginal

Brownlie, T.,IV, Utermohlen, V., Hinton, P.S. & Haas, J.D. (2004). Tissue iron deficiency

Byers, R.K. (1959). Lead poisoning: Review of the Literature and Report on 45 Cases.

iron deficiency without anemia impairs aerobic adaptation among previously

without anemia impairs adaptation in endurance capacity after aerobic training in previously untrained women. *American Journal of Clinical Nutrition,* 79: 437-443.

Related Maternal Mortality. *Journal of Nutrition* 131: 604S-615S.

in rural Bangladesh. *Archives of Diseases of Children* 65: 224-225.

untrained women. *American Journal of Clinical Nutrition* 75: 734-742.

3.3-.ANG. resolution *Biochemistry* 27 5804-5812.

the World. *Journal of Nutrition* 134 2678-2684.

*Behavioural Brain Research* 170: 224-232.

*Journal of Clinical Nutrition* 51: 381-386.

*Transfusion Medicine Reviews* 23: 103-123.

Blackwell Scientific Publication: Oxford, UK.

855-859.

237-245.

*Pediatrics* 23: 585-603.

Bangladeshi Women. *Journal of Nutrition* 131: 758-764.

61: 849- 857.

increases weight gain and psychomotor development. *Archives Disease in Children*

H., Lindley, P.F. & Mydin, A. (1988). Molecular structure of serum transferrin at

are Cost-Effective Interventions to Reduce Iron Deficiency in Four Subregions of

Moderate iron deficiency in infancy: Biology and behavior in young rats.

Weekly iron supplementation is as effective as 5 day per week iron supplementation in Bolivian school children living at high altitude. *European* 

Socioeconomic Factors Are Associated with the Hemoglobin Concentration of

Blood Iron Homeostasis: Newly Discovered Proteins and Iron Imbalance.

Brazilian Preterm Infants Fed Food Cooked in Iron Pots. *Journal of Nutrition* 128:

In 2004, the Copenhagen Consensus brought together a panel of world-renowned development economists to consider and confront the ten most pressing challenges to "global welfare" that we face today (Copenhagen Consensus, 2004). Micronutrient interventions, including iron fortification, ranked at the top of the list and offered the highest benefit: cost ratio of any development intervention. These findings were confirmed in 2008, at the most recent Consensus meeting, where iron and zinc fortification were placed within the top three global challenges (Copenhagan Consensus, 2008). This prioritization of iron and other micronutrient interventions emphasizes the need for well-designed, sustainable and effective programming efforts to combat iron deficiency anemia.

The adverse effects of anemia on mortality, morbidity and development are abundantly clear. Anemia affects how individuals participate in all areas of life, including work, school and social activities, and this limits the ability to generate income and afford iron-rich sources of food, medical treatment, and school fees. In turn, this leads to constrained social and economic development, ultimately contributing to a viscous cycle of poverty that is difficult to overcome.

The widespread prevalence of anemia, both in the developed and developing worlds, is great cause for concern. The current review highlights some of the most promising research on the etiology, prevention and control of the disorder. From this, it should be clear that although we have made strides, there is still much that we do not understand about iron deficiency and anemia, especially in relation to treatment and prevention. A renewed effort to find effective ways to combat this problem is needed, as anemia is unique and complex public health crisis that is of global proportions.

#### **9. Acknowledgements**

The author wishes to thank Dr. Alastair Summerlee, President & Vice-Chancellor of the University of Guelph, Canada and Dr. Cate Dewey, Chair of Department of Population Medicine, University of Guelph, Canada for their continued support, insight, and careful editing of the chapter. The author is supported by funding from the Canadian Institutes of Health Research and the University of Guelph, Canada.

#### **10. References**


In 2004, the Copenhagen Consensus brought together a panel of world-renowned development economists to consider and confront the ten most pressing challenges to "global welfare" that we face today (Copenhagen Consensus, 2004). Micronutrient interventions, including iron fortification, ranked at the top of the list and offered the highest benefit: cost ratio of any development intervention. These findings were confirmed in 2008, at the most recent Consensus meeting, where iron and zinc fortification were placed within the top three global challenges (Copenhagan Consensus, 2008). This prioritization of iron and other micronutrient interventions emphasizes the need for well-designed,

The adverse effects of anemia on mortality, morbidity and development are abundantly clear. Anemia affects how individuals participate in all areas of life, including work, school and social activities, and this limits the ability to generate income and afford iron-rich sources of food, medical treatment, and school fees. In turn, this leads to constrained social and economic development, ultimately contributing to a viscous cycle of poverty that is

The widespread prevalence of anemia, both in the developed and developing worlds, is great cause for concern. The current review highlights some of the most promising research on the etiology, prevention and control of the disorder. From this, it should be clear that although we have made strides, there is still much that we do not understand about iron deficiency and anemia, especially in relation to treatment and prevention. A renewed effort to find effective ways to combat this problem is needed, as anemia is unique and complex

The author wishes to thank Dr. Alastair Summerlee, President & Vice-Chancellor of the University of Guelph, Canada and Dr. Cate Dewey, Chair of Department of Population Medicine, University of Guelph, Canada for their continued support, insight, and careful editing of the chapter. The author is supported by funding from the Canadian Institutes of

ACC/SCN United Nations (2000). Fourth Report on the World Nutrition Situation. United

 http://www.ifpri.org/sites/default/files/pubs/pubs/books/4thrpt/4threport.pdf Adish, A.A., Esrey, S.A., Gyorkos, T.W., Jean-Baptiste, J. & Rojhani, A. (1999). Effect

Aisen, P., Wessling-Resnick, M. & Leibold, E.A. (1999). Iron metabolism. *Current Opinion in*

Allen, L.H. (2001). Biological Mechanisms That Might Underlie Iron's Effects on Fetal

Allen, L.H. ( 2000). Anemia and iron deficiency: effects on pregnancy outcome. *American* 

of consumption of food cooked in iron pots on iron status and growth of

Nations: Geneva. Accessed on 12th February 2012 from

young children: a randomised trial. *The Lancet* 353: 712-716.

Growth and Preterm Birth *Journal of Nutrition* 131: 581S-589S.

*Journal of Clinical Nutrition* 71: 1280S-1284S.

sustainable and effective programming efforts to combat iron deficiency anemia.

difficult to overcome.

**9. Acknowledgements** 

**10. References** 

public health crisis that is of global proportions.

Health Research and the University of Guelph, Canada.

*Chemical Biology* 3: 200-206.


Iron Deficiency Anemia: A Public Health Problem of Global Proportions 125

Douglas, C.G., Haldane, J.S. & Haldane, J.B. (1912). The laws of combination of haemoglobin with carbon monoxide and oxygen. *Journal of Physiology* 44: 275-304. Dunn, L.L., Rahmanto, Y.S. & Richardson, D.R. ( 2007). Iron uptake and metabolism in

Edgerton, V.R., Bryant, S.L., Gillespie, C.A. & Gardner, G.W. (1972). Iron Deficiency

Emanuel, R.L., Robinson, B.G., Seely, E.W., Graves, S.W., Kohane, I., Saltzman, D., Barbieri,

Faqih, A.M., Kakish, S.B. & Izzat, M. (2006). Effectiveness of intermittent iron treatment

Faura, J., Ramos, J., Reynafarje, C., English, E., Finne, P. & Finch, C.A. (1969). Effect

Felt, B.T. & Lozoff, B. (1996). Brain Iron and Behavior of Rats are Not Normalized by

Fiatarone Singh, M.A., Bernstein, M.A., Ryan, A.D., O'Neill, E.F., Clements, K.M. & Evans,

Fleming, R.E. & Bacon, B.R. (2005). Orchestration of Iron Homeostasis. *The New England*

Forget, B.G. (1979). Molecular Genetics of Human Hemoglobin Synthesis. *Annals of Internal*

Frazer, D.M. & Anderson, G.J. (2005). Iron Imports. I. Intestinal iron absorption and its

Friel, J.K., Andrews, W.L., Matthew, J.D., Long, D.R., Cornel, A.M., Cox, M. & Skinner, C.T.

Ganz, T. ( 2003). Hepcidin, a key regulator of iron metabolism and mediator of anemia

Gardner, G., Edgerton, V., Senewiratne, B., Barnard, R. & Ohira, Y. (1977). Physical

Geerligs, P.D., Brabin, B.J. & Omari, A.A. (2003a). Food prepared in iron cooking pots as

Geerligs, P.P., Brabin, B., Mkumbwa, A., Broadhead, R. & Cuevas, L.E. (2003b). The effect

of infancy. *Canadian Medical Association Journal* 143 733-737.

Anemia and Physical Performance and Activity of Rats. *Journal of Nutrition* 102:

R. & Majzoub, J.A. (1994). Corticotrophin releasing hormone levels in human plasma and amniotic fluid during gestation. *Clinical Endocrinology* 40: 257-262. Falkenberg, E.R., Davis, R.O., DuBard, M. & Parker, C.R., Jr. (1999). Effects of maternal infections on fetal adrenal steroid production. *Endocrine Research* 25: 239-249. Fandrey, J. ( 2004). Oxygen-dependent and tissue-specific regulation of erythropoietin

gene expression. *American Journal of Physiology - Regulatory, Integrative and* 

of two-to six-year-old Jordanian children with iron-deficiency anemia. *Food and*

Treatment of Iron Deficiency Anemia during Early Development. *Journal of*

W.J. (2000). The effect of oral nutritional supplements on habitual dietary quality and quantity in frail elders. *The Journal of Nutrition, Health & Aging* 4: 5-12.

regulation. *American Journal Physiology Gastrointestinal Liver Physiology* 289: G631-

(1990). Iron status of very-low-birth-weight infants during the first 15 months

work capacity and metabolic stress in subjects with iron deficiency anemia.

an intervention for reducing iron deficiency anaemia in developing countries: a systematic review. *Journal of Human Nutrition and Dietetics : the official Journal of* 

on haemoglobin of the use of iron cooking pots in rural Malawian households in

the new millennium. *Trends in Cell Biology* 17: 93-100.

*Comparative Physiology* 286: R977-988.

of Altitude on Erythropoiesis. *Blood* 33: 668-676.

*Nutrition Bulletin* 27: 220-227.

*Journal of Medicine* 352 1741-1744.

of inflammation. *Blood* 102 783-788.

*American Journal of Clinical Nutrition* 30 910-917.

*the British Dietetic Association* 16: 275-281.

*Nutrition* 126: 693-701.

*Medicine* 91: 605-616.

635.

381-399.


Campbell, J.A. (1927). Prolonged alterations of oxygen pressure in the inspired air with

Cebrian, M.E., Albores, A., Aguilar, M. & Blakely, E. (1983). Chronic Arsenic Poisoning in

Cester, N., Staffolani, R., Rabini, R.A., Magnanelli, R., Salvolini, E., Galassi, R., Mazzanti, L.

Chandra, R.K. (1973). Reduced bactericidal capacity of polymorphs in iron deficiency.",

Charlton, R.W. & Bothwell, T.H. (1983). Iron Absorption. *Annual Review of Medicine* 34: 55-

Christiansen, J., Douglas, C.G. & Haldane, J.S. (1914). The absorption and dissociation of

Clark, S.F. (2008). Iron Deficiency Anemia. *Nutrition Clinical Practicum* 23:128-141.

http://www.copenhagenconsensus.com/Projects/Copenhagen%20Consensus%

Crichton, R.R., Wilmet, S., Legssyer, R. & Ward, R.J. (2002). Molecular and cellular

Dallman, P.R., Siimes, M.A. & Manies, E.C. (1975). Brain iron: persistent deficiency

Dallman, P. (1987). Iron deficiency and the immune response. *American Journal of Clinical* 

Davies, C.T., Chukweumeka, A.C. & Van Haaren, J.P. (1973). Iron-deficiency anaemia: it

Davies, C.T.M. & Haaren, J.P.M.V. (1973). Effect of treatment on physiological responses

Davies, K.J., Donovan, C.M., Refino, C.J., Brooks, G.A., Packer, L. & Dallman, P.R.

De Benoist, B., McLean. E., Egli. I, Cogswell, M (2008). Worldwide Prevalence of Anaemia

Desai, I., Waddell, C., Dutra, S., Dutra de Oliveira, S., Duarte, E., Robazzi, M.,

Southern Brazil. *American Journal of Clinical Nutrition* 40: 135-145.

Copenhagen Consensus Centre. Copenhagen Consensus 2004. Access 12th February

mechanisms of iron homeostasis and toxicity in mammalian cells", *Journal of*

following short-term iron deprivation in the young rat. *British Journal of* 

effect on maximum aerobic power and responses to exercise in African males aged

to exercise in East African industrial workers with iron deficiency anaemia. *British* 

(1984). Distinguishing effects of anemia and muscle iron deficiency on exercise bioenergetics in the rat. *American Journal Physiology Endocrinology and* 

1993-2005. World Health Organization Press, Geneva. Accessed 12th February 2012 from: http://whqlibdoc.who.int/publications/2008/9789241596657\_eng.pdf DeMaeyer, E. & Adiels-Tegman, M. (1985). The prevalence of anaemia in the world",

*World health statistics Quarterly/Rapport trimestriel de statistiques sanitaires mondiales* 

Cevallos Romero, L., Desai, M., Vichi, F. & Bradfield, R. (1984). Marginal malnutrition and reduced physical work capacity of migrant adolescent boys in

carbon dioxide by human blood. *Journal of Physiology* 48: 244-271.

the North of Mexico. *Human and Experimental Toxicology* 2: 121-133.

hæmoglobin. *Journal of Physiology* 62: 211-231.

*Archives of the Diseases of Children* 48: 864-866.

68.

2012 from:

202004-1.aspx

*Inorganic Biochemistry* 91: 9-18.

17-40 years. *Clinical Science* 44: 555-562.

*Journal of Indian Medicine* 30: 335-340.

*Haematology* 31: 209-215.

*Metabolism* 246: E535-543.

38: 302-316.

*Nutrition* 46: 329-334.

special reference to tissue oxygen tension, tissue carbon dioxide tension and

& Romanini, C. (1994). Pregnancy induced hypertension: a role for peroxidation in microvillus plasma membranes. *Molecular and Cellular Biochemistry* 131: 151-155.


Iron Deficiency Anemia: A Public Health Problem of Global Proportions 127

Latunde-Dada, G.O., Van der Westhuizen, J., Vulpe, C.D., Anderson, G.J., Simpson, R.J. &

(Dcytb) in Iron Metabolism. *Blood Cells, Molecules, and Diseases* 29: 356-360. Layrisse, M., Cook, J.D., Martinez, C., Roche, M., Kuhn, I.N., Walker, R.B. & Finch,

Layrisse, M. & Martinez -Torres, C. (1968). Effect of Interaction of Various Foods on Iron Absorption. *American Journal of Clinical Nutrition* 21: 1175-1183. Li, R., Chen, X., Yan, H., Deurenberg, P., Garby, L. & Hautvast, J. (1994). Functional

Lin, C.C. & Santolaya-Forgas, J. (1998). Current concepts of fetal growth restriction: part I.

Linton, E.A., Behan, D.P., Saphier, P.W. & Lowry, P.J. (1990). Corticotropin-releasing

London IM, Bruns GP, Karibian D. (1964). The Regulation of Hemoglobin Synthesis and the Pathogenesis of Some Hypochromic Anemias. *Medicine* 43: 789-802. Lozoff, B., Jimenez, E. & Wolf, A. (1991). Long-term developmental outcome of infants

Lozoff, B. & Georgieff, M.K. (2006). Iron Deficiency and Brain Development. *Seminars in*

Lozoff, B., Jimenez, E., Hagen, J., Mollen, E. & Wolf, A.W. 2000, "Poorer Behavioral

Lucca, P., Hurrell, R. & Potrykus, I. (2001). Genetic engineering approaches to improve th

Lynch, S.R. (1997). Interaction of iron with other nutrients. *Nutrition Reviews* 55: 102-110. Macdougall, L.G., Anderson, R., McNab, G.M. & Katz, J (1975). The immune response

Martinez, F.E. & Vannucchi, H. (1986). Bioavailability of iron added to the diet by

McKie, A.T., Barrow, D., Latunde-Dada, G.O., Rolfs, A., Sager, G., Mudaly, E., Mudaly, M.,

McLean, M., Bisits, A., Davies, J., Woods, R., Lowry, P. & Smith, R. (1995). A placental clock controlling the length of human pregnancy. *Nature Medicine* 1: 460-463. Menendez, C., Kahigwa, E., Hirt, R., Vounatsou, P., Aponte, J.J., Font, F., Acosta, C.J.,

and Developmental Outcome More Than 10 Years After Treatment for Iron

bioavailability and the level of iron in rice grains. *Theoretical and Applied*

iniron-deficient children: Impaired cellular defense mechanisms with altered

Richardson, C., Barlow, D., Bomford, A., Peters, T.J., Raja, K.B., Shirali, S., Hediger, M.A., Garzaneh, F., & Simpson, R.J. (2001). An Iron-Regulated Ferric Reductase Associated with the Absorption of Dietary Iron. *Science* 291: 1755-1759. McLaren, G.D., Muir, W.A. & Kellermeyer, R.W. (1983). Iron overload disorders: natural

history, pathogenesis, diagnosis, and therapy. *Critical Reviews in Clinical* 

Schellenberg, D.M., Galindo, C.M., Kimario, J., Urassa, H., Brabin, B., Smith, T.A., Kitua, A.Y., Tanner, M. & Alonso, P.L. (1997). Randomised placebo-controlled

in Beijing, China. *American Journal of Clinical Nutrition* 59: 908-913.

with iron deficiency. *New England Journal of Medicine* 325: 687-694.

Deficiency in Infancy", *Pediatrics,* vol. 105, no. 4, pp. e51.

humoral components. *Journal of Pediatrics* 86: 833-843.

cooking food in an iron pot. *Nutrition Research* 6: 421-428.

*Blood* 33: 430-443.

*and Metabolism* 70: 1574-1580.

*Pediatric Neurology* 13: 158-165.

*Laboratory Sciences* 19: 205-266.

*Genetics* 102: 392-397.

1055.

McKie, A.T. (2002). Molecular and Functional Roles of Duodenal Cytochrome B

C.A. (1969). Food Iron Absorption: A Comparison of Vegetable and Animal Foods.

consequences of iron supplementation in iron-deficient female cotton mill workers

Causes, classification, and pathophysiology. *Obstetrics and Gynecology* 92: 1044-

hormone (CRH)-binding protein: reduction in the adrenocorticotropin-releasing activity of placental but not hypothalamic CRH. *Journal of Clinical Endocrinology*

an area with high malaria prevalence: a randomized trial. *Tropical Medicine & International Health* 8: 310-315.


Grantham-McGregor, S. & Ani, C. (2001). A Review of Studies on the Effect of Iron

Guggenheim, K.Y. (1995). Chlorosis: The Rise and Disappearance of a Nutritional Disease.

Haas, J.D. & Brownlie, T., IV (2001). Iron Deficiency and Reduced Work Capacity: A

Hallberg, L., Bjorn-Rasmussen, E., Rossander, L. & Suwanik, R. (1977). Iron absorption

Hallberg, L., Brune, M., Erlandsson, M., Sandberg, A. & Rossander-Hulten, L. (1991).

Hallberg, L., Brune, M. & Rossander, L. (1989). Iron absorption in man: ascorbic acid

Hallberg, L., Brune, M. & Rossander, L. (1986). Effect of ascorbic acid on iron

Hallberg, L., Garby, L., Suwanik, R. & Bjorn-Rasmussen, E (1974). Iron absorption from Southeast Asian diets. *American Journal of Clinical Nutrition* 27: 826-836. Han, O. & Kim, E.Y. (2007). Colocalization of ferroportin-1 with hephaestin on the

Harvey, P.W.J., Dexter, P.B. & Darnton Hill, I. (2000). The impact of consuming iron

Hentze, M.W., Muckenthaler, M.U. & Andrews, N.C. (2004). Balancing Acts: Molecular

Hill, A.V (1913). The Combinations of Haemoglobin with Oxygen and with Carbon

Hinton, P.S., Giordano, C., Brownlie, T. & Haas, J.D. (2000). Iron supplementation

Jorgenson, L.A., Sun, M., O'Connor, M. & Georgieff, M.K. (2005). Fetal iron deficiency

Krause, A., Neitz, S., Mägert, H., Schulz, A., Forssmann, W., Schulz-Knappe, P. &

Latham, M.C., Stephenson, L.S., Kinoti, S.N., Zaman, M.S. & Kurz, K.M. (1990).

Control of Mammalian Iron Metabolism. *Cell* 117: 285-297.

developing rat hippocampus. *Hippocampus* 15: 1094-1102.

exhibits antimicrobial activity. *FEBS letters* 480: 147-150.

Monoxide. I. *The Biochemical Journal* 7: 471-480.

absorption. *American Journal of Clinical Nutrition* 30: 539-548.

humans. *American Journal of Clinical Nutrition* 53: 112-119.

*International Health* 8: 310-315.

*Journal of Nutrition* 125: 1822-1825.

*Nutrition/Applied nutrition* 40: 97-113.

*Biochemistry* 101: 1000-1010.

Healthy Nutrition 3: 375–383.

*of Applied Physiology* 88: 1103-1111.

children. *Nutrition* 6: 159-165.

*Nutrition* 131: 676S-690S.

649S-668S.

49: 140-144.

an area with high malaria prevalence: a randomized trial. *Tropical Medicine &*

Deficiency on Cognitive Development in Children. *Journal of Nutrition* 131:

Critical Review of the Research to Determine a Causal Relationship. *Journal of* 

from Southeast Asian diets. II. Role of various factors that mightexplain low

Calcium: effect of different amounts on nonheme- and heme-iron absorption in

and dose-dependent inhibition by phytate. *American Journal of Clinical Nutrition*

absorption from different types of meals. Studies with ascorbic-acid-rich foods and synthetic ascorbic acid given in different amounts with different meals. *Human*

basolateral membrane of human intestinal absorptive cells. *Journal of Cellular* 

from non-food sources on iron status in developing countries. Publications in

improves endurance after training in iron-depleted, nonanemic women. *Journal* 

disrupts the maturation of synaptic function and efficacy in area CA1 of the

Adermann, K. (2000). LEAP-1, a novel highly disulfide-bonded human peptide,

Improvements in growth following iron supplementation in young Kenyan school


Iron Deficiency Anemia: A Public Health Problem of Global Proportions 129

Rees, J.M., Monsen, E.R. & Merrill, J.E. 91985). Iron Fortification of Infant Foods: A Decade

Rockey, D.C. & Cello, J.P. (1993). Evaluation of the Gastrointestinal Tract in Patients with Iron-Deficiency Anemia. *New England Journal of Medicine* 329: 1691-1695. Roughton, F.J. (1970). Some recent work on the interactions of oxygen, carbon dioxide

Rowland, T.W., Deisroth, M.B., Green, G.M. & Kelleher, J.F. (1988). The effect of iron

Rush, D. ( 2000). Nutrition and maternal mortality in the developing world. *American*

Schultink, W., Gross, R., Gliwitzki, M., Karyadi, D. & Matulessi, P. (1995). Effect of daily

Schwartz, H.C., Goudsmit, R., Hill, R.L., Cartwright, G.E. & Wintrobe, M.M. (1961).

Shayeghi, M., Latunde-Dada, G.O., Oakhill, J.S., Laftah, A.H., Takeuchi, K., Halliday,

Soewondo, S., Husaini, M. & Pollitt, E. (1989). Effects of iron deficiency on attention

Srikantia, S.G., Bhaskaram, C., Prasad, J.S. & Krishnamachari, K.A.V.R. (1976). Anaemia

Stoltzfus, R.J., Kvalsvig, J.D., Chwaya, H.M., Montresor, A., Albonico, M., Tielsch, J.M.,

Suharno, D., Muhilal, D., Karyadi, D., West, C.E., Hautvast, J.G.A.J. & West, C.E. (1993).

Thankachan, P., Muthayya, S., Walczyk, T., Kurpad, A.V. & Hurrell, R.F. (2007). An

Tseng, M., Chakraborty, H., Robinson, D.T., Mendez, M. & Kohlmeier, L. (1997).

Theil, E.C. (2004). Iron, ferritin and nutrition. *Annual Review of Nutrition* 24: 327-343. Tolentino, K. & Friedman, J.F. (2007). An Update on Anemia in Less Developed Countries.

women in West Java, Indonesia. *The Lancet* 342: 1325-1328.

*American Journal of Tropical Medicine and Hygiene* 77: 44-51.

Children. *Journal of Nutrition* 127: 1456-1468.

double blind, placebo controlled study. *British Medical Journal* 323: 1389. Stoltzfus RM, Mullany L, Black RE. (2005). Iron deficiency anaemia. In: *Comparative* 

therapy on the exercise capacity of nonanemic iron-deficient adolescent runners.

vs twice weekly iron supplementation in Indonesian preschool children with low

The biosynthesis of hemoglobin from iron, protoporphyrin and globin. *Journal of*

N., Khan, Y., Warley, A., McCann, F.E., Hider, R.C., Frazer, D.M., Anderson, G.J., Vulpe, C.D., Simpson, R.J. & McKie, A.T. (2005). Identification of an intestinal

and learning processes in preschool children: Bandung, Indonesia. *American* 

Savioli, L. & Pollitt, E. (2001). Effects of iron supplementation and anthelmintic treatment on motor and language development of preschool children in Zanzibar:

*Quantification of Health Risks: Global and Regional Burden of Disease Attributable to Selected Major Risk Factors. Volume 1.* World Health Organization, Geneva pp. 163-

Supplementation with vitamin A and iron for nutritional anaemia in pregnant

analysis of the etiology of anemia and iron deficiency in young women of low socioeconomic status in Bangalore, India. *Food and Nutrition Bulletin* 28: 328-336.

Adjustment of Iron Intake for Dietary Enhancers and Inhibitors in Population Studies: Bioavailable Iron in Rural and Urban Residing Russian Women and

of Change. *Clinical Pediatrics* 24: 707-710.

*Journal of Clinical Nutrition* 72: 212S-240S.

*Clinical Investigation* 40: 188-195.

heme transporter. *Cel* 122: 789-801.

209.

*Journal of Clinical Nutrition* 50: 667-674.

and immune response. *The Lancet* 307: 1307-1309.

and haemoglobin. *The Biochemical Journal* 117: 801-812.

*American Journal of Diseases of Children* 142: 165-169.

iron status. *American Journal of Clinical Nutrition* 61: 111-115.

trial of iron supplementation and malaria chemoprophylaxis for prevention of severe anaemia and malaria in Tanzanian infants. *The Lancet* 350: 844-850.


severe anaemia and malaria in Tanzanian infants. *The Lancet* 350: 844-850. Moffatt, M.E., Longstaffe, S., Besant, J. & Dureski, C. (1994). Prevention of iron

Mumtaz, Z., Shahab, S., Butt, N., Rab, M.A. & DeMuynck, A. (2000). Daily iron

Neckers, L.M. & Cossman, J. (1983). Transferrin receptor induction in mitogen-stimulated

Nelson, C.A., Bloom, F.E., Cameron, J.L., Amaral, D., Dahl, R.E. & Pine, D. (2002). An

Newhouse, I.J., Clement, D.B., Taunton, J.E. & McKenzie, D.C. (1989). The effects of

Oppenheimer, S.J. (2001). Iron and Its Relation to Immunity and Infectious Disease. *Journal* 

Pak, M., Lopez, M.A., Gabayan, V., Ganz, T. & Rivera, S. (2006). Suppression of hepcidin during anemia requires erythropoietic activity. *Blood* 108: 3730-3735. Park, C.H., Valore, E.V., Waring, A.J. & Ganz, T. (2001). Hepcidin, a Urinary

Peraza, M.A., Ayala-Fierro, F., Barber, D.S., Casarez, E. & Rael, L.T. (1998). Effects of

Perutz, M.F. (1980). Review Lecture: Stereochemical Mechanism of Oxygen Transport by Haemoglobin", *Proceedings of the Royal Society of London. Series B* 208: 135-162. Perutz, M.F. (1976). Haemoglobin: Structure, Function and Synthesis", *British Medical* 

Poranen, A.K., Ekblad, U., Uotila, P. & Ahotupa, M. (1996). Lipid peroxidation and antioxidants in normal and pre-eclamptic pregnancies. *Placenta* 17: 401-405. Prema, K., Ramalakshmi, B.A., Madhavapeddi, R. & Babu, S. (1982). Immune status of anaemic pregnant women. *British Journal of Obstetrics and Gynaecology* 89: 222-225. Preziosi, P., Prual, A., Galan, P., Daouda, H., Boureima, H. & Hercberg, S. (1997). Effect

Ramakrishnan, U. & Yip, R. (2002). Experiences and challenges in industrialized

Perutz, M.F. (1969). Structure and function of hemoglobin. *Harvey Lectures* 63: 213-261. Perutz, M.F.,Rossmann, M.G., Cullis, A.F., Muirhead H., Will, G. &North, A.C.. (1960).

resolution, obtained by X-ray analysis. *Nature* 185: 416-422.

for newborns. *American Journal of Clinical Nutrition* 66: 1178-1182.

*Nutrition* 130: 2697-2702.

14: 499-520.

7806-7810.

203-216.

*Bulletin* 32: 193-194.

*Nutrition* 132:. 820S-824S.

*United States of America* 80: 3494-3498.

*Sports and Exercise* 21: 263-268.

*of Nutrition* 131: 616S-635S.

trial of iron supplementation and malaria chemoprophylaxis for prevention of

deficiency and psychomotor decline in high-risk infants through use of ironfortified infant formula: a randomized clinical trial. *Journal of Pediatrics* 125: 527-534.

supplementation is more effective than twice weekly iron supplementation in pregnant women in Pakistan in a randomized double-blind clinical trial. *Journal of* 

human T lymphocytes is required for DNA synthesis and cell division and is regulated by interleukin 2. *Proceedings of the National Academy of Sciences of the*

integrative, multidisciplinary approach to the study of brain-behavior relations in the context of typical and atypical development. *Development and Psychopathology*

prelatent/latent iron deficiency on physical work capacity. *Medicine and Science in*

Antimicrobial Peptide Synthesized in the Liver. *Journal of Biological Chemistry* 276:

micronutrients on metal toxicity. *Environmental Health Perspectives* 106: Suppl 1.

Structure of haemoglobin: a three-dimensional Fourier synthesis at 5.5-A

of iron supplementation on the iron status of pregnant women: consequences

countries: control of iron deficiency in industrialized countries. *Journal of* 


**7** 

**Snakebite Envenoming:** 

José María Gutiérrez

*Costa Rica* 

*Universidad de Costa Rica, San José,* 

**A Public Health Perspective** 

*Instituto Clodomiro Picado, Facultad de Microbiología,* 

Envenomings by snakebites constitute a highly relevant public health problem on a world wide basis, particularly in tropical regions of Africa, Asia and Latin America (Gutiérrez et al., 2006; WHO, 2007a). It affects mostly agricultural workers and their children living in rural settings. Thus, its highest impact occurs in poor and politically underpowered people, thus representing a 'disease of poverty' (Harrison et al., 2009) which fulfils the characteristics of a truly neglected tropical disease. Accordingly, the World Health Organization (WHO) incorporated, in 2009, snakebite envenoming in its list of neglected tropical diseases (www.who.int/neglected\_disease/diseases/en). Despite the high impact of this pathology in terms of morbidity and mortality in vast regions of the world, it has received little attention from international health agencies and foundations, research agendas, and pharmaceutical companies, even when compared with other neglected diseases which have received a well deserved growing attention over the last decade (Williams et al., 2010). Such low concern for an important disease is due in part to the lack of political voice of the groups affected by snakebites, to the weakening of public health systems in many developing countries, and to the poor documentation of the actual global impact of this problem, which makes the advocacy to confront this neglected disease a difficult task. The present chapter reviews the main features associated with snakebite envenoming and its treatment, and highlights some of the most pressing tasks that need to

The actual incidence and mortality associated with snakebite envenoming is poorly known, in part due to the lack of reliable information on this disease in many regions of the world. Although health statistics, based on the reports of hospital cases to health authorities, are satisfactory in some countries (for example in Brazil, de Oliveira et al., 2009), for many countries and regions this information is largely deficitary (Gutiérrez et al., 2010b; WHO, 2007a). This is in part due to the fact that health statistics are poor in many countries, and also that many people affected by snakebites do not seek medical attention and instead rely on local traditional healers, thus remaining invisible to health authorities (Habib et al., 2001; Michael et al., 2010; Otero et al., 2000; Sharma et al., 2004). Despite these limitations, a

be undertaken to confront this public health problem.

**2. Assessing the actual impact of snakebite envenoming** 

**1. Introduction**


### **Snakebite Envenoming: A Public Health Perspective**

José María Gutiérrez

*Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica* 

#### **1. Introduction**

130 Public Health – Methodology, Environmental and Systems Issues

Turner, R.E., Langkamp-Henken, B., Littell, R.C., Lukowski, M.J. & Suarez, M.F. (2003).

Viatte, L., Lesbordes-Brion, J., Lou, D., Bennoun, M., Nicolas, G., Kahn, A., Canonne-

Viteri, F.E. (1997). Iron supplementation for the control of iron deficiency in populations

Vokurka, M., Krijt, J., Sulc, K. & Necas, E. (2006). Hepcidin mRNA levels in mouse

Walker, A.R. (1998). The remedying of iron deficiency: what priority should it have?

Walter, T., Olivares, M., Pizarro, F. & Munoz, C. (1997). Iron, anemia, and infection.

Walter, T., Dallman, P.R., Pizarro, F., Vebozo, L., Pena, G., Bartholmey, S.J., Hertrampf, E.,

Williams, J., Wolff, A., Daly, A., MacDonald, A., Aukett, A. & Booth, I.W. (1999). "Iron

http://www.who.int/nutrition/publications/en/ida\_assessment\_prevention\_

Yip, R., Binkin, N.J., Fleshood, L. & Trowbridge, F.L. (1987). Declining prevalence of

Yip, R. & Ramakrishnan, U. (2002). Experiences and challenges in developing countries.

Zhu, Y. & Haas, J. (1997). Iron depletion without anemia and physical performance in

Zoller, H., Koch, R.O., Theurl, I., Obrist, P., Pietrangelo, A., Montosi, G., Haile, D.J., Vogel,

young women. *American Journal of Clinical Nutrition* 66: 334-341.

*Journal of the American Dietetic Association* 103: 461-466.

at risk. *Nutrition Reviews* 55: 195-209.

*Scientiarum Bohemoslovaca* 55: 667-674.

*British Journal of Nutrition* 79: 227-235.

*Nutrition Reviews* 55: 111-124.

control.pdf

metabolism in hepcidin-deficient mice. *Blood* 105: 4861-4864.

in Health and Disease. *Journal of Clinical Pathology* 7: 175-200.

Switzerland. Accessed 12th February 2012 from:

*Medical Association* 258: 1619-1623.

*Journal of Nutrition* 132: 827S-830S.

*Gastroenterology* 120: 1412-1419.

Comparing nutrient intake from food to the estimated average requirements shows middle-to upper-income pregnant women lack iron and possibly magnesium.

Hergaux, F. & Vaulont, S. (2005). Deregulation of proteins involved in iron

liver respond to inhibition of erythropoiesis. *Physiological Research / Academia* 

Olivares, M., Letelier, A. & Arredondo, M. (1993). Effectiveness of Iron-Fortified Infant Cereal in Prevention of Iron Deficiency Anemia. *Pediatrics* 91: 976-982. Walter, T., De Andraca, I., Chadud, P. & Perales, C.G. (1989). Iron Deficiency Anemia: Adverse Effects on Infant Psychomotor Development. *Pediatrics* 84: 7-17. White, J.C. & Beaven, G.H. (1954). A Review of the Varieties of Human Haemoglobin

supplemented formula milk related to reduction in psychomotor decline in infants from inner city areas: randomised study. *British Medical Journal* 318: 693-697. World Health Organization (2001). Iron deficiency anaemia. Assessment, prevention and

control: A guide for programme managers. World Health Organization, Geneva,

anemia among low-income children in the United States. *Journal of the American*

W. & Weiss, G. (2001). Expression of the duodenal iron transporters divalentmetal transporter 1 and ferroportin 1 in iron deficiency and iron overload", Envenomings by snakebites constitute a highly relevant public health problem on a world wide basis, particularly in tropical regions of Africa, Asia and Latin America (Gutiérrez et al., 2006; WHO, 2007a). It affects mostly agricultural workers and their children living in rural settings. Thus, its highest impact occurs in poor and politically underpowered people, thus representing a 'disease of poverty' (Harrison et al., 2009) which fulfils the characteristics of a truly neglected tropical disease. Accordingly, the World Health Organization (WHO) incorporated, in 2009, snakebite envenoming in its list of neglected tropical diseases (www.who.int/neglected\_disease/diseases/en). Despite the high impact of this pathology in terms of morbidity and mortality in vast regions of the world, it has received little attention from international health agencies and foundations, research agendas, and pharmaceutical companies, even when compared with other neglected diseases which have received a well deserved growing attention over the last decade (Williams et al., 2010). Such low concern for an important disease is due in part to the lack of political voice of the groups affected by snakebites, to the weakening of public health systems in many developing countries, and to the poor documentation of the actual global impact of this problem, which makes the advocacy to confront this neglected disease a difficult task. The present chapter reviews the main features associated with snakebite envenoming and its treatment, and highlights some of the most pressing tasks that need to be undertaken to confront this public health problem.

#### **2. Assessing the actual impact of snakebite envenoming**

The actual incidence and mortality associated with snakebite envenoming is poorly known, in part due to the lack of reliable information on this disease in many regions of the world. Although health statistics, based on the reports of hospital cases to health authorities, are satisfactory in some countries (for example in Brazil, de Oliveira et al., 2009), for many countries and regions this information is largely deficitary (Gutiérrez et al., 2010b; WHO, 2007a). This is in part due to the fact that health statistics are poor in many countries, and also that many people affected by snakebites do not seek medical attention and instead rely on local traditional healers, thus remaining invisible to health authorities (Habib et al., 2001; Michael et al., 2010; Otero et al., 2000; Sharma et al., 2004). Despite these limitations, a

Snakebite Envenoming: A Public Health Perspective 133

Case fatality rate in snakebite envenomings, if not properly treated, can be very high, especially in bites inflicted by highly venomous species (Sharma et al., 2004; Warrell, 2010). In addition, a percentage of people that survive develop sequelae as a consequence of envenoming. In the case of bites by viperid snakes, and by some elapids (genus *Naja*) that induce local tissue necrosis, sequelae include tissue loss and dysfunction, which may lead to amputation (S.B. Abubakar et al., 2010a; Gutiérrez & Lomonte, 2009; Otero et al., 2002; Warrell, 2010). Despite the scarcity of statistics on the incidence of sequelae following snakebite, observations in sub-Saharan Africa indicate that up to 20% of the patients, perhaps more, develop permanent physical sequelae (Pugh et al., 1980; Snow et al., 1994). Bites in the hands by viperid species are more prone to leave permanent tissue damage than bites in the lower limbs (Dart et al., 1992). Moreover, people suffering snakebites also present psychological sequelae, as clearly revealed by a recent study in Sri Lanka (S.S. Williams et al., 2011). The combination of physical and psychological consequences of snakebites has a dramatic impact on the quality of life of both patients and dependants. These are mostly poor agricultural workers whose survival depends very much on their physical and emotional stability to confront everyday hardships. In many cases, a large group of people depend on them as the only source of income. Therefore, when snakebite envenomings are analyzed using the parameter of DALYs ('disability adjusted life years') lost, the actual impact of this disease becomes more evident. It is necessary to investigate the effects of this pathology from such broader perspective, through interdisciplinary research projects involving international

In order to have a more rigorous and realistic assessment of the actual dimension of snakebite envenoming worldwide, the following tasks should be implemented: (a) Introducing compulsory notification of these envenomings. (b) Implementing the use in death certification of the specific classifier T 63.0 snake venom listed in the International Statistical Classification of Diseases and Related Health Problems (WHO, 2007b). (c) Performing well-designed epidemiological research based on health statistics and community-based surveys. (d) Supporting the training of health staff for proper record keeping on snakebite envenoming in many countries. These and related efforts will contribute to the generation of a solid body of information which will help to raise awareness on the seriousness of this problem and, at the same time, will provide decisionmakers with more accurate data for the design of interventions of various sorts (Gutiérrez

Snakes capable of inducing serious envenoming in humans are classified in the families Colubridae (*sensu lato*), Atractaspididae, Elapidae and Viperidae. These families include more than 2,600 species, although a relatively reduced number of them, mostly belonging to the families Elapidae and Viperidae, are responsible for the vast majority of snakebite envenomings worldwide (Warrell, 2010). In Asia, the most relevant species belong to the elapid genera *Bungarus* (kraits) and *Naja* (cobras) (Figure 1A), and to various species of the viperid genera *Echis*, *Daboia*, *Trimeresurus* and *Hypnale* (Warrell, 1995a). In Africa, species of *Naja* and few viperids are important in the northern countries, whereas the saw-scale viper

**3. Snake species responsible for the highest toll of envenomings** 

**2.1 Beyond mortality: The impact of sequelae from snakebite envenomings** 

partnerships and networks.

et al., 2010b).

number of studies have generated valuable information on the real impact of snakebite envenoming. Snakebites affect mainly agricultural workers and their relatives, living in poor rural settings of Africa, Asia and Latin America (Alirol et al., 2010; Chippaux, 2010; Fan & Cardoso, 1995; Warrell, 2010). Thus, it is clearly an occupational hazard. Incidence is usually higher in men than women, and children are also affected mostly due to their involvement in agricultural duties. Most bites occur in lower limbs, although bites in hands are also frequent (Alirol et al., 2010; Warrell, 2010). Incidence varies along the year, associated with the rainy season and with the timing of agricultural activities (Chippaux, 2010). Natural disasters have been associated with increments in the number of snakebites, as shown in Bangladesh during the 2007 monsoon flood (Alirol et al., 2010). Some social and ethnic groups are affected to a higher extent by snakebites, as compared with other groups. In Latin America, for instance, indigenous groups present a high incidence of snakebites (Larrick et al., 1978; Pierini et al., 1996). In addition, these groups are generally more vulnerable owing to their limited access to health services, evidencing a pattern of inequity that has implications in terms of mortality and morbidity secondary to snakebite envenomings (Gutiérrez, 2011). Moreover, these accidents fuel a vicious circle of poverty, since they have a negative impact on the working performance of agricultural workers, thus affecting the already precarious source of income for their families. Thus, in addition of being a disease of the poor (Harrison et al., 2009), snakebites worsens the economic situation of victims and their families.

A pioneer study on mortality due to this pathology was conducted by Swaroop & Grab (1954) on the basis of hospital statistics. Chippaux (1998) estimated an annual total of 5,400,000 bites, over 2,500,000 envenomings and 125,000 deaths due to snakebites. A more recent study by Kasturiratne et al. (2008) estimated a global total of envenomings ranging from 421,000 to 1,841,000, with fatalities ranging from 20,000 to 94,000. These studies presented estimations of envenomings and fatalities by regions as well. South and Southeast Asia present the highest incidence of snakebites, followed by sub-Saharan Africa (Kasturiratne et al., 2008). Likewise, these three regions have the highest numbers of fatalities. However, these estimates were based on the extrapolation of data from some regions and countries and, therefore, have limitations. When community-based surveys have been performed, the picture that emerges is one of a much higher dimension, both in terms of incidence and mortality (Snow et al., 1994; Sharma et al., 2004; Trape et al., 2001). The incidence of snakebites in specific areas can be very high. Examples are the Benue valley of Nigeria (497 per 100,000 population per year, Pugh & Theakston, 1980) and in southeastern Nepal (1,162 per 100,000 population per year, Sharma et al., 2004). A metaanalysis of snakebites in Africa suggested that the actual incidence might be 3-5 times higher than that derived from hospital statistics (Chippaux, 2011). Two recent studies further illustrate this concept. A community-based survey performed in rural Bangladesh revealed an incidence of 623.4 cases per 100,000 population per year (Rahman et al., 2010), which is much higher than the incidence derived from hospital-based statistics. Moreover, a recent study on mortality in India, which was part of a large national representative mortality survey, indicates that there are 45,900 deaths due to snakebite envenoming per year in this country (Mohapatra et al., 2011). The issue of underreporting needs to be addressed by different approaches, such as by identifying regions where underreport is more likely to occur (Hansson et al., 2010), and by performing community-based surveys in countries of high incidence of snakebites.

number of studies have generated valuable information on the real impact of snakebite envenoming. Snakebites affect mainly agricultural workers and their relatives, living in poor rural settings of Africa, Asia and Latin America (Alirol et al., 2010; Chippaux, 2010; Fan & Cardoso, 1995; Warrell, 2010). Thus, it is clearly an occupational hazard. Incidence is usually higher in men than women, and children are also affected mostly due to their involvement in agricultural duties. Most bites occur in lower limbs, although bites in hands are also frequent (Alirol et al., 2010; Warrell, 2010). Incidence varies along the year, associated with the rainy season and with the timing of agricultural activities (Chippaux, 2010). Natural disasters have been associated with increments in the number of snakebites, as shown in Bangladesh during the 2007 monsoon flood (Alirol et al., 2010). Some social and ethnic groups are affected to a higher extent by snakebites, as compared with other groups. In Latin America, for instance, indigenous groups present a high incidence of snakebites (Larrick et al., 1978; Pierini et al., 1996). In addition, these groups are generally more vulnerable owing to their limited access to health services, evidencing a pattern of inequity that has implications in terms of mortality and morbidity secondary to snakebite envenomings (Gutiérrez, 2011). Moreover, these accidents fuel a vicious circle of poverty, since they have a negative impact on the working performance of agricultural workers, thus affecting the already precarious source of income for their families. Thus, in addition of being a disease of the poor (Harrison et al., 2009), snakebites worsens the economic situation

A pioneer study on mortality due to this pathology was conducted by Swaroop & Grab (1954) on the basis of hospital statistics. Chippaux (1998) estimated an annual total of 5,400,000 bites, over 2,500,000 envenomings and 125,000 deaths due to snakebites. A more recent study by Kasturiratne et al. (2008) estimated a global total of envenomings ranging from 421,000 to 1,841,000, with fatalities ranging from 20,000 to 94,000. These studies presented estimations of envenomings and fatalities by regions as well. South and Southeast Asia present the highest incidence of snakebites, followed by sub-Saharan Africa (Kasturiratne et al., 2008). Likewise, these three regions have the highest numbers of fatalities. However, these estimates were based on the extrapolation of data from some regions and countries and, therefore, have limitations. When community-based surveys have been performed, the picture that emerges is one of a much higher dimension, both in terms of incidence and mortality (Snow et al., 1994; Sharma et al., 2004; Trape et al., 2001). The incidence of snakebites in specific areas can be very high. Examples are the Benue valley of Nigeria (497 per 100,000 population per year, Pugh & Theakston, 1980) and in southeastern Nepal (1,162 per 100,000 population per year, Sharma et al., 2004). A metaanalysis of snakebites in Africa suggested that the actual incidence might be 3-5 times higher than that derived from hospital statistics (Chippaux, 2011). Two recent studies further illustrate this concept. A community-based survey performed in rural Bangladesh revealed an incidence of 623.4 cases per 100,000 population per year (Rahman et al., 2010), which is much higher than the incidence derived from hospital-based statistics. Moreover, a recent study on mortality in India, which was part of a large national representative mortality survey, indicates that there are 45,900 deaths due to snakebite envenoming per year in this country (Mohapatra et al., 2011). The issue of underreporting needs to be addressed by different approaches, such as by identifying regions where underreport is more likely to occur (Hansson et al., 2010), and by performing community-based surveys in countries of

of victims and their families.

high incidence of snakebites.

#### **2.1 Beyond mortality: The impact of sequelae from snakebite envenomings**

Case fatality rate in snakebite envenomings, if not properly treated, can be very high, especially in bites inflicted by highly venomous species (Sharma et al., 2004; Warrell, 2010). In addition, a percentage of people that survive develop sequelae as a consequence of envenoming. In the case of bites by viperid snakes, and by some elapids (genus *Naja*) that induce local tissue necrosis, sequelae include tissue loss and dysfunction, which may lead to amputation (S.B. Abubakar et al., 2010a; Gutiérrez & Lomonte, 2009; Otero et al., 2002; Warrell, 2010). Despite the scarcity of statistics on the incidence of sequelae following snakebite, observations in sub-Saharan Africa indicate that up to 20% of the patients, perhaps more, develop permanent physical sequelae (Pugh et al., 1980; Snow et al., 1994). Bites in the hands by viperid species are more prone to leave permanent tissue damage than bites in the lower limbs (Dart et al., 1992). Moreover, people suffering snakebites also present psychological sequelae, as clearly revealed by a recent study in Sri Lanka (S.S. Williams et al., 2011). The combination of physical and psychological consequences of snakebites has a dramatic impact on the quality of life of both patients and dependants. These are mostly poor agricultural workers whose survival depends very much on their physical and emotional stability to confront everyday hardships. In many cases, a large group of people depend on them as the only source of income. Therefore, when snakebite envenomings are analyzed using the parameter of DALYs ('disability adjusted life years') lost, the actual impact of this disease becomes more evident. It is necessary to investigate the effects of this pathology from such broader perspective, through interdisciplinary research projects involving international partnerships and networks.

In order to have a more rigorous and realistic assessment of the actual dimension of snakebite envenoming worldwide, the following tasks should be implemented: (a) Introducing compulsory notification of these envenomings. (b) Implementing the use in death certification of the specific classifier T 63.0 snake venom listed in the International Statistical Classification of Diseases and Related Health Problems (WHO, 2007b). (c) Performing well-designed epidemiological research based on health statistics and community-based surveys. (d) Supporting the training of health staff for proper record keeping on snakebite envenoming in many countries. These and related efforts will contribute to the generation of a solid body of information which will help to raise awareness on the seriousness of this problem and, at the same time, will provide decisionmakers with more accurate data for the design of interventions of various sorts (Gutiérrez et al., 2010b).

#### **3. Snake species responsible for the highest toll of envenomings**

Snakes capable of inducing serious envenoming in humans are classified in the families Colubridae (*sensu lato*), Atractaspididae, Elapidae and Viperidae. These families include more than 2,600 species, although a relatively reduced number of them, mostly belonging to the families Elapidae and Viperidae, are responsible for the vast majority of snakebite envenomings worldwide (Warrell, 2010). In Asia, the most relevant species belong to the elapid genera *Bungarus* (kraits) and *Naja* (cobras) (Figure 1A), and to various species of the viperid genera *Echis*, *Daboia*, *Trimeresurus* and *Hypnale* (Warrell, 1995a). In Africa, species of *Naja* and few viperids are important in the northern countries, whereas the saw-scale viper

Snakebite Envenoming: A Public Health Perspective 135

Fig. 1C. *Bothrops asper* from Costa Rica. Photo: Mahmood Sasa. From Gutiérrez et al. (2006)

In addition, some species, albeit not causing high numbers of bites, are capable of inflicting severe envenomings, such as *Lachesis* sp (bushmaster) and *Micrurus* sp (coral snakes) in the Americas (Warrell, 2004), *Atractaspis* sp (borrowing snakes) and *Dendroaspis* sp (mambas) in Africa/Middle East (WHO, 2010b), and a variety of elapid species in Australia and Papua New Guinea (White, 2010). Envenomings by colubrid species are usually not severe although fatal cases by species of the African genera *Dispholidus* and *Thelotornis* have been described (Warrell, 1995b). The taxonomy of venomous snakes is a highly dynamic field and recent modifications have been introduced in medically-relevant snake taxa (Quijada-Mascareñas & Wüster, 2010). Toxinologists, clinicians and antivenom manufacturers should be aware of these changes in taxonomy. Detailed information on the country distribution of the most important poisonous snakes is available at the WHO website

These groups of 'advanced' snakes have acquired, through a long and complex evolutionary history (Fry et al., 2006, 2009), the ability to synthesize a toxic secretion, i.e. venom, by an exocrine gland located in the maxillary region, together with a venom delivery system based on the presence of ducts and fangs (Meier & Stocker, 1995; Vonk et al., 2008). The molecular evolution of venom toxins has involved an accelerated Darwinian process, by which genes have been duplicated and recruited in venom glands, with a concomitant process of acquisition of toxic functions based on a trend to generate mutations in sequences coding predominantly for amino acid residues located in the surface of these proteins, as well as other molecular mechanisms such as domain loss and neofunctionalization, thus generating a wide versatility in their ability to interact with diverse tissue targets (Casewell et al., 2011; Fry et al., 2006; Kini & Chan, 1999; Ohno et al., 2003). In the last decade, the use of proteomic

http://apps.who.int/bloodproducts/snakeantivenoms/database/

**4. Snake venom biochemistry and toxicology** 

*PLoS Medicine* 3: e150.

(*Echis ocellatus*) (Figure 1B) inflicts a heavy toll in the sub-Saharan region, together with other viperids classified in the genera *Echis* and *Bitis*, and some cobras (*Naja* sp) (WHO, 2010b). In the Americas, species of rattlesnakes (*Crotalus*) are important in North America, whereas lance-head vipers of the genus *Bothrops*, such as *B. asper* (Figure 1C) and *B. atrox*, are responsible for most snakebites in Central and South America, in addition to a number of *Bothrops* species in South America (Fan & Cardoso, 1995; Gómez & Dart, 1995; Gutiérrez, 2010).

Fig. 1A. *Naja naja* from Sri Lanka. Photo: Mark O'Shea. Reprinted from *Journal of Proteomics* 74, 1735-1767, Williams et al., copyright 2011, with permission from Elsevier.

Fig. 1B. *Echis ocellatus* from Togo. Photo: David Williams. Reprinted from *Journal of Proteomics* 74, 1735-1767, Williams et al., copyright 2011, with permission from Elsevier.

(*Echis ocellatus*) (Figure 1B) inflicts a heavy toll in the sub-Saharan region, together with other viperids classified in the genera *Echis* and *Bitis*, and some cobras (*Naja* sp) (WHO, 2010b). In the Americas, species of rattlesnakes (*Crotalus*) are important in North America, whereas lance-head vipers of the genus *Bothrops*, such as *B. asper* (Figure 1C) and *B. atrox*, are responsible for most snakebites in Central and South America, in addition to a number of *Bothrops* species in South America (Fan & Cardoso, 1995; Gómez & Dart, 1995; Gutiérrez, 2010).

Fig. 1A. *Naja naja* from Sri Lanka. Photo: Mark O'Shea. Reprinted from *Journal of Proteomics*

74, 1735-1767, Williams et al., copyright 2011, with permission from Elsevier.

Fig. 1B. *Echis ocellatus* from Togo. Photo: David Williams. Reprinted from *Journal of Proteomics* 74, 1735-1767, Williams et al., copyright 2011, with permission from Elsevier.

Fig. 1C. *Bothrops asper* from Costa Rica. Photo: Mahmood Sasa. From Gutiérrez et al. (2006) *PLoS Medicine* 3: e150.

In addition, some species, albeit not causing high numbers of bites, are capable of inflicting severe envenomings, such as *Lachesis* sp (bushmaster) and *Micrurus* sp (coral snakes) in the Americas (Warrell, 2004), *Atractaspis* sp (borrowing snakes) and *Dendroaspis* sp (mambas) in Africa/Middle East (WHO, 2010b), and a variety of elapid species in Australia and Papua New Guinea (White, 2010). Envenomings by colubrid species are usually not severe although fatal cases by species of the African genera *Dispholidus* and *Thelotornis* have been described (Warrell, 1995b). The taxonomy of venomous snakes is a highly dynamic field and recent modifications have been introduced in medically-relevant snake taxa (Quijada-Mascareñas & Wüster, 2010). Toxinologists, clinicians and antivenom manufacturers should be aware of these changes in taxonomy. Detailed information on the country distribution of the most important poisonous snakes is available at the WHO website http://apps.who.int/bloodproducts/snakeantivenoms/database/

#### **4. Snake venom biochemistry and toxicology**

These groups of 'advanced' snakes have acquired, through a long and complex evolutionary history (Fry et al., 2006, 2009), the ability to synthesize a toxic secretion, i.e. venom, by an exocrine gland located in the maxillary region, together with a venom delivery system based on the presence of ducts and fangs (Meier & Stocker, 1995; Vonk et al., 2008). The molecular evolution of venom toxins has involved an accelerated Darwinian process, by which genes have been duplicated and recruited in venom glands, with a concomitant process of acquisition of toxic functions based on a trend to generate mutations in sequences coding predominantly for amino acid residues located in the surface of these proteins, as well as other molecular mechanisms such as domain loss and neofunctionalization, thus generating a wide versatility in their ability to interact with diverse tissue targets (Casewell et al., 2011; Fry et al., 2006; Kini & Chan, 1999; Ohno et al., 2003). In the last decade, the use of proteomic

Snakebite Envenoming: A Public Health Perspective 137

2010). Finally, the venoms of snakes of the polyphyletic family Colubridae have been studied to a lesser extent, but they also contain metalloproteinases, serine proteinases, PLA2s, CRISPs and neurotoxins (Mackessy, 2002). Snake venoms present a high variability, not only between species, but also between different populations of the same species (Alape-Girón et al., 2008; Chippaux et al., 1991; Jayanthi and Gowda, 1988). Moreover, some species present a conspicuous ontogenetic variability in the composition of their venoms, such as the Central American rattlesnake *Crotalus simus* (Calvete et al., 2010a) and the lance-head viper *Bothrops asper* (Alape-Girón et al., 2008). This high variability in venom composition has evident implications for the clinical manifestations of envenoming (Warrell, 1997) and

The large variation occurring in venom composition urges caution when classifying the clinical manifestations of snakebite envenoming, since important differences have been described in the clinical features in envenomings by closely-related species or even within a single species. However, there are general trends in the clinical picture of envenoming by the various groups of poisonous snakes. Envenomings by elapid species (sea snakes, tiger snakes and taipans in Australia, cobras and kraits in Asia, cobras and mambas in Africa, and coral snakes in the Americas) are usually characterized by progressive descending neurotoxic paralysis secondary to the action of pre- or post-synaptic neurotoxins at the neuromuscular junctions (Warrell, 1996, 2010; White, 2010). The most serious consequence of this effect is respiratory paralysis, which may lead to death if not properly and timely attended. In addition, envenomings by a number of elapid species are also characterized by rhabdomyolysis, which may lead to acute renal failure (Warrell, 1996). Patients envenomed by elapids in Australia and Papua New Guinea develop coagulation disturbances which may provoke bleeding (White, 2010). On the other hand, human envenomings by some cobras in Asia and Africa are not characterized by neurotoxic manifestations, but instead by

Viperid snake venoms provoke complex and often drastic local pathological effects, i.e. hemorrhage, dermonecrosis, blistering, myonecrosis and edema, always associated with pain (Gutiérrez & Lomonte, 2009; Warrell, 2004). These local manifestations may lead to permanent sequelae, such as tissue loss and dysfunction (Dart et al., 1992; Otero et al., 2002). After systemic venom distribution, and depending on the severity of the case, viperid snakebite envenomings are characterized by coagulopathies, bleeding, renal alterations and hemodynamic manifestations which may lead to cardiovascular shock and multisystem organ failure (Gutiérrez et al., 2009b; Warrell, 2004). Intravascular hemolysis might also occur, in some cases associated with microthrombi formation (Warrell, 1996). Exceptions to this general trend are envenomings by the South American and some populations of North American rattlesnakes, as well as some viperids in the Old World, which induce neurotoxicity (Azevedo-Marques et al., 2009; Ferquel et al., 2007). Despite the existence of these general trends, clinical studies highlight the complexity of snakebite envenoming, as demonstrated by the description of 'unusual' manifestations in cases by some elapids in Asia and South America (Faiz et al., 2010; Manock et al., 2008; Trinh et al., 2010). In addition, some venoms induce unique clinical features, such as the thrombotic effect described for the Caribbean viperid species *Bothrops lanceolatus* and *B. caribbaeus* (Thomas et al., 1996), and the acute hemorrhagic infarction of the

for the preparation of antivenoms (Gutiérrez et al., 2009a).

**5. Clinical manifestations of envenoming** 

local tissue necrosis (Warrell, 1995a, 1995b).

pituitary in envenoming by *Daboia russelli* (Tun-Pe et al., 1987).

tools based on mass spectrometric analysis and sequence determination has allowed a detailed knowledge on the composition of venoms from many species (Calvete et al., 2007; Calvete, 2010; Fox & Serrano, 2008). Understanding the snake venom proteomes ('venomes') provides valuable information for the search of novel toxins and for the design of the most appropriate mixtures of venoms for animal immunization for antivenom production, among other applications (Calvete, 2010; Gutiérrez et al., 2009a).

Venoms from snakes of the family Elapidae comprise a high percentage of proteins of the so-called 'three finger toxin' family, which are low molecular mass (6-9 kDa) polypeptides that exert a number of actions, such as the ability to block neuromuscular junctions at the post-synaptic level by binding with very high affinity to the nicotinic cholinergic receptor of the motor end-plate in skeletal muscle fibers (Hegde et al., 2010). Some three-finger toxins are membrane-disorganizing proteins, named 'cardiotoxins' or 'cytotoxins', which disrupt the integrity of cell membranes and are likely to play a role in the tissue damage associated with envenoming by some cobras (Dufton & Hider, 1988). The venoms of *Dendroaspis* sp (mambas) contain other types of neurotoxins, i.e. dendrotoxins and fasciculins, which interfere with neuromuscular junctions by various mechanisms (Harvey, 2001, 2010). Elapid venoms are also characterized by the high abundance of phospholipases A2 (PLA2s), some of which are potent neurotoxins whose mechanism of action relies in the specific binding to receptors in the presynaptic nerve terminal, followed by degradation of phospholipids at the plasma membrane of these terminals, thus affecting the normal process of neurotransmitter release (Rossetto et al., 2006). Other PLA2s induce acute muscle damage which, in the case of some sea snakes and other elapids, results in systemic myotoxicity, i.e. rhabdomyolisis, associated with myoglobinuria, hyperkalemia and acute renal failure (Gutiérrez & Ownby, 2003). Besides the predominant three-finger toxins and PLA2s, elapid venoms also contain other proteins in low concentrations, such as cysteine-rich secretory proteins (CRISPs), cobra venom factor and other hydrolases (serine proteinases, metalloproteinases, nucleotidases) (Correa-Neto et al., 2011; Kulkeaw et al., 2007; Petras et al., 2011). The clotting disturbances induced by some Australian elapid venoms are caused by procoagulant serine proteinases which are prothrombin activators (St Pierre et al., 2005)

Venoms of snakes of the family Viperidae present large variations in their composition, but nevertheless the components showing the highest concentrations correspond to zincdependent metalloproteinases, PLA2s and serine proteinases (Calvete, 2010; Fox & Serrano, 2005). In addition, these venoms contain bradykinin-potentiating peptides (BPPs), disintegrins, C-type lectin-like proteins, L-amino acid oxidase and various other enzymes (Calvete et al., 2009). Metalloproteinases are largely responsible for degradation of the basement membrane of microvessels, with the consequent hemorrhage (Escalante et al., 2011), activation of prothrombin and factor X (Kini, 2005; Tans & Rosing, 2001), thus generating the formation of microthrombi and fibrinogen depletion, i.e. defibrinogenation (Gutiérrez et al., 2010a), and degradation of the extracellular matrix (Moura-da-Silva et al., 2009), among other effects. In turn, some viperid PLA2s induce acute muscle damage at the site of venom injection (Gutiérrez & Ownby, 2003; Lomonte et al., 2003). Some viperid PLA2s also exert presynaptic neurotoxicity, such as the complex 'crotoxin', abundant in the venom of South American rattlesnakes (Bon, 1997). Serine proteinases are responsible for clotting disturbances, i.e. defibrinogenation, and hypotension (Serrano & Maroun, 2005). Venoms from species of the family Atractaspididae (burrowing asps) contain sarafotoxins, which are low molecular mass components that induce vasospasm leading to cardiac toxicity (Bdolah,

tools based on mass spectrometric analysis and sequence determination has allowed a detailed knowledge on the composition of venoms from many species (Calvete et al., 2007; Calvete, 2010; Fox & Serrano, 2008). Understanding the snake venom proteomes ('venomes') provides valuable information for the search of novel toxins and for the design of the most appropriate mixtures of venoms for animal immunization for antivenom production, among

Venoms from snakes of the family Elapidae comprise a high percentage of proteins of the so-called 'three finger toxin' family, which are low molecular mass (6-9 kDa) polypeptides that exert a number of actions, such as the ability to block neuromuscular junctions at the post-synaptic level by binding with very high affinity to the nicotinic cholinergic receptor of the motor end-plate in skeletal muscle fibers (Hegde et al., 2010). Some three-finger toxins are membrane-disorganizing proteins, named 'cardiotoxins' or 'cytotoxins', which disrupt the integrity of cell membranes and are likely to play a role in the tissue damage associated with envenoming by some cobras (Dufton & Hider, 1988). The venoms of *Dendroaspis* sp (mambas) contain other types of neurotoxins, i.e. dendrotoxins and fasciculins, which interfere with neuromuscular junctions by various mechanisms (Harvey, 2001, 2010). Elapid venoms are also characterized by the high abundance of phospholipases A2 (PLA2s), some of which are potent neurotoxins whose mechanism of action relies in the specific binding to receptors in the presynaptic nerve terminal, followed by degradation of phospholipids at the plasma membrane of these terminals, thus affecting the normal process of neurotransmitter release (Rossetto et al., 2006). Other PLA2s induce acute muscle damage which, in the case of some sea snakes and other elapids, results in systemic myotoxicity, i.e. rhabdomyolisis, associated with myoglobinuria, hyperkalemia and acute renal failure (Gutiérrez & Ownby, 2003). Besides the predominant three-finger toxins and PLA2s, elapid venoms also contain other proteins in low concentrations, such as cysteine-rich secretory proteins (CRISPs), cobra venom factor and other hydrolases (serine proteinases, metalloproteinases, nucleotidases) (Correa-Neto et al., 2011; Kulkeaw et al., 2007; Petras et al., 2011). The clotting disturbances induced by some Australian elapid venoms are caused by procoagulant serine proteinases which are prothrombin activators (St Pierre et al., 2005) Venoms of snakes of the family Viperidae present large variations in their composition, but nevertheless the components showing the highest concentrations correspond to zincdependent metalloproteinases, PLA2s and serine proteinases (Calvete, 2010; Fox & Serrano, 2005). In addition, these venoms contain bradykinin-potentiating peptides (BPPs), disintegrins, C-type lectin-like proteins, L-amino acid oxidase and various other enzymes (Calvete et al., 2009). Metalloproteinases are largely responsible for degradation of the basement membrane of microvessels, with the consequent hemorrhage (Escalante et al., 2011), activation of prothrombin and factor X (Kini, 2005; Tans & Rosing, 2001), thus generating the formation of microthrombi and fibrinogen depletion, i.e. defibrinogenation (Gutiérrez et al., 2010a), and degradation of the extracellular matrix (Moura-da-Silva et al., 2009), among other effects. In turn, some viperid PLA2s induce acute muscle damage at the site of venom injection (Gutiérrez & Ownby, 2003; Lomonte et al., 2003). Some viperid PLA2s also exert presynaptic neurotoxicity, such as the complex 'crotoxin', abundant in the venom of South American rattlesnakes (Bon, 1997). Serine proteinases are responsible for clotting disturbances, i.e. defibrinogenation, and hypotension (Serrano & Maroun, 2005). Venoms from species of the family Atractaspididae (burrowing asps) contain sarafotoxins, which are low molecular mass components that induce vasospasm leading to cardiac toxicity (Bdolah,

other applications (Calvete, 2010; Gutiérrez et al., 2009a).

2010). Finally, the venoms of snakes of the polyphyletic family Colubridae have been studied to a lesser extent, but they also contain metalloproteinases, serine proteinases, PLA2s, CRISPs and neurotoxins (Mackessy, 2002). Snake venoms present a high variability, not only between species, but also between different populations of the same species (Alape-Girón et al., 2008; Chippaux et al., 1991; Jayanthi and Gowda, 1988). Moreover, some species present a conspicuous ontogenetic variability in the composition of their venoms, such as the Central American rattlesnake *Crotalus simus* (Calvete et al., 2010a) and the lance-head viper *Bothrops asper* (Alape-Girón et al., 2008). This high variability in venom composition has evident implications for the clinical manifestations of envenoming (Warrell, 1997) and for the preparation of antivenoms (Gutiérrez et al., 2009a).

#### **5. Clinical manifestations of envenoming**

The large variation occurring in venom composition urges caution when classifying the clinical manifestations of snakebite envenoming, since important differences have been described in the clinical features in envenomings by closely-related species or even within a single species. However, there are general trends in the clinical picture of envenoming by the various groups of poisonous snakes. Envenomings by elapid species (sea snakes, tiger snakes and taipans in Australia, cobras and kraits in Asia, cobras and mambas in Africa, and coral snakes in the Americas) are usually characterized by progressive descending neurotoxic paralysis secondary to the action of pre- or post-synaptic neurotoxins at the neuromuscular junctions (Warrell, 1996, 2010; White, 2010). The most serious consequence of this effect is respiratory paralysis, which may lead to death if not properly and timely attended. In addition, envenomings by a number of elapid species are also characterized by rhabdomyolysis, which may lead to acute renal failure (Warrell, 1996). Patients envenomed by elapids in Australia and Papua New Guinea develop coagulation disturbances which may provoke bleeding (White, 2010). On the other hand, human envenomings by some cobras in Asia and Africa are not characterized by neurotoxic manifestations, but instead by local tissue necrosis (Warrell, 1995a, 1995b).

Viperid snake venoms provoke complex and often drastic local pathological effects, i.e. hemorrhage, dermonecrosis, blistering, myonecrosis and edema, always associated with pain (Gutiérrez & Lomonte, 2009; Warrell, 2004). These local manifestations may lead to permanent sequelae, such as tissue loss and dysfunction (Dart et al., 1992; Otero et al., 2002). After systemic venom distribution, and depending on the severity of the case, viperid snakebite envenomings are characterized by coagulopathies, bleeding, renal alterations and hemodynamic manifestations which may lead to cardiovascular shock and multisystem organ failure (Gutiérrez et al., 2009b; Warrell, 2004). Intravascular hemolysis might also occur, in some cases associated with microthrombi formation (Warrell, 1996). Exceptions to this general trend are envenomings by the South American and some populations of North American rattlesnakes, as well as some viperids in the Old World, which induce neurotoxicity (Azevedo-Marques et al., 2009; Ferquel et al., 2007). Despite the existence of these general trends, clinical studies highlight the complexity of snakebite envenoming, as demonstrated by the description of 'unusual' manifestations in cases by some elapids in Asia and South America (Faiz et al., 2010; Manock et al., 2008; Trinh et al., 2010). In addition, some venoms induce unique clinical features, such as the thrombotic effect described for the Caribbean viperid species *Bothrops lanceolatus* and *B. caribbaeus* (Thomas et al., 1996), and the acute hemorrhagic infarction of the pituitary in envenoming by *Daboia russelli* (Tun-Pe et al., 1987).

Snakebite Envenoming: A Public Health Perspective 139

transport of patients to health centers and, therefore, jeopardize the adequate management of these cases. First aid interventions should be focused on the immobilization of the bitten extremity and the rapid transportation to clinics or other health facilities. Communities should have strategies for rapid deployment of snakebitten people to medical treatment; an example is the use of motorcycle transportation in Nepal (Alirol et al., 2010). The interaction and communication of health staff with local healers is very important, in order to promote partnerships aimed at reducing harmful interventions and guaranteeing rapid mobilization for antivenom administration. The application of pressure-immobilization, by applying a bandage and a splint to the entire bitten limb, has been used in Australia for delaying the systemic absorption of neurotoxic venoms (Sutherland et al., 1979; White, 2010). Recently, a pharmacological intervention, based on the application of an ointment containing a nitric oxide donor, aimed at reducing the lymphatic absorption of venom, has been proposed

The parenteral administration of animal-derived antivenoms constitutes the mainstay in the therapy of snakebite envenoming (WHO, 2007a, 2010a), since the development of the first antivenoms, the *serum anti-venimeux*, during the last decade of the XIXth century (Bon, 1996). Antivenoms are preparations of immunoglobulins, or immunoglobulin fragments F(ab')2 or Fab, obtained by fractionating the plasma of animals immunized with snake venoms (Gutiérrez et al., 2011a; Lalloo & Theakston, 2003; WHO, 2010a). Antivenoms can be monospecific, when animals receive the venom of a single species, or polyspecific, when venoms from two or more species are injected. The majority of manufacturers use horses for immunization, although few use sheep and donkeys (Gutiérrez et al., 2011a, 2011b). In most cases, plasma fractionation involves the digestion of proteins with pepsin or, by few producers, with papain, followed by the purification of antibody fragments by salting-out with ammonium salts or caprylic acid fractionation and, in some cases, with chromatographic procedures (dos Santos et al., 1989; Grandgeorge et al., 1996; Raw et al., 1991; WHO, 2010a). Some producers fractionate plasma with caprylic acid to obtain whole IgG preparations (Gutiérrez et al., 2005; Rojas et al., 1994). A detailed description of the methods used in animal immunization and plasma fractionation for antivenom production can be found in the *WHO Guidelines for the Production, Control and Regulation of Snake Antivenom Immunoglobulins* (WHO, 2010a). There are antivenom-manufacturing laboratories in every continent (a complete list can be found in http://apps.who.int/bloodproducts/snakeantivenoms/database/). Following manufacture, antivenoms are subjected to a quality control protocol which involves physical, chemical and biological tests aimed at ensuring the efficacy and safety of these products

The ability of antivenoms to neutralize venom toxins is based on the capacity of antivenom antibodies, or antibody fragments, to bind and neutralize the most relevant toxins in a venom. It has been proposed that such neutralization is based on, at least, four mechanisms: (a) Binding of antibody paratopes to epitopes located at the pharmacologically-relevant molecular region, i.e. the catalytic active site in toxic enzymes such as phospholipases A2 and metalloproteinases. (b) Binding of antibodies to epitopes located close to the toxin active site, thus exerting inhibition by steric hindrance. (c) Binding of antibodies to molecular regions distant from the active/toxic site of venom components, neutralization being achieved by allosteric changes induced in the toxins, with the consequent reduction in their

(Saul et al., 2011), and its testing in the clinical setting is pending.

**6.3 Antivenoms: The key therapy of snakebite envenoming** 

(Gutiérrez & León, 2009; WHO, 2010a).

The severity of snakebite envenoming depends on a number of factors, such as the volume of venom injected, the size and physiological condition of the victim, and the region of the body where venom is delivered. A percentage of snakebites are not associated with venom injection ('dry bites') and, therefore, no clinical manifestations develop (Warrell, 2004). In general, bites in the head tend to be more severe than bites in the extremities, and envenoming in children are more prone to become severe. In the case of envenoming by pit vipers, bites in the hands are more likely to generate sequelae than bites in the lower limbs (Dart et al., 1992). Thus, a proper assessment of the clinical manifestations and severity of snakebites is a key element for the correct diagnosis and clinical management of these accidents.

#### **6. Diagnosis and treatment of snakebite envenomings**

#### **6.1 Diagnosis**

Identification of the offending snake is often difficult because in many settings there are various similar species and the bitten person is usually unable to differentiate between them. Even when the snake is killed and brought to the health facility, identification is not always correct. In Australia, kits have been developed for the immunodetection of venom in the bite site or in urine, thus allowing the identification of the offending snake (White, 2010). However, this is not the case in the vast majority of regions in the rest of the world. A 'syndromic approach' has been promoted for the diagnosis of the type of envenoming in various parts of the world (Ariaratnam et al., 2009; WHO, 2010b). For instance, in Central America, there are two predominant syndromes in snakebite envenomings: one presenting local pathological effects (swelling, pain, local tissue damage), clotting disturbances and bleeding, and another characterized by descending neuromuscular paralysis. The first syndrome is associated with envenomings inflicted by viperid species, whereas the second is due to envenomings by elapid species (*Micrurus* sp). This clinically-based diagnosis allows for the selection of the correct antivenom, i.e. polyvalent antivenom or anti-coral antivenom, respectively (Gutiérrez, 2010). Such syndromic approach has been advocated in other regions of the world as well, such as in sub-Saharan Africa (WHO, 2010b) and Sri Lanka (Ariaratnam et al., 2009). In large regions of the savannahs in sub-Saharan Africa, cases presenting clotting disturbances are associated with envenomings inflicted by the sawscale viper, *Echis ocellatus* (Warrell, 1995b). In this context, a simple laboratory test known as the '20 minute whole blood clotting test' represents a useful diagnostic tool (Warrell et al., 1974). In contrast, envenomings associated with a predominantly neurotoxic picture are caused by species of neurotoxic cobras (*Naja* sp) or mambas (*Dendroaspis* sp), and envenomings characterized by local tissue damage without coagulant disturbances are induced by species of *Bitis* or by cytotoxic cobras (WHO, 2010b).

#### **6.2 First aid in snakebite envenoming**

Snakebite cases in many regions of the world are initially attended by local healers who use a wide variety of interventions, most of which are ineffective and often exert harmful effects. Examples are the use of ligatures, incisions and suction, cryotherapy, electroshock, and the administration of synthetic or natural substances (Hardy, 2009; Warrell, 2010). Other interventions, such as application of 'black stone' or suction devices are largely ineffective for the removal of venom. In addition to their harmful effects, these actions delay the

The severity of snakebite envenoming depends on a number of factors, such as the volume of venom injected, the size and physiological condition of the victim, and the region of the body where venom is delivered. A percentage of snakebites are not associated with venom injection ('dry bites') and, therefore, no clinical manifestations develop (Warrell, 2004). In general, bites in the head tend to be more severe than bites in the extremities, and envenoming in children are more prone to become severe. In the case of envenoming by pit vipers, bites in the hands are more likely to generate sequelae than bites in the lower limbs (Dart et al., 1992). Thus, a proper assessment of the clinical manifestations and severity of snakebites is a key element for the correct diagnosis and clinical management of these

Identification of the offending snake is often difficult because in many settings there are various similar species and the bitten person is usually unable to differentiate between them. Even when the snake is killed and brought to the health facility, identification is not always correct. In Australia, kits have been developed for the immunodetection of venom in the bite site or in urine, thus allowing the identification of the offending snake (White, 2010). However, this is not the case in the vast majority of regions in the rest of the world. A 'syndromic approach' has been promoted for the diagnosis of the type of envenoming in various parts of the world (Ariaratnam et al., 2009; WHO, 2010b). For instance, in Central America, there are two predominant syndromes in snakebite envenomings: one presenting local pathological effects (swelling, pain, local tissue damage), clotting disturbances and bleeding, and another characterized by descending neuromuscular paralysis. The first syndrome is associated with envenomings inflicted by viperid species, whereas the second is due to envenomings by elapid species (*Micrurus* sp). This clinically-based diagnosis allows for the selection of the correct antivenom, i.e. polyvalent antivenom or anti-coral antivenom, respectively (Gutiérrez, 2010). Such syndromic approach has been advocated in other regions of the world as well, such as in sub-Saharan Africa (WHO, 2010b) and Sri Lanka (Ariaratnam et al., 2009). In large regions of the savannahs in sub-Saharan Africa, cases presenting clotting disturbances are associated with envenomings inflicted by the sawscale viper, *Echis ocellatus* (Warrell, 1995b). In this context, a simple laboratory test known as the '20 minute whole blood clotting test' represents a useful diagnostic tool (Warrell et al., 1974). In contrast, envenomings associated with a predominantly neurotoxic picture are caused by species of neurotoxic cobras (*Naja* sp) or mambas (*Dendroaspis* sp), and envenomings characterized by local tissue damage without coagulant disturbances are

Snakebite cases in many regions of the world are initially attended by local healers who use a wide variety of interventions, most of which are ineffective and often exert harmful effects. Examples are the use of ligatures, incisions and suction, cryotherapy, electroshock, and the administration of synthetic or natural substances (Hardy, 2009; Warrell, 2010). Other interventions, such as application of 'black stone' or suction devices are largely ineffective for the removal of venom. In addition to their harmful effects, these actions delay the

**6. Diagnosis and treatment of snakebite envenomings** 

induced by species of *Bitis* or by cytotoxic cobras (WHO, 2010b).

**6.2 First aid in snakebite envenoming** 

accidents.

**6.1 Diagnosis** 

transport of patients to health centers and, therefore, jeopardize the adequate management of these cases. First aid interventions should be focused on the immobilization of the bitten extremity and the rapid transportation to clinics or other health facilities. Communities should have strategies for rapid deployment of snakebitten people to medical treatment; an example is the use of motorcycle transportation in Nepal (Alirol et al., 2010). The interaction and communication of health staff with local healers is very important, in order to promote partnerships aimed at reducing harmful interventions and guaranteeing rapid mobilization for antivenom administration. The application of pressure-immobilization, by applying a bandage and a splint to the entire bitten limb, has been used in Australia for delaying the systemic absorption of neurotoxic venoms (Sutherland et al., 1979; White, 2010). Recently, a pharmacological intervention, based on the application of an ointment containing a nitric oxide donor, aimed at reducing the lymphatic absorption of venom, has been proposed (Saul et al., 2011), and its testing in the clinical setting is pending.

#### **6.3 Antivenoms: The key therapy of snakebite envenoming**

The parenteral administration of animal-derived antivenoms constitutes the mainstay in the therapy of snakebite envenoming (WHO, 2007a, 2010a), since the development of the first antivenoms, the *serum anti-venimeux*, during the last decade of the XIXth century (Bon, 1996). Antivenoms are preparations of immunoglobulins, or immunoglobulin fragments F(ab')2 or Fab, obtained by fractionating the plasma of animals immunized with snake venoms (Gutiérrez et al., 2011a; Lalloo & Theakston, 2003; WHO, 2010a). Antivenoms can be monospecific, when animals receive the venom of a single species, or polyspecific, when venoms from two or more species are injected. The majority of manufacturers use horses for immunization, although few use sheep and donkeys (Gutiérrez et al., 2011a, 2011b). In most cases, plasma fractionation involves the digestion of proteins with pepsin or, by few producers, with papain, followed by the purification of antibody fragments by salting-out with ammonium salts or caprylic acid fractionation and, in some cases, with chromatographic procedures (dos Santos et al., 1989; Grandgeorge et al., 1996; Raw et al., 1991; WHO, 2010a). Some producers fractionate plasma with caprylic acid to obtain whole IgG preparations (Gutiérrez et al., 2005; Rojas et al., 1994). A detailed description of the methods used in animal immunization and plasma fractionation for antivenom production can be found in the *WHO Guidelines for the Production, Control and Regulation of Snake Antivenom Immunoglobulins* (WHO, 2010a). There are antivenom-manufacturing laboratories in every continent (a complete list can be found in http://apps.who.int/bloodproducts/snakeantivenoms/database/). Following manufacture, antivenoms are subjected to a quality control protocol which involves physical, chemical and biological tests aimed at ensuring the efficacy and safety of these products (Gutiérrez & León, 2009; WHO, 2010a).

The ability of antivenoms to neutralize venom toxins is based on the capacity of antivenom antibodies, or antibody fragments, to bind and neutralize the most relevant toxins in a venom. It has been proposed that such neutralization is based on, at least, four mechanisms: (a) Binding of antibody paratopes to epitopes located at the pharmacologically-relevant molecular region, i.e. the catalytic active site in toxic enzymes such as phospholipases A2 and metalloproteinases. (b) Binding of antibodies to epitopes located close to the toxin active site, thus exerting inhibition by steric hindrance. (c) Binding of antibodies to molecular regions distant from the active/toxic site of venom components, neutralization being achieved by allosteric changes induced in the toxins, with the consequent reduction in their

Snakebite Envenoming: A Public Health Perspective 141

the consequent neutralization of neurotoxins in the circulation before reaching neuromuscular junctions. However, neutralization is more difficult when neurotoxins are bound to receptors at the synapse. In the case of post-synaptic neurotoxins, their binding can be reverted (Alape-Girón et al., 1996; Boulain & Ménez, 1982), but presynaptically-acting toxins are known to destroy the nerve terminal, thus precluding neutralization and generating a more prolonged pattern of nerve damage (Prasarnpun et al., 2005). Thus, the clinical efficacy of antivenoms is intimately related to the ability of these products to bind with high affinity and neutralize relevant venom toxins located in tissues or in the bloodstream, as well as to the toxicokinetics of toxins and the pharmacokinetics of antivenom antibodies or antibody fragments (Gutiérrez et al., 2003; Scherrmann, 1994; WHO, 2010a). For instance, low molecular mass neurotoxins characteristic of elapid snake venoms are rapidly distributed and readily reach their targets in the neuromuscular junctions; in these cases, there is a mismatch between the toxicokinetics of these neurotoxins and the pharmacokinetics of antivenom antibodies (Gutiérrez et al., 2003; Ismail et al., 1998). On the other hand, low molecular mass Fab fragments have a relatively short half-life, thus resulting in the phenomenon of recurrence of envenoming, i.e. the reappearance of signs and symptoms of envenoming several hours after antivenom therapy (Ariaratnam et al., 1999; Boyer et al., 2001; Meyer et al., 1997). Careful clinical following up of patients is

The rapid access to effective antivenoms constitutes a key issue in the proper management of snakebite envenoming. If the envenoming is potentially severe, and if the access to antivenom is delayed, due to reasons that range from hesitation to use antivenoms to prolonged transportation times to health facilities and lack of antivenoms in health posts, the efficacy of antivenoms is jeopardized and various pathophysiological complications might ensue. Another factor that determines the efficacy of antivenom treatment has to do with the use of a correct dose of this immunobiological, and to the assessment of whether the patient needs an additional dose of antivenom, based on the evolution of clinical and laboratory parameters. These considerations demand that the health staff in charge of treating these envenomings have an adequate knowledge of the basic elements of

Administration of antivenom is associated, in a variable percentage of cases, with early and late adverse reactions. Early adverse reactions (EARs) can be, in few cases, truly anaphylactic reactions, i.e. IgE-mediated, or, alternatively, anaphylactoid reactions, which occur more frequently, and are *de novo* reactions not mediated by previous exposure to horse proteins (Warrell, 1995a). The mechanisms of these reactions are not well understood, but are likely to depend on (a) complement activation by antibody aggregates present in antivenom (Sutherland, 1977); (b) formation of complexes between human heterophylic antibodies against antivenom antibodies, with consequent complement activation (León et al., 2008); or (c) presence of antibodies in antivenoms that react with cells, such as erythrocytes (León et al., 2007), leukocytes or endothelial cells, thus provoking adverse reactions. Such EARs can be mild, characterized by urticaria and itching only, or severe, involving angioedema, bronchospasm and hypotension (Warrell, 1995a). The incidence of EARs varies significantly among different antivenoms, from as low as 5% to higher than 70% of the cases with some products (Chippaux et al., 1998; Gawarammana et al., 2004;

necessary to determine the need of an additional dose of antivenom.

antivenom usage.

**6.3.2 Antivenom safety** 

ability to bind to tissue or cellular targets and to cause damage. (d) Formation of immunocomplexes between antibodies and toxins, with the subsequent removal by phagocytic cells; this last mechanism does not operate in the case of antivenoms made of monovalent Fab fragments, since they do not form complexes (Gutiérrez & León, 2009; Gutiérrez et al., 2011b).

#### **6.3.1 Clinical performance of antivenoms: Efficacy**

Antivenoms are administered parenterally, mostly by the intravenous route, and preferably diluted in physiological solution. Intradermal hypersentivity tests are not recommended since they have a very poor predictive value (Cupo et al., 1991; Malasit et al., 1986). The clinical performance of antivenoms depends on several factors associated with the immunological and physico-chemical characteristics of these products, as well as with the circumstances of their use in the clinical setting. At the preclinical level, antivenoms should be effective in the neutralization of the most relevant toxic activities of the venoms of medically-relevant snakes in a particular country or region. In some cases, this is achieved by using antivenoms raised against the venoms of the species that provoke the bite. In other cases, antivenoms are able to neutralize the venoms of species not used in the immunization of animals, but being phylogenetically related (WHO, 2010a). This phenomenon of immunological cross-reactivity has been clearly demonstrated, for instance, in the case of antivenoms raised against *Bothrops* sp venoms in Latin America (Otero et al., 1995; Segura et al., 2010a). In other cases, however, the cross-reactivity of antivenoms is low and, therefore, the efficacy of some products to neutralize venoms of medically-relevant species not included in immunization mixtures is limited, as occurs with venoms of some rattlesnakes and coral snakes in the Americas (Saravia et al., 2002; Tanaka et al., 2010). This issue of low cross-reactivity of some antivenoms may have potentially serious implications, when some products are used in the treatment of envenomings by species whose venoms are immunologically different from the ones used in immunization. One example has been the use of antivenoms manufactured in India for the treatment of envenomings in sub-Saharan Africa (Visser et al., 2008). This problem is complicated by the frequent lack of regulation and quality control of imported antivenoms in many countries, thus precluding the proper assessment of their neutralizing ability. This issue urges upgrading the regulatory capacities of countries in Asia, Africa and Latin America, as to ensure that antivenoms being introduced in these regions are evaluated with standard preclinical tests, such as those recommended by the WHO (2010a).

Antivenoms have demonstrated to be highly effective, when administered timely, at halting the most relevant systemic manifestations of snakebite envenoming (Gutiérrez & León, 2009; Lalloo & Theakston, 2003; Warrell, 1992). In the case of bites by viperids, systemic bleeding, hemodynamic manifestations and coagulation disturbances are controlled within hours after antivenom infusion. In contrast, toxins responsible for local pathological effects (edema, dermonecrosis, local hemorrhage and myonecrosis) are more difficult to neutralize by antivenoms, basically because the early onset of these effects upon venom injection, thus precluding an effective blockade by antivenom antibodies (Gutiérrez et al., 1998), a problem that is worsened by the occurrence of venom-induced vascular alterations, which affect the distribution of antivenom to the affected tissue (Battellino et al., 2003). In the case of neurotoxic venoms, characteristic of most elapid and some viperid species, the development of neurotoxic manifestations is prevented by the timely administration of antivenoms, with

ability to bind to tissue or cellular targets and to cause damage. (d) Formation of immunocomplexes between antibodies and toxins, with the subsequent removal by phagocytic cells; this last mechanism does not operate in the case of antivenoms made of monovalent Fab fragments, since they do not form complexes (Gutiérrez & León, 2009;

Antivenoms are administered parenterally, mostly by the intravenous route, and preferably diluted in physiological solution. Intradermal hypersentivity tests are not recommended since they have a very poor predictive value (Cupo et al., 1991; Malasit et al., 1986). The clinical performance of antivenoms depends on several factors associated with the immunological and physico-chemical characteristics of these products, as well as with the circumstances of their use in the clinical setting. At the preclinical level, antivenoms should be effective in the neutralization of the most relevant toxic activities of the venoms of medically-relevant snakes in a particular country or region. In some cases, this is achieved by using antivenoms raised against the venoms of the species that provoke the bite. In other cases, antivenoms are able to neutralize the venoms of species not used in the immunization of animals, but being phylogenetically related (WHO, 2010a). This phenomenon of immunological cross-reactivity has been clearly demonstrated, for instance, in the case of antivenoms raised against *Bothrops* sp venoms in Latin America (Otero et al., 1995; Segura et al., 2010a). In other cases, however, the cross-reactivity of antivenoms is low and, therefore, the efficacy of some products to neutralize venoms of medically-relevant species not included in immunization mixtures is limited, as occurs with venoms of some rattlesnakes and coral snakes in the Americas (Saravia et al., 2002; Tanaka et al., 2010). This issue of low cross-reactivity of some antivenoms may have potentially serious implications, when some products are used in the treatment of envenomings by species whose venoms are immunologically different from the ones used in immunization. One example has been the use of antivenoms manufactured in India for the treatment of envenomings in sub-Saharan Africa (Visser et al., 2008). This problem is complicated by the frequent lack of regulation and quality control of imported antivenoms in many countries, thus precluding the proper assessment of their neutralizing ability. This issue urges upgrading the regulatory capacities of countries in Asia, Africa and Latin America, as to ensure that antivenoms being introduced in these regions are evaluated with standard preclinical tests, such as those

Antivenoms have demonstrated to be highly effective, when administered timely, at halting the most relevant systemic manifestations of snakebite envenoming (Gutiérrez & León, 2009; Lalloo & Theakston, 2003; Warrell, 1992). In the case of bites by viperids, systemic bleeding, hemodynamic manifestations and coagulation disturbances are controlled within hours after antivenom infusion. In contrast, toxins responsible for local pathological effects (edema, dermonecrosis, local hemorrhage and myonecrosis) are more difficult to neutralize by antivenoms, basically because the early onset of these effects upon venom injection, thus precluding an effective blockade by antivenom antibodies (Gutiérrez et al., 1998), a problem that is worsened by the occurrence of venom-induced vascular alterations, which affect the distribution of antivenom to the affected tissue (Battellino et al., 2003). In the case of neurotoxic venoms, characteristic of most elapid and some viperid species, the development of neurotoxic manifestations is prevented by the timely administration of antivenoms, with

Gutiérrez et al., 2011b).

recommended by the WHO (2010a).

**6.3.1 Clinical performance of antivenoms: Efficacy** 

the consequent neutralization of neurotoxins in the circulation before reaching neuromuscular junctions. However, neutralization is more difficult when neurotoxins are bound to receptors at the synapse. In the case of post-synaptic neurotoxins, their binding can be reverted (Alape-Girón et al., 1996; Boulain & Ménez, 1982), but presynaptically-acting toxins are known to destroy the nerve terminal, thus precluding neutralization and generating a more prolonged pattern of nerve damage (Prasarnpun et al., 2005). Thus, the clinical efficacy of antivenoms is intimately related to the ability of these products to bind with high affinity and neutralize relevant venom toxins located in tissues or in the bloodstream, as well as to the toxicokinetics of toxins and the pharmacokinetics of antivenom antibodies or antibody fragments (Gutiérrez et al., 2003; Scherrmann, 1994; WHO, 2010a). For instance, low molecular mass neurotoxins characteristic of elapid snake venoms are rapidly distributed and readily reach their targets in the neuromuscular junctions; in these cases, there is a mismatch between the toxicokinetics of these neurotoxins and the pharmacokinetics of antivenom antibodies (Gutiérrez et al., 2003; Ismail et al., 1998). On the other hand, low molecular mass Fab fragments have a relatively short half-life, thus resulting in the phenomenon of recurrence of envenoming, i.e. the reappearance of signs and symptoms of envenoming several hours after antivenom therapy (Ariaratnam et al., 1999; Boyer et al., 2001; Meyer et al., 1997). Careful clinical following up of patients is necessary to determine the need of an additional dose of antivenom.

The rapid access to effective antivenoms constitutes a key issue in the proper management of snakebite envenoming. If the envenoming is potentially severe, and if the access to antivenom is delayed, due to reasons that range from hesitation to use antivenoms to prolonged transportation times to health facilities and lack of antivenoms in health posts, the efficacy of antivenoms is jeopardized and various pathophysiological complications might ensue. Another factor that determines the efficacy of antivenom treatment has to do with the use of a correct dose of this immunobiological, and to the assessment of whether the patient needs an additional dose of antivenom, based on the evolution of clinical and laboratory parameters. These considerations demand that the health staff in charge of treating these envenomings have an adequate knowledge of the basic elements of antivenom usage.

#### **6.3.2 Antivenom safety**

Administration of antivenom is associated, in a variable percentage of cases, with early and late adverse reactions. Early adverse reactions (EARs) can be, in few cases, truly anaphylactic reactions, i.e. IgE-mediated, or, alternatively, anaphylactoid reactions, which occur more frequently, and are *de novo* reactions not mediated by previous exposure to horse proteins (Warrell, 1995a). The mechanisms of these reactions are not well understood, but are likely to depend on (a) complement activation by antibody aggregates present in antivenom (Sutherland, 1977); (b) formation of complexes between human heterophylic antibodies against antivenom antibodies, with consequent complement activation (León et al., 2008); or (c) presence of antibodies in antivenoms that react with cells, such as erythrocytes (León et al., 2007), leukocytes or endothelial cells, thus provoking adverse reactions. Such EARs can be mild, characterized by urticaria and itching only, or severe, involving angioedema, bronchospasm and hypotension (Warrell, 1995a). The incidence of EARs varies significantly among different antivenoms, from as low as 5% to higher than 70% of the cases with some products (Chippaux et al., 1998; Gawarammana et al., 2004;

Snakebite Envenoming: A Public Health Perspective 143

2010b). The complexity of snake venoms and the corresponding variability in the clinical presentation of these envenomings complicates the management of the cases and demands an adequate training of the health staff in charge of treating these emergencies, in order to

The poor efficacy of antivenoms to neutralize local tissue damage induced by viperid and some elapid venoms brings the need to find alternative therapies. A very promising avenue is the possibility of using natural or synthetic inhibitors of venom toxins, such as inhibitors of phospholipases A2, metalloproteinases and hyaluronidases, for blocking the action of tissue-damaging toxins by rapidly administering these inhibitors directly on the site of venom injection (Gutiérrez et al., 2007; Lomonte et al., 2009; Perales et al., 2005). Such possibility has been tested, with excellent results, at the preclinical level in mouse models (Borkow et al., 1997; Lomonte et al., 2009; Rucavado et al., 2000; Yingprasertchai et al., 2003). It is necessary to identify and develop inhibitors, some of which may be already in use for other pathologies, and to test them at the preclinical and clinical levels. The therapy of snakebite envenoming in the future will likely involve, in addition to intravenous antivenom administration, the local injection of toxin inhibitors, as well as other ancillary interventions aimed at controlling the systemic aspects of envenoming and to modulate the deleterious aspects of the inflammatory response of the organism to snake venoms

The large intra- and interspecies variability in the composition of snake venoms poses a problem for antivenom efficacy, since cross-neutralization of antivenoms against venoms not used in the immunizing mixture might not occur. Therefore, the distribution of antivenoms to countries or regions where medically-relevant snakes are different from those used in immunization schemes needs to be carefully evaluated in order to ensure that these antivenoms are indeed effective. This issue gets complicated by the fact that, quite often, regulatory agencies in developing countries do not have the facilities and expertise to perform adequate preclinical testing of the antivenoms being imported (D. Williams et al., 2011). A proper assessment of antivenom efficacy should be based on preclinical and clinical testing. At the preclinical level, it is necessary to assess the capacity of antivenoms to neutralize the lethal, as well as other relevant toxic activities, of the most important venoms in a country or region. This demands, in the first place, the establishment of local facilities to collect and keep medically-relevant snakes. These snake colonies should provide pools of venoms, which could then be used in preclinical testing of antivenoms. Precise indications on how to built and run these facilities are included in the *WHO Guidelines for Antivenom Production, Control and Regulation of Antivenoms* (WHO, 2010a). In the case of viperid venoms, a battery of preclinical tests usually includes the evaluation of the neutralization of lethal, hemorrhagic, coagulant, defibrinogenating and myotoxic activities (Gutiérrez et al., 2011b; Theakston, 1986; WHO, 2010a). In the case of elapid snakes, antivenom preclinical efficacy should be assessed by the neutralization of lethality and, in the case of elapid venoms that induce necrosis or coagulopathy, by the neutralization of dermonecrosis and coagulant activities, respectively (Gutiérrez et al., 2011b; WHO, 2010a). These methods involve simple laboratory procedures that need to be implemented in countries where antivenoms are being produced or imported. In addition, international collaborative

guarantee the implementation of effective therapeutic interventions.

(Gutiérrez et al., 2007).

**7. Preclinical and clinical testing of antivenoms** 

Otero-Patiño et al., 1998). Such high variability is due to the different physicochemical quality of antivenoms, since some products have high protein concentration and high amounts of protein aggregates. Therefore, the physicochemical features of antivenoms greatly determine their safety profile, an issue that demands renewed efforts at the technological and regulatory levels. Another type of reaction observed in some antivenoms are pyrogenic reactions, associated with chills and fever (WHO, 2010b), but these should be avoided by a proper quality control, i.e. pyrogenicity testing, of these products. In the event of EARs, antivenom infusion should be stopped, and the reaction treated with adrenaline, anti-histamines and steroids (Warrell, 1995a). Once the reaction is controlled, antivenom infusion should be continued. Pretreatment with adrenaline has been advocated for reducing the incidence of EARs (de Silva et al., 2011). Late adverse reactions (LARs) to antivenoms occur 5-24 days after treatment, and are characterized by itching, fever, urticaria, arthralgia and proteinuria (Warrell, 1995a). This corresponds to a typical type III hypersensitivity reaction, i.e. serum sickness, due to the formation of immune complexes between antivenom antibodies and antibodies generated in the patient against antivenom proteins. The incidence of serum sickness after antivenom administration correlates with the amount of foreign protein, i.e. antivenom, administered (LoVecchio et al., 2003). LARs are treated with anti-histamines and steroids. Another aspect of antivenom safety that has to be considered is microbial safety, which is guaranteed by sterile filtration of the final product and the use of viral inactivation/removal steps (Burnouf et al., 2004; WHO, 2010a). Some of the manufacturing steps currently used in antivenom production inactivate or remove viruses, thus contributing to the microbial safety of these products (Burnouf et al., 2004; WHO, 2010a).

Such high heterogeneity in the safety of antivenoms, in terms of incidence of adverse reactions, calls for international cooperative efforts aimed at improving the technological platform of many antivenom producers, in order to increase the physicochemical quality of antivenoms on a world wide basis (Gutiérrez et al., 2011a). A number of antivenom producers in Asia, Africa and Latin America need to upgrade their facilities and protocols. The experience gained by well-developed antivenom manufacturing laboratories in various parts of the world should contribute to the improvement of less developed antivenom producers, through a variety of activities such as technology transfer programs, workshops, training and exchanges of various sorts. Such networking scenario should be promoted by the WHO and its regional offices, and by organizations such as the Global Snake Bite Initiative (www.snakebiteinitiative.org/).

#### **6.4 Ancillary treatments**

The therapy of snakebite envenoming includes a series of interventions in addition to antivenom administration. In the case of viperid venoms, hemodynamic and renal disturbances demand careful control of fluid therapy, monitoring of central venous pressure, and use of diuretics (Warrell, 1995a; WHO, 2010b). Infection often develops in viperid snakebites and requires the use of antibiotics. Moreover, local tissue damage by viperid and some elapid snakebites calls for debridement of necrotic tissue and care of the bitten limb. In some cases, when muscle intracompartmental pressures increase beyond 45 mm Hg, compartment syndrome ensues and fasciotomy is indicated (WHO, 2010b). In the case of neurotoxic envenomings caused by elapid and some viperid species, mechanical ventilation should be provided in the event of respiratory paralysis (Warrell, 1995a; WHO,

Otero-Patiño et al., 1998). Such high variability is due to the different physicochemical quality of antivenoms, since some products have high protein concentration and high amounts of protein aggregates. Therefore, the physicochemical features of antivenoms greatly determine their safety profile, an issue that demands renewed efforts at the technological and regulatory levels. Another type of reaction observed in some antivenoms are pyrogenic reactions, associated with chills and fever (WHO, 2010b), but these should be avoided by a proper quality control, i.e. pyrogenicity testing, of these products. In the event of EARs, antivenom infusion should be stopped, and the reaction treated with adrenaline, anti-histamines and steroids (Warrell, 1995a). Once the reaction is controlled, antivenom infusion should be continued. Pretreatment with adrenaline has been advocated for reducing the incidence of EARs (de Silva et al., 2011). Late adverse reactions (LARs) to antivenoms occur 5-24 days after treatment, and are characterized by itching, fever, urticaria, arthralgia and proteinuria (Warrell, 1995a). This corresponds to a typical type III hypersensitivity reaction, i.e. serum sickness, due to the formation of immune complexes between antivenom antibodies and antibodies generated in the patient against antivenom proteins. The incidence of serum sickness after antivenom administration correlates with the amount of foreign protein, i.e. antivenom, administered (LoVecchio et al., 2003). LARs are treated with anti-histamines and steroids. Another aspect of antivenom safety that has to be considered is microbial safety, which is guaranteed by sterile filtration of the final product and the use of viral inactivation/removal steps (Burnouf et al., 2004; WHO, 2010a). Some of the manufacturing steps currently used in antivenom production inactivate or remove viruses, thus contributing to the microbial safety of these products (Burnouf et al., 2004;

Such high heterogeneity in the safety of antivenoms, in terms of incidence of adverse reactions, calls for international cooperative efforts aimed at improving the technological platform of many antivenom producers, in order to increase the physicochemical quality of antivenoms on a world wide basis (Gutiérrez et al., 2011a). A number of antivenom producers in Asia, Africa and Latin America need to upgrade their facilities and protocols. The experience gained by well-developed antivenom manufacturing laboratories in various parts of the world should contribute to the improvement of less developed antivenom producers, through a variety of activities such as technology transfer programs, workshops, training and exchanges of various sorts. Such networking scenario should be promoted by the WHO and its regional offices, and by organizations such as the Global Snake Bite

The therapy of snakebite envenoming includes a series of interventions in addition to antivenom administration. In the case of viperid venoms, hemodynamic and renal disturbances demand careful control of fluid therapy, monitoring of central venous pressure, and use of diuretics (Warrell, 1995a; WHO, 2010b). Infection often develops in viperid snakebites and requires the use of antibiotics. Moreover, local tissue damage by viperid and some elapid snakebites calls for debridement of necrotic tissue and care of the bitten limb. In some cases, when muscle intracompartmental pressures increase beyond 45 mm Hg, compartment syndrome ensues and fasciotomy is indicated (WHO, 2010b). In the case of neurotoxic envenomings caused by elapid and some viperid species, mechanical ventilation should be provided in the event of respiratory paralysis (Warrell, 1995a; WHO,

WHO, 2010a).

Initiative (www.snakebiteinitiative.org/).

**6.4 Ancillary treatments** 

2010b). The complexity of snake venoms and the corresponding variability in the clinical presentation of these envenomings complicates the management of the cases and demands an adequate training of the health staff in charge of treating these emergencies, in order to guarantee the implementation of effective therapeutic interventions.

The poor efficacy of antivenoms to neutralize local tissue damage induced by viperid and some elapid venoms brings the need to find alternative therapies. A very promising avenue is the possibility of using natural or synthetic inhibitors of venom toxins, such as inhibitors of phospholipases A2, metalloproteinases and hyaluronidases, for blocking the action of tissue-damaging toxins by rapidly administering these inhibitors directly on the site of venom injection (Gutiérrez et al., 2007; Lomonte et al., 2009; Perales et al., 2005). Such possibility has been tested, with excellent results, at the preclinical level in mouse models (Borkow et al., 1997; Lomonte et al., 2009; Rucavado et al., 2000; Yingprasertchai et al., 2003). It is necessary to identify and develop inhibitors, some of which may be already in use for other pathologies, and to test them at the preclinical and clinical levels. The therapy of snakebite envenoming in the future will likely involve, in addition to intravenous antivenom administration, the local injection of toxin inhibitors, as well as other ancillary interventions aimed at controlling the systemic aspects of envenoming and to modulate the deleterious aspects of the inflammatory response of the organism to snake venoms (Gutiérrez et al., 2007).

#### **7. Preclinical and clinical testing of antivenoms**

The large intra- and interspecies variability in the composition of snake venoms poses a problem for antivenom efficacy, since cross-neutralization of antivenoms against venoms not used in the immunizing mixture might not occur. Therefore, the distribution of antivenoms to countries or regions where medically-relevant snakes are different from those used in immunization schemes needs to be carefully evaluated in order to ensure that these antivenoms are indeed effective. This issue gets complicated by the fact that, quite often, regulatory agencies in developing countries do not have the facilities and expertise to perform adequate preclinical testing of the antivenoms being imported (D. Williams et al., 2011). A proper assessment of antivenom efficacy should be based on preclinical and clinical testing. At the preclinical level, it is necessary to assess the capacity of antivenoms to neutralize the lethal, as well as other relevant toxic activities, of the most important venoms in a country or region. This demands, in the first place, the establishment of local facilities to collect and keep medically-relevant snakes. These snake colonies should provide pools of venoms, which could then be used in preclinical testing of antivenoms. Precise indications on how to built and run these facilities are included in the *WHO Guidelines for Antivenom Production, Control and Regulation of Antivenoms* (WHO, 2010a). In the case of viperid venoms, a battery of preclinical tests usually includes the evaluation of the neutralization of lethal, hemorrhagic, coagulant, defibrinogenating and myotoxic activities (Gutiérrez et al., 2011b; Theakston, 1986; WHO, 2010a). In the case of elapid snakes, antivenom preclinical efficacy should be assessed by the neutralization of lethality and, in the case of elapid venoms that induce necrosis or coagulopathy, by the neutralization of dermonecrosis and coagulant activities, respectively (Gutiérrez et al., 2011b; WHO, 2010a). These methods involve simple laboratory procedures that need to be implemented in countries where antivenoms are being produced or imported. In addition, international collaborative

Snakebite Envenoming: A Public Health Perspective 145

neutralize the venoms of several species of viperids and spitting cobras from sub-Saharan Africa (Calvete et al., 2010b; Segura et al., 2010b; Petras et al., 2011). Similarly, the ideal venom mixtures for antivenoms to be used in some parts of Asia need to be re-assessed on the basis of recent clinical evidence of the existence of medically-relevant species whose venoms are not routinely used in antivenom manufacture, such as that of the viperid *Hypnale hypnale* (Ariaratnam et al., 2008). Likewise, the decision on whether to prepare monospecific or polyspecific antivenoms has to be based on sound epidemiological, clinical, biochemical and immunological evidence. Consequently, the design and re-design of venom mixtures for immunization requires a multidisciplinary approach. On the other hand, there are other aspects of antivenom technological development that should be considered, such as stability and improved immunization schemes. Liquid antivenoms have to be stored at 2-8 ºC (WHO, 2010a). However, the quality of the cold chain in many regions of the world is poor, thus complicating the distribution of antivenoms, especially to rural settings where most snakebites occur. This problem can be overcome by producing freeze-dried antivenoms, but this increases the production cost and, therefore, the price. Alternatives are being explored aimed at formulating liquid antivenoms stable at room temperature (Rodrigues-Silva et al., 1999; Segura et al., 2009). Likewise, the design of immunization protocols based on multi-site injection of small volumes containing low amounts of venom has resulted in higher neutralizing titers with very little damage to the immunized animals (Chotwiwatthanakun et al., 2001). Furthermore, the search for novel adjuvants is a relevant

task in the efforts to improve antivenom antibody titers (Gutiérrez et al., 2011a).

Despite the widespread demonstration of antivenom efficacy for the treatment of snakebite envenoming, and the fact that many aspects of the know-how required to produce antivenoms are freely available (WHO, 2010a), there is a current deficit in antivenom accessibility in various regions of the world, most notably in sub-Saharan Africa and some countries of south-east Asia (Chippaux, 2010; Theakston et al., 2003; D. Williams et al., 2011; WHO, 2007a). This phenomenon has multiple causes, such as: (a) Withdrawal of some manufacturers from these markets due to profit considerations. (b) Privatization of former public laboratories, with the consequent increments in the prices of antivenoms. (c) The impact of international policies designed to reduce the size of the public sector, including a reduction in the provision of public health services and their privatization. (d) Weakening of antivenom manufacturing laboratories of the public sector in many developing countries, associated with lack of investment in facilities and technology, and reduction in training programs for the staff. (e) Lack of financial support for antivenom purchase by ministries of health, due to economic constraints and to prioritization on other health issues perceived as more pressing needs. (f) Loss of confidence in antivenom treatment in some regions due to the use of antivenoms of poor efficacy or safety. (g) Poor advocacy for promoting greater attention to snakebite envenoming as a neglected tropical disease. (h) Low profile of snakebite envenoming in the international public health agenda. As a result, antivenom accessibility is deficient in vast regions of Asia and Africa (WHO, 2007a; D. Williams et al., 2011). The solution to this complex problem demands concerted actions at various levels, from the technological and manufacturing realm to the public health arena (Chippaux, 2010;

**9. The accessibility and correct use of antivenoms** 

Gutiérrez et al., 2010b; D. Williams et al., 2010, 2011).

projects, involving well-developed laboratories, could be implemented in order to test antivenoms (D. Williams et al., 2011). More recently, a proteomic approach, named 'antivenomics', has been adapted for the evaluation of immune reactivity of antivenoms against particular toxins in venoms (Calvete, 2010; Gutiérrez et al., 2009a; Lomonte et al., 2008). This methodology allows for the identification of the toxins recognized by antivenom antibodies.

The preclinical assessment of antivenoms should be followed by clinical evaluation of antivenom safety and efficacy (WHO, 2010a). Since phase I clinical trials in healthy volunteers are ethically unacceptable in the case of antivenoms, because they might induce adverse reactions, a substitution of phase I clinical trial, by a protocol known as '3 + 3 dose escalation design', has been proposed for antivenoms (S.B. Abubakar et al., 2010b). This is then followed by phase III clinical trials in which a new antivenom is compared with an existing antivenom of known efficacy and safety (see for example the studies of I.S. Abubakar et al., 2010; Cardoso et al., 1993; Otero et al., 1999; Otero-Patiño et al., 1998; Smalligan et al., 2004; Warrell et al., 1974). Clinical trials should use robust clinical and laboratory end points for the assessment of therapeutic success. Furthermore, postmarketing surveillance (pharmacovigilance) is required to detect possible adverse reactions not reported in the clinical trials and to follow up efficacy (WHO, 2010a).

#### **8. Technological aspects for antivenom improvement**

The need to have antivenoms of wide cross-reactivity, able to neutralize venoms from as many snake species as possible, demands a careful revision of the design of venom mixtures used for immunization of animals. There is a large body of knowledge in the biochemistry, toxicology and immunology of snake venoms, especially of venoms from species having a heavy medical impact, which should be used for the re-evaluation of the immunizing mixtures and for the design of novel mixtures for new antivenoms (Gutiérrez et al., 2009a; D. Williams et al., 2011). Proteomics technologies, together with neutralization tests, constitute valuable tools to analyze venom composition and effects, and to assess the neutralizing profile of current and new antivenoms. The Global Snake Bite Initiative has proposed a strategy to structure an international collaborative effort to evaluate current antivenoms and to design improved antivenoms (D. Williams et al., 2011). One aspect of this strategy is based on the development of regional polyspecific antivenoms for use in sub-Saharan Africa and Asia using clinical, phylogenetic, proteomic and antivenomic analyses for the selection of the best venom mixtures for immunization. These antivenoms, manufactured by several laboratories, will then be evaluated by independent preclinical assessment, followed by clinical trials in various countries, performed by local medical personnel. In parallel, international expert committees will validate production facilities for prequalification, in a process aimed at ensuring the manufacture of the volume of antivenom needed in those regions (D. Williams et al., 2011).

One example of the potential usefulness of such an approach has to do with the design of immunizing mixtures for antivenoms to be used in sub-Saharan Africa. Several antivenoms use a mixture of venoms from many species; however, a recently developed antivenom was produced by using a mixture of venoms from only three species (Gutiérrez et al., 2005). Neutralization and antivenomic studies have shown that this new antivenom is able to

projects, involving well-developed laboratories, could be implemented in order to test antivenoms (D. Williams et al., 2011). More recently, a proteomic approach, named 'antivenomics', has been adapted for the evaluation of immune reactivity of antivenoms against particular toxins in venoms (Calvete, 2010; Gutiérrez et al., 2009a; Lomonte et al., 2008). This methodology allows for the identification of the toxins recognized by antivenom

The preclinical assessment of antivenoms should be followed by clinical evaluation of antivenom safety and efficacy (WHO, 2010a). Since phase I clinical trials in healthy volunteers are ethically unacceptable in the case of antivenoms, because they might induce adverse reactions, a substitution of phase I clinical trial, by a protocol known as '3 + 3 dose escalation design', has been proposed for antivenoms (S.B. Abubakar et al., 2010b). This is then followed by phase III clinical trials in which a new antivenom is compared with an existing antivenom of known efficacy and safety (see for example the studies of I.S. Abubakar et al., 2010; Cardoso et al., 1993; Otero et al., 1999; Otero-Patiño et al., 1998; Smalligan et al., 2004; Warrell et al., 1974). Clinical trials should use robust clinical and laboratory end points for the assessment of therapeutic success. Furthermore, postmarketing surveillance (pharmacovigilance) is required to detect possible adverse reactions

The need to have antivenoms of wide cross-reactivity, able to neutralize venoms from as many snake species as possible, demands a careful revision of the design of venom mixtures used for immunization of animals. There is a large body of knowledge in the biochemistry, toxicology and immunology of snake venoms, especially of venoms from species having a heavy medical impact, which should be used for the re-evaluation of the immunizing mixtures and for the design of novel mixtures for new antivenoms (Gutiérrez et al., 2009a; D. Williams et al., 2011). Proteomics technologies, together with neutralization tests, constitute valuable tools to analyze venom composition and effects, and to assess the neutralizing profile of current and new antivenoms. The Global Snake Bite Initiative has proposed a strategy to structure an international collaborative effort to evaluate current antivenoms and to design improved antivenoms (D. Williams et al., 2011). One aspect of this strategy is based on the development of regional polyspecific antivenoms for use in sub-Saharan Africa and Asia using clinical, phylogenetic, proteomic and antivenomic analyses for the selection of the best venom mixtures for immunization. These antivenoms, manufactured by several laboratories, will then be evaluated by independent preclinical assessment, followed by clinical trials in various countries, performed by local medical personnel. In parallel, international expert committees will validate production facilities for prequalification, in a process aimed at ensuring the manufacture of the volume of antivenom needed in those regions (D. Williams et al.,

One example of the potential usefulness of such an approach has to do with the design of immunizing mixtures for antivenoms to be used in sub-Saharan Africa. Several antivenoms use a mixture of venoms from many species; however, a recently developed antivenom was produced by using a mixture of venoms from only three species (Gutiérrez et al., 2005). Neutralization and antivenomic studies have shown that this new antivenom is able to

not reported in the clinical trials and to follow up efficacy (WHO, 2010a).

**8. Technological aspects for antivenom improvement** 

antibodies.

2011).

neutralize the venoms of several species of viperids and spitting cobras from sub-Saharan Africa (Calvete et al., 2010b; Segura et al., 2010b; Petras et al., 2011). Similarly, the ideal venom mixtures for antivenoms to be used in some parts of Asia need to be re-assessed on the basis of recent clinical evidence of the existence of medically-relevant species whose venoms are not routinely used in antivenom manufacture, such as that of the viperid *Hypnale hypnale* (Ariaratnam et al., 2008). Likewise, the decision on whether to prepare monospecific or polyspecific antivenoms has to be based on sound epidemiological, clinical, biochemical and immunological evidence. Consequently, the design and re-design of venom mixtures for immunization requires a multidisciplinary approach. On the other hand, there are other aspects of antivenom technological development that should be considered, such as stability and improved immunization schemes. Liquid antivenoms have to be stored at 2-8 ºC (WHO, 2010a). However, the quality of the cold chain in many regions of the world is poor, thus complicating the distribution of antivenoms, especially to rural settings where most snakebites occur. This problem can be overcome by producing freeze-dried antivenoms, but this increases the production cost and, therefore, the price. Alternatives are being explored aimed at formulating liquid antivenoms stable at room temperature (Rodrigues-Silva et al., 1999; Segura et al., 2009). Likewise, the design of immunization protocols based on multi-site injection of small volumes containing low amounts of venom has resulted in higher neutralizing titers with very little damage to the immunized animals (Chotwiwatthanakun et al., 2001). Furthermore, the search for novel adjuvants is a relevant task in the efforts to improve antivenom antibody titers (Gutiérrez et al., 2011a).

#### **9. The accessibility and correct use of antivenoms**

Despite the widespread demonstration of antivenom efficacy for the treatment of snakebite envenoming, and the fact that many aspects of the know-how required to produce antivenoms are freely available (WHO, 2010a), there is a current deficit in antivenom accessibility in various regions of the world, most notably in sub-Saharan Africa and some countries of south-east Asia (Chippaux, 2010; Theakston et al., 2003; D. Williams et al., 2011; WHO, 2007a). This phenomenon has multiple causes, such as: (a) Withdrawal of some manufacturers from these markets due to profit considerations. (b) Privatization of former public laboratories, with the consequent increments in the prices of antivenoms. (c) The impact of international policies designed to reduce the size of the public sector, including a reduction in the provision of public health services and their privatization. (d) Weakening of antivenom manufacturing laboratories of the public sector in many developing countries, associated with lack of investment in facilities and technology, and reduction in training programs for the staff. (e) Lack of financial support for antivenom purchase by ministries of health, due to economic constraints and to prioritization on other health issues perceived as more pressing needs. (f) Loss of confidence in antivenom treatment in some regions due to the use of antivenoms of poor efficacy or safety. (g) Poor advocacy for promoting greater attention to snakebite envenoming as a neglected tropical disease. (h) Low profile of snakebite envenoming in the international public health agenda. As a result, antivenom accessibility is deficient in vast regions of Asia and Africa (WHO, 2007a; D. Williams et al., 2011). The solution to this complex problem demands concerted actions at various levels, from the technological and manufacturing realm to the public health arena (Chippaux, 2010; Gutiérrez et al., 2010b; D. Williams et al., 2010, 2011).

Snakebite Envenoming: A Public Health Perspective 147

effectiveness of interventions (WHO, 2007c). The compulsory report of snakebite envenoming (WHO, 2010a) and the use of geographical information systems to identify high risk areas (Hansson et al., 2010; Leynaud & Reati, 2009) would greatly contribute to generate a solid basis of information on the actual magnitude of the problem. Furthermore, the awareness of national and regional health authorities on the impact of snakebite envenoming should be promoted by academic, public health and civil society organizations, in order to ensure the acquisition and distribution of the required volumes of antivenom. Likewise, antivenom distribution strategies should benefit from the use of the cold chain system already developed for vaccine distribution. Also, the provision of antivenom access to rural settings and the training of rural health staff in the correct administration of antivenoms should be prioritized. Interventions tailored to the conditions of each country and region should be promoted, in order to optimize the available resources and guarantee

The distribution of antivenoms to regions where snakebites occur should be complemented by a proper training of health staff in the correct usage of this product and in the proper treatment of snakebite envenomings. There is evidence of poor knowledge of medical and nursing staff in various regions of the world on how to diagnose and treat snakebite envenomings, how to use antivenoms, and how to treat possible adverse reactions to their administration (Gutiérrez et al., 2009c; Simpson, 2008). This requires concerted efforts at medical and nursing schools in the universities, as well as the implementation of permanent educational programs on this subject, particularly aimed at rural health facilities. Likewise, the implementation of teaching material and the development of guidelines for the diagnosis and treatment of snakebite envenomings should be actively promoted, both at regional (WHO, 2010b) and national levels. These tasks should involve not only teaching institutions, but also public health authorities, local organizations of the civil society, and antivenom manufacturers. The critical revision of antivenom prospects, on the basis of current knowledge on the taxonomy of snakes and on the clinics of snakebite envenomings, are of great relevance, in the light of evident misconceptions included in the prospects of

Prevention programs aimed at reducing the impact and incidence of snakebite envenomings should be a priority in the international efforts required to confront this problem. The design of these programs should be tailored to the cultural, social, economic and institutional characteristics of the populations, and should involve the active participation of the communities in their design and implementation. Impoverished and excluded groups, such as indigenous communities in many parts of the world, should receive particular attention. The design of these programs should be also based on sound social science research aimed at understanding the particularities and needs of each region and context, with the participation of the communities. It is highly relevant, for instance, to understand how the problem is perceived in the community and what types of preventive interventions are suited for each particular context. Likewise, specific strategies should be designed for situations involving natural disasters, as snakebites have been reported to increase in such

a rapid access to treatment (see for example Otero et al., 1992).

**9.3 Promoting the correct use of antivenoms** 

some antivenoms (Simpson & Norris, 2007).

**10. Prevention of snakebites** 

#### **9.1 How to enhance the accessibility of antivenoms**

Economic and political constraints constitute one of the main causes of poor accessibility of antivenoms in many countries. It is evident that the sole drive of the market forces will not solve this problem and, instead, well-designed strategies with a strong participation of governments and non-governmental organizations have to be implemented. This is a critical aspect that needs to be addressed by a variety of interventions such as: (a) Increasing the technological capacity of manufacturing laboratories in developing countries, both in the public and private realms, and introduction of cost-effective methodologies for antivenom production. One example is the manufacture of whole IgG antivenoms by caprylic acid fractionation of plasma (Gutiérrez et al., 2005; Rojas et al., 1994). This procedure generates antivenoms of high quality and high yield, at reduced production costs, thus constituting an excellent alternative for low-income countries (Brown & Landon, 2010). (b) Increased recognition of governments of low-income countries on the impact of snakebite envenoming as a public health problem, with the consequent political and financial decisions for the acquisition of adequate volumes of antivenom. (c) Using the capacity of large antivenom producers in order to manufacture antivenoms for other regions of the world at reasonable prices. This could be accomplished by promoting international partnerships between manufacturers, public health authorities, organizations of the civil society, and donors, similarly to what has been done for other neglected tropical diseases (Hotez et al., 2006). (d) Promoting strategies for price reduction, such as differential pricing arrangements or large scale 'pooled' purchases for various countries (Gutiérrez et al., 2010b).

#### **9.2 Distribution of antivenoms: guaranteeing access to regions where snakebites occur**

Even if governments purchase adequate volumes of antivenom, this does not guarantee that these drugs will reach the rural health posts where most snakebite envenomings occur. This problem has diverse roots, such as: (a) Incomplete information on the epidemiology of snakebites. In countries where official statistics of snakebite incidence are lacking or incomplete, the decision on where to distribute antivenoms cannot be taken on a rigorous base. This is another reason for underscoring the relevance of proper epidemiological register of this pathology. (b) Antivenom acquisition procedures by the ministries of health in many countries are slow and cumbersome; moreover, due to budgetary constraints, the volumes of antivenom purchased are often insufficient; both of these factors preclude the distribution of adequate volumes of this drug to rural settings. (c) Antivenoms are often distributed only to hospitals and clinics in large cities, distant from the regions where the majority of snakebites occur, thus affecting the timely treatment of patients. (d) As discussed previously, the lack of an adequate cold chain system in many rural settings of the world precludes the effective distribution of antivenoms. (e) Many rural regions are devoid of healthcare facilities, thus affecting the access to antivenom and other medical interventions and forcing people to travel large distances to receive medical attention.

This complex scenario demands the design of well-structured and effective strategies of antivenom distribution, on the basis of sound epidemiological information on snakebite incidence. An intersectorial and interprogrammatic approach should be promoted, in conjunction with other efforts being performed in the public health realm, in order to favour a synergy with other actors and projects, with the consequent impact in the cost-

Economic and political constraints constitute one of the main causes of poor accessibility of antivenoms in many countries. It is evident that the sole drive of the market forces will not solve this problem and, instead, well-designed strategies with a strong participation of governments and non-governmental organizations have to be implemented. This is a critical aspect that needs to be addressed by a variety of interventions such as: (a) Increasing the technological capacity of manufacturing laboratories in developing countries, both in the public and private realms, and introduction of cost-effective methodologies for antivenom production. One example is the manufacture of whole IgG antivenoms by caprylic acid fractionation of plasma (Gutiérrez et al., 2005; Rojas et al., 1994). This procedure generates antivenoms of high quality and high yield, at reduced production costs, thus constituting an excellent alternative for low-income countries (Brown & Landon, 2010). (b) Increased recognition of governments of low-income countries on the impact of snakebite envenoming as a public health problem, with the consequent political and financial decisions for the acquisition of adequate volumes of antivenom. (c) Using the capacity of large antivenom producers in order to manufacture antivenoms for other regions of the world at reasonable prices. This could be accomplished by promoting international partnerships between manufacturers, public health authorities, organizations of the civil society, and donors, similarly to what has been done for other neglected tropical diseases (Hotez et al., 2006). (d) Promoting strategies for price reduction, such as differential pricing arrangements or large

**9.1 How to enhance the accessibility of antivenoms** 

scale 'pooled' purchases for various countries (Gutiérrez et al., 2010b).

and forcing people to travel large distances to receive medical attention.

**occur** 

**9.2 Distribution of antivenoms: guaranteeing access to regions where snakebites** 

Even if governments purchase adequate volumes of antivenom, this does not guarantee that these drugs will reach the rural health posts where most snakebite envenomings occur. This problem has diverse roots, such as: (a) Incomplete information on the epidemiology of snakebites. In countries where official statistics of snakebite incidence are lacking or incomplete, the decision on where to distribute antivenoms cannot be taken on a rigorous base. This is another reason for underscoring the relevance of proper epidemiological register of this pathology. (b) Antivenom acquisition procedures by the ministries of health in many countries are slow and cumbersome; moreover, due to budgetary constraints, the volumes of antivenom purchased are often insufficient; both of these factors preclude the distribution of adequate volumes of this drug to rural settings. (c) Antivenoms are often distributed only to hospitals and clinics in large cities, distant from the regions where the majority of snakebites occur, thus affecting the timely treatment of patients. (d) As discussed previously, the lack of an adequate cold chain system in many rural settings of the world precludes the effective distribution of antivenoms. (e) Many rural regions are devoid of healthcare facilities, thus affecting the access to antivenom and other medical interventions

This complex scenario demands the design of well-structured and effective strategies of antivenom distribution, on the basis of sound epidemiological information on snakebite incidence. An intersectorial and interprogrammatic approach should be promoted, in conjunction with other efforts being performed in the public health realm, in order to favour a synergy with other actors and projects, with the consequent impact in the costeffectiveness of interventions (WHO, 2007c). The compulsory report of snakebite envenoming (WHO, 2010a) and the use of geographical information systems to identify high risk areas (Hansson et al., 2010; Leynaud & Reati, 2009) would greatly contribute to generate a solid basis of information on the actual magnitude of the problem. Furthermore, the awareness of national and regional health authorities on the impact of snakebite envenoming should be promoted by academic, public health and civil society organizations, in order to ensure the acquisition and distribution of the required volumes of antivenom. Likewise, antivenom distribution strategies should benefit from the use of the cold chain system already developed for vaccine distribution. Also, the provision of antivenom access to rural settings and the training of rural health staff in the correct administration of antivenoms should be prioritized. Interventions tailored to the conditions of each country and region should be promoted, in order to optimize the available resources and guarantee a rapid access to treatment (see for example Otero et al., 1992).

#### **9.3 Promoting the correct use of antivenoms**

The distribution of antivenoms to regions where snakebites occur should be complemented by a proper training of health staff in the correct usage of this product and in the proper treatment of snakebite envenomings. There is evidence of poor knowledge of medical and nursing staff in various regions of the world on how to diagnose and treat snakebite envenomings, how to use antivenoms, and how to treat possible adverse reactions to their administration (Gutiérrez et al., 2009c; Simpson, 2008). This requires concerted efforts at medical and nursing schools in the universities, as well as the implementation of permanent educational programs on this subject, particularly aimed at rural health facilities. Likewise, the implementation of teaching material and the development of guidelines for the diagnosis and treatment of snakebite envenomings should be actively promoted, both at regional (WHO, 2010b) and national levels. These tasks should involve not only teaching institutions, but also public health authorities, local organizations of the civil society, and antivenom manufacturers. The critical revision of antivenom prospects, on the basis of current knowledge on the taxonomy of snakes and on the clinics of snakebite envenomings, are of great relevance, in the light of evident misconceptions included in the prospects of some antivenoms (Simpson & Norris, 2007).

#### **10. Prevention of snakebites**

Prevention programs aimed at reducing the impact and incidence of snakebite envenomings should be a priority in the international efforts required to confront this problem. The design of these programs should be tailored to the cultural, social, economic and institutional characteristics of the populations, and should involve the active participation of the communities in their design and implementation. Impoverished and excluded groups, such as indigenous communities in many parts of the world, should receive particular attention. The design of these programs should be also based on sound social science research aimed at understanding the particularities and needs of each region and context, with the participation of the communities. It is highly relevant, for instance, to understand how the problem is perceived in the community and what types of preventive interventions are suited for each particular context. Likewise, specific strategies should be designed for situations involving natural disasters, as snakebites have been reported to increase in such

Snakebite Envenoming: A Public Health Perspective 149

within distribution channels already developed for other immunobiologicals, such as vaccines. (b) Incorporating antivenoms in integrated drug purchasing schemes in developing countries on a regional basis. (c) Strengthening the development of public health systems in remote rural areas where snakebite envenomings are frequent. (d) Promoting partnerships of diverse sorts with groups involved in the combat of neglected tropical diseases, such as foundations, nongovernmental organizations and other advocacy groups. (e) Including snakebite envenoming in the agenda of organizations that provide financial support for research and intervention programs for neglected tropical diseases in developing countries. (f) Incorporating the subject of snakebite prevention, diagnosis and treatment within the context of educational packages on neglected tropical diseases in teaching institutions, public health facilities and communities. (g) Promoting the inclusion of the subject of snakebite envenoming within the agenda of community organizations for the promotion of health in rural areas of countries in Africa, Asia

1. Acquisition of reliable information on snakebite incidence and mortality

7. Accessibility of antivenoms at affordable prices in low-income countries 8. Preclinical and clinical assessment of antivenom efficacy and safety

3. Strengthening the capacity of laboratories in low-income countries to manufacture

4. Commitment of manufacturers to produce antivenoms for regions devoid of local

5. Implementation of economic strategies to ensure the sustainable production of

6. Improvement of the national regulatory expertise and quality control of

9. Development of effective antivenom distribution programs to regions of high

10. Permanent training programs for health staff on snakebite envenomings and their

11. Development of programs to support people suffering from sequelae of snakebite

12. Preventive and educational programs at the community level with involvement of

Table 1. Summary of some of the most important tasks for an integrated strategy to confront the problem of snakebite envenoming from a global perspective. Adapted from Gutiérrez et

In the long term, the reduction of the impact of snakebite morbidity and mortality, with its drastic effects on the quality of human life, should involve a global partnership incorporating many different actors at various levels in our societies, such as: (a) The scientific ('epistemic') community of toxinologists, represented by the International Society on Toxinology (IST) and researchers in every continent. (b) Groups working on technological development and technology transfer activities, both in the pharmaceutical industry and in university departments. (c) Antivenom manufacturers. (d) International health organizations, especially the WHO and its regional offices. (e) National public health

2. Innovation in the technology for the production of antivenoms

and Latin America.

and control antivenoms

incidence of snakebites

antivenoms in low-income countries

production

antivenoms

treatment

al. (2010b)

envenomings

local organizations

circumstances. In addition, the natural history of envenomings should be considered, including the distribution of snakes in various types of crops and the behaviour of snakes. In some regions of Asia, bites by kraits (genus *Bungarus*) often occur at nights inside human dwellings while people are asleep on the ground (Sharma et al., 2004). The use of mosquito nets has reduced the incidence of envenoming by kraits in Nepal (Chapuis et al., 2007). The majority of viperid snakebites occur in the feet; thus, a preventive measure should be the use of footware (Alirol et al., 2010; Gutiérrez, 2010; Warrell, 2010).

#### **11. Final remarks: The need for an integrated approach and for the promotion of partnerships**

The world wide efforts required to reduce the impact of snakebite envenoming should be conceptualized within the frame of the Millennium Development Goals (MDGs) (http://www.un.org/millenniumgoals/global.shtml), particularly regarding the provision of access to essential drugs (WHO, 2011), in this case antivenoms, to developing countries. The access to adequate health services is a human right, and states and other international instances have the obligation to ensure the access to health facilities, goods and services on a non-discriminatory basis, especially to vulnerable and marginalized groups, and to provide education and access to information to the communities on relevant health issues, such as snakebite envenoming. Therefore, interventions aimed at ameliorating the impact of this pathology should be viewed within a frame of human rights and social responsibility of states, international organizations and non-governmental groups.

Snakebite envenoming is a 'tool-ready' disease, in the sense that the basic technological therapeutic tools to treat this pathology, i.e. antivenoms, are available. However, there is a need to implement renewed efforts to improve the quality of some antivenoms, to design new antivenoms for various regions in the world, and to increase the volume of production as to fulfil the world wide needs to these immunobiologicals. Scientific, technological and public health tasks include acquisition of more rigorous data on the incidence of snakebite incidence and mortality, assessment of preclinical and clinical performance of currently available antivenoms, and development of novel antivenoms on the basis of epidemiological, biochemical, toxicological and immunological knowledge on venoms. Moreover, the strengthening of antivenom manufacture on a global basis should involve an active process of technology transfer and training aimed at improving the current technological platform of many antivenom producers, especially those located in developing countries. Finally, renewed efforts should be undertaken to guarantee the deployment and effective distribution and use of antivenoms to the regions of the world where this pathology has its highest impact. Table 1 summarizes some of the most pressing tasks that need to be promoted as part of a global strategy to reduce the impact of snakebite envenomings.

The design of effective strategies to confront this problem should be also integrated with the more general efforts in the arena of neglected tropical diseases (WHO, 2007c). Such strategies should be conceived from an intersectorial and interprogrammatic perspective (see WHO, 2007c), with a synergistic approach involving the control of other neglected tropical diseases; such an approach will significantly increase the cost-effectiveness of interventions. Areas of possible interprogrammatic cooperation include: (a) The collaborative delivery of antivenoms

circumstances. In addition, the natural history of envenomings should be considered, including the distribution of snakes in various types of crops and the behaviour of snakes. In some regions of Asia, bites by kraits (genus *Bungarus*) often occur at nights inside human dwellings while people are asleep on the ground (Sharma et al., 2004). The use of mosquito nets has reduced the incidence of envenoming by kraits in Nepal (Chapuis et al., 2007). The majority of viperid snakebites occur in the feet; thus, a preventive measure should be the

**11. Final remarks: The need for an integrated approach and for the promotion of** 

The world wide efforts required to reduce the impact of snakebite envenoming should be conceptualized within the frame of the Millennium Development Goals (MDGs) (http://www.un.org/millenniumgoals/global.shtml), particularly regarding the provision of access to essential drugs (WHO, 2011), in this case antivenoms, to developing countries. The access to adequate health services is a human right, and states and other international instances have the obligation to ensure the access to health facilities, goods and services on a non-discriminatory basis, especially to vulnerable and marginalized groups, and to provide education and access to information to the communities on relevant health issues, such as snakebite envenoming. Therefore, interventions aimed at ameliorating the impact of this pathology should be viewed within a frame of human rights and social responsibility of

Snakebite envenoming is a 'tool-ready' disease, in the sense that the basic technological therapeutic tools to treat this pathology, i.e. antivenoms, are available. However, there is a need to implement renewed efforts to improve the quality of some antivenoms, to design new antivenoms for various regions in the world, and to increase the volume of production as to fulfil the world wide needs to these immunobiologicals. Scientific, technological and public health tasks include acquisition of more rigorous data on the incidence of snakebite incidence and mortality, assessment of preclinical and clinical performance of currently available antivenoms, and development of novel antivenoms on the basis of epidemiological, biochemical, toxicological and immunological knowledge on venoms. Moreover, the strengthening of antivenom manufacture on a global basis should involve an active process of technology transfer and training aimed at improving the current technological platform of many antivenom producers, especially those located in developing countries. Finally, renewed efforts should be undertaken to guarantee the deployment and effective distribution and use of antivenoms to the regions of the world where this pathology has its highest impact. Table 1 summarizes some of the most pressing tasks that need to be promoted as part of a global strategy to reduce the impact of snakebite

The design of effective strategies to confront this problem should be also integrated with the more general efforts in the arena of neglected tropical diseases (WHO, 2007c). Such strategies should be conceived from an intersectorial and interprogrammatic perspective (see WHO, 2007c), with a synergistic approach involving the control of other neglected tropical diseases; such an approach will significantly increase the cost-effectiveness of interventions. Areas of possible interprogrammatic cooperation include: (a) The collaborative delivery of antivenoms

use of footware (Alirol et al., 2010; Gutiérrez, 2010; Warrell, 2010).

states, international organizations and non-governmental groups.

**partnerships** 

envenomings.

within distribution channels already developed for other immunobiologicals, such as vaccines. (b) Incorporating antivenoms in integrated drug purchasing schemes in developing countries on a regional basis. (c) Strengthening the development of public health systems in remote rural areas where snakebite envenomings are frequent. (d) Promoting partnerships of diverse sorts with groups involved in the combat of neglected tropical diseases, such as foundations, nongovernmental organizations and other advocacy groups. (e) Including snakebite envenoming in the agenda of organizations that provide financial support for research and intervention programs for neglected tropical diseases in developing countries. (f) Incorporating the subject of snakebite prevention, diagnosis and treatment within the context of educational packages on neglected tropical diseases in teaching institutions, public health facilities and communities. (g) Promoting the inclusion of the subject of snakebite envenoming within the agenda of community organizations for the promotion of health in rural areas of countries in Africa, Asia and Latin America.


Table 1. Summary of some of the most important tasks for an integrated strategy to confront the problem of snakebite envenoming from a global perspective. Adapted from Gutiérrez et al. (2010b)

In the long term, the reduction of the impact of snakebite morbidity and mortality, with its drastic effects on the quality of human life, should involve a global partnership incorporating many different actors at various levels in our societies, such as: (a) The scientific ('epistemic') community of toxinologists, represented by the International Society on Toxinology (IST) and researchers in every continent. (b) Groups working on technological development and technology transfer activities, both in the pharmaceutical industry and in university departments. (c) Antivenom manufacturers. (d) International health organizations, especially the WHO and its regional offices. (e) National public health

Snakebite Envenoming: A Public Health Perspective 151

supported by Vicerrectoría de Investigación (University of Costa Rica), the International

Abubakar, I.S.; Abubakar, S.B.; Habib, A.G.; Nasidi, A.; Durfa, N.; Yusuf, P.O.; Larnyang, S.;

Abubakar, S.B.; Habib, A.G. & Mathew, J. (2010a). Amputation and disability following

Abubakar, S.B.; Abubakar, I.S.; Habib, A.G.; Nasidi, A.; Durfa, N.; Yusuf, P.O.; Larnyang, S.;

Alape-Girón, A.; Stiles, B.G. & Gutiérrez, J.M. (1996). Antibody-mediated neutralization and

Alape-Girón, A.; Sanz, L.; Escolano, J.; Flores-Díaz, M.; Madrigal, M.; Sasa, M. & Calvete, J.J.

Alirol, E.; Sharma, S.K.; Bawaskar, H.S.; Kuch, U. & Chappuis, F. (2010). Snake bite in South Asia: A review. *PLoS Neglected Tropical Diseases*, Vol.4, No.1, pp. e603 Ariaratnam, C.A.; Meyer, W.P.; Perera, G.; Addleston, M.; Kuleratne, S.A.; Attapattu, W.;

Ariaratnam, C.A.; Thuraisingam, V.; Kularatne, S.A.; Sheriff, M.H.; Theakston, R.D.G.; de

Ariaratnam, C.A.; Sheriff, M.H.; Arambepola, C.; Theakston, R.D.G. & Warrell, D.A. (2009).

Battellino, C.; Piazza, R.; da Silva, A.M.; Cury, Y. & Farsky, S.H.P. (2003). Assessment of the

*Bothrops jararaca* snake venom. *Toxicon*, Vol.41, No.5, pp. 583-593

Garnvwa, J.; Sokomba, E.; Salako, L.; Theakston, R.D.G.; Juszczak, E.; Alder, N. & Warrell, D.A. (2010). Randomised controlled double-blind non-inferiority trial of two antivenoms for saw-scaled or carpet viper (*Echis ocellatus*) envenoming in

Garnvwa, J.; Sokomba, E.; Salako, L.; Laing, G.D.; Theakston, R.D.G.; Juszczak, E.; Alder, N. & Warrell, D.A. (2010b). Pre-clinical and preliminary dose-finding and safety studies to identify candidate antivenoms for treatment of envenoming by saw-scaled or carpet vipers (*Echis ocellatus*) in northern Nigeria. *Toxicon*, Vol.55,

binding-reversal studies on α-neurotoxins from *Micrurus nigrocinctus nigrocinctus*

(2008). Snake venomics of the lancehead pitviper *Bothrops asper*: geographic, individual, and ontogenetic variations. *Journal of Proteome Research*, Vol.7, No.8, pp.

Sheriff, R.; Richards, A.M.; Theakston, R.D.G. & warrell, D.A. (1999). A new monospecific ovine Fab fragment antivenom for treatment of envenoming by the Sri Lankan Russell's viper (*Daboia russelli russelli*): a preliminary dose-finding and pharmacokinetic study. *American Journal of Tropical Medicine and Hygiene*, Vol. 61,

Silva, A. & Warrell, D.A. (2008). Frequent and potentially fatal envenoming by hump-nosed pit vipers (*Hypnale hypnale* and *H. nepa*) in Sri Lanka: lack of effective antivenom. *Transactions of the Royal Society of Tropical Medicine and Hygiene*, Vol.102,

Syndromic approach to treatment of snake bite in Sri Lanka based on results of a prospective national hospital-based survey of patients envenomed by identified snakes. *American Journal of Tropical Medicine and Hygiene*, Vol.81, No.4, pp. 725-731 Azevedo-Marques, M.M.; Hering, S.E. & Cupo, P. (2009). Acidente crotálico. In: *Animais* 

*Peçonhentos no Brasil. Biologia, Clínica e Terapêutica dos Acidentes*, *2nd. Edition*, J.L.C. Cardoso; F.O.S. França; H.W. Fan; C.M.S. Málaque & V. Haddad, (Eds), Sarvier,

efficacy of bothropic antivenom therapy on microcirculatory effects induced by

Foundation for Science (IFS), CRUSA-CSIC, NeTropica and the program CYTED.

Nigeria. *PLoS Neglected Tropical Diseases*, Vol.4, No.7, pp. e767

snakebite in Nigeria. *Tropical Doctor*, Vol.40, No.2, pp. 114-116

(coral snake) venom. *Toxicon*, Vol.34, No.3, pp. 369-380

108-115, ISBN 978-85-7378-194-6, São Paulo, Brazil.

**13. References** 

No.4, pp. 719-723

3556-3571

No.2, pp. 259-265

No.11, pp. 1120-1126

authorities, i.e. Ministries of Health and other organizations of the public health sector. (f) National regulatory bodies, responsible for ensuring the safety and efficacy of antivenoms being distributed. (g) Non-governmental organizations (NGOs) and advocacy groups that promote a public health agenda and the access of essential drugs to developing countries. (h) Organizations of the civil society of countries having a high burden of snakebite envenoming (Figure 2). The current tasks of generating a growing international awareness on the magnitude of this problem, establishing partnerships to ensure the development, availability and accessibility to antivenoms, and promoting prevention and an effective clinical management of this pathology, are being promoted by the Global Snake Bite Initiative (GSI), the WHO, and a number of national and regional projects in various parts of the world.

Fig. 2. Some of the participants that should be involved in a global partnership aimed at the reduction of the impact of snakebite envenoming in the world.

#### **12. Acknowledgements**

The author thanks his colleagues of Instituto Clodomiro Picado, University of Costa Rica, and of other groups in Latin America and elsewhere, for long-standing collaborations in this subject. Special thanks are due to David A. Warrell, David Williams, Fan Hui Wen, João Luiz Costa Cardoso, Rafael Otero-Patiño, Robert Harrison, Kenneth D. Winkel, R. David G. Theakston, Juan José Calvete, Jean Philippe Chippaux, Abdusalam Nasidi, Abdulrazaq Habib, Ulrich Kuch, and Thierry Burnouf for valuable discussions and cooperation in the field of snakebite envenoming and treatment. David Williams and Mark O'Shea kindly provided photographs of snakes. Many of the studies cited in this review have been supported by Vicerrectoría de Investigación (University of Costa Rica), the International Foundation for Science (IFS), CRUSA-CSIC, NeTropica and the program CYTED.

#### **13. References**

150 Public Health – Methodology, Environmental and Systems Issues

authorities, i.e. Ministries of Health and other organizations of the public health sector. (f) National regulatory bodies, responsible for ensuring the safety and efficacy of antivenoms being distributed. (g) Non-governmental organizations (NGOs) and advocacy groups that promote a public health agenda and the access of essential drugs to developing countries. (h) Organizations of the civil society of countries having a high burden of snakebite envenoming (Figure 2). The current tasks of generating a growing international awareness on the magnitude of this problem, establishing partnerships to ensure the development, availability and accessibility to antivenoms, and promoting prevention and an effective clinical management of this pathology, are being promoted by the Global Snake Bite Initiative (GSI), the WHO, and a number of national and regional projects in various parts of

Fig. 2. Some of the participants that should be involved in a global partnership aimed at the

The author thanks his colleagues of Instituto Clodomiro Picado, University of Costa Rica, and of other groups in Latin America and elsewhere, for long-standing collaborations in this subject. Special thanks are due to David A. Warrell, David Williams, Fan Hui Wen, João Luiz Costa Cardoso, Rafael Otero-Patiño, Robert Harrison, Kenneth D. Winkel, R. David G. Theakston, Juan José Calvete, Jean Philippe Chippaux, Abdusalam Nasidi, Abdulrazaq Habib, Ulrich Kuch, and Thierry Burnouf for valuable discussions and cooperation in the field of snakebite envenoming and treatment. David Williams and Mark O'Shea kindly provided photographs of snakes. Many of the studies cited in this review have been

reduction of the impact of snakebite envenoming in the world.

**12. Acknowledgements** 

the world.


Snakebite Envenoming: A Public Health Perspective 153

Chappuis, F.; Sharma, S.K.; Jha, N.; Loutan, L. & Bovier, P.A. (2007). Protection against

Chippaux, J.P.; Williams, V. & White, J. (1991). Snake venom variability: methods of study,

Chippaux, J.P. (1998). Snake-bites: appraisal of the global situation. *Bulletin of the World* 

Chippaux, J.P.; Lang, J.; Eddine, S.A.; Fagot, P.; Rage, V.; Peyrieux, J.C. & Le Mener, V.

Chippaux, J.P. (2010). Snakebite in Africa. Current situation and urgent needs, In: *Handbook* 

Chippaux, J.P. (2011). Estimate of the burden of snakebites in sub-Saharan Africa: a meta-

Chotwiwatthanakun, C.; Pratanaphon, R.; Akesowan, S.; Sriprapat, S. & Ratanabanangkoon,

Corrêa-Netto, C.; Junqueira-de-Azevedo, I.; Silva, D.A.; Ho, P.L.; Leitão-de-Araújo, M.;

*altirostris* and *M. corallinus*. *Journal of Proteomics*, Vol.74, No.9, pp. 1795-1809 Cupo, P.; Azevedo-Marques, M.M.; de Menezes, J.B. & Hering, S.E. (1991). Reações de

Dart, R.C.; Mcnally, J.T.; Spaite, D.W. & Gustafson, R. (1992). The sequelae of pitviper

de Oliveira, R.C.; Fan, H.W. & Sifuentes, D.N. (2009). Epidemiologia dos accidentes por

dos Santos, M.C.; D'Imperio-Lima, M.R.; Furtado, G.C.; Colletto, G.M.; Kipnis, T.L & Dias

Dufton, M.J. & Hider, R.C. (1988). Structure and pharmacology of elapid cytotoxins.

Brodie, (Eds.), 395-404, Selva, ISBN 0-9630537-0-1, Texas, USA.

*Tropical Medicine and Hygiene*, Vol.77, No.1, pp. 197-199

*Health Organization*, Vol.76, No.5, pp. 515-524

*Medicine and Hygiene*, Vol.92, No.6, pp. 657-662

*Tropical de São Paulo*, Vol.33, No.2, pp. 115-122

trial. *PLoS Medicine*, Vol.8, No.5, pp. e1000435

*Pharmacology and Therapy*, Vol.36, No.1, pp. 1-40

yield. *Toxicon*, Vol.27, No.3, pp. 297-303.

analytic approach. *Toxicon*, Vol.57, No.4, pp. 586-599

0-8493-9165-1, Boca Raton, USA

No.10, pp. 1487-1494

results and interpretation. *Toxicon*, Vol.29, No.11, pp. 1279-1303

2637-2649.

metalloproteinase toxin genes. *Molecular Biology and Evolution*, Vol.28, No.9, pp.

snake bites by sleeping under a bed net in southeastern Nepal. *American Journal of* 

(1998). Clinical safety of a polyvalent F(ab')2 equine antivenom in 223 African snake envenomations: a field trial in Cameroon. *Transactions of the Royal Society of Tropical* 

*of Venoms and Toxins of Reptiles*, S.P. Mackessy, (Ed.), 453-473, CRC Press, ISBN 978-

K. (2001). Production of potent polyvalent antivenom against three elapid venoms using a low dose, low volume, multi-site immunization protocol. *Toxicon*, Vol.39,

Alves, M.L.; Sanz, L.; Foguel, D.; Zingali, R.B. & Calvete, J.J. (2011). Snake venomics and venom gland transcriptomic analysis of Brazilian coral snakes, *Micrurus* 

hipersensibilidade imediatas após uso intravenoso de soros antivenenos: valor prognóstico dos testes de sensibilidade intradérmicos. *Revista Instituto de Medicina* 

poisoning in the United States. In: Biology of the Pitvipers, J.A. Cambpell & E.D.

animais peçonhentos. In: *Animais Peçonhentos no Brasil. Biologia, Clínica e Terapêutica dos Acidentes*, *2nd Edition*, J.L.C. Cardoso; F.O.S. França; H.W. Fan; C.M.S. Málaque & V. Haddad, (Eds), Sarvier, 6-21, ISBN 978-85-7378-194-6, São Paulo, Brazil. de Silva, H.A.; Pathmeswaran, A.; Ranasinha, C.D.; Jayamanne, S.; Samarakoon, S.B.;

Hittharage, A.; Kalupahana, R.; Ratnatilaka, G.A.; Uluwatthage, W.; Aronson, J.K.; Armitage, J.M.; Lalloo, D.G & de Silva, H.J. (2011). Low-dose adrenaline, promethazine, and hydrocortisone in the prevention of acute adverse reactions to antivenom following snakebite: a randomised, double-blind, placebo-controlled

da Silva, W. (1989). Purification of F(ab')2 anti-snake venom by caprylic acid: a fast method for obtaining IgG fragments with high neutralization activity, purity and


Bdolah, A. (2010). Sarafotoxins, the snake venom homologs of the endothelins, In: *Handbook* 

Bon, C. (1996). Serum therapy was discovered 100 years ago. In: *Envenomings and Their* 

Bon, C. (1997). Multicomponent neurotoxic phospholipases A2. In: *Venom Phospholipase A2*

Borkow, G.; Gutiérrez, J.M. & Ovadia, M. (1997). Inhibition of toxic activities of *Bothrops* 

Boulain, J.C.; Ménez, A. (1982). Neurotoxin-specific immunoglobulins accelerate dissociation of the neurotoxin-acetylcholine receptor complex. *Science*, Vol.217, pp. 732-733 Boyer, L.V.; Seifert, S.A. & Cain, J.S. (2001). Recurrence phenomena after immunoglobulin

Burnouf, T.; Griffiths, E.; Padilla, A.; Seddick, S.; Stephano, M.A. & Gutiérrez, J.M. (2004).

Calvete, J.J.; Juárez, P. & Sanz, L. (2007). Snake venomics. Strategy and applications. *Journal* 

Calvete, J.J.; Sanz, L.; Angulo, Y.; Lomonte, B. & Gutiérrez, J.M. (2009). Venoms, venomics,

Calvete, J.J. (2010). Antivenomics and venom phenotyping: A marriage of convenience to

Calvete, J.J.; Sanz, L.; Cid, P.; de la Torre, P.; Flores-Díaz, M.; Dos Santos, M.C.; Borges, A.;

Calvete, J.J.; Cid, P.; Sanz, L.; Segura, A.; Villalta, M.; Herrera, M.; León, G.; Harrison, R.;

*American Journal of Tropical Medicine and Hygiene*, Vol.82, No.6, pp 1194-1201 Cardoso, J.L.C.; Fan, H.W.; França, F.O.S.; Jorge, M.T.; Leite, R.P.; Nishioka, S.A.; Avila, A.;

Casewell, N.R.; Wagstaff, S.C.; Harrison, R.A.; Renjifo, C. & Wüster, W. (2011). Domain loss

Brazil. *Quarterly Journal of Medicine*, Vol.86, No.5, pp. 315-325

*Toxicology and Applied Pharmacology*, Vol.147, No.2, pp. 442-447

0-8493-9165-1, Boca Raton, USA

0-471-96189-2, Chichester, United Kingdom.

*Toxicon*, Vol.55, No.7, pp. 1405-1407

*Biologicals*, Vol.32, No.3, pp. 115-128

*Research*, Vol.9, No.1, pp. 528-544

No.7, pp. 1284-1291

*of Mass Spectrometry*, Vol.42, No.11, pp. 1405-1414

antivenomics. *FEBS Letters*, Vol.583, No.11, pp. 1736-1743

France.

*of Venoms and Toxins of Reptiles*, S.P. Mackessy, (Ed.), 303-315, CRC Press, ISBN 978-

*Treatments*, C. Bon & M. Goyffon, (Eds), 3-9, Fondation Marcel Mérieux, Lyon,

*Enzymes: Structure, Function and Mechanism*, R.M. Kini, (Ed.), 269-285, Wiley, ISBN

*asper* venom and other crotalid snake venoms by a novel neutralizing mixture.

therapy for snake envenomations: Part 2. Guidelines for clinical management with crotaline Fab antivenom. *Annals of Emergency Medicine*, Vol.37, No.2, pp. 196-201 Brown, N. & Landon, J. (2010). Antivenom: the most cost-effective treatment in the world?

Assessment of the viral safety of antivenoms fractionated from equine plasma.

address the performance and range of clinical use of antivenoms. *Toxicon*, Vol.56,

Bremo, A.; Angulo, Y.; Lomonte, B.; Alape-Girón, A. & Gutiérrez, J.M. (2010a). Snake venomics of the Central American rattlesnake *Crotalus simus* and the South American *Crotalus durissus* complex points to neurotoxicity as an adaptive paedomorphic trend along Crotalus dispersal in South America. *Journal of Proteome* 

Durfa, N.; Nasidi, A.; Theakston, R.D.G.; Warrell, D.A. & Gutiérrez, J.M. (2010b). Antivenomic assessment of the immunological reactivity of EchiTAb-Plus-ICP, an antivenom for the treatment of snakebite envenoming in sub-Saharan Africa.

Sano-Martins, I.S.; Tomy, S.C.; Santoro, M.L.; Chudzinski, A.M.; Castro, S.C.B.; Kamiguti, A.S.; Kelen, E.M.A.; Hirata, M.H.; Mirandola, R.M.S.; Theakston, R.D.G. & Warrell, D.A. (1993). Randomized comparative trial of three antivenoms in the treatment of envenoming by lance-headed vipers (*Bothrops jararaca*) in São Paulo,

facilitates accelerated evolution and neofunctionalization of duplicate snake venom

metalloproteinase toxin genes. *Molecular Biology and Evolution*, Vol.28, No.9, pp. 2637-2649.


Snakebite Envenoming: A Public Health Perspective 155

Gutiérrez, J.M.; Theakston, R.D.G. & Warrell, D.A. (2006). Confronting the neglected

Gutiérrez, J.M.; Lomonte, B.; León, G.; Rucavado, A.; Chaves, F. & Angulo, Y. (2007). Trends

considerations. *Current Pharmaceutical Design*, Vol.13, No.28, pp. 2935-2950 Gutiérrez, J.M. & León, G. (2009). Snake antivenoms. Technological, clinical and public

Gutiérrez, J.M. & Lomonte, B. (2009). Efectos locales en el envenenamiento ofídico en

Gutiérrez, J.M.; Lomonte, B.; León, G.; Alape-Girón, A.; Flores-Díaz, M.; Sanz, L.; Angulo, Y.

Gutiérrez, J.M.; Escalante, T. & Rucavado, A. (2009b). Experimental pathophysiology of

Gutiérrez, J.M.; Fan, H.W.; Silvera, C.L. & Angulo, Y. (2009c). Stability, distribution and use

Gutiérrez, J.M. (2010). Snakebite envenomation in Central America, In: *Handbook of Venoms* 

Gutiérrez, J.M.; Rucavado, A. & Escalante, T. (2010a). Snake venom metalloproteinases.

Gutiérrez, J.M.; Williams, D.; Fan, H.W. & Warrell, D.A. (2010b). Snakebite envenoming

Gutiérrez, J.M. (2011). Envenenamientos por mordeduras de serpientes en América Latina y

Gutiérrez, J.M.; León, G. & Burnouf, T. (2011a). Antivenoms for the treatment of snakebite

Gutiérrez, J.M.; León, G.; Lomonte, B. & Angulo, Y. (2011b). Antivenoms for snakebite envenomings. *Inflammation & Allergy-Drug Targets*, Vol. 10, No.5, pp. 369-380 Habib, A.G.; Gebi, U.I. & Onyemelukwe, G.C. (2001). Snake bite in Nigeria. *African Journal of* 

Hansson, E.; Cuadra, S.; Oudin, A.; de Jong, K.; Stroh, E.; Torén, K. & Albin, M. (2010).

envenomings: the road ahead. *Biologicals*, Vol.39, No.3, pp. 129-142

(Eds), Sarvier, 352-365, ISBN 978-85-7378-194-6, São Paulo, Brazil.

*Journal of Proteomics*, Vol.72, No.2, pp. 165-182

*Toxicon*, Vol.53, No.6, pp. 625-630

*Ambiental*, Vol.51, No.1, pp. 1-16

ISBN 978-0-8493-9165-1, Boca Raton, USA

*Medicine and Medical Sciences*, Vol.30, pp. 171-178

*PLoS Neglected Tropical Diseases*, Vol.4, No.11, pp. e896

9165-1, Boca Raton, USA

*Tropical Medicine and Hygiene*, Vol. 99, No.6, pp. 468-475

*Medicine*, Vol. 3, No.6, pp. e150

Brazil.

pp. 976-987

pp. 1223-1235

alternative to the antivenom crisis in Africa. *Transactions of the Royal Society of* 

problem of snake bite envenoming: the need for a global partnership. *PLoS* 

in snakebite envenomation therapy: scientific, technological and public health

health issues. In: *Animal Toxins: State of the Art. Perspectives in Health and Biotechnology*, M.E. de Lima; A.M.C. Pimenta; M.F. Martin-Euclaire; R.B. Zingali & H. Rochat, (Eds.), 393-421, Editora UFMG, ISBN 978-85-7041-735-0, Belo Horizonte,

América Latina. In: *Animais Peçonhentos no Brasil. Biologia, Clínica e Terapêutica dos Acidentes*, J.L.C. Cardoso; F.O.S. França; H.W. Fan; C.M.S. Málaque & V. Haddad,

& Calvete, J.J. (2009a). Snake venomics and antivenomics: Proteomic tools in the design and control of antivenoms for the treatment of snakebite envenoming.

systemic alterations induced by *Bothrops asper* snake venom. *Toxicon*, Vol.54, No.7,

of antivenoms for snakebite envenomation in Latin America: report of a workshop.

*and Toxins of Reptiles*, S.P. Mackessy, (Ed.), 491-507, CRC Press, ISBN 978-0-8493-

Biological roles and participation in the pathophysiology of envenomation, In: *Handbook of Venoms and Toxins of Reptiles*, S.P. Mackessy, (Ed.), 115-138, CRC Press,

from a global perspective: Towards an integrated approach. *Toxicon*, Vol.56, No.7,

el Caribe: Una visión integral de carácter regional. *Boletín de Malariología y Salud* 

Mapping snakebite epidemiology in Nicaragua-Pitfalls and possible solutions.


Escalante, T.; Rucavado, A.; Fox, J.W. & Gutiérrez, J.M. (2011). Key events in microvascular

Faiz, A.; Ghose, A.; Ahsan, F.; Rahman, R.; Amin, R.; Hassan, M.U.; Chowdhury, A.W.;

snake bite envenoming in Bangladesh. *Brain*, Vol.133, No.11, pp. 3181-3193 Fan, H.W. & Cardoso J.L. (1995). Clinical toxicology of snake bites in South America, In:

Ferquel, E.; de Haro, L.; Jan, V.; Guillemin, I.; Jourdain, S.; Teynié, A.; d'Alayer, J. &

Fox, J.W. & Serrano, S.M.T. (2005). Structural considerations of the snake venom

Fox, J.W. & Serrano S.M.T. (2008). Exploring snake venom proteomes: multifaceted analyses

Fry, B.G.; Vidal, N.; Norman, J.A.; Vonk, F.J.; Scheib, H.; Ramjan, S.F.; Kuruppu, S.; Fung, K.;

Fry, B.G.; Vidal, N.; van der Weerd, L.; Kochva, E. & Renjifo, C. (2009) Evolution and

Gawarammana, I.B.; Kularatne, S.A.; Dissanayake, W.P.; Kumarasiri, R.P.; Senanayake, N. &

Gómez, H.F. & Dart, R.C. (1995). Clinical toxicology of snakebite in North America, In:

Grandgeorge, M.; Véron, J.L.; Lutsch, C.; Makula, M.F.; Riffard, P.; Pépin, S. & Scherrmann,

Gutiérrez, J.M.; León, G.; Rojas, G.; Lomonte, B.; Rucavado, A. & Chaves, F. (1998).

Gutiérrez, J.M. & Ownby, C.L. (2003). Skeletal muscle degeneration induced by venom

Gutiérrez, J.M.; León, G. & Lomonte, B. (2003). Pharmacokinetic-pharmacodynamic

Gutiérrez, J.M.; Rojas, E.; Quesada, L.; León, G.; Núñez, J.; Laing, G.D.; Sasa, M.; Renjifo,

(Eds.), 619-644, CRC Press, ISBN 0-8493-4489-1, Boca Raton, USA

(Ed.), 667-688, CRC Press, ISBN 0-8493-4489-1, Boca Raton, USA

for complex toxin mixtures. *Proteomics*, Vol.8, No.4, pp. 909-920

metalloproteinases. *Toxicon*, Vol.45, No.8, pp. 969-985

*Medical Journal of Australia*, Vol.180, No.1, pp. 20-23

venom. *Toxicon*, Vol.36, No.11, pp. 1529-1538

*Pharmacokinetics*, Vol.42, No.8, pp. 721-741.

*Toxicon*, Vol.42, No.8, pp. 915-931

*Proteomics*, Vol.74, No.9, pp. 1781-1794

Vol.2, No.11, pp. e1194

*Nature*, Vol.439, pp. 584-588

No.2, pp. 127-136

France

damage induced by snake venom hemorrhagic metalloproteinases. *Journal of* 

Kuch, U.; Rocha, T.; Harris, J.B.; Theakston, R.D.G. & Warrell, D.A. (2010). The greater black krait (*Bungarus niger*), a newly recognized cause of neuro-myotoxic

*Handbook of Clinical Toxicology of Animal Venoms and Poisons*, J. Meier & J. White,

Choumet, V. (2007). Reappraisal of *Vipera aspis* venom neurotoxicity. *PLoS ONE*,

metalloproteinases, key members of the M12 reprolysin family of

Hedges, S.B.; Richardson, M.K.; Hodgson, W.C.; Ignjativoc, V.; Summerhayes, R. & Kochva, E. (2006). Early evolution of the venom system in lizards and snakes.

diversification of the Toxicofera reptile venom system. *Journal of Proteomics*, Vol.72,

Ariyasena, H. (2004). Parallel infusion of hydrocortisone +/- chlorpheniramine bolus injection to prevent acute adverse reactions to antivenom for snakebites.

*Handbook of Clinical Toxicology of Animal Venoms and Poisons*, J. Meier & J. White,

J.M. (1996). Preparation of improved F(ab')2 antivenoms. An example: new polyvalent European viper antivenom (equine). In: *Envenomings and Their Treatments*, C. Bon & M. Goyffon, (Eds), 161-172, Fondation Marcel Mérieux, Lyon,

Neutralization of local tissue damage induced by *Bothrops asper* (terciopelo) snake

phospholipases A2: insights into the mechanisms of local and systemic myotoxicity.

relationships of immunoglobulin therapy for envenomation. *Clinical* 

J.M.; Nasidi, A.; Warrell, D.A.; Theakston, R.D.G. & Rojas, G. (2005). Pan-African polyspecific antivenom produced by caprylic acid purification of horse IgG: an alternative to the antivenom crisis in Africa. *Transactions of the Royal Society of Tropical Medicine and Hygiene*, Vol. 99, No.6, pp. 468-475


Snakebite Envenoming: A Public Health Perspective 157

Leynaud, G.C. & Reati, G.J. (2009). Identificación de las zonas de riesgo ofídico en Córdoba,

Lomonte, B.; Angulo, Y. & Calderón, L. (2003). An overview of lysine-49 phospholipase A2

Lomonte, B.; Escolano, J.; Fernández, J.; Sanz, L.; Angulo, Y.; Gutiérrez, J.M. & Calvete, J.J.

Lomonte, B.; León, G.; Angulo, Y.; Rucavado, A. & Núñez, V. (2009). Neutralization of

LoVecchio, F.; Klemens, J.; Roundy, E.B. & Klemens, A. (2003). Serum sickness following

Mackessy, S.P. (2002). Biochemistry and pharmacology of colubrid snake venoms. *Journal of* 

Malasit, P.; Warrell, D.A.; Chanthavanich, P.; Viravan, C.; Mongkolsapaya, J.; Singhthong, B.

Manock, S.R.; Suarez, G.; Graham, D.; Avila-Agüero, M.L. & Warrell, D.A. (2008).

Meyer, W.P.; Habib, A.G.; Onayade, A.A.; Yakubu, A.; Smith, D.C.; Nasidi, A.; Daudu, I.J.;

Michael, G.C.; Thacher, T.D. & Shehu, M.I.L. (2010). The effect of pre-hospital care for

Mohapatra, B.; Warrell, D.A.; Suraweera, W.; Bhatia, P.; Dhingra, N.; Jotkar, R.M.;

Moura-da-Silva, A.M.; Serrano, S.M.T.; Fox, J.W. & Gutiérrez, J.M. (2009) Snake venom

*Society of Tropical Medicine and Hygiene*, Vol.102, No.11, pp. 1127-1132 Meier, J. & Stocker, K.F. (1995). Biology and distribution of venomous snakes of medical

myotoxic action. *Toxicon*, Vol.42, No.8, pp. 885-901.

*Toxicology-Toxin Reviews*, Vol.21, No. 1-2, pp. 43-83.

Press, ISBN 0-8493-4489-1, Boca Raton, USA

Vol.5, No.4, pp. e1018

*Tropical Medicine and Hygiene*, Vol.56, No.3, pp. 291-300

*Tropical Medicine and Hygiene*, Vol.105, No.2, pp. 95-101

*Toxicon*, Vol.54, No.7, pp. 1012-1028

1115-1119

pp. 2445-2457.

220-221

17-20

Vol.26, No.1, pp. 64-69

*Transactions of the Royal Society of Tropical Medicine and Hygiene*, Vol.102, No.11, pp.

Argentina, mediante el programa SIGEpi. *Revista Panamericana de Salud Pública*,

myotoxins from crotalid snake venoms and their structural determinants of

(2008). Snake venomics and antivenomics of the arboreal neotropical pitvipers *Bothriechis lateralis* and *Bothriechis schlegelii*. *Journal of Proteome Research*, Vol.7, No.6,

*Bothrops asper* venom by antibodies, natural products and synthetic drugs: contributions to understanding snakebite envenomings and their treatment.

administration of Antivenin (Crotalidae) Polyvalent in 181 cases of presumed rattlesnake envenomation. *Wilderness and Environmental Medicine*, Vol. 14, No.4, pp.

& Supich, C. (1986). Prediction, prevention, and mechanism of early (anaphylactic) antivenom reactions in victims of snake bites. *British Medical Journal*, Vol. 292, pp.

Neurotoxic envenoming by South American coral snake (*Micrurus lemniscatus helleri*): case report from eastern Ecuador and review. *Transactions of the Royal* 

importance and the composition of snake venoms, In: *Handbook of Clinical Toxicology of Animal Venoms and Poisons*, J. Meier & J. White, (Eds.), 367-412, CRC

Warrell, D.A. & Theakston, R.D.G. (1997). First clinical experiences with a new ovine Fab *Echis ocellatus* snake bite antivenom in Nigeria: randomized comparative trial with Institute Pasteur Serum (IPSER) Africa Antivenom. *American Journal of* 

venomous snake bite on outcome in Nigeria. *Transactions of the Royal Society of* 

Rodriguez, P.S.; Mishra, K.; Whitaker, R. & Jha, P. (2011). Snakebite mortality in India: a nationally representative mortality survey. *PLoS Neglected Tropical Diseases*,

metalloproteinases. Structure, function and effects on snake bite pathology. In: *Animal Toxins: State of the Art. Perspectives in Health and Biotechnology*, M.E. de Lima;


Hardy, D.L. (2009). Alternatives in the field management of venomous snakebite. In:

Harrison, R.A.; Hargreaves, A.; Wagstaff, S.C.; Faragher, B. & Lalloo, D.G. (2009). Snakebite

Harvey, A.L. (2010). Fasciculins. Toxins from mamba venoms that inhibit

Hotez, P.J.; Molyneux, D.H.; Fenwick, A.; Ottesen, E.; Ehrlich-Sachs, S. & Sachs, J.D. (2006).

Ismail, M.; Abd-Elsalam, M.A. & Al-Ahaidib, M.S. (1998). Pharmacokinetics of 125I-labelled

Kasturiratne, A.; Wickremasinghe, A.R.; de Silva, N.; Gunawardena, N.K.; Pathmeswaran,

Kini R.M & Chan, Y.M. (1999). Accelerated evolution and molecular surface of venom phospholipase A2 enzymes. *Molecular Evolution*, Vol.48, No.2, pp. 125-132 Kini, R.M. (2005). The intriguing world of prothrombin activators from snake venom.

Kulkeaw, K.; Chaicumpa, W.; Sakolvaree, Y.; Tongtawe, P. & Tapchaisiri, P. (2007).

Lalloo, D.G. & Theakston, R.D.G. (2003). Snake antivenoms. *Journal of Toxicology-Clinical* 

Larrick, J.W.; Yost, J.A. & Kaplan, J. (1978). Snake bite among the Waorani Indians of Eastern

León, G.; Rodríguez, M.A.; Rucavado, A.; Lomonte, B. & Gutiérrez, J.M. (2007). Anti-human

León, G.; Segura, A.; Herrera, M.; Otero, R.; França, F.O.S.; Barbaro, K.C.; Cardoso, J.L.C.;

snakebite envenomations. *Biologicals*, Vol.35, No.1, pp. 5-11

IgG, F(ab')2 and Fab of the antivenin. *Toxicon*, Vol.36, No.1, pp. 93-114 Jayanthi G.P. & Gowda, T.V. (1988). Geographical variation in India in the composition and

of envenoming and deaths. *PLoS Medicine*, Vol.5, No.11, pp. e218

*Toxicon*, Vol.45, No.8, pp. 1133-1145

*Toxicology*, Vol. 41, No.3, pp. 277-290

Vol.49, No.7, pp. 1026-104

No.5, pp. 542-543

Sarvier, 454-468, ISBN 978-85-7378-194-6, São Paulo, Brazil.

CRC Press, ISBN 978-0-8493-9165-1, Boca Raton, USA

Harvey, A.L. (2001). Twenty years of dendrotoxins. *Toxicon*, Vol.39, No.1, pp. 15-26

(Ed.), 317-324, CRC Press, ISBN 978-0-8493-9165-1, Boca Raton, USA Hegde, R.P.; Rajagopalan, N.; Doley, R. & Kini, R.M. (2010). Snake venom three-finger

e569

e102

257-264

*Animais Peçonhentos no Brasil. Biologia, Clínica e Terapêutica dos Acidentes*, *2nd Edition,* J.L.C. Cardoso; F.O.S. França; H.W. Fan; C.M.S. Málaque & V. Haddad, (Eds),

envenoming: a disease of poverty. *PLoS Neglected Tropical Diseases*, Vol.3, No.12, pp.

acetylcholinesterase, In: *Handbook of Venoms and Toxins of Reptiles*, S.P. Mackessy,

toxins, In: *Handbook of Venoms and Toxins of Reptiles*, S.P. Mackessy, (Ed.), 287-301,

Incorporating a rapid-impact package for neglected tropical diseases with programs for HIV/AIDS, tuberculosis, and malaria. *PLoS Medicine*, Vol.3, No.5, pp.

*Walterinnesia aegyptia* venom and its specific antivenins: flash absorption and distribution of the venom and its toxin versus slow absorption and distribution of

lethal potency of Russell's viper (*Vipera russelli*) venom. *Toxicon*, Vol.26, No.3, pp.

A.; Premaratna, R.; Savioli, L.; Lalloo, D.G. & de Silva, H.J. (2008). The global burden of snakebite: a literature analysis and modeling based on regional estimates

Proteome and immunome of the venom of the Thai cobra, *Naja kaouthia*. *Toxicon*,

Ecuador. *Transactions of the Royal Society of Tropical Medicine and Hygiene*, Vol.72,

erythrocyte antibodies in horse-derived antivenoms used in the treatment of

Wen, F.H.; de Medeiros, C.R.; Prado, J.C.; Málaque, C.M.; Lomonte, B. & Gutiérrez, J.M. (2008). Human heterophylic antibodies against equine immunoglobulins: assessment of their role in the early adverse reactions to antivenom administration. *Transactions of the Royal Society of Tropical Medicine and Hygiene*, Vol.102, No.11, pp. 1115-1119


Snakebite Envenoming: A Public Health Perspective 159

Prasarnpun, S.; Walsh, J.; Awad, S.S. & Harris, J.B. (2005). Envenoming bites by kraits: the

Pugh, R.N. & Theakston, R.D.G. (1980). Incidence and mortality on snake bite in savanna

Pugh, R.N.; Theakston, R.D.G. & Reid, H.A. (1980). Malumfashi Endemic Diseases Research

Quijada-Mascareñas, A. & Wüster, W. (2010). Recent advances in venomous snake

Rahman, R.; Faiz, M.A.; Selim, S.; Rahman, B.; Basher, A.; Jones, A.; d'Este, C.; Hossain, M.;

Raw, I.; Guidolin, R.; Higashi, H.G & Kelen, E.M.A. (1991). Antivenins in Brazil:

Rodrigues-Silva, R.; Antunes, G.F.; Velarde, D.T. & Santoro, M.M. (1999). Thermal stability studies of hyperimmune horse antivenoms. *Toxicon*, Vol.37, No.1, pp. 33-45 Rojas, G.; Jiménez, J.M & Gutiérrez, J.M. (1994). Caprylic acid fractionation of hyperimmune

Rossetto, O.; Morbiato, L.; Caccin, P.; Rigoni, M. & Montecucco, C. (2006). Presynaptic enzymatic neurotoxins. *Journal of Neurochemistry*, Vol.97, No.6, pp. 1534-1545 Rucavado, A.; Escalante, T.; Franceschi, A.; Chaves, F.; León, G.; Cury, Y.; Ovadia, M. &

Saravia, P.; Rojas, E.; Arce, V.; Guevara, C.; López, J.C.; Chaves, E.; Velásquez, R.; Rojas, G.

Saul, M.E.; Thomas, P.A.; Dosen, P.J.; Isbister, G.K.; O'Leary, M.A.; Whyte, I.M.; McFadden,

Scherrmann, J.M. (1994). Antibody treatment of toxin poisoning-recent advances. *Journal of* 

Segura, A.; Herrera, M.; González, E.; Vargas, M.; Solano, G.; Gutiérrez, J.M & León, G.

Segura, A.; Castillo, M.C.; Núñez, V.; Yarlequé, A.; Gonçalves, L.R.; Villalta, M.; Bonilla, C.;

implications. *Revista de Biología Tropical*, Vol. 50, No.1, pp. 337-346

for snakebite. *Nature Medicine*, Vol.17, No.7, pp. 809-811

*Toxicology-Clinical Toxicology*, Vol.32, No.4, pp. 363-375

Bangladesh. *PLoS Neglected Tropical Diseases*, Vol.4, No.10, pp. e860

No.12, pp. 2987-2996

Vol.32, No.3, pp. 351-363

313-319.

No.6, pp. 609-615

Nigeria. *Lancet*, Vol.2, pp. 1181-1183

*and Parasitology*, Vol.74, No.5, pp. 523-530

(Ed,), 557-581, Marcel Dekker, New York, USA.

64, CRC Press, ISBN 978-0-8493-9165-1, Boca Raton, USA

biological basis of treatment-resistant neuromuscular paralysis. *Brain*, Vol. 128,

Project, XIII. Epidemiology of human encounters with the spitting cobra, *Naja nigricollis*, in the Malumfashi area of northern Nigeria. *Annals of Tropical Medicine* 

systematics, In: *Handbook of Venoms and Toxins of Reptiles*, S.P. Mackessy, (Ed.), 25-

Islam, Z.; Ahmed, H. & Milton, A.H. (2010). Annual incidence of snake bite in rural

Preparation. In: *Handbook of Natural Toxins, Vol 5, Reptile Venoms and Toxins,* A.T. Tu

horse plasma: description of a simple procedure for antivenom production. *Toxicon*,

Gutiérrez, J.M. (2000). Inhibition of local hemorrhage and dermonecrosis induced by *Bothrops asper* snake venom: effectiveness of early *in situ* administration of the peptidomimetic metalloproteinase inhibitor batimastat and the chelating agent CaNa2EDTA. *American Journal of Tropical Medicine and Hygiene*, Vol.63, No.5-6, pp.

& Gutiérrez, J.M. (2002). Geographic and ontogenetic variability in the venom of the neotropical rattlesnake *Crotalus durissus*: pathophysiological and therapeutic

S.A. & van Heyden, D.F. (2011). A pharmacological approach to first aid treatment

(2009). Stability of equine IgG antivenoms obtained by caprylic acid precipitation: towards a liquid formulation stable at tropical room temperature. *Toxicon*, Vol.53,

Herrera, M.; Vargas, M.; Fernández, M.; Yano, M.Y.; Araújo, H.P.; Boller, M.A.; León, P.; Tintaya, B.; Sano-Martins, I.S.; Gómez, A.; Fernández, G.P.; Geoghegan, P.; Higashi, H.G., León, G. & Gutiérrez, J.M. (2010a). Preclinical assessment of the neutralizing capacity of antivenoms produced in six Latin American countries

A.M.C. Pimenta; M.F. Martin-Euclaire; R.B. Zingali & H. Rochat, (Eds.), 525-546, Editora UFMG, ISBN 978-85-7041-735-0, Belo Horizonte, Brazil.


Ohno, M.; Chijiwa, T.; Oda-Ueda, N.; Ogawa, T. & Hattori, S. (2003). Molecular evolution of myotoxic phospholipases A2 from snake venom. *Toxicon*, Vol.42, No.8, pp. 841-854 Otero, R.; Valderrama, R.; Osorio, R.G. & Posada, L.E. (1992). Programa de atención

Otero, R.; Núñez, V.; Osorio, R.G.; Gutiérrez, J.M.; Giraldo, C.A. & Posada, L.E. (1995).

Otero, R.; Gutiérrez, J.M.; Rojas, G.; Núñez, V.; Díaz, A.; Miranda, E.; Urige, A.F.; Silva, J.F.;

Otero, R.; Fonnegra, R.; Jiménez, S.L.; Núñez, V.; Evans, N.; Alzate, S.P.; García, M.E.;

Otero, R.; Gutiérrez, J.; Mesa, M.B.; Duque, E.; Rodríguez, O.; Arango, J.L.; Gómez, F.; Toro,

Otero-Patiño, R.; Cardoso, J.L.C.; Higashi, H.G.; Núñez, V.; Díaz, A.; Toro, M.F.; García,

Perales, J.; Neves-Ferreira, A.G.; Valente, R.H. & Domont, G.B. (2005). Natural inhibitors of

Petras, D.; Sanz, L.; Segura, A.; Herrera, M.; Villalta, M.; Solano, D.; Vargas, M.; León, G.;

Pierini, S.V.; Warrell, D.A.; de Paulo, A. & Theakston, R.G.D. (1996). High incidence of bites

J*ournal of Proteome Research*, Vol.10, No.3, pp. 1266-1280

Editora UFMG, ISBN 978-85-7041-735-0, Belo Horizonte, Brazil.

antivenoms. *Toxicon*, Vol.37, No.6, pp. 895-908

*and Hygiene*, Vol. 58, No.2, pp. 183-189

pp. 96-102

809-815

No.3, pp. 493-504

1114

A.M.C. Pimenta; M.F. Martin-Euclaire; R.B. Zingali & H. Rochat, (Eds.), 525-546,

primaria del accidente ofídico. Una propuesta para Colombia. *Iatreia*, Vol.5, No.2,

Ability of six Latin American antivenoms to neutralize the venom of mapaná equis (*Bothrops atrox*) from Antioquia and Chocó (Colombia). Toxicon, Vol.33, No.6, pp.

Ospina, J.G.; Medina, Y.; Toro, M.F.; García, M.E.; León, G.; García, M.; Lizano, S.; de la Torre, J.; Márquez, J.; Mena, Y.; González, N.; Arenas, L.C.; Puzón, A.; Blanco, N.; Sierra, A.; Espinal, M.E.; Arboleda, M.; Jiménez, J.C.; Ramírez, P.; Díaz, M.; Guzmán, M.C.; Barros, J.; Henao, S.; Ramírez, A.; Macea, U. & Lozano, R. (1999). A randomized blinded clinical trial of two antivenoms, prepared by caprylic acid or ammonium sulphate fractionation of IgG, in *Bothrops* and *Porthidium* snake bites in Colombia: correlation between safety and biochemical characteristics of

Saldarriaga, M.; del Valle, G.; Osorio, R.G.; Díaz, A.; Valderrama, R.; Duque, A. & Vélez, H.N. (2000). Snakebites and ethnobotany in the northwestern region of Colombia: Part I: traditional use of plants. *Journal of Ethnopharmacology*, Vol.71,

A.; Cano, F.; Rodríguez, L.M.; Caro, E.; Martínez, J.; Cornejo, W.; Gómez, L.M.; Uribe, F.L.; Cárdenas, S.; Núñez, V. & Díaz, A. (2002). Complications of *Bothrops*, *Porthidium*, and *Bothriechis* snakebites in Colombia. A clinical and epidemiological study of 39 cases attended in a university hospital. *Toxicon*, Vol.40, No.8, pp. 1107-

M.E.; Sierra, A.; García, L.F.; Moreno, A.M.; Medina, M.C.; Castañeda, N.; Silva-Díaz, J.F.; Murcia, M.; Cárdenas, S.Y. & Dias-da-Silva, W. (1998). A randomized, blinded, comparative trial of one pepsin-digested and two whole IgG antivenoms for *Bothrops* snake bites in Urabá, Colombia. *American Journal of Tropical Medicine* 

snake venom hemorrhagic metalloproteinases. *Toxicon*, Vol.45, No.8, pp. 1013-1020

Warrell, D.A.; Theakston, R.D.G.; Harrison, R.A.; Durfa, N.; Nasidi, A.; Gutiérrez, J.M. & Calvete, J.J. (2011). Snake venomics of African spitting cobras: toxin composition and assessment of congeneric cross-reactivity of the pan-African EchiTAb-Plus-ICP antivenom by antivenomics and neutralization approaches.

and stings by snakes and other animals among rubber tappers and Amazonian Indians of the Juruá Valley, Acre State, Brazil. *Toxicon*, Vol.34, No.2, pp. 225-236


Snakebite Envenoming: A Public Health Perspective 161

Thomas, L.; Tyburn, B. & the Research Group on Snake Bite in Martinique (1996). *Bothrops* 

Trape, J.F.; Pison, G.; Guyavarch, E. & Mane, Y. (2001). High mortality from snakebite in

Trinh, K.X.; Khac, Q.L.; Trinh, L.X. & Warrell, D.A. (2010). Hyponatremia, rhabdomyolysis,

Tun-Pe; Phillips, R.E.; Warrell, D.A.; Moore, R.A.; Tin-Un-Swe; Myint-Lwin & Burke, C.W.

Vonk, F.J.; Admiraal, J.F.; Jackson, K.; Reshef, R.; de Bakker, M.A.; Vanderschoot, K.; vanden

Warrell, D.A.; Davidson, N.M.; Omerod, L.D.; Pope, H.M.; Watkins, B.J.; Greenwood, B.M.

Warrell, D.A. (1992) The global problem of snake bite: its prevention and treatment. In:

Warrell, D.A. (1995a). Clinical toxicology of snakebite in Asia, In: *Handbook of Clinical* 

Warrell, D.A. (1995b). Clinical toxicology of snakebite in Africa and the Middle East /

Warrell, D.A. (1996). Clinical features of envenoming from snake bites. In: *Envenomings and* 

Warrell, D.A. (1997). Geographical and intraspecies variation in the clinical manifestationsof

Warrell, D.A. (2004). Snakebites in Central and South America: epidemiology, clinical

two specific antivenoms. *British Medical Journal*, Vol.4, pp. 437-440

following bites by Russell's viper in Burma. *Lancet*, Vol.2, pp. 763-767 Visser, L.E.; Kyed-Faried, S.; Belcher, D.W.; Geelhoed, D.W.; van Leeuwen, J.S. & van

*of Tropical Medicine and Hygiene*, Vol. 102, No.5, pp. 445-450

development of snake fangs. *Nature*, Vol.454, pp. 630-633

121-153, National University of Singapore, Singapore.

Press, ISBN 0-8493-4489-1, Boca Raton, USA

Lyon, France.

1070-1075

USA

Lyon, France.

United Kingdom.

ISBN 0-8014-4141-2, Ithaca, USA Warrell, D.A. (2010). Snake bite. *Lancet*, Vol.375, pp. 77-88

*Hygiene*, Vol.95, No.4, pp. 420-423

*lanceolatus* bites in Martinique: Clinical aspects and treatment. In: *Envenomings and Their Treatments*, C. Bon & M. Goyffon, (Eds), 255-265, Fondation Marcel Mérieux,

south-eastern Senegal. *Transactions of the Royal Society of Tropical Medicine and* 

alterations in blood pressure and persistent mydriasis in patients envenomed by Malayan kraits (*Bungarus candidus*) in southern Viet Nam. *Toxicon*, Vol.56, No.6, pp.

(1987). Acute and chronic pituitary failure resembling Shehan's syndrome

Roosmalen, J. (2008). Failure of a new antivenom to treat *Echis ocellatus* snake bite in rural Ghana: the importance of quality surveillance. *Transactions of the Royal Society* 

Berge, I.; van Atten, M.; Burgerhout, E.; Beck, A.; Mirtschin, P.J.; Kochva, E.; Witte, F.; Fry, B.G.; Woods, A.E. & Richardson, M.K. (2008). Evolutionary origin and

& Ried, H.A. (1974). Bites by the saw-scaled or carpet viper (*Echis carinatus*): trial of

*Advances in Toxinology Research, Vol. 1,* P. Gopalakrishnakone & C.K. Tan, (Eds),

*Toxicology of Animal Venoms and Poisons*, J. Meier & J. White, (Eds.), 493-594, CRC

Arabian Peninsula, In: *Handbook of Clinical Toxicology of Animal Venoms and Poisons*, J. Meier & J. White, (Eds.), 433-492, CRC Press, ISBN 0-8493-4489-1, Boca Raton,

*Their Treatments*, C. Bon & M. Goyffon, (Eds), 63-76, Fondation Marcel Mérieux,

envenoming by snakes. In: *Venomous Snakes. Ecology, Evolution and Snakebite*, R.S.Thorpe, W. Wüster & A. Malhotra, (Eds.), 189-203, Clarendon Press, Oxford,

features and clinical management, In: *The Venomous Reptiles of the Western Hemisphere*, J.A. Campbell & W.W. Lamar, (Eds.), 709-761, Cornell University Press,

against medically-relevant *Bothrops* snake venoms. *Toxicon*, Vol.56, No.6, pp. 980- 989


Segura, A.; Villalta, M.; Herrera, M.; León, G.; Harrison, R.; Durfa, N.; Nasidi, A.; Calvete,

Serrano, S.M.T. & Maroun, R.C. (2005). Snake venom serine proteinases: sequence homology

Sharma, S.K.; Chappuis, F.; Jha, N.; Bovier, P.A.; Loutan, L. & Koirala, S. (2004). Impact of

Simpson, I.D. & Norris, R.L. (2007). Snake antivenom product guidelines in India: "the devil is in the details". *Wilderness and Environmental Medicine*, Vol.18, No.3, pp. 163-168 Simpson, I.D. (2008). A study of the current knowledge base in treating snake bite amongst

Smalligan, R.; Cole, J.; Brito, N.; Laing, G.D.; Mertz, B.L.; Manock, S.; Maudin, J.; Quist, B.;

American polyspecific antivenoms. *British Medical Journal*, Vol.328, pp. 1129 Snow, R.W.; Bronzan, R.; Roques, T.; Nyamawi, C.; Murphy, S. & Marsh, K. (1994). The

St Pierre, L.; Masci, P.P.; Filippovich, I.; Sorokina, N.; Marsh, N.; Miller, D.J. & Lavin, M.F.

Sutherland, S.K.; Coulter, A.R. & Harris, R.D. (1979). Rationalisation of first-aid measures for

Swaroop, S. & Grab, B. (1954). Snakebite mortality in the world. *Bulletin of the World Health* 

Tanaka, G.D.; Furtado, M.F.; Portaro, F.C.; Sant'Anna, O.A. & Tambourgi, D.V. (2010).

Tans, G. & Rosing, J. (2001). Snake venom activators of factor X: an overview. *Haemostasis*,

Theakston, R.D.G. (1986). Characterization of venoms and standardization of antivenoms.

Theakston, R.D.G.; Warrell, D.A. & Griffiths, E. (2003). Report of a WHO workshop on the standardization and control of antivenoms. *Toxicon*, Vol.41, No.5, pp. 541-557

antitoxins. *Medical Journal of Australia*, Vol.1,No.17, pp. 613-615

elapid snakebite. *Lancet*, Vol.1, pp. 183-186

*Organization*, Vol.10, No.1, pp. 35-76

*Journal of Tropical Medicine and Hygiene*, Vol.21, No.2, pp. 234-238

*Medicine and Hygiene*, Vol. 102, No.11, pp. 1108-1114

989

369-374

1132.

pp. 665-671

No.3, pp. e622

Vol.31, No.3-6, pp. 225-233

Press, Okford, United Kingdom.

against medically-relevant *Bothrops* snake venoms. *Toxicon*, Vol.56, No.6, pp. 980-

J.J.; Theakston, R.D.G.; Warrell, D.A. & Gutiérrez, J.M. (2010b). Preclinical assessment of the efficacy of a new antivenom (EchiTAb-Plus-ICP) for the treatment of viper envenoming in sub-Saharan Africa. *Toxicon*, Vol.55, No.2-3, pp.

vs. substrate specificity, a paradox to be solved. *Toxicon*, Vol.45, No.8, pp. 1115-

snake bites and determinants of fatal outcomes in southeastern Nepal. *American* 

doctors in the high-risk countries of India and Pakistan: does snake bite treatment training reflect local requirements? *Transactions of the Royal Society of Tropical* 

Holland, G.; Nelson, S.; Lalloo, D.G.; Rivadeneira, G.; Barragan, M.E.; Dolley, D.; Addleston, M.; Warrell, D.A. & Theakston, R.D.G. (2004). Crotaline snake bite in the Ecuadorian Amazon: randomised double blind comparative trial of three South

prevalence and morbidity of snake bite and treatment-seeking behavior among a rural Kenyan population. *Annals of Tropical Medicine and Parasitology*, Vol.88, No.6,

(2005). Comparative analysis of prothrombin activators from the venom of Australian elapids. *Molecular Biology and Evolution*, Vol.22, No.9, pp. 1853-1864 Sutherland, S.K. (1977). Serum reactions. An analysis of commercial antivenoms and the

possible role of anticomplementary activity in de-novo reactions to antivenoms and

Diversity of *Micrurus* snake species related to their venom toxic effects and the prospective of antivenom neutralization. *PLoS Neglected Tropical Diseases*, Vol.4,

In: *Natural Toxins. Animal, Plant and Microbial*, J.B. Harris, (Ed.), 287-303, Clarendon


**8** 

*Colombia* 

**Chemical Residues in Animal** 

María Constanza Lozano and Mary Trujillo

**Food Products: An Issue of Public Health** 

*Pharmacy Department, Faculty of Sciences, National University of Colombia* 

Human beings consume protein-rich foods to supply their nutritional requirements, mainly of animal origin, such origin lying in meat from different species (cattle, sheep, caprines, birds, pigs, fish and seafood/shellfish), milk and eggs. With the exception of some products derived from fishing, these foods are obtained from financial exploitations in which the animals' health must be guaranteed, thereby ensuring that food is harmless. In several countries the safety of such food has mainly been focused on avoiding the transmission of zoonotic diseases, less attention thus being paid to potentially present chemical residues, perhaps due to the course of the resulting disease. Whilst infectious processes are frequently of the acute type, toxicosis caused by contaminants in foods (more than acute) may be

The primary production of such food involves the animals' interaction with their setting from which they may become exposed to undesirable chemical substances which may generate residuality. The chemical substances to which animals may become exposed during their production cycle which have been identified to date could come from drugs and growth promoters aimed at treating diseases and improving production parameters, biologically-derived toxins (mycotoxins, phycotoxins, phytotoxins) and/or environmental contaminants linked to atmospheric pollution, from the soil and/or water. This chapter will be orientated towards dealing with residues from chemicals substances in foodstuffs of animal origin caused by drugs and growth promoters, as well as by toxins having a biological origin. It will also deal with general concepts such as toxic agent's target in an organism, the regulation of residues in food and the analytical methods used for detecting them. The contamination of food by chemical risks is a worldwide public health matter

Living beings continuously are being exposed to external substances, generically called xenobiotics, which can have adverse effects according to their chemical characteristics. Oral, dermal and inhalation routes represent the commonest means of exposure to these substances, the first being of interest as it deals with risks to human health due to the consumption of foodstuffs contaminated by potentially toxic substances. On the other hand, animals (representing a readily available source of food for humans) are exposed to

chronic, silent and often lacking a known aetiological agent.

**2. The destination for toxic agents in an organism** 

which may also hamper international trade.

**1. Introduction** 


http://apps.who.int/classifications/apps/icd/icd10online/


http://www.who.int/bloodproducts/snake\_antivenoms/snakeantivenomguide/en/


### **Chemical Residues in Animal Food Products: An Issue of Public Health**

María Constanza Lozano and Mary Trujillo *Pharmacy Department, Faculty of Sciences, National University of Colombia Colombia* 

#### **1. Introduction**

162 Public Health – Methodology, Environmental and Systems Issues

White, J. (2010). Envenomation. Prevention and treatment in Australia. In: *Handbook of* 

Williams, D.; Gutiérrez, J.M.; Harrison, R.A.; Warrell, D.A., White, J.; Winkel, K.D. &

Williams, D.J.; Gutiérrez, J.M.; Calvete, J.J.; Wüster, W.; Ratanabanangkoon, K.; Paiva, O.;

Williams, S.S.; Wijesinghe, C.A.; Jayamanne, S.F.; Buckley, N.A.; Dawson, A.H.; Lalloo, D.G.

World Health Organization (2007c). *Global Plan to Combat Neglected Tropical Diseases 2008- 2015*, World Health Organization, Geneva, Switzerland, Available from

World Health Organization (2010a). *WHO Guidelines for the Production, Control and Regulation* 

 http://www.who.int/bloodproducts/snake\_antivenoms/snakeantivenomguide/en/ World Health Organization (2010b). *Guidelines for the Prevention and Clinical Management of Snakebite in Africa*, World Health Organization, Geneva, Switzerland, Available from

World Health Organization (2011). *WHO Model List of Essential Medicines*, World Health

 http://www.who.int/medicines/publications/essentialmedicines/en/index.html Yingprasertchai, S.; Bunyasrisawat, S. & Ratanabanangkoon, K. (2003). Hyaluronidase

and *Calloselasma rhodostoma* venoms. *Toxicon*, Vol.42, No.6, pp. 635-646

inhibitors (sodium chromoglycate and sodium auro-thiomalate) reduce the local tissue damage and prolong the survival time of mice injected with *Naja kaouthia*

http://www.afro.who.int/en/clusters-a-programmes/hss/essentialmedicines/highlights/2731-guidelines-for-the-prevention-and-clinical-

http://whqlibdoc.who.int/hq/2007/WHO\_CDS\_NTD\_2007.3\_eng.pdf

envenoming. *PLoS Neglected Tropical Diseases*, Vol.5, No.8, pp. e1255 World Health Organization (2007a). *Rabies and Envenomings. A Neglected Public Health Issue*, World Health Organization, ISBN 978 92 4 156348 2, Geneva, Switzerland World Health Organization (2007b). *International Statistical Classification of Diseases and* 

http://apps.who.int/classifications/apps/icd/icd10online/

8493-9165-1, Boca Raton, USA

snake bite. *Lancet,* Vol.375, pp. 89-91

*Proteomics*, Vol.74, No.9, pp. 1735-1767

Switzerland, Available from

Switzerland, Available from

management-of-snakebite-in-africa.html

Organization, Geneva, Switzerland. Available from

*Venoms and Toxins of Reptiles*, S.P. Mackessy, (Ed.), 423-451, CRC Press, ISBN 978-0-

Gopalakrishnakone, P. (2010). The Global Snake Bite Initiative: an antidote for

Brown, N.I.; Casewell, N.R.; Harrison, R.A.; Rowley, P.D.; O'Shea, M., Jensen, S.D.; Winkel, K.D. & Warrell, D.A. (2011). Ending the drought: new strategies for improving the flow of affordable, effective antivenoms in Asia and Africa. *Journal of* 

& de Silva, H.J. (2011). Delayed psychological morbidity associated with snakebite

*Related Health Problems*, *10th Revision*, World Health Organization, Geneva,

*of Snake Antivenom Immunoglobulins,* World Health Organization, Geneva,

Human beings consume protein-rich foods to supply their nutritional requirements, mainly of animal origin, such origin lying in meat from different species (cattle, sheep, caprines, birds, pigs, fish and seafood/shellfish), milk and eggs. With the exception of some products derived from fishing, these foods are obtained from financial exploitations in which the animals' health must be guaranteed, thereby ensuring that food is harmless. In several countries the safety of such food has mainly been focused on avoiding the transmission of zoonotic diseases, less attention thus being paid to potentially present chemical residues, perhaps due to the course of the resulting disease. Whilst infectious processes are frequently of the acute type, toxicosis caused by contaminants in foods (more than acute) may be chronic, silent and often lacking a known aetiological agent.

The primary production of such food involves the animals' interaction with their setting from which they may become exposed to undesirable chemical substances which may generate residuality. The chemical substances to which animals may become exposed during their production cycle which have been identified to date could come from drugs and growth promoters aimed at treating diseases and improving production parameters, biologically-derived toxins (mycotoxins, phycotoxins, phytotoxins) and/or environmental contaminants linked to atmospheric pollution, from the soil and/or water. This chapter will be orientated towards dealing with residues from chemicals substances in foodstuffs of animal origin caused by drugs and growth promoters, as well as by toxins having a biological origin. It will also deal with general concepts such as toxic agent's target in an organism, the regulation of residues in food and the analytical methods used for detecting them. The contamination of food by chemical risks is a worldwide public health matter which may also hamper international trade.

#### **2. The destination for toxic agents in an organism**

Living beings continuously are being exposed to external substances, generically called xenobiotics, which can have adverse effects according to their chemical characteristics. Oral, dermal and inhalation routes represent the commonest means of exposure to these substances, the first being of interest as it deals with risks to human health due to the consumption of foodstuffs contaminated by potentially toxic substances. On the other hand, animals (representing a readily available source of food for humans) are exposed to

Chemical Residues in Animal Food Products: An Issue of Public Health 165

During absorption, the xenobiotics cross cell membranes and reach the systemic circulation. Many toxic agents enter with foodstuff and are absorbed by the same routes as the other substances present in them. The chemicals cross the cell membrane's lipid bilayer through two basic processes: diffusion (favouring their concentration gradients) and active transport (against their concentration gradients). Most liposoluble xenobiotics are transported by simple diffusion through the cell membranes. Organic acids and/or bases thus tend to become absorbed when they are in their most liposoluble form (non-ionised), which is determined by surrounding pH. Weak acids will become more easily absorbed in the stomach whilst this will happen to bases in the intestines. Hydrophilic substances having a small molecular weight become diffused through aqueous pores formed by proteins (facilitated diffusion). Active transport, against concentration gradients requiring an expenditure of energy, occurs through proteins present on the membrane mobilising a

It should be born in mind that certain factors may sometimes alter xenobiotics' absorption; for example, the flora present in the gastrointestinal tract may transform them and leave them less available for being absorbed. This is why ruminants are resistant to some mycotoxins. Pre-systemic elimination of a toxic agent may occur with enterohepatic

Once it has been absorbed, a toxic agent becomes distributed throughout the whole body; during its initial phase this distribution is dominated by the blood flow. The penetration of toxic agent into the cells depends on passive diffusion or specialised transport; however, certain toxicants do not cross the membranes and become distributed via the blood flow. Some become accumulated in determined parts of the organism as a result of their binding to proteins or their high solubility in fat. When a toxicant becomes stored, then equilibrium is reached with the free fraction which is in the plasma. Thus, when the chemical becomes metabolised or is excreted, then substance is released from the storage site, thereby meaning

Albumin is the main plasmatic protein transporting xenobiotics. This protein may also be a toxicant reservoir since it impedes transport through the membranes due to its high molecular weight. The presence of toxic agents in the blood could be exploited for

Many organic compounds are very stable and lipophilic, becoming accumulated in the environment, becoming rapidly absorbed and concentrated in body fat. The toxicants become accumulated in fat because they are dissolved in it. A substance stored in fat is not toxic for the carrier, but there is rapid lipid mobilisation, for example poisoning may occur during long periods of fasting. Animal fat, a potential reserve of liposoluble toxicants, could

The liver and the kidneys have a large capacity for proteins to bind a broad range of chemicals. These organs important function lying in the metabolism and elimination of xenobiotics makes them concentrate more toxic agents than all the rest combined. Thus consuming such viscera may represent a risk for the end consumer. There is a lower

substance from one side to another (Lehman-McKeeman, 2008).

circulation, thereby minimising its potential adverse effect.

that the xenobiotic half-life could become very long.

recognising exposure, whether in humans or animals.

be consumed by human beings.

**2.1 Absorption** 

**2.2 Distribution** 

xenobiotics in multiple ways which could be present in their products. If one is dealing with veterinary drugs then the route of exposure could be oral (for example, coccidiostatics in poultry), dermal (e.g. external antiparasitic agents in ruminants), parenteral (e.g. antibiotic treatment in large animals) and even inhalation (if the animals are given anaesthesia before surgical procedures). Biologically-derived toxins mainly enter food-producing animals by the oral route (e.g. forage contaminated with mycotoxins or fish consuming toxic algae).

Xenobiotics in an organism go through a series of stages including absorption, distribution, metabolism and excretion, forming part of the pharmacokinetics or toxicokinetics according to the effects produced by a particular substance (pharmacological or toxicological). Xenobiotics enter a food-producing animals' organism and, according to its kinetics, reach the tissues which will become food for human beings (Figure 1). These concepts will be dealt with below, approaching them from the perspective of potential residuality which different substances can cause in an animal's organism.

Fig. 1. Destination of toxicants in foodstuffs animal organism and risk for humans

#### **2.1 Absorption**

164 Public Health – Methodology, Environmental and Systems Issues

xenobiotics in multiple ways which could be present in their products. If one is dealing with veterinary drugs then the route of exposure could be oral (for example, coccidiostatics in poultry), dermal (e.g. external antiparasitic agents in ruminants), parenteral (e.g. antibiotic treatment in large animals) and even inhalation (if the animals are given anaesthesia before surgical procedures). Biologically-derived toxins mainly enter food-producing animals by the oral route (e.g. forage contaminated with mycotoxins or fish consuming toxic algae).

Xenobiotics in an organism go through a series of stages including absorption, distribution, metabolism and excretion, forming part of the pharmacokinetics or toxicokinetics according to the effects produced by a particular substance (pharmacological or toxicological). Xenobiotics enter a food-producing animals' organism and, according to its kinetics, reach the tissues which will become food for human beings (Figure 1). These concepts will be dealt with below, approaching them from the perspective of potential residuality which different

Fig. 1. Destination of toxicants in foodstuffs animal organism and risk for humans

substances can cause in an animal's organism.

During absorption, the xenobiotics cross cell membranes and reach the systemic circulation. Many toxic agents enter with foodstuff and are absorbed by the same routes as the other substances present in them. The chemicals cross the cell membrane's lipid bilayer through two basic processes: diffusion (favouring their concentration gradients) and active transport (against their concentration gradients). Most liposoluble xenobiotics are transported by simple diffusion through the cell membranes. Organic acids and/or bases thus tend to become absorbed when they are in their most liposoluble form (non-ionised), which is determined by surrounding pH. Weak acids will become more easily absorbed in the stomach whilst this will happen to bases in the intestines. Hydrophilic substances having a small molecular weight become diffused through aqueous pores formed by proteins (facilitated diffusion). Active transport, against concentration gradients requiring an expenditure of energy, occurs through proteins present on the membrane mobilising a substance from one side to another (Lehman-McKeeman, 2008).

It should be born in mind that certain factors may sometimes alter xenobiotics' absorption; for example, the flora present in the gastrointestinal tract may transform them and leave them less available for being absorbed. This is why ruminants are resistant to some mycotoxins. Pre-systemic elimination of a toxic agent may occur with enterohepatic circulation, thereby minimising its potential adverse effect.

#### **2.2 Distribution**

Once it has been absorbed, a toxic agent becomes distributed throughout the whole body; during its initial phase this distribution is dominated by the blood flow. The penetration of toxic agent into the cells depends on passive diffusion or specialised transport; however, certain toxicants do not cross the membranes and become distributed via the blood flow. Some become accumulated in determined parts of the organism as a result of their binding to proteins or their high solubility in fat. When a toxicant becomes stored, then equilibrium is reached with the free fraction which is in the plasma. Thus, when the chemical becomes metabolised or is excreted, then substance is released from the storage site, thereby meaning that the xenobiotic half-life could become very long.

Albumin is the main plasmatic protein transporting xenobiotics. This protein may also be a toxicant reservoir since it impedes transport through the membranes due to its high molecular weight. The presence of toxic agents in the blood could be exploited for recognising exposure, whether in humans or animals.

Many organic compounds are very stable and lipophilic, becoming accumulated in the environment, becoming rapidly absorbed and concentrated in body fat. The toxicants become accumulated in fat because they are dissolved in it. A substance stored in fat is not toxic for the carrier, but there is rapid lipid mobilisation, for example poisoning may occur during long periods of fasting. Animal fat, a potential reserve of liposoluble toxicants, could be consumed by human beings.

The liver and the kidneys have a large capacity for proteins to bind a broad range of chemicals. These organs important function lying in the metabolism and elimination of xenobiotics makes them concentrate more toxic agents than all the rest combined. Thus consuming such viscera may represent a risk for the end consumer. There is a lower

Chemical Residues in Animal Food Products: An Issue of Public Health 167

as being the, "reasonable certainty of no harm," and the aforementioned agencies regulate which additives are allowed in food and what levels of unavoidable contaminants are acceptable. The US Department of Agriculture's (USDA) Food Safety Inspection Service (FSIS) is responsible for the safety of meat, poultry, and egg products in the USA (Lodovico et al., 2008). The European Food Safety Authority (EFSA) is the keystone of the European Union's (EU) risk assessment regarding food and animal feed safety. The Codex Alimentarius Commission (created by the FAO and WHO) develops food standards, guidelines and related texts such as codes of practice under the Joint FAO/WHO Food Standards Programme (JECFA). The main purposes of this programme are protecting consumers' health, ensuring fair trade practices in the food trade and promoting the coordination of all food standards' work undertaken by international governmental and

Health authorities recommend maximum acceptable or tolerable levels for chemicals which are neither genotoxic nor carcinogenic, such as acceptable daily intake (ADI), reference dose (RfD), especially for pesticides, tolerable daily intake (TDI) and provisional tolerable weekly intake (PTWI) for contaminants which may accumulate in the body. The responsible agencies conduct risk assessment to determine such levels; this consists of hazard identification and characterisation, exposure assessment and subsequent risk

Hazards are identified and characterised from human epidemiological observations and animal-based toxicity testing supported by *in vitro* mechanistic studies which can make extrapolation from animals to humans become more realistic. Structure–activity relationships-based indications and the increased use of novel molecular biology techniques

Dose–response information is essential for quantifying an adverse health effect. This may be graphically presented as being the relationship between the increase of a dose and the increase of a pertinent biological response. Such dose–response curve is essential for identifying a non-active dose taken as being the no observed adverse effect level (NOAEL), the highest dose of a substance which causes no detectable adverse alteration in line with defined treatment conditions. Interspecies differences should be taken into account as well as the fact that humans may exhibit substantial differences in their sensitivity to certain toxins due to differences regarding metabolic pathways and other factors. Uncertainty factors are thus applied when extrapolating from the toxicity observed in laboratory animals to health risks in humans, this usually being a factor of 10 for interspecies difference and a factor of up to 10 for human variability (depending on the extent and quality of available

The resulting value (equation 1) provides an estimate of the amount of a substance in food, expressed on a body weight basis, that can be ingested daily over a lifetime without appreciable risk (standard human= 60 kg). The ADI is then used for determining the maximum allowable levels of a particular chemical in a specific food, depending on the extent to which this food contributes towards the overall intake of such chemical (Lodovico et al., 2008). These are called maximum limits for some chemicals and maximum residue limits (MRLs) for substances such as veterinary drugs and hormone

non-governmental organisations.

characterisation.

are also very valuable.

human data).

residues.

presence of residues in animals' musculature (meat) compared to the viscera (kidneys, liver) and fat. Their accumulation at an injection site is feasible in cases where there has been exposure to the intramuscular drug route, this being important in animals which are to be consumed by humans.

The distribution of some chemicals in eggs, as well as reducing palatability, could represent a risk for the end consumer.

#### **2.3 Metabolism**

The object of xenobiotics' metabolism is to increase characteristics regarding an increase in substances' hydrosolubility so that they can be more easily excreted. This process occurs in two phases; hydrolysis, reduction and oxidation reactions are presented during phase 1, most of them being enzyme-mediated. Cytochrome P450 (CYP450) oxidation enzymes being of particular importance during this phase due to their catalytic versatility and the great number of xenobiotics constituted in their substrate. Conjugation reactions occur during phase 2, mainly with glucuronic acid, glutathione conjugates and sulphates; such reactions are enzymatically mediated by protein superfamilies called, respectively, uridine diphosphate glucuronosyltransferase, glutathione S-transferases and sulphotransferases. In spite of the initial purpose of xenobiotics' metabolism (or biotransformation) being detoxification, substances can occasionally acquire their true toxic power on being biotransformed; such reaction is called bioactivation or metabolic activation. Aflatoxins and pyrrolizidine alkaloids are bioactivated substances of interest regarding the residuality which they represent in food of animal origin.

#### **2.4 Excretion**

Toxicants are eliminated from the body by various routes, the kidneys being the most important organ for excreting xenobiotics since it is the main elimination route. The biliary route involving the faeces is the other elimination route for toxic substances which have been consumed.

Milk is an important elimination route due to the risk of contamination which it represents; this liquid is a lipid emulsion in an aqueous protein solution and may thus contain whatever toxicant which is in solution in an animal's body water. Simple chemicals arrive at the mammary glands by diffusion in their free form, bound to proteins or dissolved in lipids. The percentage of the total amount of compounds eliminated in milk is very low because the other elimination routes are more efficient. However, the main problem lies in chronic exposure and/or liposoluble compounds (Panter & James, 1990).

The concept of withdrawal time has been established to avoid the accumulation of drug residues in animals; it is defined as being the time required after a drug has been administered to an animal to ensure that drug residues in marketable products (meat, eggs, viscera or other edible products) are below a determined maximum residue limit (MRL).

#### **3. Regulating and evaluating risk**

There can never be an absolute guarantee that our food is safe; it is simply impossible to test every contaminant. Every country has an agency which oversees food safety; this is defined

presence of residues in animals' musculature (meat) compared to the viscera (kidneys, liver) and fat. Their accumulation at an injection site is feasible in cases where there has been exposure to the intramuscular drug route, this being important in animals which are to be

The distribution of some chemicals in eggs, as well as reducing palatability, could represent

The object of xenobiotics' metabolism is to increase characteristics regarding an increase in substances' hydrosolubility so that they can be more easily excreted. This process occurs in two phases; hydrolysis, reduction and oxidation reactions are presented during phase 1, most of them being enzyme-mediated. Cytochrome P450 (CYP450) oxidation enzymes being of particular importance during this phase due to their catalytic versatility and the great number of xenobiotics constituted in their substrate. Conjugation reactions occur during phase 2, mainly with glucuronic acid, glutathione conjugates and sulphates; such reactions are enzymatically mediated by protein superfamilies called, respectively, uridine diphosphate glucuronosyltransferase, glutathione S-transferases and sulphotransferases. In spite of the initial purpose of xenobiotics' metabolism (or biotransformation) being detoxification, substances can occasionally acquire their true toxic power on being biotransformed; such reaction is called bioactivation or metabolic activation. Aflatoxins and pyrrolizidine alkaloids are bioactivated substances of interest regarding the residuality

Toxicants are eliminated from the body by various routes, the kidneys being the most important organ for excreting xenobiotics since it is the main elimination route. The biliary route involving the faeces is the other elimination route for toxic substances which have

Milk is an important elimination route due to the risk of contamination which it represents; this liquid is a lipid emulsion in an aqueous protein solution and may thus contain whatever toxicant which is in solution in an animal's body water. Simple chemicals arrive at the mammary glands by diffusion in their free form, bound to proteins or dissolved in lipids. The percentage of the total amount of compounds eliminated in milk is very low because the other elimination routes are more efficient. However, the main problem lies in chronic

The concept of withdrawal time has been established to avoid the accumulation of drug residues in animals; it is defined as being the time required after a drug has been administered to an animal to ensure that drug residues in marketable products (meat, eggs, viscera or other edible products) are below a determined maximum residue limit (MRL).

There can never be an absolute guarantee that our food is safe; it is simply impossible to test every contaminant. Every country has an agency which oversees food safety; this is defined

exposure and/or liposoluble compounds (Panter & James, 1990).

consumed by humans.

**2.3 Metabolism** 

**2.4 Excretion** 

been consumed.

a risk for the end consumer.

which they represent in food of animal origin.

**3. Regulating and evaluating risk** 

as being the, "reasonable certainty of no harm," and the aforementioned agencies regulate which additives are allowed in food and what levels of unavoidable contaminants are acceptable. The US Department of Agriculture's (USDA) Food Safety Inspection Service (FSIS) is responsible for the safety of meat, poultry, and egg products in the USA (Lodovico et al., 2008). The European Food Safety Authority (EFSA) is the keystone of the European Union's (EU) risk assessment regarding food and animal feed safety. The Codex Alimentarius Commission (created by the FAO and WHO) develops food standards, guidelines and related texts such as codes of practice under the Joint FAO/WHO Food Standards Programme (JECFA). The main purposes of this programme are protecting consumers' health, ensuring fair trade practices in the food trade and promoting the coordination of all food standards' work undertaken by international governmental and non-governmental organisations.

Health authorities recommend maximum acceptable or tolerable levels for chemicals which are neither genotoxic nor carcinogenic, such as acceptable daily intake (ADI), reference dose (RfD), especially for pesticides, tolerable daily intake (TDI) and provisional tolerable weekly intake (PTWI) for contaminants which may accumulate in the body. The responsible agencies conduct risk assessment to determine such levels; this consists of hazard identification and characterisation, exposure assessment and subsequent risk characterisation.

Hazards are identified and characterised from human epidemiological observations and animal-based toxicity testing supported by *in vitro* mechanistic studies which can make extrapolation from animals to humans become more realistic. Structure–activity relationships-based indications and the increased use of novel molecular biology techniques are also very valuable.

Dose–response information is essential for quantifying an adverse health effect. This may be graphically presented as being the relationship between the increase of a dose and the increase of a pertinent biological response. Such dose–response curve is essential for identifying a non-active dose taken as being the no observed adverse effect level (NOAEL), the highest dose of a substance which causes no detectable adverse alteration in line with defined treatment conditions. Interspecies differences should be taken into account as well as the fact that humans may exhibit substantial differences in their sensitivity to certain toxins due to differences regarding metabolic pathways and other factors. Uncertainty factors are thus applied when extrapolating from the toxicity observed in laboratory animals to health risks in humans, this usually being a factor of 10 for interspecies difference and a factor of up to 10 for human variability (depending on the extent and quality of available human data).

The resulting value (equation 1) provides an estimate of the amount of a substance in food, expressed on a body weight basis, that can be ingested daily over a lifetime without appreciable risk (standard human= 60 kg). The ADI is then used for determining the maximum allowable levels of a particular chemical in a specific food, depending on the extent to which this food contributes towards the overall intake of such chemical (Lodovico et al., 2008). These are called maximum limits for some chemicals and maximum residue limits (MRLs) for substances such as veterinary drugs and hormone residues.

Chemical Residues in Animal Food Products: An Issue of Public Health 169

The scientific community focuses on developing reliable, economic and rapid methods (and whenever possible automated) which could be applied to evaluating the safety of foodstuffs bearing in mind the broad range of existing chemicals and matrices (Botsoglou & Fletouris, 2000). A unified procedure would eliminate the need for using separate methods for detecting multiple residues in the same product; however, such methods are rarely found in real life. There is an immense variety of methods for identifying, confirming and quantifying analytes which could be used individually or coupled to each other in a suitable way. These methods can be grouped into bioassays, microbiology assays, immunochemical

Biological methods for determining toxic residues in foodstuffs can be used both *in vivo* and *in vitro* (FAO, 2004) and have been particularly developed for detecting and quantifying the phycotoxins present in shellfish. The mouse bioassay is the most used one and is even

A toxin extract is intraperitoneally injected into mice having around 20 g body weight in the mouse bioassay and their survival is monitored from 24 to 48 hours. One mouse unit (MU) is defined as being the minimum quantity of toxin needed to kill a mouse within 24 hours. Sample toxicity (MU/g whole tissue) is determined from the smallest dose at which two mice or more in a group of three die within 24 hours. The regulatory level is set at 0.05 MU/g whole tissues in many countries; this assay's major disadvantages are therefore a lack of specificity (no differentiation between various toxin components), subjectivity regarding the animals' time of death as well as maintaining and killing laboratory animals. This assay may also give false positives because of interference which can be very toxic for mice. The EU has issued directions on how to perform this assay in an attempt to standardise the

Other bioassays which are also used would include the suckling mouse assay for detecting marine toxins which determin the weight of the intestine regarding body weight, the rat bioassay which is based on inducing diarrhoea in rats, the *Daphnia magna* assay which is used for detecting okadaic acid, the intestinal loop assay which determines the accumulation of fluids in rabbit intestine and mice and cytotoxicity assays which are based

The microbiological methods used for detecting antimicrobial residues in foodstuffs are based on inhibiting microbial growth, microbial receptor activity and enzymatic reactions and could be applied to all types of matrices, usually milk, meat, eggs and honey. Microbial inhibition assays involve culturing a microorganism from a standard strain, usually *Bacillus stearothermophilus, Bacillus subtilis, Bacillus cereus, Micrococcus luteus, Escherichia coli, Bacillus* 

Immunochemical methods represent an important tool for determining drug residues, given their high specificity, they lead to analytes being determined in samples having had very

*megatherium, Sarcina lutea* and/or *Streptococcus thermophilus* (AEFI, 2001).

assays and physical-chemical assays.

accepted by regulating entities.

mouse bioassay methodology.

**4.2 Microbiological assays** 

**4.3 Immunochemical assays** 

on observing morphological changes in cells.

**4.1 Bioassays** 


Equation 1. Acceptable daily intake calculation

The regulatory approach (not strictly scientific) for genotoxic and carcinogenic compounds is based on the assumption that there is no threshold dose (it is assumed that one genotoxic molecule is sufficient to hit a single DNA base, thereby inducing damage). The aim in this case is to keep the exposure level as low as technologically achievable (Lodovico et al., 2008).

#### **4. Analytical methodologies**

Analytical data quality is a key factor in the success of a control programme dealing with residues in foodstuffs. The analytical results of methods regarding official standards offer the necessary information for developing and managing programmes responding to a population's public health needs. It is very important that sanitary authorities have readily available practical analysis methods which will reliably detect and quantify (without ambiguity) a drug's residues which could be present in meat, milk, or eggs at a suitable concentration level. Unfortunately, methods having these attributes are not available for all residues, partly due to the large amount of possible substances which may be found in animals' food chains.

Chemical residues in food of animal origin, such as meat, milk or eggs, are frequently present in very low concentrations or trace levels, thereby representing an important challenge for a chemical analyst, given that the analytical methods developed must be highly sensitive and selective.

The prior treatment which a sample has received is very important for ensuring that methods reach desired detection levels as well as an acceptable level of exactitude and precision, thereby enabling the factors responsible for analyte loss to be controlled during such procedure (The Spanish Industrial Pharmaceutics Association – Asociación Española de Farmacéuticos de la Industria [AEFI], 2001); this would include the presence of functional reactive groups which can interfere with such determination (LoBrutto & Patel, 2007).

Analytical methods will always have to be tested/proved on material from each animal species since differences in composition (fat, specifics proteins, eg: myoglobin) can influence both analyte extraction and separation. Another important consideration concerns the treatment which biotransformation enzyme-rich tissues such as the liver should receive as this may induce post-mortem metabolism thereby altering real results. Special management musts also be used for determining analytes in eggs because eggs consist of two distinct compartments (the white and the yolk) whose chemical composition is different, as well as depending on the components of chicken's diet.

The scientific community focuses on developing reliable, economic and rapid methods (and whenever possible automated) which could be applied to evaluating the safety of foodstuffs bearing in mind the broad range of existing chemicals and matrices (Botsoglou & Fletouris, 2000). A unified procedure would eliminate the need for using separate methods for detecting multiple residues in the same product; however, such methods are rarely found in real life. There is an immense variety of methods for identifying, confirming and quantifying analytes which could be used individually or coupled to each other in a suitable way. These methods can be grouped into bioassays, microbiology assays, immunochemical assays and physical-chemical assays.

#### **4.1 Bioassays**

168 Public Health – Methodology, Environmental and Systems Issues

NOAEL

UF

UF : Uncertainty Factor (10, 100, n)

ADI : Acceptable Daily Intake

The regulatory approach (not strictly scientific) for genotoxic and carcinogenic compounds is based on the assumption that there is no threshold dose (it is assumed that one genotoxic molecule is sufficient to hit a single DNA base, thereby inducing damage). The aim in this case is to keep the exposure level as low as technologically achievable (Lodovico et al., 2008).

NOAEL : Not Observed Adverse Effect Level

Analytical data quality is a key factor in the success of a control programme dealing with residues in foodstuffs. The analytical results of methods regarding official standards offer the necessary information for developing and managing programmes responding to a population's public health needs. It is very important that sanitary authorities have readily available practical analysis methods which will reliably detect and quantify (without ambiguity) a drug's residues which could be present in meat, milk, or eggs at a suitable concentration level. Unfortunately, methods having these attributes are not available for all residues, partly due to the large amount of possible substances which may be found in

Chemical residues in food of animal origin, such as meat, milk or eggs, are frequently present in very low concentrations or trace levels, thereby representing an important challenge for a chemical analyst, given that the analytical methods developed must be

The prior treatment which a sample has received is very important for ensuring that methods reach desired detection levels as well as an acceptable level of exactitude and precision, thereby enabling the factors responsible for analyte loss to be controlled during such procedure (The Spanish Industrial Pharmaceutics Association – Asociación Española de Farmacéuticos de la Industria [AEFI], 2001); this would include the presence of functional

Analytical methods will always have to be tested/proved on material from each animal species since differences in composition (fat, specifics proteins, eg: myoglobin) can influence both analyte extraction and separation. Another important consideration concerns the treatment which biotransformation enzyme-rich tissues such as the liver should receive as this may induce post-mortem metabolism thereby altering real results. Special management musts also be used for determining analytes in eggs because eggs consist of two distinct compartments (the white and the yolk) whose chemical composition is different, as

reactive groups which can interfere with such determination (LoBrutto & Patel, 2007).

well as depending on the components of chicken's diet.

Equation 1. Acceptable daily intake calculation

ADI =

**4. Analytical methodologies** 

animals' food chains.

highly sensitive and selective.

Biological methods for determining toxic residues in foodstuffs can be used both *in vivo* and *in vitro* (FAO, 2004) and have been particularly developed for detecting and quantifying the phycotoxins present in shellfish. The mouse bioassay is the most used one and is even accepted by regulating entities.

A toxin extract is intraperitoneally injected into mice having around 20 g body weight in the mouse bioassay and their survival is monitored from 24 to 48 hours. One mouse unit (MU) is defined as being the minimum quantity of toxin needed to kill a mouse within 24 hours. Sample toxicity (MU/g whole tissue) is determined from the smallest dose at which two mice or more in a group of three die within 24 hours. The regulatory level is set at 0.05 MU/g whole tissues in many countries; this assay's major disadvantages are therefore a lack of specificity (no differentiation between various toxin components), subjectivity regarding the animals' time of death as well as maintaining and killing laboratory animals. This assay may also give false positives because of interference which can be very toxic for mice. The EU has issued directions on how to perform this assay in an attempt to standardise the mouse bioassay methodology.

Other bioassays which are also used would include the suckling mouse assay for detecting marine toxins which determin the weight of the intestine regarding body weight, the rat bioassay which is based on inducing diarrhoea in rats, the *Daphnia magna* assay which is used for detecting okadaic acid, the intestinal loop assay which determines the accumulation of fluids in rabbit intestine and mice and cytotoxicity assays which are based on observing morphological changes in cells.

#### **4.2 Microbiological assays**

The microbiological methods used for detecting antimicrobial residues in foodstuffs are based on inhibiting microbial growth, microbial receptor activity and enzymatic reactions and could be applied to all types of matrices, usually milk, meat, eggs and honey. Microbial inhibition assays involve culturing a microorganism from a standard strain, usually *Bacillus stearothermophilus, Bacillus subtilis, Bacillus cereus, Micrococcus luteus, Escherichia coli, Bacillus megatherium, Sarcina lutea* and/or *Streptococcus thermophilus* (AEFI, 2001).

#### **4.3 Immunochemical assays**

Immunochemical methods represent an important tool for determining drug residues, given their high specificity, they lead to analytes being determined in samples having had very

Chemical Residues in Animal Food Products: An Issue of Public Health 171

levels depend on withdrawal time. In spite of most drugs representing a relatively low risk for the general public, when used responsibly and in line with instructions approved by the laboratories making veterinary drugs, adverse reactions have been frequently reported for some compounds; these would include antibacterial, antihelminthic, anticoccidial and

Residues from antibacterial drugs in food products of animal origin can represent a danger for consumers. The poisonous effects are not very probable since the residues are present in very low concentrations. Some substances must receive particular attention due allergic reactions. The main hazardous effect is likely to be the development of resistant bacterial strains following sub-therapeutic doses of antimicrobials being ingested; such resistance could be transferred to other bacteria. This could include resistance being transferred from non-pathogenic organisms to pathogenic ones which would then no longer respond to standard drug treatment (the Institute of Food Technologists [IFT], 2006). However, the

Differences in substitutions in the basic ring structures between the various aminoglycosides account for the relatively minor differences in antimicrobial spectra and resistance and toxicity patterns. Aminoglycosides given in therapeutic dosages mainly cause ototoxicosis,

Chloramphenicol (an antibacterial belonging to the amphenicol group) has been used in treatment and prophylactically in food-producing animals for several years now (i.e. poultry, calves, pigs, sheep and fish). Chloramphenicol's most serious toxic effect is bone marrow depression which is generally dose-related and reversible but can sometimes be fatal in patients who are probably genetically predisposed. A toxic syndrome has been reported in newborn infants receiving large doses of chloramphenicol which is characterised by vomiting, hypothermia, cyanosis and circulatory collapse followed by death; this syndrome rarely occurs in adults. Chloramphenicol may also cause neuritis, encephalopathy with dementia and ototoxicity; its use is restricted in many countries, while it is totally banned for use in food-producing animals within the European Union and the USA.

Differences in substitutions in the basic ring structures between the various aminoglycosides account for the relatively minor differences in antimicrobial spectra and resistance and toxicity patterns. Aminoglycosides given in therapeutic dosages mainly cause ototoxicosis,

Chloramphenicol (an antibacterial belonging to the amphenicol group) has been used in treatment and prophylactically in food-producing animals for several years now (i.e. poultry, calves, pigs, sheep and fish). Chloramphenicol's most serious toxic effect is bone marrow depression which is generally dose-related and reversible but can sometimes be fatal in patients who are probably genetically predisposed. A toxic syndrome has been reported in newborn infants receiving large doses of chloramphenicol which is characterised by vomiting, hypothermia, cyanosis and circulatory collapse followed by death; this syndrome rarely occurs in adults. Chloramphenicol may also cause neuritis, encephalopathy with dementia and ototoxicity; its use is restricted in many countries, while it is totally

but may also cause nephrotoxicosis, allergy and neuromuscular disturbances.

Chloramphenicol and its metabolites could be genotoxic (Lozano & Arias, 2008).

but may also cause nephrotoxicosis, allergy and neuromuscular disturbances.

antiprotozoal drugs and growth promoters.

generally used antibacterial drugs are presented in Table 1.

**5.1 Antibacterial drugs** 

reduced prior cleaning treatment. These assays are based on the reaction of an antigen binding to a specific primary antibody or for each antigen, analogously to an enzymesubstrate reaction. The most common immunochemical methods would include the enzyme-linked immunosorbent assay (ELISA), direct and indirect competitive enzymelinked immunosorbent assays, immunoaffinity chromatography (IAC), radioimmunoassay (RIA), the enzyme-monitored immunotest (EMIT), the fluorescent immunoassay (FIA) and the chemiluminescence immunoassay.

#### **4.4 Physical-chemical assays**

Physical-chemical methods are mainly used for isolating, separating, quantifying and confirming the presence of dangerous residues in samples; this requires that the sensitivity of a particular selection method and the determinative or confirmation method are similar. Numerous procedures based on the analytes'different physicochemical properties have been developed for achieving this objective. Even though a drug's chemical structure greatly determines the most suitable method for its determination, different methods are usually available for the same analyte due to the large amount of possibilities and by coupling different methods to obtain optimum analyte separation and detection.

Separation methods are based on the principles of chromatography and are generally coupled to high sensitivity and selectivity detection techniques leading to quantifying an analyte with a high level of precision and exactitude and also its unequivocal identification at very low concentration levels. The chromatographic methods used for determining analytes in complex matrices would be gas chromatography (GC), high performance liquid chromatography (HPLC), ionic chromatography (IC), size exclusion chromatography (SEC), supercritical fluid chromatography (SFC), affinity chromatography (AC).

Spectrometric methods are also used either alone or coupled to chromatographic or immunochemical methods such as ultraviolet-visible absorption spectrometry, absorption spectrometry in the near and middle infrared sections, fluorescence and chemiluminescence spectrometry, X-ray fluorescence spectrometry, atomic absorption spectrometry, atomic emission spectrometry (AES), inductively-coupled plasma atomic emission spectrometry (ICP-AES), nuclear magnetic resonance (NMR), mass spectrometry (MS) and mass spectrometry in tandem (MS/MS) (Mastovska, 2011).

Other separations methods are used in determined analysis such as capillary electrophoresis (CE), electro capillary chromatography (ECC) and polarimetry (Rouessac & Rouessac, 2003).

#### **5. Veterinary drugs and growth promoters in food of animal origin**

Currently, rearing animals aimed at feeding the human population mainly depends on using pharmacologically-active compounds. Using drugs in the animals is fundamental for animal health and wellbeing and for the economy of agribusiness. The reported benefits are mainly derived from keeping animals in good health, thereby reducing the possibility of a disease becoming transmitted from animals to humans. However, residues from drugs used in producing food of animal origin could increase the risk of disease in the people who consume products from treated animals.

In principle, all pharmaceutical preparations administered to animals producing foodstuffs can give rise to residues in edible tissue, milk or eggs. In addition to drug dose, residue levels depend on withdrawal time. In spite of most drugs representing a relatively low risk for the general public, when used responsibly and in line with instructions approved by the laboratories making veterinary drugs, adverse reactions have been frequently reported for some compounds; these would include antibacterial, antihelminthic, anticoccidial and antiprotozoal drugs and growth promoters.

#### **5.1 Antibacterial drugs**

170 Public Health – Methodology, Environmental and Systems Issues

reduced prior cleaning treatment. These assays are based on the reaction of an antigen binding to a specific primary antibody or for each antigen, analogously to an enzymesubstrate reaction. The most common immunochemical methods would include the enzyme-linked immunosorbent assay (ELISA), direct and indirect competitive enzymelinked immunosorbent assays, immunoaffinity chromatography (IAC), radioimmunoassay (RIA), the enzyme-monitored immunotest (EMIT), the fluorescent immunoassay (FIA) and

Physical-chemical methods are mainly used for isolating, separating, quantifying and confirming the presence of dangerous residues in samples; this requires that the sensitivity of a particular selection method and the determinative or confirmation method are similar. Numerous procedures based on the analytes'different physicochemical properties have been developed for achieving this objective. Even though a drug's chemical structure greatly determines the most suitable method for its determination, different methods are usually available for the same analyte due to the large amount of possibilities and by coupling

Separation methods are based on the principles of chromatography and are generally coupled to high sensitivity and selectivity detection techniques leading to quantifying an analyte with a high level of precision and exactitude and also its unequivocal identification at very low concentration levels. The chromatographic methods used for determining analytes in complex matrices would be gas chromatography (GC), high performance liquid chromatography (HPLC), ionic chromatography (IC), size exclusion chromatography (SEC),

Spectrometric methods are also used either alone or coupled to chromatographic or immunochemical methods such as ultraviolet-visible absorption spectrometry, absorption spectrometry in the near and middle infrared sections, fluorescence and chemiluminescence spectrometry, X-ray fluorescence spectrometry, atomic absorption spectrometry, atomic emission spectrometry (AES), inductively-coupled plasma atomic emission spectrometry (ICP-AES), nuclear magnetic resonance (NMR), mass spectrometry (MS) and mass

Other separations methods are used in determined analysis such as capillary electrophoresis (CE), electro capillary chromatography (ECC) and polarimetry (Rouessac & Rouessac, 2003).

Currently, rearing animals aimed at feeding the human population mainly depends on using pharmacologically-active compounds. Using drugs in the animals is fundamental for animal health and wellbeing and for the economy of agribusiness. The reported benefits are mainly derived from keeping animals in good health, thereby reducing the possibility of a disease becoming transmitted from animals to humans. However, residues from drugs used in producing food of animal origin could increase the risk of disease in the people who

In principle, all pharmaceutical preparations administered to animals producing foodstuffs can give rise to residues in edible tissue, milk or eggs. In addition to drug dose, residue

**5. Veterinary drugs and growth promoters in food of animal origin** 

different methods to obtain optimum analyte separation and detection.

supercritical fluid chromatography (SFC), affinity chromatography (AC).

spectrometry in tandem (MS/MS) (Mastovska, 2011).

consume products from treated animals.

the chemiluminescence immunoassay.

**4.4 Physical-chemical assays** 

Residues from antibacterial drugs in food products of animal origin can represent a danger for consumers. The poisonous effects are not very probable since the residues are present in very low concentrations. Some substances must receive particular attention due allergic reactions. The main hazardous effect is likely to be the development of resistant bacterial strains following sub-therapeutic doses of antimicrobials being ingested; such resistance could be transferred to other bacteria. This could include resistance being transferred from non-pathogenic organisms to pathogenic ones which would then no longer respond to standard drug treatment (the Institute of Food Technologists [IFT], 2006). However, the generally used antibacterial drugs are presented in Table 1.

Differences in substitutions in the basic ring structures between the various aminoglycosides account for the relatively minor differences in antimicrobial spectra and resistance and toxicity patterns. Aminoglycosides given in therapeutic dosages mainly cause ototoxicosis, but may also cause nephrotoxicosis, allergy and neuromuscular disturbances.

Chloramphenicol (an antibacterial belonging to the amphenicol group) has been used in treatment and prophylactically in food-producing animals for several years now (i.e. poultry, calves, pigs, sheep and fish). Chloramphenicol's most serious toxic effect is bone marrow depression which is generally dose-related and reversible but can sometimes be fatal in patients who are probably genetically predisposed. A toxic syndrome has been reported in newborn infants receiving large doses of chloramphenicol which is characterised by vomiting, hypothermia, cyanosis and circulatory collapse followed by death; this syndrome rarely occurs in adults. Chloramphenicol may also cause neuritis, encephalopathy with dementia and ototoxicity; its use is restricted in many countries, while it is totally banned for use in food-producing animals within the European Union and the USA. Chloramphenicol and its metabolites could be genotoxic (Lozano & Arias, 2008).

Differences in substitutions in the basic ring structures between the various aminoglycosides account for the relatively minor differences in antimicrobial spectra and resistance and toxicity patterns. Aminoglycosides given in therapeutic dosages mainly cause ototoxicosis, but may also cause nephrotoxicosis, allergy and neuromuscular disturbances.

Chloramphenicol (an antibacterial belonging to the amphenicol group) has been used in treatment and prophylactically in food-producing animals for several years now (i.e. poultry, calves, pigs, sheep and fish). Chloramphenicol's most serious toxic effect is bone marrow depression which is generally dose-related and reversible but can sometimes be fatal in patients who are probably genetically predisposed. A toxic syndrome has been reported in newborn infants receiving large doses of chloramphenicol which is characterised by vomiting, hypothermia, cyanosis and circulatory collapse followed by death; this syndrome rarely occurs in adults. Chloramphenicol may also cause neuritis, encephalopathy with dementia and ototoxicity; its use is restricted in many countries, while it is totally

Chemical Residues in Animal Food Products: An Issue of Public Health 173

dermatitis caused by contact and urticaria (hives) caused by consuming residues present in meat and/or milk (Medina et al., 2008). The allergic reactions caused by penicillin and its derivatives have been considered by the JECFA committee as being determinant factors for evaluating and establishing safe residue levels in foodstuffs. The adverse effects associated

Lincomycine-macrolide is used for the initial treatment of mild to moderate staphylococcal infections in calves, sheep, goats and pigs and it is also added in feed for growth-promoting purposes. Lincomycine is reported to cause gastrointestinal disturbances including diarrhoea, vomiting and nausea which that may prove fatal. Other adverse effects include skin rashes, urticaria, polyarthritis, hepatic damage and haematological disturbances (WHO,

All nitrofurans have been widely used in the prophylactic and therapeutic treatment of infections caused by bacteria and protozoa in pigs, cattle, poultry, rabbits and fish. The use of nitrofurans in food-producing animals has been controversial because residues from these drugs may be mutagenic and tumorigenic. Toxicological studies have shown that nitrofurazone is a carcinogenic but not genotoxic agent, whereas furazolidone has exhibited both carcinogenic and genotoxic properties (WHO, 1993). The metabolites from nitrofurans can remain stored for weeks or m animal proteins, including eggs from farmyard birds, species in which this compound has been used as an anticoccidial. The systemic use of nitrofurans in food producing animals has thus been prohibited in the USA and Europe,

Quinolones are synthetic antibiotics which are very effective in combating various diseases in animal husbandry and aquaculture. The most frequent adverse affects of quinolone antibiotics most frequently occurring adverse affects are gastrointestinal disturbances including nausea, vomiting, diarrhoea, headache, visual disturbances and insomnia. Rashes, pruritus and epidermal necrolysis have sometimes also occurred (Jimenez et al., 2011).

The residuality of sulphonamides used in treating coccidial and bacterial infections and also as growth-promoting agents may cause hypersensitivity reactions, mainly cutaneous rash;

There is sufficient evidence indicating that ingesting the antibiotics in sub-therapeutic doses makes a significant contribution towards the appearance of resistant microorganisms in animals which can become transmitted to humans, thereby provoking difficult to treat infections. Some sample studies have found antibiotic-resistant coliform microorganisms in raw and cooked meat. Likewise, antibiotics consumed by human beings from residues present in food of animal origin lead to an alteration of intestinal flora and consequently a reduction of bacteria competing with pathogenous microorganisms, thereby increasing the

Tetracyclines can generate bacterial resistance; oxytetracycline particularly induces antibiotic resistance in coliform microorganisms present in the human intestine. Recognition of this effect has been used by the JECFA committee as the point of reference for defining

The problem of resistance is not the only motive for the medical community's preoccupation. Farmers and veterinarians are worried because bacterial resistance in farm

acceptable consumption levels for different antibiotics.

however, no anaphylactic manifestations caused by this type of residues is known.

with cephalosporins are similar to those described for penicillins.

1989).

(EMEA, 1997).

risk of disease.

banned for use in food-producing animals within the European Union and the USA. Chloramphenicol and its metabolites could be genotoxic (Lozano & Arias, 2008).


Table 1. Drugs administrated in treatment and prophylactically in food-producing animals

Penicillins have low toxicity; hypersensitivity reactions, especially skin rashes, are by far their most common adverse effects. Gastrointestinal disturbances including diarrhoea, nausea and vomiting may also sometimes appear. No teratogenic effects have been reported. Some studies have indicated that sensitive people have experienced allergic reactions, such as genera pruritis (itching), difficulty in swallowing and talking, dyspnoea,

banned for use in food-producing animals within the European Union and the USA.

Amphenicols Chloramphenicol, thiamphenicol, florfenicol

Macrolides Erythromycin, spiramycin, kitasamycin, josamycin,

Tetracyclines Chlortetracycline, oxytetracycline, demeclocycline,

Salicylanilides Closantel, niclosamide, oxyclozanide, rafoxanide

Quinolonederivates Buquinolate, decoquinate, methylbenzoquate

Anabolic hormones Estradiol-17, progesterone, testosterone

Antibiotics Monensin, salinomycin, bambermycin, avilamycin

Nitroimidazoles Ronidazole, dimetridazole, metronidazole, ipronidazole Polyether ionophore Monensin, narasin, lasalocid, salinomycin, maduramicin

Peptide antibiotics Avoparcin, bacitracin, efrotomycin, enramycin, thiopeptin

Benzamidazoles Thiabendazol, flubendazol, fenbendazol, mebendazol, albendazol, oxfendazol, febantel

Nitrofurans Furazolidone, nitrofurazone, furaltadone, nitrofurantoin Quinolones Ciprofloxacin, danofloxacin, difloxacin, enrofloxacin,

Aminoglicosides Streptomycin, kanamycin, amikacin, neomycin, apramycin

desmycosin, mirosamycin, tilmicosin, leucomycin, tylosin

flumequine, marbofloxacin, norfloxacin, ofloxacin

Sulfadiazine, sulfadimethoxine, sulfamethazine, sulfadoxine, sulfaethoxypyridazine, sulfaguanidine, sulfamerazine, sulfamethoxazole, sulfapyridine, sulfamethoxydiazine, sulfamethoxypyridazine, sulfamonomethoxine, sulfathiazole, sulfaquinoxaline

Boldenone, chlormadinone acetate, ethylenestrol, fluoxymesterone, medroxyprogesterone acetate, megestrol

Bambuterol, bromobuterol, carbuterol, cimaterol, clenbuterol, dobutamine, fenoterol, isoproterenol, mabuterol, mapenterol, metaproterenol, pirbuterol, ractopamine, reproterol, rimiterol, ritodrine, salbutamol

acetate, methandienone, methylboldenone, methyltestosterone, drostanolone, norethandrolone, norgestomet, norgestrel, nortestosterone oxymetholone

doxycycline, methacycline, minocycline

Chloramphenicol and its metabolites could be genotoxic (Lozano & Arias, 2008).

Beta-lactams Penicilins, cephalosporins

Imidazotiazoles Levamisole

Tetrahydropyrimidines Morantel, pyrantel

Carbanilides Nicarbazin, imidocarb

Organic arsenicals Arsanilic acid

Quinoxaline-1,4-dioxides Carbadox, olaquindox

Table 1. Drugs administrated in treatment and prophylactically in food-producing animals

Penicillins have low toxicity; hypersensitivity reactions, especially skin rashes, are by far their most common adverse effects. Gastrointestinal disturbances including diarrhoea, nausea and vomiting may also sometimes appear. No teratogenic effects have been reported. Some studies have indicated that sensitive people have experienced allergic reactions, such as genera pruritis (itching), difficulty in swallowing and talking, dyspnoea,

Organophosphates Haxolon, coumaphos, dichlorvos

Sustituted phenols Dichlorophen, hexachlorophen Macrocycliclactones Abamectin, ivermectin, moxidectin Piperazinederivates Piperazine, diethylcarbamazine

Benzamides Aklomide, nitromide, dinitolmide

Triazines Clazuril, diclazuril, toltrazuril

Antibacterials

Antihelmintics

Antiprotozoals

Growth promoters Sulphonamides

Synthetic steroidal

Beta-adrenergic agonists

dermatitis caused by contact and urticaria (hives) caused by consuming residues present in meat and/or milk (Medina et al., 2008). The allergic reactions caused by penicillin and its derivatives have been considered by the JECFA committee as being determinant factors for evaluating and establishing safe residue levels in foodstuffs. The adverse effects associated with cephalosporins are similar to those described for penicillins.

Lincomycine-macrolide is used for the initial treatment of mild to moderate staphylococcal infections in calves, sheep, goats and pigs and it is also added in feed for growth-promoting purposes. Lincomycine is reported to cause gastrointestinal disturbances including diarrhoea, vomiting and nausea which that may prove fatal. Other adverse effects include skin rashes, urticaria, polyarthritis, hepatic damage and haematological disturbances (WHO, 1989).

All nitrofurans have been widely used in the prophylactic and therapeutic treatment of infections caused by bacteria and protozoa in pigs, cattle, poultry, rabbits and fish. The use of nitrofurans in food-producing animals has been controversial because residues from these drugs may be mutagenic and tumorigenic. Toxicological studies have shown that nitrofurazone is a carcinogenic but not genotoxic agent, whereas furazolidone has exhibited both carcinogenic and genotoxic properties (WHO, 1993). The metabolites from nitrofurans can remain stored for weeks or m animal proteins, including eggs from farmyard birds, species in which this compound has been used as an anticoccidial. The systemic use of nitrofurans in food producing animals has thus been prohibited in the USA and Europe, (EMEA, 1997).

Quinolones are synthetic antibiotics which are very effective in combating various diseases in animal husbandry and aquaculture. The most frequent adverse affects of quinolone antibiotics most frequently occurring adverse affects are gastrointestinal disturbances including nausea, vomiting, diarrhoea, headache, visual disturbances and insomnia. Rashes, pruritus and epidermal necrolysis have sometimes also occurred (Jimenez et al., 2011).

The residuality of sulphonamides used in treating coccidial and bacterial infections and also as growth-promoting agents may cause hypersensitivity reactions, mainly cutaneous rash; however, no anaphylactic manifestations caused by this type of residues is known.

There is sufficient evidence indicating that ingesting the antibiotics in sub-therapeutic doses makes a significant contribution towards the appearance of resistant microorganisms in animals which can become transmitted to humans, thereby provoking difficult to treat infections. Some sample studies have found antibiotic-resistant coliform microorganisms in raw and cooked meat. Likewise, antibiotics consumed by human beings from residues present in food of animal origin lead to an alteration of intestinal flora and consequently a reduction of bacteria competing with pathogenous microorganisms, thereby increasing the risk of disease.

Tetracyclines can generate bacterial resistance; oxytetracycline particularly induces antibiotic resistance in coliform microorganisms present in the human intestine. Recognition of this effect has been used by the JECFA committee as the point of reference for defining acceptable consumption levels for different antibiotics.

The problem of resistance is not the only motive for the medical community's preoccupation. Farmers and veterinarians are worried because bacterial resistance in farm

Chemical Residues in Animal Food Products: An Issue of Public Health 175

A number of nitroimidazoles have already been banned within the European Union, even for therapeutic purposes, since they are mutagens and suspected carcinogens. The use of ronidazole has been banned by Council Regulation 3426/93/EEC (Official Journal of the European Communities, 1993) whereas dimetridazole use is banned by Council Regulation 1798/95/EEC (Official Journal of the European Communities, 1995). Their antibacterial and mutagenic activity is closely related to the reduction of the 5-nitro group, which is common to all nitroimidazole drugs. Metronidazole is used for treating bovine trichomoniasis by topical application or intravenous injection but it is a genotoxic carcinogen in animals.

Polyether antibiotics are produced by various actinomyces, mostly *Streptomyces* species, and constitute the agents most widely used by the poultry industry over the last two decades. They provide excellent disease control and are refractory for the development of resistance. They have a low therapeutic index but may be very toxic in certain species; salinomycin and

The problem of residues from antihelminthic, anticoccidial and antiprotozoal drugs may be easily controlled by imposing obligatory withdrawal times, generally 7-10 days, but this is unfortunately not always respected. On the other hand, given the large number of drugs which may be easily obtained on the market, many producers change one compound for another to avoid resistance becoming developed to drugs; however, this increases the degree of exposure to thema the misms, which may lead to yet another problem if it is taken

Growth promoters are substances which produce improvements in growth rate when added to animal feed in sub-therapeutic dosages over an extended period of time. Table 1 shows the compounds most commonly used for this purpose. The anabolic hormonal-type growth promoters can be classified according to their chemical structure or origin into endogenous sex steroids, steroidal compounds, not naturally occurring non-steroidal compounds and

Anabolic hormones (estradiol-17 and progesterone - two female sex hormones, and testosterone - one male sex hormone) are used for increasing body mass in livestock rearing. Synthetic steroidal compounds have only been approved for therapy regarding reproductive behaviour and disorders in non-food-producing animals; however, they are used illegally around the world. Boldenone, chlormadinone acetate, ethylenestrol, fluoxymesterone, medroxyprogesterone acetate, megestrol acetate, methandienone, methylboldenone, methylthisterone, drostanolone, norethandrolone, norgestomet, norgestrel, norethisterone (nandrolone), norethisterone decanoate, oxymetholone, and stanozolol would be examples of synthetic steroidal compounds which have only been approved for therapy regarding reproductive behaviour and disorders in non-food-

Zeranol and stilbene estrogens, including diethylstilbestrol, hexestrol and dienestrol, are the major non-steroidal not naturally occurring compounds included in the class of anabolic drugs and somatropin is the most common polypeptide compound affecting growth. Diethylstilbestrol, hexestrol and dienestrol are all stilbene estrogens which are currently banned worldwide for use in food-producing animals. They are genotoxic, not easily

narasin can be fatally toxic in turkeys, for example (Weissinger, 1994).

into account that these drugs are also used as growth promoters.

**5.3 Growth promoters** 

polypeptide hormones.

producing animals.

animals is interfering with drug efficacy thereby leading to the use of greater concentrations than those initially established as being therapeutic. However, in spite of antibiotics being the type of veterinary drugs most used in the agribusiness industry, there are few options to choose from due to the limited offer of drugs which have been approved for use in animals producing foodstuffs compared to those regarding therapeutic use in humans.

#### **5.2 Antihelminthic, anticoccidial and antiprotozoal drugs**

Parasitic diseases constitute an ever present threat in rearing birds and livestock, but they may be controlled by adding low levels of drugs to daily rations. The drugs generally against internal parasites affecting animals collectively called helminths are shown in Table 1. Such drugs are used at levels which do not allow resistant strains to develop and also become rapidly metabolised in an animal's organism so that the residues in edible tissues are minimal.

Benzimidazoles, like thiabendazole, are used in sheep, cattle, horses, pigs and poultry. They become rapidly eliminated from the organism due to their high solubility; however, some studies has shown that these compounds are teratogenic and nephrotoxic in mice and ewes (Danaher et al., 2007). Mebendazole metabolits (hydroxylmebendazole and aminomebendazole) belonging to the benzimidazole group and which are widely used as an antinematode in horses, sheep, pigs and poultry, have also been shown to have teratogenic effects (Buchmann et al., 1992).

Levamisol is the most well-known drug from the imidazothiazole group, which has a broad spectrum of activity against nematodes; however, it has been found that it induces idiosyncratic organulocytosis in some individuals. Levamisol's toxic effects have caused preoccupation in the regulatory bodies and, given that these effects the original compound than in its metabolites, this is the analyte of interest in tissue samples.

Organophosphates represent one of the alternatives for treating benzimidazole-resistant nematodes; haloxon (being one of them) is the safest and has been approved by the US Food and Drug Administration (FDA) for use in sheep, cattle and goats. By contrast, dichlorvos has an acceptable antihelminthic spectrum in cattle and sheep, but it does not have FDA approval for use in ruminants due to its suspected carcinogenic effects and narrow safety margin (Botsoglou & Fletouris, 2000).

Ivermectin, a macrocyclic lactone, is exceptionally effective in very low dosages against nematodes and arthropod parasites in cattle and has been widely used for treating endoand ecto-parasites in cattle, sheep, goats and pigs; however, ivermectin has had a teratogenic effect in rats, rabbits and mice (Moreno et al., 2008).

Anticoccidial and antiprotozoal drugs are generally used in the poultry industry against protozoan infections caused by pathogenic species of *Eimeria.* Some compounds used as antibacterial drugs are also used as coccidiostats, including sulphaquinoxaline, sulphadimethoxine, sulphamethoxypyridazine, sulphachlorpyrazine, sulphamethazine, sulphaguanidine, furazolidone, nitrofurazone, tetracycline and chlortetracycline. Table 1 shows compounds whose primary function and use are related to antiprotozoal drugs.

A number of nitroimidazoles have already been banned within the European Union, even for therapeutic purposes, since they are mutagens and suspected carcinogens. The use of ronidazole has been banned by Council Regulation 3426/93/EEC (Official Journal of the European Communities, 1993) whereas dimetridazole use is banned by Council Regulation 1798/95/EEC (Official Journal of the European Communities, 1995). Their antibacterial and mutagenic activity is closely related to the reduction of the 5-nitro group, which is common to all nitroimidazole drugs. Metronidazole is used for treating bovine trichomoniasis by topical application or intravenous injection but it is a genotoxic carcinogen in animals.

Polyether antibiotics are produced by various actinomyces, mostly *Streptomyces* species, and constitute the agents most widely used by the poultry industry over the last two decades. They provide excellent disease control and are refractory for the development of resistance. They have a low therapeutic index but may be very toxic in certain species; salinomycin and narasin can be fatally toxic in turkeys, for example (Weissinger, 1994).

The problem of residues from antihelminthic, anticoccidial and antiprotozoal drugs may be easily controlled by imposing obligatory withdrawal times, generally 7-10 days, but this is unfortunately not always respected. On the other hand, given the large number of drugs which may be easily obtained on the market, many producers change one compound for another to avoid resistance becoming developed to drugs; however, this increases the degree of exposure to thema the misms, which may lead to yet another problem if it is taken into account that these drugs are also used as growth promoters.

#### **5.3 Growth promoters**

174 Public Health – Methodology, Environmental and Systems Issues

animals is interfering with drug efficacy thereby leading to the use of greater concentrations than those initially established as being therapeutic. However, in spite of antibiotics being the type of veterinary drugs most used in the agribusiness industry, there are few options to choose from due to the limited offer of drugs which have been approved for use in animals producing foodstuffs compared to those regarding

Parasitic diseases constitute an ever present threat in rearing birds and livestock, but they may be controlled by adding low levels of drugs to daily rations. The drugs generally against internal parasites affecting animals collectively called helminths are shown in Table 1. Such drugs are used at levels which do not allow resistant strains to develop and also become rapidly metabolised in an animal's organism so that the residues in edible tissues

Benzimidazoles, like thiabendazole, are used in sheep, cattle, horses, pigs and poultry. They become rapidly eliminated from the organism due to their high solubility; however, some studies has shown that these compounds are teratogenic and nephrotoxic in mice and ewes (Danaher et al., 2007). Mebendazole metabolits (hydroxylmebendazole and aminomebendazole) belonging to the benzimidazole group and which are widely used as an antinematode in horses, sheep, pigs and poultry, have also been shown to have teratogenic

Levamisol is the most well-known drug from the imidazothiazole group, which has a broad spectrum of activity against nematodes; however, it has been found that it induces idiosyncratic organulocytosis in some individuals. Levamisol's toxic effects have caused preoccupation in the regulatory bodies and, given that these effects the original compound

Organophosphates represent one of the alternatives for treating benzimidazole-resistant nematodes; haloxon (being one of them) is the safest and has been approved by the US Food and Drug Administration (FDA) for use in sheep, cattle and goats. By contrast, dichlorvos has an acceptable antihelminthic spectrum in cattle and sheep, but it does not have FDA approval for use in ruminants due to its suspected carcinogenic effects and narrow safety

Ivermectin, a macrocyclic lactone, is exceptionally effective in very low dosages against nematodes and arthropod parasites in cattle and has been widely used for treating endoand ecto-parasites in cattle, sheep, goats and pigs; however, ivermectin has had a

Anticoccidial and antiprotozoal drugs are generally used in the poultry industry against protozoan infections caused by pathogenic species of *Eimeria.* Some compounds used as antibacterial drugs are also used as coccidiostats, including sulphaquinoxaline, sulphadimethoxine, sulphamethoxypyridazine, sulphachlorpyrazine, sulphamethazine, sulphaguanidine, furazolidone, nitrofurazone, tetracycline and chlortetracycline. Table 1 shows compounds whose primary function and use are related to antiprotozoal drugs.

than in its metabolites, this is the analyte of interest in tissue samples.

teratogenic effect in rats, rabbits and mice (Moreno et al., 2008).

therapeutic use in humans.

effects (Buchmann et al., 1992).

margin (Botsoglou & Fletouris, 2000).

are minimal.

**5.2 Antihelminthic, anticoccidial and antiprotozoal drugs** 

Growth promoters are substances which produce improvements in growth rate when added to animal feed in sub-therapeutic dosages over an extended period of time. Table 1 shows the compounds most commonly used for this purpose. The anabolic hormonal-type growth promoters can be classified according to their chemical structure or origin into endogenous sex steroids, steroidal compounds, not naturally occurring non-steroidal compounds and polypeptide hormones.

Anabolic hormones (estradiol-17 and progesterone - two female sex hormones, and testosterone - one male sex hormone) are used for increasing body mass in livestock rearing. Synthetic steroidal compounds have only been approved for therapy regarding reproductive behaviour and disorders in non-food-producing animals; however, they are used illegally around the world. Boldenone, chlormadinone acetate, ethylenestrol, fluoxymesterone, medroxyprogesterone acetate, megestrol acetate, methandienone, methylboldenone, methylthisterone, drostanolone, norethandrolone, norgestomet, norgestrel, norethisterone (nandrolone), norethisterone decanoate, oxymetholone, and stanozolol would be examples of synthetic steroidal compounds which have only been approved for therapy regarding reproductive behaviour and disorders in non-foodproducing animals.

Zeranol and stilbene estrogens, including diethylstilbestrol, hexestrol and dienestrol, are the major non-steroidal not naturally occurring compounds included in the class of anabolic drugs and somatropin is the most common polypeptide compound affecting growth. Diethylstilbestrol, hexestrol and dienestrol are all stilbene estrogens which are currently banned worldwide for use in food-producing animals. They are genotoxic, not easily

Chemical Residues in Animal Food Products: An Issue of Public Health 177

In spite of the aforementioned effects, the Codex Alimentarius considers it unnecessary to establish an LMR for anabolic hormones as it is improbable that residues arising from the correct used of these substances as growth stimulators represent a danger for human health. It has also been demonstrated that the endogenous concentration of these hormones is greater when they are administered exogenously. Another reason negating the potential risk of this type of substance is the availability of metabolic routes which become rapidly degraded, meaning that the residues which the meat of treated animals may contain do not affect a consumer's endocrine system. However, dispositions in Europe regarding these substances are stricter and do not allow any residual level of

Peptide antibiotics are compounds usually containing D-amino acids. They are usually added to animal feeds in low concentrations and produce residues in tissues at very low or undetectable levels. Unfortunately, most peptide antibiotics' metabolic pathways have not yet been elucidated. These antibiotics are regulated under separate legislation within the

Quinoxaline-1,4-dioxides and their possible residues in edible animal products have caused much debate regarding their mutagenic and carcinogenic potency. Carbadox was initially the main drug in use, but suspicion as to its safety arose because this compound exhibited both genotoxic and mutagenic activity. Olaquindox is also a strongly mutagenic agent but

Toxins have a biological origin, mycotoxins, phycotoxins and phytotoxins having attracted most attention due to their potential residuality in foodstuffs, including animal subproducts.

Mycotoxins are secondary metabolites from fungi, mainly from the species *Aspergillus*, *Fusarium* and *Penicillium*, aflatoxins, ochratoxins, zearalenone, trichothecenes and fumonisins having been the most studied to date. The foodstuffs fundamentally contaminated by these toxins are grains and cereals constituting the main source of contamination for human beings. However, farm animals consuming contaminated foodstuffs may generate residues in meat, viscera, milk and eggs. Residuality is determined by contamination by high concentrations in foodstuffs ingested by animals, this being very uncommon, and also by theway in which the xenobiotic becomes metabolised in the organism. Mycotoxins do not become totally destroyed during cooking or industrialisation

The types of mycotoxicosis (disease resulting from consuming mycotoxins) in human beings are mainly chronic. These would include Balkan endemic neuropathy in Russia caused by the consumption of ochratoxin A which generates nephrotoxicosis, alimentary toxic aleukia in the former Soviet Union associated with dermatitis, vomiting and hematopoietic alterations caused by trichothecenes (diacetoxiscirpenol and T-2 toxin), possible endocrinal alterations related to reduced masculine fertility caused by consuming zearalenone (such toxin acting as an xenoestrogen), hepatic cancer caused by aflatoxin B1 and possible esophagic and renal cancer caused by fumonisin and ochratoxin A, respectively. The IARC

anabolizant drugs in meats.

European Union (Brogden et al., 2003).

seemingly devoid of carcinogenic activity.

**6. Toxins in food of animal origin** 

of foodstuffs due to their heat-stability.

**6.1 Mycotoxins** 

metabolised compounds, which are considered capable of irreversibly initiating the carcinogenic process even in small residue concentrations. Diethylstilbestrol and hexestrol have been legally permitted for use as anabolics for quite some time in many countries, whilst the use of dienestrol, which is a diethylstilbestrol metabolite, has been restricted to illegal practice (Dickson, 2003).

Using these compounds, either natural or synthetic, as growth promoters in meat-producing animals has not been allowed in the European Union since 1988, due to potential adverse effects to human health, unlike in the United States where some anabolic hormonal-type growth promoters are permitted.

*In vivo* studies have demonstrated DNA strand breaks and oxidative damage being triggered by desencadenados por the 17-β estradiol, thereby leading to this hormone being considered as triggering a genotoxic effect (for example, the proliferation of carcinogenic mammary cells); however, the dosage at which these alterations occur is greater than that at which endocrine effects are produced in animals (Mikus et al., 2001).

Testosterone's adverse effects are due to its hormonal activity, particularly in the prostate gland. Testosterone is also considered to be potentially embryotoxic and its consumption in therapeutic doses has led to the induction of hepatic cystitis (Durlinger et al., 2002).

Following the ban of stilbene and other hormonal-type growth promoters, interest has focused on alternative compounds for promoting live weight gain in food-producing animals. The beta-adrenergic agonists constitute such group of compounds, clembuterol being the main one. It has been reported that consuming calf liver in Spain and France containing clenbuterol residues has induced muscular tremors, tachycardia, muscular pain, nervousness, headache, vertigo, nausea, vomiting and fever. It has also been used as an anabolizant steroid; clenbuterol is used as a tocolytic in cows, thereby supposing an additional risk.

A controversy has arisen around these events regarding whether to accept or prohibit using clenbuterol in animal production. This drug increases channel performance, it is not potentially oncogenic or mutagenic and is only embryotoxic in large doses whilst its adverse effects on consumers becomes evident when recommended withdrawal times are not respected and when excessive doses are used, whether through inadequate management or aimed at increasing animals' weight gain even more (Brambilla et al., 2007).

The foregoing has led to clenbuterol being a highly controlled drug today in many countries which have developed programmes and mechanisms for monitoring it and its follow-up. However, in spite of these controls and warning signs, unfortunate events involving adverse reactions continue to be presented, as happened in November 2005 in Jalisco, México, when about 225 people experienced trembling, headaches and discomfort after having consumed beef containing residues of this type (Gojmerac, 2002).

Arsanylic acid, peptide antibiotics and quinoxaline-1,4-dioxides are non-steroidal compounds used as growth promoters in different animal species. Arsanylic acid and its sodium salt are most commonly used, particularly in pigs. They are also efficacious in the egg-producing industry and were previously approved for use in egg-laying hens. However, their use in animals is generally rather limited and the risk–benefit ratio is questionable because these drugs can produce toxicosis, known as peripheral nerve demyelination.

In spite of the aforementioned effects, the Codex Alimentarius considers it unnecessary to establish an LMR for anabolic hormones as it is improbable that residues arising from the correct used of these substances as growth stimulators represent a danger for human health. It has also been demonstrated that the endogenous concentration of these hormones is greater when they are administered exogenously. Another reason negating the potential risk of this type of substance is the availability of metabolic routes which become rapidly degraded, meaning that the residues which the meat of treated animals may contain do not affect a consumer's endocrine system. However, dispositions in Europe regarding these substances are stricter and do not allow any residual level of anabolizant drugs in meats.

Peptide antibiotics are compounds usually containing D-amino acids. They are usually added to animal feeds in low concentrations and produce residues in tissues at very low or undetectable levels. Unfortunately, most peptide antibiotics' metabolic pathways have not yet been elucidated. These antibiotics are regulated under separate legislation within the European Union (Brogden et al., 2003).

Quinoxaline-1,4-dioxides and their possible residues in edible animal products have caused much debate regarding their mutagenic and carcinogenic potency. Carbadox was initially the main drug in use, but suspicion as to its safety arose because this compound exhibited both genotoxic and mutagenic activity. Olaquindox is also a strongly mutagenic agent but seemingly devoid of carcinogenic activity.

#### **6. Toxins in food of animal origin**

Toxins have a biological origin, mycotoxins, phycotoxins and phytotoxins having attracted most attention due to their potential residuality in foodstuffs, including animal subproducts.

#### **6.1 Mycotoxins**

176 Public Health – Methodology, Environmental and Systems Issues

metabolised compounds, which are considered capable of irreversibly initiating the carcinogenic process even in small residue concentrations. Diethylstilbestrol and hexestrol have been legally permitted for use as anabolics for quite some time in many countries, whilst the use of dienestrol, which is a diethylstilbestrol metabolite, has been restricted to

Using these compounds, either natural or synthetic, as growth promoters in meat-producing animals has not been allowed in the European Union since 1988, due to potential adverse effects to human health, unlike in the United States where some anabolic hormonal-type

*In vivo* studies have demonstrated DNA strand breaks and oxidative damage being triggered by desencadenados por the 17-β estradiol, thereby leading to this hormone being considered as triggering a genotoxic effect (for example, the proliferation of carcinogenic mammary cells); however, the dosage at which these alterations occur is greater than that at

Testosterone's adverse effects are due to its hormonal activity, particularly in the prostate gland. Testosterone is also considered to be potentially embryotoxic and its consumption in

Following the ban of stilbene and other hormonal-type growth promoters, interest has focused on alternative compounds for promoting live weight gain in food-producing animals. The beta-adrenergic agonists constitute such group of compounds, clembuterol being the main one. It has been reported that consuming calf liver in Spain and France containing clenbuterol residues has induced muscular tremors, tachycardia, muscular pain, nervousness, headache, vertigo, nausea, vomiting and fever. It has also been used as an anabolizant steroid; clenbuterol is used as a tocolytic in cows, thereby supposing an

A controversy has arisen around these events regarding whether to accept or prohibit using clenbuterol in animal production. This drug increases channel performance, it is not potentially oncogenic or mutagenic and is only embryotoxic in large doses whilst its adverse effects on consumers becomes evident when recommended withdrawal times are not respected and when excessive doses are used, whether through inadequate management or

The foregoing has led to clenbuterol being a highly controlled drug today in many countries which have developed programmes and mechanisms for monitoring it and its follow-up. However, in spite of these controls and warning signs, unfortunate events involving adverse reactions continue to be presented, as happened in November 2005 in Jalisco, México, when about 225 people experienced trembling, headaches and discomfort after having consumed

Arsanylic acid, peptide antibiotics and quinoxaline-1,4-dioxides are non-steroidal compounds used as growth promoters in different animal species. Arsanylic acid and its sodium salt are most commonly used, particularly in pigs. They are also efficacious in the egg-producing industry and were previously approved for use in egg-laying hens. However, their use in animals is generally rather limited and the risk–benefit ratio is questionable because these drugs can produce toxicosis, known as peripheral nerve

aimed at increasing animals' weight gain even more (Brambilla et al., 2007).

beef containing residues of this type (Gojmerac, 2002).

therapeutic doses has led to the induction of hepatic cystitis (Durlinger et al., 2002).

which endocrine effects are produced in animals (Mikus et al., 2001).

illegal practice (Dickson, 2003).

growth promoters are permitted.

additional risk.

demyelination.

Mycotoxins are secondary metabolites from fungi, mainly from the species *Aspergillus*, *Fusarium* and *Penicillium*, aflatoxins, ochratoxins, zearalenone, trichothecenes and fumonisins having been the most studied to date. The foodstuffs fundamentally contaminated by these toxins are grains and cereals constituting the main source of contamination for human beings. However, farm animals consuming contaminated foodstuffs may generate residues in meat, viscera, milk and eggs. Residuality is determined by contamination by high concentrations in foodstuffs ingested by animals, this being very uncommon, and also by theway in which the xenobiotic becomes metabolised in the organism. Mycotoxins do not become totally destroyed during cooking or industrialisation of foodstuffs due to their heat-stability.

The types of mycotoxicosis (disease resulting from consuming mycotoxins) in human beings are mainly chronic. These would include Balkan endemic neuropathy in Russia caused by the consumption of ochratoxin A which generates nephrotoxicosis, alimentary toxic aleukia in the former Soviet Union associated with dermatitis, vomiting and hematopoietic alterations caused by trichothecenes (diacetoxiscirpenol and T-2 toxin), possible endocrinal alterations related to reduced masculine fertility caused by consuming zearalenone (such toxin acting as an xenoestrogen), hepatic cancer caused by aflatoxin B1 and possible esophagic and renal cancer caused by fumonisin and ochratoxin A, respectively. The IARC

Chemical Residues in Animal Food Products: An Issue of Public Health 179

ZEA is frequently implicated in reproductive disorders in animals and occasionally in hyperestrogenism syndromes in humans. ZEA becomes biotransformed in the intestine by the mucosa or bacterial flora and involves the formation of α- and β-zearalenol and α- and β-zearalanol. Alpha-zearalanol and β-zearalenol have greater estrogenic power than ZEA since they bind with greater force to their corresponding receptors (Zinedine et al., 2007). Alpha-zearalanol has been employed as growth promoter in cattle. Studies orientated towards determining residuality through experimentation have suggested that residues are not present in meat or eggs, even at high doses. However, a recent study has show that the presence of α-zearalenol in meat-based foodstuffs for infants reached levels of 30.5 µg/kg; the same study demonstrated the presence of mycoestrogens (ZEA, α-zearalenole and β-

FUM properties suggest that their presence in animal meat does not represent an important source of contamination, since they are poorly absorbed. FUM produce liquefaction of the brain in horse and pulmonary oedema in pigs; ruminants and birds are more resistant. They have been correlated with oesophageal cancer in humans in some parts of the world and it has also been presumed that they may cause neural tube alterations. Their presence has been demonstrated and in the liver and kidneys of turkeys fed with the maximum levels

The main TCT of interest in producing animals are T-2 toxin, HT-2 toxin, diacetoxiscirpenol and deoxinivalenol. The TCT do not usually represent a risk of contamination in food of

Around 75 species of marine micro-algae, belonging to the dinoflagellate group, produce secondary metabolites which represent potent toxins, generically called phycotoxins. These organisms form part of the marine plankton and therefore the aquatic food chain leading to filtrator mollusks, gastropods, crustaceans and fish which can accumulate toxins being

The microalgae population may increase suddenly and generate an algal bloom which has increased in frequency, intensity and geographical distribution during recent years. Amongst the explanations put forward to explain for this phenomena has been the increased use of coastal waters for aquaculture, eutrophication caused by domestic, industrial and agricultural residues, the mobility of trace metals and humic substances due to deforestation and/or acid rain and changes in climatic conditions (Erdner et al., 2008). Reports of phycotoxins poisoning have increased during the last few years, perhaps due to the scientific community's greater knowledge and interest in the matter or due to the increase in algal bloom. Such poisoning is mainly acute course; however, there is interest in evaluating the effects being triggered by chronic consumption. The lack of studies on animals which are continuously exposed to phycotoxins and the scarce availability of certified reference materials have led to difficulties in evaluating risk, developing analytical

Studying toxins produced by algae has classically been approached according to the type of poisoning which they have caused. Four groups of toxins can thus be distinguished, causing paralytic shellfish poisoning, diarrhoeic shellfish poisoning, amnesic shellfish

zearalenole) in infants' cow milk formulas (Meucci et al., 2011).

animal origin due to their rapid metabolism (Bailly & Guerre, 2009).

permitted in Europe (Tardieu et al., 2008).

methodologies and regulating these substances.

**6.2 Phycotoxins** 

consumed (FAO, 2004).

has classified aflatoxin B1 within group 1 (proven carcinogenic effect on humans) and fumonisin B1 and ochratoxin A within group 2B (possibly carcinogenic to humans) (IARC, 1993). The evidence from *in vitro* studies has shown that zearalenone is a probably implicated in cancer of the reproductive system (Khosrokhavar et al., 2009).

Aflatoxins and ochratoxin A are the main mycotoxins which can generate residuality and attention concerning them as being animal subproduct contaminants has mainly been focused on their presence in milk; however, it has been demonstrated that they can also generate residuality in meat and eggs.

#### **6.1.1 Aflatoxins**

Aflatoxins (AF) B1 B2 G1 and G2 are produced by fungi from the genera *Aspergillus*. AFB1 may be bioactivated through CYP450 enzymes to become an epoxide which is able to form adducts with DNA, meaning that it has been considered that AFB1 undergoes bioactivation in the organism. AFB1 may also become hydroxylised to AFM1 and be excreted in milk. It has been estimated that 1% to 6% of AFB1 ingested by a milk-producing cow could be excreted as AFM1 in milk, depending on bovine productivity. AFM1, like AFB1, may form an epoxide and alter DNA sequences. IARC is considered to be an AFM1 in group 2B (IARC, 1993). MRL regulated in different countries ranges from 0.05 to 0.5 µg/L; MRL has also been established for AF consumed by ruminants (FAO, 2003). Experimental studies have shown that when animals consume foodstuffs contaminated by high levels of AF, that it is difficult to find naturally, the liver and kidneys are the organs where most toxins become accumulated, and their presence in muscle is scarce (Bailly & Guerre, 2009). These types of studies have also demonstrated the presence of AF in eggs from different avian species.

#### **6.1.2 Ochratoxin A**

Ochratoxin A (OA) is produced by fungi from the genera *Aspergillus* and *Pencillium*, the former being from tropical regions and the latter from temperate regions. OA may thus be widely distributed throughout the world. OA may become biotransformed through hydrolysis reactions in which metabolites become less toxic by the opening of the lactone ring which occurs during bioactivation. Detoxification may occur in ruminants through digestive flora action before absorption, thereby limiting the possibility that OA might be found in milk and/or beef (Bailly & Guerre, 2009). However, a recent study evaluating the presence of OA in cows' milk formulas for infants found contamination in 72% of the samples analysed, levels around 690 ng/L being found (Meucci et al., 2010). It has been shown that OA may become accumulated in pigs' kidneys. In countries such as Denmark, OA levels in these organs are regulated since porcine ochratoxicosis is common.

#### **6.1.3 Fusariotoxins**

The fusariotoxins are mycotoxins which are produced by fungi from the genera *Fusarium*, zearalenone (ZEA), the fumonisins (FUM) and the trichothecenes (TCT) being the most important for public health.

ZEA is frequently implicated in reproductive disorders in animals and occasionally in hyperestrogenism syndromes in humans. ZEA becomes biotransformed in the intestine by the mucosa or bacterial flora and involves the formation of α- and β-zearalenol and α- and β-zearalanol. Alpha-zearalanol and β-zearalenol have greater estrogenic power than ZEA since they bind with greater force to their corresponding receptors (Zinedine et al., 2007). Alpha-zearalanol has been employed as growth promoter in cattle. Studies orientated towards determining residuality through experimentation have suggested that residues are not present in meat or eggs, even at high doses. However, a recent study has show that the presence of α-zearalenol in meat-based foodstuffs for infants reached levels of 30.5 µg/kg; the same study demonstrated the presence of mycoestrogens (ZEA, α-zearalenole and βzearalenole) in infants' cow milk formulas (Meucci et al., 2011).

FUM properties suggest that their presence in animal meat does not represent an important source of contamination, since they are poorly absorbed. FUM produce liquefaction of the brain in horse and pulmonary oedema in pigs; ruminants and birds are more resistant. They have been correlated with oesophageal cancer in humans in some parts of the world and it has also been presumed that they may cause neural tube alterations. Their presence has been demonstrated and in the liver and kidneys of turkeys fed with the maximum levels permitted in Europe (Tardieu et al., 2008).

The main TCT of interest in producing animals are T-2 toxin, HT-2 toxin, diacetoxiscirpenol and deoxinivalenol. The TCT do not usually represent a risk of contamination in food of animal origin due to their rapid metabolism (Bailly & Guerre, 2009).

#### **6.2 Phycotoxins**

178 Public Health – Methodology, Environmental and Systems Issues

has classified aflatoxin B1 within group 1 (proven carcinogenic effect on humans) and fumonisin B1 and ochratoxin A within group 2B (possibly carcinogenic to humans) (IARC, 1993). The evidence from *in vitro* studies has shown that zearalenone is a probably

Aflatoxins and ochratoxin A are the main mycotoxins which can generate residuality and attention concerning them as being animal subproduct contaminants has mainly been focused on their presence in milk; however, it has been demonstrated that they can also

Aflatoxins (AF) B1 B2 G1 and G2 are produced by fungi from the genera *Aspergillus*. AFB1 may be bioactivated through CYP450 enzymes to become an epoxide which is able to form adducts with DNA, meaning that it has been considered that AFB1 undergoes bioactivation in the organism. AFB1 may also become hydroxylised to AFM1 and be excreted in milk. It has been estimated that 1% to 6% of AFB1 ingested by a milk-producing cow could be excreted as AFM1 in milk, depending on bovine productivity. AFM1, like AFB1, may form an epoxide and alter DNA sequences. IARC is considered to be an AFM1 in group 2B (IARC, 1993). MRL regulated in different countries ranges from 0.05 to 0.5 µg/L; MRL has also been established for AF consumed by ruminants (FAO, 2003). Experimental studies have shown that when animals consume foodstuffs contaminated by high levels of AF, that it is difficult to find naturally, the liver and kidneys are the organs where most toxins become accumulated, and their presence in muscle is scarce (Bailly & Guerre, 2009). These types of studies have also demonstrated the presence of AF in eggs from different avian

Ochratoxin A (OA) is produced by fungi from the genera *Aspergillus* and *Pencillium*, the former being from tropical regions and the latter from temperate regions. OA may thus be widely distributed throughout the world. OA may become biotransformed through hydrolysis reactions in which metabolites become less toxic by the opening of the lactone ring which occurs during bioactivation. Detoxification may occur in ruminants through digestive flora action before absorption, thereby limiting the possibility that OA might be found in milk and/or beef (Bailly & Guerre, 2009). However, a recent study evaluating the presence of OA in cows' milk formulas for infants found contamination in 72% of the samples analysed, levels around 690 ng/L being found (Meucci et al., 2010). It has been shown that OA may become accumulated in pigs' kidneys. In countries such as Denmark, OA levels in these organs are regulated since porcine ochratoxicosis is

The fusariotoxins are mycotoxins which are produced by fungi from the genera *Fusarium*, zearalenone (ZEA), the fumonisins (FUM) and the trichothecenes (TCT) being the most

implicated in cancer of the reproductive system (Khosrokhavar et al., 2009).

generate residuality in meat and eggs.

**6.1.1 Aflatoxins** 

species.

common.

**6.1.3 Fusariotoxins** 

important for public health.

**6.1.2 Ochratoxin A** 

Around 75 species of marine micro-algae, belonging to the dinoflagellate group, produce secondary metabolites which represent potent toxins, generically called phycotoxins. These organisms form part of the marine plankton and therefore the aquatic food chain leading to filtrator mollusks, gastropods, crustaceans and fish which can accumulate toxins being consumed (FAO, 2004).

The microalgae population may increase suddenly and generate an algal bloom which has increased in frequency, intensity and geographical distribution during recent years. Amongst the explanations put forward to explain for this phenomena has been the increased use of coastal waters for aquaculture, eutrophication caused by domestic, industrial and agricultural residues, the mobility of trace metals and humic substances due to deforestation and/or acid rain and changes in climatic conditions (Erdner et al., 2008). Reports of phycotoxins poisoning have increased during the last few years, perhaps due to the scientific community's greater knowledge and interest in the matter or due to the increase in algal bloom. Such poisoning is mainly acute course; however, there is interest in evaluating the effects being triggered by chronic consumption. The lack of studies on animals which are continuously exposed to phycotoxins and the scarce availability of certified reference materials have led to difficulties in evaluating risk, developing analytical methodologies and regulating these substances.

Studying toxins produced by algae has classically been approached according to the type of poisoning which they have caused. Four groups of toxins can thus be distinguished, causing paralytic shellfish poisoning, diarrhoeic shellfish poisoning, amnesic shellfish

Chemical Residues in Animal Food Products: An Issue of Public Health 181

This poisoning is also known as domoic acid (DA) poisoning since memory loss is not always present. It was described for the first time in Canada (Prince Edward Island) in 1987 when 105 people became poisoned after consuming blue mussels. There have also been several reports of poisoning involving effects on wild life, demonstrating that the toxin forms part of the food chain; the toxin responsible for this has been DA which is produced

The DA mechanism of action acts on excitatory amino acid receptors (L-glutamate, Laspartate) and/or synaptic transmission. DA activates specific excitatory amino acid L glutamate receptors producing an excessive accumulation of calcium resulting in cell death. The kainate receptor is DA's primary target. Recent interest in DA has been centred on recognising that effects can result following chronic exposure to it at low concentrations,

Intestinal absorption is limited (5%-10% of the dose administered to experimental animals). It has high distribution in the blood compartment and scarcely penetrates the hematoencephalic barrier. There is no evidence that DA may become metabolised. Elimination occurs via the kidneys. Poisoning in humans produces gastroenteritis which may be accompanied by headache, confusion and permanent loss of short-term memory

Neurotoxic shellfish poisoning, which is endemic on the Gulf of México and the eastern coast of Florida, is caused by brevetoxin (BTX) produced by the dinoflagellate *Gymnodinium breve* (synonyms: *Ptychodiscus breve, Karenia brevis*) present in red-tides. This alga has the special feature of being able to form aerosols due to wave action thereby constituting a risk

BTX are liposolube toxins consisting of around 14 different substances, leading to depolarisation opening sodium channels in cell membranes and increasing the inflow of sodium causing persistent and repetitive activation. Symptoms caused by oral exposure to BTX occur within the first 30 minutes to 3 hours after consuming contaminated organisms and include vomiting, diarrhoea, shivering, sweating, conflicting perception of temperature, hypotension, arrhythmias, numbness, paraesthesia of the lips, face and extremities, cramps, bronchoconstriction, paralysis, convulsions and coma. There have been no reports of lethality. Respiratory difficulty and irritation of the mucosa are the most common symptoms

Ciguatera fish poisoning is caused by ciguatoxin (CTX) which is produced by dinoflagellates from the genera *Gambierdiscus.* CTX becomes accumulated through the food chain, from small herbivorous fish up to large carnivorous fish. This poisoning has passed from being a problem limited to insular regions which affected local communities to being a global matter, given the worldwide consumption of seafood and international tourism. This is the most common poisoning caused by seafood and may affect up to 50,000 people annually

given the discovery that chemical route alteration leads to neurological disturbances.

**6.2.3 Amnesic shellfish poisoning** 

(FAO, 2004).

of aerial exposure.

by diatoms from the genera *Pseudo-nitzschia*.

**6.2.4 Neurotoxic shellfish poisoning** 

when inhalatory exposure occurs.

**6.2.5 Ciguatera fish poisoning** 

poisoning and neurotoxic shellfish poisoning (FAO, 2004). Another group of phycotoxins of interest due to their accumulation in sea fish are the ciguatoxins causing ciguatera fish poisoning.

#### **6.2.1 Paralytic shellfish poisoning**

The toxins responsible for this poisoning are mainly produced by algae from the genera *Alexandrium, Gymnodinium* and *Pyrodinium*. Chemically, they correspond to tetrahydropurin molecules, saxitoxin (STX) having the most importance. PSP incidence and geographical distribution has increased since the 1970s; this poisoning was initially confined to temperate waters in Europe, North America and Japan, but is now considered to be worldwide problem (FAO, 2004).

STX becomes rapidly absorbed through the gastrointestinal tract and equally has rapid distribution, metabolism and excretion. STX selectively blocks (and with high affinity) sodium-dependent channels present in nerves, skeletal muscular fibres and most cardiac muscular fibres, thereby reducing or eliminating the action of propagation potential (Etheridge, 2010).

Paralytic shellfish poisoning symptoms begin in human beings within the first 30 minutes following consumption of contaminated foodstuff and the onset of numbness and/or pins and needles around the lips, gradually extending to the face, neck, arms and legs. Headaches, nausea, lack of muscular coordination and, occasionally, temporary blindness occur. There may be paraesthesia in the arms and legs, motor inability and difficulty in talking in moderate cases and paralysis of respiratory muscles leading to death may occur in severe cases.

#### **6.2.2 Diarrhoeic shellfish poisoning**

Diarrhoeic shellfish poisoning toxins are produced by dinoflagellates from the genera *Dinophysis* and *Protoceratium*, having worldwide distribution. Okadaic acid (OA) and its analogues (dinophysis toxins , pectenotoxin and yessotoxin are included within this group. However, yessotoxin and pectenotoxin produce different toxicological effects in experimental animals. Yessotoxin is related to lesions in the cardiac muscle, liver, pancreas and cerebral neurons and pectenotoxin is clearly hepatotoxic. The effect which yessotoxin and pectenotoxin may have on human beings remains unknown (Dominguez et al., 2010).

OA was initially reported in Japan and Europe, areas in which diarrhoeic shellfish poisoning has had greater importance. OA and its analogues are potent protein phosphatase inhibitors (serine/threonine phosphatases PP1 and PP2A) which dephosphorylate molecules closely related to metabolic processes. It has been postulated that OA induces diarrhoea due to an alteration in hydric balance in the intestines via one of the two following mechanisms: stimulating the phosphorylation of proteins controlling sodium secretion in enterocytes or promoting the phosphorylation of intercell binding proteins regulating solute permeability. Diarrhoeic shellfish poisoning is characterised by diarrhoea, nausea, vomiting and, in some cases, abdominal pain which can begin within the first 3 or 12 hours after having consumed contaminated organisms. No lethal effects have been reported concerning human as having been caused by OA or its analogues.

#### **6.2.3 Amnesic shellfish poisoning**

180 Public Health – Methodology, Environmental and Systems Issues

poisoning and neurotoxic shellfish poisoning (FAO, 2004). Another group of phycotoxins of interest due to their accumulation in sea fish are the ciguatoxins causing ciguatera fish

The toxins responsible for this poisoning are mainly produced by algae from the genera *Alexandrium, Gymnodinium* and *Pyrodinium*. Chemically, they correspond to tetrahydropurin molecules, saxitoxin (STX) having the most importance. PSP incidence and geographical distribution has increased since the 1970s; this poisoning was initially confined to temperate waters in Europe, North America and Japan, but is now considered to be worldwide

STX becomes rapidly absorbed through the gastrointestinal tract and equally has rapid distribution, metabolism and excretion. STX selectively blocks (and with high affinity) sodium-dependent channels present in nerves, skeletal muscular fibres and most cardiac muscular fibres, thereby reducing or eliminating the action of propagation potential

Paralytic shellfish poisoning symptoms begin in human beings within the first 30 minutes following consumption of contaminated foodstuff and the onset of numbness and/or pins and needles around the lips, gradually extending to the face, neck, arms and legs. Headaches, nausea, lack of muscular coordination and, occasionally, temporary blindness occur. There may be paraesthesia in the arms and legs, motor inability and difficulty in talking in moderate cases and paralysis of respiratory muscles leading to death may occur in

Diarrhoeic shellfish poisoning toxins are produced by dinoflagellates from the genera *Dinophysis* and *Protoceratium*, having worldwide distribution. Okadaic acid (OA) and its analogues (dinophysis toxins , pectenotoxin and yessotoxin are included within this group. However, yessotoxin and pectenotoxin produce different toxicological effects in experimental animals. Yessotoxin is related to lesions in the cardiac muscle, liver, pancreas and cerebral neurons and pectenotoxin is clearly hepatotoxic. The effect which yessotoxin and pectenotoxin may have on human beings remains unknown (Dominguez

OA was initially reported in Japan and Europe, areas in which diarrhoeic shellfish poisoning has had greater importance. OA and its analogues are potent protein phosphatase inhibitors (serine/threonine phosphatases PP1 and PP2A) which dephosphorylate molecules closely related to metabolic processes. It has been postulated that OA induces diarrhoea due to an alteration in hydric balance in the intestines via one of the two following mechanisms: stimulating the phosphorylation of proteins controlling sodium secretion in enterocytes or promoting the phosphorylation of intercell binding proteins regulating solute permeability. Diarrhoeic shellfish poisoning is characterised by diarrhoea, nausea, vomiting and, in some cases, abdominal pain which can begin within the first 3 or 12 hours after having consumed contaminated organisms. No lethal effects have been reported concerning human as having

poisoning.

problem (FAO, 2004).

(Etheridge, 2010).

severe cases.

et al., 2010).

**6.2.1 Paralytic shellfish poisoning** 

**6.2.2 Diarrhoeic shellfish poisoning** 

been caused by OA or its analogues.

This poisoning is also known as domoic acid (DA) poisoning since memory loss is not always present. It was described for the first time in Canada (Prince Edward Island) in 1987 when 105 people became poisoned after consuming blue mussels. There have also been several reports of poisoning involving effects on wild life, demonstrating that the toxin forms part of the food chain; the toxin responsible for this has been DA which is produced by diatoms from the genera *Pseudo-nitzschia*.

The DA mechanism of action acts on excitatory amino acid receptors (L-glutamate, Laspartate) and/or synaptic transmission. DA activates specific excitatory amino acid L glutamate receptors producing an excessive accumulation of calcium resulting in cell death. The kainate receptor is DA's primary target. Recent interest in DA has been centred on recognising that effects can result following chronic exposure to it at low concentrations, given the discovery that chemical route alteration leads to neurological disturbances.

Intestinal absorption is limited (5%-10% of the dose administered to experimental animals). It has high distribution in the blood compartment and scarcely penetrates the hematoencephalic barrier. There is no evidence that DA may become metabolised. Elimination occurs via the kidneys. Poisoning in humans produces gastroenteritis which may be accompanied by headache, confusion and permanent loss of short-term memory (FAO, 2004).

#### **6.2.4 Neurotoxic shellfish poisoning**

Neurotoxic shellfish poisoning, which is endemic on the Gulf of México and the eastern coast of Florida, is caused by brevetoxin (BTX) produced by the dinoflagellate *Gymnodinium breve* (synonyms: *Ptychodiscus breve, Karenia brevis*) present in red-tides. This alga has the special feature of being able to form aerosols due to wave action thereby constituting a risk of aerial exposure.

BTX are liposolube toxins consisting of around 14 different substances, leading to depolarisation opening sodium channels in cell membranes and increasing the inflow of sodium causing persistent and repetitive activation. Symptoms caused by oral exposure to BTX occur within the first 30 minutes to 3 hours after consuming contaminated organisms and include vomiting, diarrhoea, shivering, sweating, conflicting perception of temperature, hypotension, arrhythmias, numbness, paraesthesia of the lips, face and extremities, cramps, bronchoconstriction, paralysis, convulsions and coma. There have been no reports of lethality. Respiratory difficulty and irritation of the mucosa are the most common symptoms when inhalatory exposure occurs.

#### **6.2.5 Ciguatera fish poisoning**

Ciguatera fish poisoning is caused by ciguatoxin (CTX) which is produced by dinoflagellates from the genera *Gambierdiscus.* CTX becomes accumulated through the food chain, from small herbivorous fish up to large carnivorous fish. This poisoning has passed from being a problem limited to insular regions which affected local communities to being a global matter, given the worldwide consumption of seafood and international tourism. This is the most common poisoning caused by seafood and may affect up to 50,000 people annually

Chemical Residues in Animal Food Products: An Issue of Public Health 183

probable risk of exposure for humans; however, food of animal origin can also contain pyrrolizidine alkaloids. It has been presumed that more than 6,000 plant species contain AP, mainly those belonging to the families *Asteraceae* or *Compositae* (genera *Senecio* and *Eupatorium*), *Boraginaceae* (genera *Heliotropium* and *Echium*) and *Fabaceae* or *Leguminosae* 

APs are heterocyclic compounds which are mainly derived from four necine bases: retronecine, heliotridine, otonecine and platynecine. AP can become enzimatically hydrolised or oxidised; the resulting N-oxide is slightly toxic and may also be found in plants. AP becomes bioactivated to a toxin pyrrole through CYP450 which is electrophilic and unstable and reacts rapidly with endogenous macromolecules (particularly with DNA forming adducts). DNA adducts could be a continuous source of carbon ions originating new adducts, meaning that the total elimination of AP derivates may take months (Edgar et

AP levels in foodstuffs are rarely so significant that they can cause acute diseases; however, low levels and continuous exposure could lead to the presentation of chronic diseases which could hardly be attributable to the toxin in foodstuffs. Pyrroles cause thickening and occlusion of the hepatic vessels resulting in veno-occlusive disease and cirrhosis. They can also alter the pulmonary vessels, causing pulmonary hypertension and congestive cardiac failure. The IARC has classified lasiocarpine, monocrotaline and riddelliine AP within group 2B (probably carcinogenic for humans, given the pertinent evidence regarding how they are

It has been demonstrated that 0.1% to 4% of AP in foodstuffs for lactating cows and sheep could be excreted in milk (Hoogenboom, 2011). The meat and viscera of cattle fed on APrich plants may contain the toxin and its derivates at levels reaching 250 mg/kg in muscle and 2,500 mg/kg in liver (Fletcher et al., 2011). AP can potentially be present in eggs when fowl are fed on AP-rich diets, thereby constituting a risk for human health (Eröksüz et al., 2008). In spite of potential contamination in milk, meat, viscera and eggs, honey is the only foodstuff of animal origin which has been shown to be naturally contaminated with AP; a large number of plants habitually used in apiaries could be a source of significant levels of

Other alkaloids of interest due to their potential residual effect on animal subproducts are the indolizidinics (especially swainsonine) producing lysosomal storage disease and piperidine (coniin and gamma-conicein) which in acute form can produce muscular paralysis and (chronically) teratogenesis (Panter & James, 1990). These last named alkaloids seem to be responsible for coturnism, a human disease which occurs following the consumption of migrating wild European quail; it is characterised by weakness, muscular pain, paralysis of the legs, vomiting and myoglobinuria. It has been postulated that these symptoms occur due to the accumulation of coniin in birds' tissues following the

Glucosinolates, known as mustard oil glucosides as they confer the characteristic flavour on black mustard (*Brassica nigra*), are secondary metabolites produced by plants belonging to the order *Brassicales*, mainly in the family *Brassicaceae (*or *Cruciferae)*. Many plants which are

(genera *Crotalaria*).

al., 2011).

the toxins.

**6.3.2 Glucosinolates** 

cancerigens in animals) (Edgar et al., 2011).

consumption of *Conium maculatum* (López & Bianchini, 1999).

(FAO, 2004). CTX are liposoluble toxins having 13 to 14 rings fused in a rigid structure. CTX bind to sodium channels causing them to open during cell membranes' potential repose altering bioenergetic mechanisms. CTX acts on the same receptor as BTX does but with greater affinity (Lehane & Lewis, 2000).

*Gambierdiscus toxicus*, the alga specie most commonly related to CTX production, is distributed throughout the tropical region of the Pacific Ocean, western Indian Ocean and the Caribbean where CFP is endemic. Many coral fish species are involved, including herbivores and carnivores. The latter constitute the main vector for poisoning in humans, particularly *Muraenidae* (moray eels), *Lutjanidae* (snappers), *Carrangidae* (carrangs), *Scombridae* (mackerels) and *Sphyraenidae* (barracuda) (FAO, 2004).

CTX become rapidly absorbed through the intestine and are mainly excreted in the faeces via the bile. The symptoms are gastrointestinal or neurological in nature, the former include vomiting, diarrhoea, nausea and abdominal pain. The neurological symptoms which can begin later include pins and needles in the lips, hand and feet, disturbances in perception of temperature, severe pruritus and fatigue. Some patients can experience pain (muscular, articular and dental) and anxiety. There may be hypotension and bradycardia in severe cases and death may occur, though this is not very common. The neurological symptoms can persist for years in some cases; it seems that the toxin may become accumulated in fat and be released in certain circumstances or also produce an immunological response (Shoemaker et al., 2010).

Other phycotoxins of interest due to their residuality are the azaspiracida*,* discovered in 1998, and whose symptoms resemble those of diarrhoeic shellfish poisoning; the cyclic imins (gymnodimine, spirolids, pinnatoxins, prorocentrolide, spirocentrimine and pteriatoxin), still have no associated effects in humans, but their residuality does interfere with the analytical methodologies used for determining the presence of marine toxins and cianotoxins (nodularin and cylindrospermin) by producing hepatoxicity and inhibiting protein synthesis.

#### **6.3 Phytotoxins**

Plants have secondary metabolites with which they defend themselves from the aggression of herbivorous animals. Many of these are toxic for humans and animals, causing numerous pathologies. Given that the diverse compounds present in plants are degraded during digestion and/or the metabolism of xenobiotics in animals of livestock interest, only some manage to contaminate products of animal origin. Milk is the main subproduct which has been studied in which toxins from plants may be present; however, their presence has also been demonstrated in muscle, viscera and eggs. Regulations regarding these toxins are scarce and are mainly orientated towards their presence in botanical products having pharmacological uses; however, there is growing interest in making advances in this field. The regulating entities have shown the greatest interest in pyrrolizidine alkaloids amongst the plant toxins due to their abundance and proven toxic effects.

#### **6.3.1 Pyrrolizidine alkaloids**

Pyrrolizidine alkaloids (AP) are present in a large variety of plants and are perhaps the most widely distributed toxins. Foodstuffs or botanically-based remedies represent the most

(FAO, 2004). CTX are liposoluble toxins having 13 to 14 rings fused in a rigid structure. CTX bind to sodium channels causing them to open during cell membranes' potential repose altering bioenergetic mechanisms. CTX acts on the same receptor as BTX does but with

*Gambierdiscus toxicus*, the alga specie most commonly related to CTX production, is distributed throughout the tropical region of the Pacific Ocean, western Indian Ocean and the Caribbean where CFP is endemic. Many coral fish species are involved, including herbivores and carnivores. The latter constitute the main vector for poisoning in humans, particularly *Muraenidae* (moray eels), *Lutjanidae* (snappers), *Carrangidae* (carrangs),

CTX become rapidly absorbed through the intestine and are mainly excreted in the faeces via the bile. The symptoms are gastrointestinal or neurological in nature, the former include vomiting, diarrhoea, nausea and abdominal pain. The neurological symptoms which can begin later include pins and needles in the lips, hand and feet, disturbances in perception of temperature, severe pruritus and fatigue. Some patients can experience pain (muscular, articular and dental) and anxiety. There may be hypotension and bradycardia in severe cases and death may occur, though this is not very common. The neurological symptoms can persist for years in some cases; it seems that the toxin may become accumulated in fat and be released in certain circumstances or also produce an immunological response

Other phycotoxins of interest due to their residuality are the azaspiracida*,* discovered in 1998, and whose symptoms resemble those of diarrhoeic shellfish poisoning; the cyclic imins (gymnodimine, spirolids, pinnatoxins, prorocentrolide, spirocentrimine and pteriatoxin), still have no associated effects in humans, but their residuality does interfere with the analytical methodologies used for determining the presence of marine toxins and cianotoxins (nodularin and cylindrospermin) by producing hepatoxicity and inhibiting

Plants have secondary metabolites with which they defend themselves from the aggression of herbivorous animals. Many of these are toxic for humans and animals, causing numerous pathologies. Given that the diverse compounds present in plants are degraded during digestion and/or the metabolism of xenobiotics in animals of livestock interest, only some manage to contaminate products of animal origin. Milk is the main subproduct which has been studied in which toxins from plants may be present; however, their presence has also been demonstrated in muscle, viscera and eggs. Regulations regarding these toxins are scarce and are mainly orientated towards their presence in botanical products having pharmacological uses; however, there is growing interest in making advances in this field. The regulating entities have shown the greatest interest in pyrrolizidine alkaloids amongst

Pyrrolizidine alkaloids (AP) are present in a large variety of plants and are perhaps the most widely distributed toxins. Foodstuffs or botanically-based remedies represent the most

*Scombridae* (mackerels) and *Sphyraenidae* (barracuda) (FAO, 2004).

the plant toxins due to their abundance and proven toxic effects.

greater affinity (Lehane & Lewis, 2000).

(Shoemaker et al., 2010).

protein synthesis.

**6.3 Phytotoxins** 

**6.3.1 Pyrrolizidine alkaloids** 

probable risk of exposure for humans; however, food of animal origin can also contain pyrrolizidine alkaloids. It has been presumed that more than 6,000 plant species contain AP, mainly those belonging to the families *Asteraceae* or *Compositae* (genera *Senecio* and *Eupatorium*), *Boraginaceae* (genera *Heliotropium* and *Echium*) and *Fabaceae* or *Leguminosae*  (genera *Crotalaria*).

APs are heterocyclic compounds which are mainly derived from four necine bases: retronecine, heliotridine, otonecine and platynecine. AP can become enzimatically hydrolised or oxidised; the resulting N-oxide is slightly toxic and may also be found in plants. AP becomes bioactivated to a toxin pyrrole through CYP450 which is electrophilic and unstable and reacts rapidly with endogenous macromolecules (particularly with DNA forming adducts). DNA adducts could be a continuous source of carbon ions originating new adducts, meaning that the total elimination of AP derivates may take months (Edgar et al., 2011).

AP levels in foodstuffs are rarely so significant that they can cause acute diseases; however, low levels and continuous exposure could lead to the presentation of chronic diseases which could hardly be attributable to the toxin in foodstuffs. Pyrroles cause thickening and occlusion of the hepatic vessels resulting in veno-occlusive disease and cirrhosis. They can also alter the pulmonary vessels, causing pulmonary hypertension and congestive cardiac failure. The IARC has classified lasiocarpine, monocrotaline and riddelliine AP within group 2B (probably carcinogenic for humans, given the pertinent evidence regarding how they are cancerigens in animals) (Edgar et al., 2011).

It has been demonstrated that 0.1% to 4% of AP in foodstuffs for lactating cows and sheep could be excreted in milk (Hoogenboom, 2011). The meat and viscera of cattle fed on APrich plants may contain the toxin and its derivates at levels reaching 250 mg/kg in muscle and 2,500 mg/kg in liver (Fletcher et al., 2011). AP can potentially be present in eggs when fowl are fed on AP-rich diets, thereby constituting a risk for human health (Eröksüz et al., 2008). In spite of potential contamination in milk, meat, viscera and eggs, honey is the only foodstuff of animal origin which has been shown to be naturally contaminated with AP; a large number of plants habitually used in apiaries could be a source of significant levels of the toxins.

Other alkaloids of interest due to their potential residual effect on animal subproducts are the indolizidinics (especially swainsonine) producing lysosomal storage disease and piperidine (coniin and gamma-conicein) which in acute form can produce muscular paralysis and (chronically) teratogenesis (Panter & James, 1990). These last named alkaloids seem to be responsible for coturnism, a human disease which occurs following the consumption of migrating wild European quail; it is characterised by weakness, muscular pain, paralysis of the legs, vomiting and myoglobinuria. It has been postulated that these symptoms occur due to the accumulation of coniin in birds' tissues following the consumption of *Conium maculatum* (López & Bianchini, 1999).

#### **6.3.2 Glucosinolates**

Glucosinolates, known as mustard oil glucosides as they confer the characteristic flavour on black mustard (*Brassica nigra*), are secondary metabolites produced by plants belonging to the order *Brassicales*, mainly in the family *Brassicaceae (*or *Cruciferae)*. Many plants which are

Chemical Residues in Animal Food Products: An Issue of Public Health 185

contaminated by chemical substances could lead to chronic exposure leading to the presentation of diseases lacking an apparent cause and being difficult to diagnose. Foods of animal origin presuppose the risk of contamination, whether from drugs and growth promoters used for optimising livestock production systems, or with biological toxins present in food ingested by animals. It is thus necessary to control these substances in foods,

Sanitary authorities must thus promulgate and ensure compliance with standards and guidelines concerning the production of harmless foodstuffs. Achieving such objective represents a great challenge for underdeveloped and developing countries due to institutional difficulties and the limited availability of equipment and qualified personnel. All nations must make it a priority to try to ensure the safe consumption of foodstuffs by their populations, exercising strict sanitary control aimed to avoid problems of health in the population and preventing the appearance of new problems affecting the development of

Alonso-Amelot, ME.; Castillo, U.; Smith, BL. & Lauren, DR. (1996). Bracken ptaquiloside in

Asociación Española de Farmacéuticos de la Industria. (March 2001). *Validación de Métodos* 

Bailly, JD. & Guerre P. (2009). Mycotoxins in Meat and Processed Meat Products In: *Safety of* 

Botsoglou, N. & Fletouris, D. (2000). *Drug Residues in Foods, Pharmacology, Food Safety and Analysis,* (First Edition), Marcel Dekker Inc., ISBN: 0-8247-8959-8, New York, USA Blondin, P. & Sirard, M. (1998). Oocyte quality and embryo production in cattle. *Canadian* 27 *Journal of Animal Science,* Vol. 78, (October 1998), pp. 513–516, ISSN 0008-3984 Brambilla, G.; Di Beza, S.; Pietraforte, D.; Minetti, M.; Campanella, L. & Loizzo, A. (2007). *Ex* 

Brogden, KA.; Ackermann, M.; McCray, PB Jr. & Tack, BF. ( 2003). Antimicrobial peptides in

Buchmann, K.; Roepstorff, A. & Waller, P.J. (1992). Experimental selection of mebendazole

Dickson, L. C.; Macneil, J.D.; Reid J. & Fesser A. (2003). Validation of Screening Method for

*Diseases,* Vol. 15 No. 5, (September 1992), pp. 393-400, ISSN 1365-2761 Danaher, M.; De Ruyckb, H.; Crooks, S.; Dowling, G. & O'Keeffe, M. (2007). Review of

*Analíticos* (First Edition), Monografías de A.E.F.I., ISBN 84-89602-33-6, Barcelona,

*meat and processed meat,* Fidel Toldrá, pp (1-699) Springer, ISBN 978-0-387-89025-8,

*vivo formation* of gastric metabolites of clenbuterol: Preliminary characterisation of their chemical structure. *Analytica Chimica Acta*, Vol. 586, (July 2006), pp. 426–431,

animals and their role in host defences. *International Journal of Antimicrobial Agents*,

resistant gill monogeneans from European eel *Anguilla anguilla*. *Journal of Fish* 

methodology for the determination of benzimidazole residues in biological matrices. *Journal of Chromatography B*, Vol. 845 No. 1, (July 2006), pp. 1–37, ISSN

Residues of Diethylstilbestrol, Dienestrol, Hexestrol, and Zeranol, in Bovine Urine Using Immunoaffinity Chromatography and Gas Chromatography/Mass

milk. *Nature*, Vol. 382, No.6592, pp 587, ISSN 0028-0836

Vol. 22, (February 2003), pp. 465-478, ISSN 0924-8579

thereby supposing technological and institutional efforts.

the agro-food industry and global trade in foodstuffs.

**8. References** 

España

New York, USA

ISSN 0003-2670

1570-0232

common in the human diet belong to this family (broccoli, Brussels sprouts, cabbage and cauliflower) and several genera (*Brassica*, *Crambe*, *Sinapis* and *Raphanus*) including crops used for producing vegetal oils. The plant *Camelina sativa* (false flax) has recently aroused interest due to biofuel production. When the oils are extracted, the glucosinolates remain in the seed; the resulting press cake is used in animals diet (EFSA, 2008).

The glucosinolates become hydrolysed by enzymatic action giving place to isothiocyanates, thiocyanates, oxazolidinethiones and nitriles. The thiocyanates interfere with iodine capture and the oxazolidinethiones with thyroid (T3 and T4) hormone synthesis, leading to hypothyroidism and thyroid gland enlargement. Consequently, metabolism in all tissues, including the reproductive organs, may become affected.

It has been shown that high glucosinolate consumption in lactating cows reduces iodine levels and increases thiocyanates in milk, the liver and the kidneys. The residues in milk account for around 0.1% of the dose received by animals; the residues in muscles and viscera are even lower. Residuality has also been found in eggs after rapeseed has been administered to egg-laying birds; these also acquire a disagreeable flavour (EFSA, 2008).

#### **6.3.3 Ptaquiloside**

Bracken fern (genus *Pteridium)*, considered one of the five most abundant plants in the world, contains a norsesquiterpene glucoside called ptaquiloside. It has been proved that ptaquiloside may cause tumours in the urinary bladder, mammary glands, intestine and other organs in laboratory rodents. It causes degeneration of the retina in sheep and causes urinary bladder cancer known as bovine enzootic haematuria in cattle. There is epidemiological evidence relating the consumption of bracken fern in humans (Japanese population) suffering from esophagic and gastric cancer, possibly caused by ptaquiloside. Around 8.6% of the ptaquiloside present in *P. aquilinum*, consumed by lactating cows, is excreted in milk, thereby making contaminated raw milk a risk for human health (Alonso-Amelot et al., 1996).

#### **6.3.4 Tremetol**

This is a liposoluble compound mixture of terpene, sterols, tremetone, hydroxytremetone and dehydrotremetone; the last three are present in a ketonic fraction. It is present in the perennial plants white snakeroot (*Ageratina altissima*, previously called *Eupatorium rugosum*) and rayless goldenrod (*Haploppapus heterophyllus*). This poisoning reached an epidemic proportion during the 18th and 19th centuries in the USA (Indiana, Illinois and Ohio), producing high mortality, and its aetiological agent was only discovered in 1910. Cows accumulate the toxin in fat and excrete it in milk. The toxin becomes diluted in milk reception tanks, making such poisoning not very common in modern milk production systems. Tremetol produces acidosis, hyperglycaemia and ketonaemia, as a consequence in the Krebs cycle inhibitor. The symptoms of poisoning are anorexia, listlessness, weakness, stiffness of muscles, vomiting, constipation and coma. Marked acidosis and ketosis may lead to death (Lewis & Elvin-Lewis, 2003).

#### **7. Conclusions**

Interest in diseases caused by food has mainly been orientated towards the acute presentation principally produced by microbiological agents; however, consuming food contaminated by chemical substances could lead to chronic exposure leading to the presentation of diseases lacking an apparent cause and being difficult to diagnose. Foods of animal origin presuppose the risk of contamination, whether from drugs and growth promoters used for optimising livestock production systems, or with biological toxins present in food ingested by animals. It is thus necessary to control these substances in foods, thereby supposing technological and institutional efforts.

Sanitary authorities must thus promulgate and ensure compliance with standards and guidelines concerning the production of harmless foodstuffs. Achieving such objective represents a great challenge for underdeveloped and developing countries due to institutional difficulties and the limited availability of equipment and qualified personnel. All nations must make it a priority to try to ensure the safe consumption of foodstuffs by their populations, exercising strict sanitary control aimed to avoid problems of health in the population and preventing the appearance of new problems affecting the development of the agro-food industry and global trade in foodstuffs.

#### **8. References**

184 Public Health – Methodology, Environmental and Systems Issues

common in the human diet belong to this family (broccoli, Brussels sprouts, cabbage and cauliflower) and several genera (*Brassica*, *Crambe*, *Sinapis* and *Raphanus*) including crops used for producing vegetal oils. The plant *Camelina sativa* (false flax) has recently aroused interest due to biofuel production. When the oils are extracted, the glucosinolates remain in

The glucosinolates become hydrolysed by enzymatic action giving place to isothiocyanates, thiocyanates, oxazolidinethiones and nitriles. The thiocyanates interfere with iodine capture and the oxazolidinethiones with thyroid (T3 and T4) hormone synthesis, leading to hypothyroidism and thyroid gland enlargement. Consequently, metabolism in all tissues,

It has been shown that high glucosinolate consumption in lactating cows reduces iodine levels and increases thiocyanates in milk, the liver and the kidneys. The residues in milk account for around 0.1% of the dose received by animals; the residues in muscles and viscera are even lower. Residuality has also been found in eggs after rapeseed has been administered to egg-laying birds; these also acquire a disagreeable flavour (EFSA, 2008).

Bracken fern (genus *Pteridium)*, considered one of the five most abundant plants in the world, contains a norsesquiterpene glucoside called ptaquiloside. It has been proved that ptaquiloside may cause tumours in the urinary bladder, mammary glands, intestine and other organs in laboratory rodents. It causes degeneration of the retina in sheep and causes urinary bladder cancer known as bovine enzootic haematuria in cattle. There is epidemiological evidence relating the consumption of bracken fern in humans (Japanese population) suffering from esophagic and gastric cancer, possibly caused by ptaquiloside. Around 8.6% of the ptaquiloside present in *P. aquilinum*, consumed by lactating cows, is excreted in milk, thereby

This is a liposoluble compound mixture of terpene, sterols, tremetone, hydroxytremetone and dehydrotremetone; the last three are present in a ketonic fraction. It is present in the perennial plants white snakeroot (*Ageratina altissima*, previously called *Eupatorium rugosum*) and rayless goldenrod (*Haploppapus heterophyllus*). This poisoning reached an epidemic proportion during the 18th and 19th centuries in the USA (Indiana, Illinois and Ohio), producing high mortality, and its aetiological agent was only discovered in 1910. Cows accumulate the toxin in fat and excrete it in milk. The toxin becomes diluted in milk reception tanks, making such poisoning not very common in modern milk production systems. Tremetol produces acidosis, hyperglycaemia and ketonaemia, as a consequence in the Krebs cycle inhibitor. The symptoms of poisoning are anorexia, listlessness, weakness, stiffness of muscles, vomiting, constipation and coma. Marked acidosis and ketosis may lead

Interest in diseases caused by food has mainly been orientated towards the acute presentation principally produced by microbiological agents; however, consuming food

making contaminated raw milk a risk for human health (Alonso-Amelot et al., 1996).

the seed; the resulting press cake is used in animals diet (EFSA, 2008).

including the reproductive organs, may become affected.

**6.3.3 Ptaquiloside** 

**6.3.4 Tremetol** 

**7. Conclusions** 

to death (Lewis & Elvin-Lewis, 2003).


Chemical Residues in Animal Food Products: An Issue of Public Health 187

Hoogenboom, LA.; Mulder, PP.; Zeilmaker MJ.; Van Den Top HJ.; Remmelink, J.; Brandon,

International Agency for Research on Cancer, IARC. (1993). *Some Naturally Occurring* 

Jiménez, V.; Companyó, R. & Guiteras, J. (2011). Validation of a method for the analysis of

Khosrokhavar, R.; Rahimifard, N.; Shoeibi S.; Pirali, M. & Hosseini, M. (2009). Effects of

Lehane, L. & Lewis, RJ. (2000). Review Ciguatera: recent advances but the risk remains.

Lehman-McKeeman, LD. (2008). Absorption, distribution and excretion of toxicants, In:

LoBrutto, R. & Patel, T. (2007). Method Validation, In: *HPLC for Pharmaceutical Scientists,* Y.

Lodovico, C.; Marinovicha M. & Lotti M. (2008). Is the acceptable daily intake as presently

López, TA. & Bianchini, ML. (1999). Biochemistry of hemlock (*Conium maculatum* L.)

Lozano, MC., & Arias, DC. (2008). Residuos de fármacos de origen animal: panorama actual

Mastovska, K. (2011). Multiresidue analysis of antibiotics in food of animal origin using

Medina, M.; González D. & Ramírez A. (2008). Detection of antimicrobial residues in animal

Meucci, V.; Razzuoli, E.; Soldani, G. & Massart, F. (2010). Mycotoxin detection in infant

pp. (1-1309) McGraw-Hill, ISBN 978-0-07-147051-3, New York, USA Lewis, WH. & Elvin-Lewis, PF. (2003). *Medical botany: plants affecting human health. 2nd Ed.* 

Wiley Press, pp (1-832), ISBN: 978-0-471-62882-8

Vol.37, No.6, (June, 1999), pp 841-865, ISSN 1879-3150

Vol.28, No.3, (March, 2011), pp 359-372, ISSN 1944-0049

(1- 599) ISBN 92 832 1256 8 Lyon, France

596-606. ISSN 0039-9140

68162-5, New Jersey, USA

pp. 121-135, ISSN 0120-0690

( May 2011), pp. 267-307, ISSN 1940-6029

2, (May 2008), pp. 110-115, ISSN 0253-570

99, ISSN 0378-4274

1537-6516

ISSN 1879-3460

EF.; Klijnstra, M.; Meijer, GA.; Schothorst, R. and Van Egmond, HP. (2011). Carryover of pyrrolizidine alkaloids from feed to milk in dairy cows. Food Additives and Contaminants. Part A Chemistry, analysis, control, exposure and risk assessment,

*Substances: Food Items and Constituents, Heterocyclic Aromatic Amines and Mycotoxins*. IARC Monographs on the evaluation of carcinogenic risk to humans 56. IARC, pp.

nine quinolones in eggs by pressurized liquid extraction and liquid chromatography with fluorescence detection. *Talanta*, Vol. 85 No. 1, (July 2011), pp.

zearalenone and *α*-Zearalenol in comparison with Raloxifene on T47D cells. *Toxicology mechanisms and methods,* Vol. 19, No. 3, (March, 2009), pp 245-250, ISSN

*International Journal of Food Microbioly,* Vo.61, No.2-3, (November, 2000) pp 91-125,

*Casarett &Doul's Toxicology. The basic science of poisons 7h Edition*, Curtis Klaassen,

Kazakevich & R. Lobrutto, pp. 455-502, Wiley Interscience, ISBN-13: 978-0-471-

used an axiom or a dogma?. *ToxicologyLetters*, Vol.180, No.2, (August, 2008), pp 93-

alkaloids and their acute and chronic toxicity in livestock. A review. *Toxicon,* 

en Colombia. *Revista Colombiana de Ciencias Pecuarias*, Vol. 21 No. 1, (March 2008),

liquid chromatography-mass spectrometry. *Methods in Molecular Biology*, Vol. 747,

tissues and tetracyclines in bones of pigs. *Revista de Salud Animal*, Vol. 30 No.

formula milks in Italy. *Food Addittives and Contaminants. Part A chemistry, analysis,* 

Spectrometry. *Journal of AOAC International*, Vol. 86, No. 4, (April 2003), pp. 631-639 ISSN 1060-3271


Dominguez, HJ.; Paz, B.; Daranas, A; Norte, M.; Franco, J. & Fernández,JJ. (2010)

Durlinger, A.; Visser, J. & Axel T. (2002). Regulation of ovarian function: the role of anti-

Edgar, JA.; Colegate, SM.; Boppré, M. & Molyneux RJ. (2011). Pyrrolizidine alkaloids in

Erdner,DL.; Dyble, J.; Parsons,ML.; Stevens, RC.; Hubbard K.; Wrabel, ML.; Moore, S.;

*access science source*,Vol.7, Suppl.2, (November, 2008), ISSN 1476-069X Etheridge, SM. (2010). Paralytic shellfish poisoning: Sea food safety and human health perspectives. *Toxicon,* Vol. 56, No.2, (August, 2010), pp 108–122, ISSN 1879-3150 Eroksuz, Y.; Ceribasi, A.; Cevik, A.; Eroksuz, H.; Tosun, F. & Tamer, U. (2008). Toxicity of

*and Animal Sciences*, Vol.32, No. 6, pp 475-482, ISSN 1303-6181

No.2, (August, 2010), pp 191–217, ISSN 1879-3150

No.3, (March 2011), pp 308–324, ISSN: 1944-0049

ISSN 1060-3271

1-76, ISSN 1831-4732

9781845938338, USA.

ISBN 92-5-105162-32004, Rome, Italy

London, UK

1330-9862

1626

Spectrometry. *Journal of AOAC International*, Vol. 86, No. 4, (April 2003), pp. 631-639

Dinoflagellate polyether within the yessotoxin, pectenotoxin and okadaic acid toxin groups: Characterization, analysis and human health implications. *Toxicon,* Vol. 56,

Müllerian hormone. *Reproduction,* Vol. 124, (April 2002), pp. 601–609 ISSN: 1470-

food: a spectrum of potential health consequences. *Food Addittives and Contaminants. Part A chemistry, analysis, control, exposure and risk assessment*, Vol.28,

Lefebvre, K.; Anderson, D.; Bienfang, P.; Bidigare, R.; Parker, MS.; Moeller, P.; Brand, L. & Trainer VL.(2008). Centers for Oceans and Human Health: a unified approach to the challenge of harmful algal blooms. *Environmental Health : a global* 

*Heliotropium dolosum*, *Heliotropium circinatum*, and *Senecio vernalis* in Parental Quail and Their Progeny, with Residue Evaluation of Eggs. *Turkish Journal of Veterinary* 

Contaminants in the Food Chain on a request from the European Commission on glucosinolates as undesirable substances in animal feed, *The EFSA Journal* 590, pp

Summary Report, Committee for Veterinary Medicinal Products, EMEA/MRL,

containing pyrrolizidine alkaloids for livestock and meat quality in Northern Australia*.* In: *Poisoning by plants, mycotoxins and related toxins,* Riet-Correa, F.; Pfister, J.; Schild, AL. and Wierenga, TL. pp (1-660) CABI Publishing, ISBN

*regulations for mycotoxins in food and feed in 2003*. FAO Food and nutrition paper 81.

Liver Following Prolonged Administration of a Growth-Promoting Dose. *Food Technology and Biotechnology,* Vol. 40, No. 4, (November 2002), pp. 343–346, ISSN

European Food Safety Authority, EFSA. (2008). Opinion of the Scientific Panel on

European Agency for the Evaluation of Medicinal Products, EMEA. (1997). In Furazolidone,

Fletcher, MT.; McKenzie, RA.; Reichmann, KG. & Blaney, BJ. (2011) Risks from plants

Food and Agriculture Organization of the United Nations, FAO. (2003). *Worldwide* 

Food and Agriculture Organization of the United Nations. (2004). *Marine biotoxins*, FAO

Gojmerac, T.; Mandi, B.; Pleadin, K. & Mitak, M. (2002). Determination of Clenbuterol in Pig

Food and Nutrition Paper 80, ISNN 0254-4725 Rome, Italy


**9** 

*University of Messina* 

 *Italy* 

**Viable but Nonculturable Bacteria in Food** 

Marco Sebastiano Nicolò and Salvatore Pietro Paolo Guglielmino

"Dis-moi ce que tu manges, je te dirai ce que tu es." (Tell me what you eat and I will tell you what you are - Anthelme Brillat-Savarin, *Physiologie du Goût, ou Méditations de* 

"Ninety per cent of the diseases known to man are caused by cheap foodstuffs. You are

In every time, availability of food has been a struggle for human survival. In particular, food storage techniques and maintaining have represented one of the most important milestones in the evolution and development of human society. In that sense, the use of fire for cooking and salt and spices for conservation have been important discoveries in food processing,

Nowadays, food resources remain a primary objective for human society, specifically in

Infectious foodborne diseases are caused by the consumption of food contaminated by

Contamination may occur as consequence of incorrect practices at different steps of food

The rapid globalization and trade among countries with different hygienic standards have increased the possibility of food contamination. Hence, outbreaks of foodborne diseases that were once contained within a small community may now take place on global dimensions. The fundamental strategy for outbreaks monitoring is the traceability, that is the possibility

One of the most critical problems in traceability of bacterial pathogens is represented by a state of latency of most foodborne bacterial species, called "viable but nonculturable (VBNC) state", induced by environmental stresses, such as low temperature, high osmolarity, and

Following environmental stimuli, as temperature shift or replenishment of nutrients, VBNC bacteria can "resuscitate", so restoring their ability to grow on common culture media.

In such a state, bacteria show a discrete metabolic activity, but are not able to replicate.

processing, such as handling of feedstock, decontamination, packaging and storage.

to identify the pathogen(s), the ways of food contamination and the spreading.

In fact, up to date more than 250 foodborne diseases have been described.

**1. Introduction** 

*Gastronomie Transcendante,* 1826)

terms of safety and control.

nutrient starvation.

what you eat." (Victor Lindlahr, 1923)

with immediate consequences on human habits and life.

pathogen bacteria, viruses, parasites and prions.

*control, exposure and risk assessment,* Vol. 27, No. 1*,* (January, 2010), pp 94-71, ISSN: 1944-0049


### **Viable but Nonculturable Bacteria in Food**

Marco Sebastiano Nicolò and Salvatore Pietro Paolo Guglielmino *University of Messina Italy* 

#### **1. Introduction**

188 Public Health – Methodology, Environmental and Systems Issues

Mikus, JH., Duff, GC., Krehbie, C.; Hallford, DM.; Walker, DA.; Graham, JD., & Ralphs, M

Moreno, L.; Alvarez, L.; Ceballos, L.; Sánchez Bruni S. & Lanusse C. (2008). Pattern of

Panter, KE. & James LF.(1990). Natural plant toxicants in milk: a review. *Journal of Animal* 

Rouessac, F. & Rouessac, A. (2003). *Análisis Químico. Métodos y Técnicas Instrumentales Modernas* (First edition), McGraw Hill, ISBN: 9788448137854, Madrid, España The Institute of Food Technologists. (2006). Comprehensive reviews Food Science and Food

Shoemaker, RC.; House, D. & Ryan, JC. (2010). Defining the neurotoxin derived

Tardieu, D.; Bailly, JD.; Skiba, F.; Grosjean, F. & Guerre, P. (2008). Toxicokinetics of

Weissinger, J. (1994). *Animal Drugs and Human Health (*fourth edition*)*, L.M. Crawford, and D.A. Franco, Technomic Publishing Co, ISBN: 9781566761024, Lancaster, USA World Health Organization, WHO. (1989). In Evaluation of Certain Veterinary Drug

World Health Organization,WHO.(1993). In Evaluation of Certain Veterinary Drug Residues

Zinedine, A.; Soriano, JM.; Moltó JC. & Mañes J. (2007). Review on the toxicity, occurrence,

*Science*, Vo.68, No.3, (March, 1990), pp 892-904, ISSN 0021-8812

1944-0049

1541-4337

2001), pp. 109-11, ISSN 1080-7446

2008), pp. 406-412, ISSN 1944-0049

(December, 2010), pp 633-639, ISSN 1872-9738

on Food Additives, Technical Report Series 788

Additives, Technical Report Series 832

ISSN 0278-6915

*control, exposure and risk assessment,* Vol. 27, No. 1*,* (January, 2010), pp 94-71, ISSN:

H. (2001). Effects of an Estradiol Implant on Locoweed Consumption, Toxicity, and Recovery in Growing Beef Steers, *Professional Animal Scientist,* Vol. 17, No. 2, (June

ivermectin (sheep) and doramectin (cattle) residues in muscular tissue from various anatomical locations. *Food Additives & Contaminants: Part A*, Vol. 25, No. 4, (April

Safety, *Institute of Food Technologist*,Vol. 5. No. 3 (August 2006) pp. 71-137 ISNN

illness chronic ciguatera using markers of chronic systemic inflammatory disturbances: a case/control study. *Neurotoxicology and Teratology*, Vol.32, No.6,

fumonisin B1 in turkey poults and tissue persistence after exposure to a diet containing the maximum European tolerance for fumonisins in avian feeds. *Food and Chemical Toxicology*, Vol. 4, No. 9, (January, 2008), pp 3213-3218, ISSN 0278-6915

Residues in Food, Thirty-fourth Report of the Joint FAO/WHO Expert Committee

in Food, Fortieth Report of the Joint FAO/WHO Expert Committee on Food

metabolism, detoxification, regulations and intake of zearalenone: an oestrogenic mycotoxin. *Food and Chemical Toxicology*,Vol. 45, No. 1, (January, 2007), pp 1-18 "Dis-moi ce que tu manges, je te dirai ce que tu es." (Tell me what you eat and I will tell you what you are - Anthelme Brillat-Savarin, *Physiologie du Goût, ou Méditations de Gastronomie Transcendante,* 1826)

"Ninety per cent of the diseases known to man are caused by cheap foodstuffs. You are what you eat." (Victor Lindlahr, 1923)

In every time, availability of food has been a struggle for human survival. In particular, food storage techniques and maintaining have represented one of the most important milestones in the evolution and development of human society. In that sense, the use of fire for cooking and salt and spices for conservation have been important discoveries in food processing, with immediate consequences on human habits and life.

Nowadays, food resources remain a primary objective for human society, specifically in terms of safety and control.

In fact, up to date more than 250 foodborne diseases have been described.

Infectious foodborne diseases are caused by the consumption of food contaminated by pathogen bacteria, viruses, parasites and prions.

Contamination may occur as consequence of incorrect practices at different steps of food processing, such as handling of feedstock, decontamination, packaging and storage.

The rapid globalization and trade among countries with different hygienic standards have increased the possibility of food contamination. Hence, outbreaks of foodborne diseases that were once contained within a small community may now take place on global dimensions.

The fundamental strategy for outbreaks monitoring is the traceability, that is the possibility to identify the pathogen(s), the ways of food contamination and the spreading.

One of the most critical problems in traceability of bacterial pathogens is represented by a state of latency of most foodborne bacterial species, called "viable but nonculturable (VBNC) state", induced by environmental stresses, such as low temperature, high osmolarity, and nutrient starvation.

In such a state, bacteria show a discrete metabolic activity, but are not able to replicate.

Following environmental stimuli, as temperature shift or replenishment of nutrients, VBNC bacteria can "resuscitate", so restoring their ability to grow on common culture media.

Viable but Nonculturable Bacteria in Food 191

For example, in 2005, in South Wales an outbreak of *Escherichia coli* O157 occurred, with identification of 157 cases, 31 people hospitalized, and one 5-year old child died, as well as

Recently, in October 2010 an outbreak of cholera occurred in Haiti. On 18 August 2011, the total number of reported cholera cases was 419,511, with 222,359 hospitalized. Overall, data

Moreover, actually it has been recognized that outbreaks may spread on wider geographic areas. In fact, the rapid globalization and trade among countries with different hygienic standards has increased the risk and entity of food contamination. As consequence, many outbreaks that were once contained within a small community may now take place on wider

The Bovine spongiform encephalopathy (BSE), commonly known as **mad-cow disease**, was firstly detected in United Kingdom and represented a world-wide crisis. The cause of global infection was attributed to the use of contaminated bone meal, used in livestock feeding. Only in the United Kingdom, 4.4 millions of cattle were slaughtered in the attempt to eradicate the pathology and limit its spreading; moreover, the European Union banned

In 2011, an outbreak of gastroenteritis caused by *E. coli* O104:H4 in Germany had several

Often, such kind of outbreaks are very difficult to identify while occurring. An example is represented by an outbreak of *Salmonella* which simultaneously happened in Europe, North America and Israel, following the importation of contaminated snack food. The outbreak identification was accidental, because it was caused by a rare strain of *Salmonella* and the

Therefore, there is a strong probability that contaminated foodstuffs distributed in different countries could origin outbreaks which are not readily pointed out by national health authorities if the spreading in each single country is limited. Many sporadic cases

Contamination may occur as consequence of incorrect practices at different steps of food processing, such as feedstock production and handling, food preparation, packaging and

Use of raw manure or sewage for fertilization and lack of hygiene and health controls in fish and cattle breeding are the first cause of contamination. Spinach and lettuce have been linked to *E. coli* O157:H7 outbreaks (Erickson et al., 2010). Untreated food, such as fresh fruit juice and milk, carry risks because they are not subjected to any decontaminating procedure. Similarly, incorrect slaughterhouse practices can lead to contamination, especially when

happening in single countries may be, then, part of a common global outbreak.

in 2008, an outbreak of listeriosis in Canada (57 clinical cases) killed 23 people.

from health facilities indicate that 5,968 people have died (case fatality rate 1.4%).

geographic areas and several examples can be considered.

exports of British beef from March 1996 to May 2006.

number of cases in each geographical entity was not high.

storage. Each stage of food processing shows critical points.

faecal or intestinal matter from cattle mixes with the meat.

**3. The causes of food contamination** 

**3.1 Feedstock production** 

social, political and economic implications throughout whole Europe.

Still open is the debate about the possibility, for pathogen bacteria in VBNC state, to maintain pathogenicity and trigger disease in their hosts.

The evidence of contrasting results, in fact, indicates the need for a better understanding of such complex phenomenon, particularly about the underlying molecular network.

Several studies have shown that the most of human pathogens, especially foodborne bacteria, may enter VBNC state as survival strategy against environmental stress.

Many chemo-physical characteristics in food (as acidic pH, low content in carbohydrates, etc.), as well as processing, decontamination and storage, may induce VBNC state.

The observation of VBNC forms of foodborne bacteria and the lack of a ultimate information about the possibility for VBNC bacteria to retain their virulence has raised the problem about the necessity of new procedures for VBNC detection in food.

Many systems have been proposed for VBNC detection in water, but their application on food seems to be quite difficult. In fact, factors as food texture and pH, as well as presence of free lytic enzymes and other compounds, may interfere with the chemical reaction(s) required for the assay.

Then, traceability of foodborne VBNC pathogens strongly requires the design of new detecting systems.

#### **2. The causes of infectious foodborne disease outbreaks**

Infectious foodborne diseases are caused by the consumption of food or beverages contaminated by many pathogenic bacteria, viruses, parasites and prions. In addition, contamination can also be due to molecules produced by bacteria, called **toxins**. Toxins can be derived from cell structure, such as lipopolysaccharide (LPS) of Gram negative bacteria (**endotoxins**), as well as they are synthesized inside the cell and secreted in the surrounding environment (**exotoxins**), as botulinum, cholera and Shiga toxins.

After food contamination, the microbe or its toxin enters the organism via the digestive tract, triggering the illness.

In general, an outbreak of foodborne disease occurs when a group of people eats the same contaminated food and two or more of them develop the same illness.

Many outbreaks consist of sporadic cases and are self-limiting, in that they are related to a small quantity of contaminated food which, usually, is totally eaten by few people and involve a specific geographic area. A sporadic outbreak may follow a catered meal or eating a meal at a restaurant on a particularly busy day.

The number of people affected and the extension of geographic areas in which a foodborne outbreak occurs have considerably increased as consequence of the variations in social habits. Commonly, workers and students may have meals at large-scale retail trade structures as canteens, highway stops, fast food chains and refectories. Such structures, usually, look to suppliers which, from the place where food is produced, distribute their products on national scale. Then, contaminated food may be transported in different places, causing many distinct outbreaks at the same time within a country.

Still open is the debate about the possibility, for pathogen bacteria in VBNC state, to

The evidence of contrasting results, in fact, indicates the need for a better understanding of

Several studies have shown that the most of human pathogens, especially foodborne

Many chemo-physical characteristics in food (as acidic pH, low content in carbohydrates,

The observation of VBNC forms of foodborne bacteria and the lack of a ultimate information about the possibility for VBNC bacteria to retain their virulence has raised the problem

Many systems have been proposed for VBNC detection in water, but their application on food seems to be quite difficult. In fact, factors as food texture and pH, as well as presence of free lytic enzymes and other compounds, may interfere with the chemical reaction(s)

Then, traceability of foodborne VBNC pathogens strongly requires the design of new

Infectious foodborne diseases are caused by the consumption of food or beverages contaminated by many pathogenic bacteria, viruses, parasites and prions. In addition, contamination can also be due to molecules produced by bacteria, called **toxins**. Toxins can be derived from cell structure, such as lipopolysaccharide (LPS) of Gram negative bacteria (**endotoxins**), as well as they are synthesized inside the cell and secreted in the surrounding

After food contamination, the microbe or its toxin enters the organism via the digestive

In general, an outbreak of foodborne disease occurs when a group of people eats the same

Many outbreaks consist of sporadic cases and are self-limiting, in that they are related to a small quantity of contaminated food which, usually, is totally eaten by few people and involve a specific geographic area. A sporadic outbreak may follow a catered meal or eating

The number of people affected and the extension of geographic areas in which a foodborne outbreak occurs have considerably increased as consequence of the variations in social habits. Commonly, workers and students may have meals at large-scale retail trade structures as canteens, highway stops, fast food chains and refectories. Such structures, usually, look to suppliers which, from the place where food is produced, distribute their products on national scale. Then, contaminated food may be transported in different places,

such complex phenomenon, particularly about the underlying molecular network.

bacteria, may enter VBNC state as survival strategy against environmental stress.

etc.), as well as processing, decontamination and storage, may induce VBNC state.

about the necessity of new procedures for VBNC detection in food.

**2. The causes of infectious foodborne disease outbreaks** 

environment (**exotoxins**), as botulinum, cholera and Shiga toxins.

contaminated food and two or more of them develop the same illness.

causing many distinct outbreaks at the same time within a country.

a meal at a restaurant on a particularly busy day.

required for the assay.

tract, triggering the illness.

detecting systems.

maintain pathogenicity and trigger disease in their hosts.

For example, in 2005, in South Wales an outbreak of *Escherichia coli* O157 occurred, with identification of 157 cases, 31 people hospitalized, and one 5-year old child died, as well as in 2008, an outbreak of listeriosis in Canada (57 clinical cases) killed 23 people.

Recently, in October 2010 an outbreak of cholera occurred in Haiti. On 18 August 2011, the total number of reported cholera cases was 419,511, with 222,359 hospitalized. Overall, data from health facilities indicate that 5,968 people have died (case fatality rate 1.4%).

Moreover, actually it has been recognized that outbreaks may spread on wider geographic areas. In fact, the rapid globalization and trade among countries with different hygienic standards has increased the risk and entity of food contamination. As consequence, many outbreaks that were once contained within a small community may now take place on wider geographic areas and several examples can be considered.

The Bovine spongiform encephalopathy (BSE), commonly known as **mad-cow disease**, was firstly detected in United Kingdom and represented a world-wide crisis. The cause of global infection was attributed to the use of contaminated bone meal, used in livestock feeding. Only in the United Kingdom, 4.4 millions of cattle were slaughtered in the attempt to eradicate the pathology and limit its spreading; moreover, the European Union banned exports of British beef from March 1996 to May 2006.

In 2011, an outbreak of gastroenteritis caused by *E. coli* O104:H4 in Germany had several social, political and economic implications throughout whole Europe.

Often, such kind of outbreaks are very difficult to identify while occurring. An example is represented by an outbreak of *Salmonella* which simultaneously happened in Europe, North America and Israel, following the importation of contaminated snack food. The outbreak identification was accidental, because it was caused by a rare strain of *Salmonella* and the number of cases in each geographical entity was not high.

Therefore, there is a strong probability that contaminated foodstuffs distributed in different countries could origin outbreaks which are not readily pointed out by national health authorities if the spreading in each single country is limited. Many sporadic cases happening in single countries may be, then, part of a common global outbreak.

#### **3. The causes of food contamination**

Contamination may occur as consequence of incorrect practices at different steps of food processing, such as feedstock production and handling, food preparation, packaging and storage. Each stage of food processing shows critical points.

#### **3.1 Feedstock production**

Use of raw manure or sewage for fertilization and lack of hygiene and health controls in fish and cattle breeding are the first cause of contamination. Spinach and lettuce have been linked to *E. coli* O157:H7 outbreaks (Erickson et al., 2010). Untreated food, such as fresh fruit juice and milk, carry risks because they are not subjected to any decontaminating procedure.

Similarly, incorrect slaughterhouse practices can lead to contamination, especially when faecal or intestinal matter from cattle mixes with the meat.

Viable but Nonculturable Bacteria in Food 193

Bacteria are single-cell microrganisms colonizing any environment. From an ecological point of view, they play a pivotal role in the biogeochemical cycles, by which chemical elements move from living to non-living matter and vice versa. Some of them produce molecules which improve the quality of human life, such as antibiotics, vitamins, probiotics and other

In food industry, several *Lactobacillus* species are very important for production of cheese,

Several bacteria inhabit the skin and the intestine of animals and humans, protecting them

Pathogen bacteria are responsible for infectious diseases, by colonization of tissues and

Foodborne pathogen bacteria are a group of microrganisms which can contaminate food

Common symptoms of foodborne illness are diarrhoea and/or vomiting, abdominal

A description of the most common known foodborne pathogen bacteria and related illness(es) follows. Intriguingly, for the most of them, the VBNC state has been described.

Gram positive rod-shaped bacterium, able to form endospores under negative conditions, commonly found in soil and vegetation. After pasteurisation or heating, endospores survive, whereas competing microflora is eliminated. During food cooling, endospores germinate and vegetative cells proliferate, producing several toxins, one of which is highly resistant to heat and to pH between 2 and 11. The infection may be almost diarrhoeal, but some cases

Genus of gram negative, aerobic, rod-spherical shaped bacteria which infect animals and humans. Four species are recognised as harmful, that is *B. abortus* (from cattle), *B. melitensis*

It can contaminate raw milk and cheese, but may be acquired also by inhalation, causing **brucellosis**, also known as Malta fever, undulant fever, Rock of Gibraltar fever, and Bang's

The symptoms are non-specific and systemic, with fever, sweats, headache, anorexia, back pain. The chronic form causes suppurative lesions in the liver, spleen, and bone, with a

Gram negative, microaerophilic, spirally curved bacterium, commonly living in the bowel of

(from goats, sheep, and camels), *B. suis* (from pigs), and *B. canis* (from dogs).

yogurt, beer, cider, wine, bread and chocolate, as well as in functional food.

from contamination by hazardous microbes and playing a role in metabolism.

organs, whose physiology is altered by bacterial metabolism and reproduction.

**4. Foodborne bacteria** 

nutritional factors.

*Bacillus cereus* 

*Brucella spp.* 

disease.

*Campylobacter jejuni* 

and, after swallowing, cause illness.

cramps, nausea, fever, joint/back aches, and fatigue.

described nausea and vomiting. The illness is self-limiting.

animals, especially in poultry, and spread by faeces and milk.

mortality of 5% in untreated individuals.

#### **3.2 Feedstock handling**

Handling and washing of feedstock before processing are very important sources of contamination. Handling by ill people which have cuts, open sores or skin infections causes spreading of *Staphylococcus aureus*, which is often found on skin with boils and blisters. That is why food handling should be performed by personnel wearing gloves. However, touching contaminated surfaces, coughing into a gloved hand or handling money before food preparation can still spread germs, which is why gloves should be changed often.

#### **3.3 Food preparation**

Beyond the causes of contamination above discussed, in this step cross contamination is a crucial factor. This can happen during food preparation and storage. For example, juices from raw beef and poultry could mix with ready-to-eat food or when kitchen tools are used without distinction for both raw beef and fresh vegetables. Cleaning tools with soap and hot water strongly reduces contamination.

Also undercooking can be cause of food contamination, because heat drastically reduces the presence of bacteria in food.

#### **3.4 Food packaging**

In food packaging, hygiene of handlers and materials employed are critical parameters.

Historically, canned food, if prepared under unsafe conditions, can be susceptible to contamination by toxinogenic microbes, such as *Clostridium botulinum*.

Nowadays, food packaging is mostly based on plastics, employed for vegetables, cheese, fruit. However, even if a correct packaging assures food safety from microbe contamination and remarkably prolongs shelf-life, much attention has to be paid about hygienic requirements. Bacteria can colonize and adhere on plastics, producing biofilms which may reduce shelf life or, for pathogenic bacteria, they may secrete toxins or proliferate on food.

#### **3.5 Food storage**

This is a critical step for perishable food which requires refrigeration or freezing, because low temperature slows down bacterial reproduction, even if some species, called **psycrophiles**, such as *Listeria monocytogenes*, *Yersinia enterocolitica* and *Pseudomonas* sp., are able to develop also at refrigerator temperature.

Food storage has become increasingly important in the perspective of globalization, in that different food tipologies may be transported together. Physical contact and dripping of juices to other foodstuffs are causes of crosscontamination. In this way, one kind of food can be contaminated by unusual bacteria, that become difficult to be identified. Also changes in livestock farming and industrialization of slaughtering of pigs have played an important role. Transportation of live animals for slaughtering has in some studies been proved to be an important factor in dissemination of *Y. enterocolitica* from farm to farm (Nesbakken, 2007*).*

#### **4. Foodborne bacteria**

192 Public Health – Methodology, Environmental and Systems Issues

Handling and washing of feedstock before processing are very important sources of contamination. Handling by ill people which have cuts, open sores or skin infections causes spreading of *Staphylococcus aureus*, which is often found on skin with boils and blisters. That is why food handling should be performed by personnel wearing gloves. However, touching contaminated surfaces, coughing into a gloved hand or handling money before food preparation can still spread germs, which is why gloves should be

Beyond the causes of contamination above discussed, in this step cross contamination is a crucial factor. This can happen during food preparation and storage. For example, juices from raw beef and poultry could mix with ready-to-eat food or when kitchen tools are used without distinction for both raw beef and fresh vegetables. Cleaning tools with soap and hot

Also undercooking can be cause of food contamination, because heat drastically reduces the

In food packaging, hygiene of handlers and materials employed are critical parameters.

contamination by toxinogenic microbes, such as *Clostridium botulinum*.

Historically, canned food, if prepared under unsafe conditions, can be susceptible to

Nowadays, food packaging is mostly based on plastics, employed for vegetables, cheese, fruit. However, even if a correct packaging assures food safety from microbe contamination and remarkably prolongs shelf-life, much attention has to be paid about hygienic requirements. Bacteria can colonize and adhere on plastics, producing biofilms which may reduce shelf life or, for pathogenic bacteria, they may secrete toxins or

This is a critical step for perishable food which requires refrigeration or freezing, because low temperature slows down bacterial reproduction, even if some species, called **psycrophiles**, such as *Listeria monocytogenes*, *Yersinia enterocolitica* and *Pseudomonas* sp., are

Food storage has become increasingly important in the perspective of globalization, in that different food tipologies may be transported together. Physical contact and dripping of juices to other foodstuffs are causes of crosscontamination. In this way, one kind of food can be contaminated by unusual bacteria, that become difficult to be identified. Also changes in livestock farming and industrialization of slaughtering of pigs have played an important role. Transportation of live animals for slaughtering has in some studies been proved to be an important factor in dissemination of *Y. enterocolitica* from farm to farm

**3.2 Feedstock handling** 

changed often.

**3.3 Food preparation** 

water strongly reduces contamination.

able to develop also at refrigerator temperature.

presence of bacteria in food.

**3.4 Food packaging** 

proliferate on food.

**3.5 Food storage** 

(Nesbakken, 2007*).*

Bacteria are single-cell microrganisms colonizing any environment. From an ecological point of view, they play a pivotal role in the biogeochemical cycles, by which chemical elements move from living to non-living matter and vice versa. Some of them produce molecules which improve the quality of human life, such as antibiotics, vitamins, probiotics and other nutritional factors.

In food industry, several *Lactobacillus* species are very important for production of cheese, yogurt, beer, cider, wine, bread and chocolate, as well as in functional food.

Several bacteria inhabit the skin and the intestine of animals and humans, protecting them from contamination by hazardous microbes and playing a role in metabolism.

Pathogen bacteria are responsible for infectious diseases, by colonization of tissues and organs, whose physiology is altered by bacterial metabolism and reproduction.

Foodborne pathogen bacteria are a group of microrganisms which can contaminate food and, after swallowing, cause illness.

Common symptoms of foodborne illness are diarrhoea and/or vomiting, abdominal cramps, nausea, fever, joint/back aches, and fatigue.

A description of the most common known foodborne pathogen bacteria and related illness(es) follows. Intriguingly, for the most of them, the VBNC state has been described.

#### *Bacillus cereus*

Gram positive rod-shaped bacterium, able to form endospores under negative conditions, commonly found in soil and vegetation. After pasteurisation or heating, endospores survive, whereas competing microflora is eliminated. During food cooling, endospores germinate and vegetative cells proliferate, producing several toxins, one of which is highly resistant to heat and to pH between 2 and 11. The infection may be almost diarrhoeal, but some cases described nausea and vomiting. The illness is self-limiting.

#### *Brucella spp.*

Genus of gram negative, aerobic, rod-spherical shaped bacteria which infect animals and humans. Four species are recognised as harmful, that is *B. abortus* (from cattle), *B. melitensis* (from goats, sheep, and camels), *B. suis* (from pigs), and *B. canis* (from dogs).

It can contaminate raw milk and cheese, but may be acquired also by inhalation, causing **brucellosis**, also known as Malta fever, undulant fever, Rock of Gibraltar fever, and Bang's disease.

The symptoms are non-specific and systemic, with fever, sweats, headache, anorexia, back pain. The chronic form causes suppurative lesions in the liver, spleen, and bone, with a mortality of 5% in untreated individuals.

#### *Campylobacter jejuni*

Gram negative, microaerophilic, spirally curved bacterium, commonly living in the bowel of animals, especially in poultry, and spread by faeces and milk.

Viable but Nonculturable Bacteria in Food 195

*C. burnetii* secretes a toxin inside the phagolysosome which inhibits its fusion with the cell

In humans it causes the **Q fever**, with fever, severe headache, muscle and joint pains, upper respiratory problems and gastro-intestinal symptoms such as nausea, vomiting and diarrhoea. Life threatening complications are acute respiratory distress syndrome (ARDS),

The chronic form of Q fever is virtually identical to endocarditis. It is usually fatal if

Gram negative, rod-shaped bacterium, inhabitant of mammal bowel. Among the pathogenic

• Enterotoxigenic (ETEC) *E. coli* produces and releases two exotoxins, LT (heat-labile) enterotoxin, similar to cholera toxin, and ST enterotoxin, which causes cGMP accumulation in the target cells and a subsequent secretion of fluid and electrolytes into the intestinal lumen. Both toxins induce watery diarrhoea, similarly to cholera. It is responsible of the

• Enteroinvasive (EIEC) *E. coli* can invade the intestinal wall, giving inflammation, fever

• Among the enterohaemorrhagic (EHEC) *E. coli* strains, the most important is the well known serotype O157:H7. EHEC strains produce Shiga-like toxins, which induce haemorrhagic colitis, resulting in bloody diarrhoea. Sometimes the toxin may spread in kidney, causing a very dangerous complication called **haemolytic uraemic syndrome**.

• Enteroaggregative (EAEC) strains have fimbriae which aggregate tissue culture cells. EAEC strains bind to the intestinal mucosa to cause watery diarrhoea without fever and are not invasive. They produce a haemolysin and a ST enterotoxin similar to that of ETEC

Gram negative, spiral-shaped microaerophilic bacterium, normally colonising the stomach

The ways of transmission are likely to be oral and oro-faecal routes, even if it has been quite

In the stomach, the bacterium secretes the enzyme urease, which degrades urea to ammonia.

It is responsible of chronic gastritis, peptic ulcer disease and stomach cancer. Actually, it is also associated with the gastric MALT lymphoma and its role on other several illnesses, as

majority of "traveller's diarrhoea" and infant diarrhoea in developing countries.

The first symptom is the presence of blood in urine leading to kidney failure. • Enteropathogenic (EPEC) strains use an adhesin known as **intimin** to bind host intestinal cells. Adherence to the intestinal mucosa causes a rearrangement of actin in the host cell, causing significant deformation. EPEC cells are moderately invasive and elicit an inflammatory response. Changes in intestinal cell ultrastructure are likely the

untreated; however, with appropriate treatment the mortality falls to around 10%.

degradation endosomes.

*Escherichia coli* 

strains. *Helicobacter pylori* 

granulomatous hepatitis and retinal vasculitis.

strains, distinct groups can be identified.

and diarrhoea similar to *Shigella*-like dysentery.

prime cause of diarrhoea in those afflicted with EPEC.

of 30-50% of the human population in developed countries.

difficult its isolation by the faeces (Thomas et al., 1992; Kelly et al., 1994).

Sjögren, Prader-Willy and Raynaud syndromes, is matter of discussion.

Ammonia lowers the pH locally, so permitting the development of the infection.

It can also contaminate incorrectly prepared meat and poultry.

It is one of the first cause of foodborne illness (**campylobacteriosis**) in United States and in United Kingdom. Symptoms of campylobacteriosis are fever, cramping abdominal pain and diarrhoea. Remission follows within a week.

#### *Clostridium botulinum*

Gram positive rod-shaped bacterium, obligate anaerobe, which forms endospores in presence of oxygen or other environmental stresses. Food contamination can occur due to improperly preserved or home-canned, low-acid food, which allows endospore germination and subsequent toxin secretion. Among the toxins, seven are neurotoxins, that cause the **flaccid muscular paralysis** by inhibition of neuromuscular transmission through decreased acetylcholine release. Death occurs when respiratory mechanics is compromised.

#### *Clostridium perfringens*

Gram positive rod-shaped bacterium, obligate anaerobe, which forms endospores in presence of oxygen or other environmental stresses. Preferentially, it contaminates meatbased food, because aminoacid content satisfies its nutritional requirements. After cooking, oxygen concentration is lowered and endospores germinate during cooling. Storage time and refrigeration are critical, in that the vegetative cell can duplicate in 20 minutes. After ingestion, in the bowel the bacterium produces an exotoxin which causes abdominal pain and diarrhoea. In the most of cases, the illness is self-limiting.

#### *Corynebacterium ulcerans*

Gram-positive, nonmotile, straight to slightly curved rod-shaped bacterium that causes subacute bovine mastitis, but *C. ulcerans* has increasingly been isolated from domestic animals such as dogs and cats.

Consumption of raw milk and dairy products or contact with cattle may cause infection of humans, causing a disease very similar to diphtheria by secretion of a toxin which inhibits protein synthesis of epithelial cells, leading them to death.

Symptoms are sore throat, low fever, and a pseudomembrane on the upper respiratory tract.

Toxin may spread through the bloodstream and can lead to life-threatening complications in heart and kidneys. It can also cause nerve damage, eventually leading to paralysis. 40% to 50% of those left untreated can die.

#### *Coxiella burnetii*

*C. burnetii* is an obligate intracellular, small Gram-negative bacterium highly resistant to high temperature, osmotic pressure, ultraviolet light.

It can contaminate people after ingestion of raw milk or contact with infected animals.

*C. burnetii* is highly infectious via the respiratory route, but the infectivity via the oral route is poorly understood. It has been proposed that *C. burnetii* can escape the gastrointestinal tract and produce infection sufficient to stimulate systemic immunity (Loftis et al., 2010).

*C. burnetii* secretes a toxin inside the phagolysosome which inhibits its fusion with the cell degradation endosomes.

In humans it causes the **Q fever**, with fever, severe headache, muscle and joint pains, upper respiratory problems and gastro-intestinal symptoms such as nausea, vomiting and diarrhoea. Life threatening complications are acute respiratory distress syndrome (ARDS), granulomatous hepatitis and retinal vasculitis.

The chronic form of Q fever is virtually identical to endocarditis. It is usually fatal if untreated; however, with appropriate treatment the mortality falls to around 10%.

#### *Escherichia coli*

194 Public Health – Methodology, Environmental and Systems Issues

It is one of the first cause of foodborne illness (**campylobacteriosis**) in United States and in United Kingdom. Symptoms of campylobacteriosis are fever, cramping abdominal pain and

Gram positive rod-shaped bacterium, obligate anaerobe, which forms endospores in presence of oxygen or other environmental stresses. Food contamination can occur due to improperly preserved or home-canned, low-acid food, which allows endospore germination and subsequent toxin secretion. Among the toxins, seven are neurotoxins, that cause the **flaccid muscular paralysis** by inhibition of neuromuscular transmission through decreased acetylcholine release. Death occurs when respiratory mechanics is

Gram positive rod-shaped bacterium, obligate anaerobe, which forms endospores in presence of oxygen or other environmental stresses. Preferentially, it contaminates meatbased food, because aminoacid content satisfies its nutritional requirements. After cooking, oxygen concentration is lowered and endospores germinate during cooling. Storage time and refrigeration are critical, in that the vegetative cell can duplicate in 20 minutes. After ingestion, in the bowel the bacterium produces an exotoxin which causes abdominal pain

Gram-positive, nonmotile, straight to slightly curved rod-shaped bacterium that causes subacute bovine mastitis, but *C. ulcerans* has increasingly been isolated from domestic

Consumption of raw milk and dairy products or contact with cattle may cause infection of humans, causing a disease very similar to diphtheria by secretion of a toxin which inhibits

Symptoms are sore throat, low fever, and a pseudomembrane on the upper respiratory tract. Toxin may spread through the bloodstream and can lead to life-threatening complications in heart and kidneys. It can also cause nerve damage, eventually leading to paralysis. 40% to

*C. burnetii* is an obligate intracellular, small Gram-negative bacterium highly resistant to

*C. burnetii* is highly infectious via the respiratory route, but the infectivity via the oral route is poorly understood. It has been proposed that *C. burnetii* can escape the gastrointestinal tract and produce infection sufficient to stimulate systemic immunity

It can contaminate people after ingestion of raw milk or contact with infected animals.

It can also contaminate incorrectly prepared meat and poultry.

and diarrhoea. In the most of cases, the illness is self-limiting.

protein synthesis of epithelial cells, leading them to death.

high temperature, osmotic pressure, ultraviolet light.

diarrhoea. Remission follows within a week.

*Clostridium botulinum* 

compromised.

*Clostridium perfringens* 

*Corynebacterium ulcerans* 

animals such as dogs and cats.

50% of those left untreated can die.

*Coxiella burnetii* 

(Loftis et al., 2010).

Gram negative, rod-shaped bacterium, inhabitant of mammal bowel. Among the pathogenic strains, distinct groups can be identified.


#### *Helicobacter pylori*

Gram negative, spiral-shaped microaerophilic bacterium, normally colonising the stomach of 30-50% of the human population in developed countries.

The ways of transmission are likely to be oral and oro-faecal routes, even if it has been quite difficult its isolation by the faeces (Thomas et al., 1992; Kelly et al., 1994).

In the stomach, the bacterium secretes the enzyme urease, which degrades urea to ammonia. Ammonia lowers the pH locally, so permitting the development of the infection.

It is responsible of chronic gastritis, peptic ulcer disease and stomach cancer. Actually, it is also associated with the gastric MALT lymphoma and its role on other several illnesses, as Sjögren, Prader-Willy and Raynaud syndromes, is matter of discussion.

Viable but Nonculturable Bacteria in Food 197

headache, then diarrhoea appears and fever declines. Differently from salmonellosis, bacteria multiply into phagocytic cells and disseminate in the body. In severe cases,

Today, the mortality rate of typhoid fever is about 1-2%; in the past it exceeded also 10%.

The genus *Shigella* is composed of Gram negative, rod-shaped bacteria, which are normally

Food contamination occurs via handling by unhealthy operators. Four species are pathogenic: *S. sonnei*, *S. dysenteriae*, *S. flexneri* and *S. boydii*. Many cases of "traveller's

*S. dysenteriae* causes severe dysentery and prostration by secreting the "Shiga toxin", which inhibits protein synthesis in epithelial cells of intestinal wall, killing them. Bacteria multiply in the small intestine and spread in the large intestin, entering the epithelial cells. Infection proceeds towards neighbouring cells, via a "cell-to-cell" mechanism, thus avoiding immune system response. As consequence, intestinal mucosa is damaged, causing severe diarrhoea with blood and mucus in the faeces. Additional symptoms may be abdominal cramps and

Only *S. dysenteriae* may reach the bloodstream, causing septicaemia. Its mortality rate is

Gram positive, spherical-shaped bacterium, it is a frequent inhabitant of respiratory tract from which it can contaminate hands and skin. Typically, *S. aureus* and others staphylococcal species are resistant to various stresses. They can survive 60°C for 30 minutes, drying and high osmotic pressures. Such characteristics allow their survival under conditions that, generally, eliminate the most of bacteria. In absence of competitors, then, it

It is able to produce several toxins that improve the virulence or damage tissues, but the toxin produced by serogroup A is responsible for most of the cases. The toxin is heat-stable,

Symptoms of intoxication are vomiting and abdominal cramps followed by diarrhoea.

Genus of gram positive, microaerophilic, spherical-shaped bacteria which occur in chains or pairs. The genus is defined by a combination of antigenic, haemolytic, and physiological

Group A is formed by a single species, *S. pyogenes*, with 40 antigenic types, while Group D

quite significant and may reach 20% in tropical areas, where it is prevalent.

in that maintains its virulence up to 30 minutes of boiling.

perforation of bowel mucosa may occur.

diarrhoea" are supported by *Shigella* spp.

present in the bowel of humans, apes and monkeys.

*Shigella* **spp.**

fever.

*Staphylococcus aureus* 

can rapidly proliferate.

*Streptococcus* **spp.**

Remission is reached within 24 hours.

characteristics into Groups A, B, C, D, F, and G.

is represented by the new genus *Enterococcus*.

Groups A and D can be transmitted to humans via food.

#### *Listeria monocytogenes*

Gram positive, facultative anaerobe, rod-shaped intracellular bacterium, commonly spread in soil and water. Vegetables are contaminated by the soil or by manure used as fertilizer. Animals can carry the bacterium asymptomatically and can contaminate food, such as uncooked meat, raw milk and soft cheeses, that are usually prohibited to pregnant women.

It is the causative agent of **listeriosis**. After having entered immune system cells, it becomes septicemic and can grow. The intracellular state in phagocytic cells may permit access to the brain and probably to reach the fetus in pregnant women by crossing the placenta.

Listeriosis may arise as septicaemia, meningoencephalitis, corneal ulcer, pneumonia and during pregnancy (causing abortion within 6-9 months or stillbirth).

When no internalization in cells occurs, the disease is presented as a febrile gastroenteritis.

*L. monocytogenes* is the leading cause of death among foodborne bacterial pathogens, with 20 to 30 percent of clinical infections resulting in death.

#### *Plesiomonas shigelloides*

Gram-negative, rod-shaped bacterium, which has been isolated from freshwater, freshwater fish, and shellfish and from many types of animals including cattle in tropical and subtropical areas.

Common symptoms are fever, shivers, abdominal pain, nausea, diarrhoea (which, in severe cases, may be greenish-yellow, foamy, and bloody) or vomiting and appear 20-24 hours after consumption of contaminated food or water.

*P. shigelloides* gastroenteritis is usually a mild self-limiting disease with remission in healthy people within 1-7 days, described in African countries, but sporadic cases have been reported also in Europe and North America. Severe forms may include sepsis and meningoencephalitis in newborns, arthritis, cholecystitis and osteomyelitis (Terpeluk, 1992).

#### *Salmonella* **spp.**

The genus *Salmonella* consists of Gram negative, rod-shaped facultatively anaerobe bacteria, which live in the bowel of humans and animals.

Meat, poultry and eggs are the most common food contaminated by *Salmonella*. However, food is contaminated by low loads of *Salmonella*, which are quite difficult to be appreciated by standard detection methods.

All species are considered pathogenic and responsible for a gastroenteritis known as **salmonellosis**. Bacteria reach the bowel and duplicate. Sometimes, they may cross the mucosa, entering lymphatic and cardiovascular systems; from there, they may infect other organs, causing septicaemia.

Symptoms are moderate fever, nausea, abdominal pain and cramps, diarrhoea. Average mortality rate is below 1%, but increases in children and old people, occurring as septicaemia.

Among the species, *S. typhi* is the most virulent. It is the aetiological agent of **typhoid fever** and is spread only by human faeces. Symptoms are high fever (40°C) and continued headache, then diarrhoea appears and fever declines. Differently from salmonellosis, bacteria multiply into phagocytic cells and disseminate in the body. In severe cases, perforation of bowel mucosa may occur.

Today, the mortality rate of typhoid fever is about 1-2%; in the past it exceeded also 10%.

#### *Shigella* **spp.**

196 Public Health – Methodology, Environmental and Systems Issues

Gram positive, facultative anaerobe, rod-shaped intracellular bacterium, commonly spread in soil and water. Vegetables are contaminated by the soil or by manure used as fertilizer. Animals can carry the bacterium asymptomatically and can contaminate food, such as uncooked meat, raw milk and soft cheeses, that are usually prohibited to pregnant women. It is the causative agent of **listeriosis**. After having entered immune system cells, it becomes septicemic and can grow. The intracellular state in phagocytic cells may permit access to the

Listeriosis may arise as septicaemia, meningoencephalitis, corneal ulcer, pneumonia and

When no internalization in cells occurs, the disease is presented as a febrile gastroenteritis. *L. monocytogenes* is the leading cause of death among foodborne bacterial pathogens, with 20

Gram-negative, rod-shaped bacterium, which has been isolated from freshwater, freshwater fish, and shellfish and from many types of animals including cattle in tropical and sub-

Common symptoms are fever, shivers, abdominal pain, nausea, diarrhoea (which, in severe cases, may be greenish-yellow, foamy, and bloody) or vomiting and appear 20-24 hours

*P. shigelloides* gastroenteritis is usually a mild self-limiting disease with remission in healthy people within 1-7 days, described in African countries, but sporadic cases have been reported also in Europe and North America. Severe forms may include sepsis and meningoencephalitis in newborns, arthritis, cholecystitis and osteomyelitis (Terpeluk, 1992).

The genus *Salmonella* consists of Gram negative, rod-shaped facultatively anaerobe bacteria,

Meat, poultry and eggs are the most common food contaminated by *Salmonella*. However, food is contaminated by low loads of *Salmonella*, which are quite difficult to be appreciated

All species are considered pathogenic and responsible for a gastroenteritis known as **salmonellosis**. Bacteria reach the bowel and duplicate. Sometimes, they may cross the mucosa, entering lymphatic and cardiovascular systems; from there, they may infect other

Symptoms are moderate fever, nausea, abdominal pain and cramps, diarrhoea. Average mortality rate is below 1%, but increases in children and old people, occurring as

Among the species, *S. typhi* is the most virulent. It is the aetiological agent of **typhoid fever** and is spread only by human faeces. Symptoms are high fever (40°C) and continued

brain and probably to reach the fetus in pregnant women by crossing the placenta.

during pregnancy (causing abortion within 6-9 months or stillbirth).

to 30 percent of clinical infections resulting in death.

after consumption of contaminated food or water.

which live in the bowel of humans and animals.

by standard detection methods.

organs, causing septicaemia.

*Listeria monocytogenes* 

*Plesiomonas shigelloides* 

tropical areas.

*Salmonella* **spp.** 

septicaemia.

The genus *Shigella* is composed of Gram negative, rod-shaped bacteria, which are normally present in the bowel of humans, apes and monkeys.

Food contamination occurs via handling by unhealthy operators. Four species are pathogenic: *S. sonnei*, *S. dysenteriae*, *S. flexneri* and *S. boydii*. Many cases of "traveller's diarrhoea" are supported by *Shigella* spp.

*S. dysenteriae* causes severe dysentery and prostration by secreting the "Shiga toxin", which inhibits protein synthesis in epithelial cells of intestinal wall, killing them. Bacteria multiply in the small intestine and spread in the large intestin, entering the epithelial cells. Infection proceeds towards neighbouring cells, via a "cell-to-cell" mechanism, thus avoiding immune system response. As consequence, intestinal mucosa is damaged, causing severe diarrhoea with blood and mucus in the faeces. Additional symptoms may be abdominal cramps and fever.

Only *S. dysenteriae* may reach the bloodstream, causing septicaemia. Its mortality rate is quite significant and may reach 20% in tropical areas, where it is prevalent.

#### *Staphylococcus aureus*

Gram positive, spherical-shaped bacterium, it is a frequent inhabitant of respiratory tract from which it can contaminate hands and skin. Typically, *S. aureus* and others staphylococcal species are resistant to various stresses. They can survive 60°C for 30 minutes, drying and high osmotic pressures. Such characteristics allow their survival under conditions that, generally, eliminate the most of bacteria. In absence of competitors, then, it can rapidly proliferate.

It is able to produce several toxins that improve the virulence or damage tissues, but the toxin produced by serogroup A is responsible for most of the cases. The toxin is heat-stable, in that maintains its virulence up to 30 minutes of boiling.

Symptoms of intoxication are vomiting and abdominal cramps followed by diarrhoea. Remission is reached within 24 hours.

#### *Streptococcus* **spp.**

Genus of gram positive, microaerophilic, spherical-shaped bacteria which occur in chains or pairs. The genus is defined by a combination of antigenic, haemolytic, and physiological characteristics into Groups A, B, C, D, F, and G.

Groups A and D can be transmitted to humans via food.

Group A is formed by a single species, *S. pyogenes*, with 40 antigenic types, while Group D is represented by the new genus *Enterococcus*.

Viable but Nonculturable Bacteria in Food 199

For the most of the above described foodborne bacteria, the VBNC state has been proven by several studies. Therefore, the meaning of VBNC state as well as inducing and resuscitating factors have to be well understood, in order to plan how to face the difficulties of detection

In their natural environments, bacteria undergo fluctuating chemo-physical conditions, such as nutrient availability, temperature, osmolarity, and pH, which may interfere with their growth and survival. In such a situation, they modulate their gene expression, in order to survive, by activating the **bacterial stress response**, which consists in variations of cell morphology, dimensions, energetic levels, directly related to cell survival. The final effect is an increased global resistance of surviving cells against further stresses, such as exposure to antibiotics, hydrogen peroxide and high osmolarity (Matin, 1991; Nyström et al., 1992).

In such a situation, some genera of Gram positive bacteria, as *Bacillus*, *Clostridium* and few

Endospore is a differentiated bacterial cell in which new structures, required for mechanical and physical resistance, are synthesised. In the endospore, metabolism has been abolished by a controlled process of dehydration. The lack of metabolism confers a global resistance extended for prolonged periods of time. When environmental conditions become favourable, endospore undergoes **germination**, a process by which the metabolic activity is

The most of Gram positive and all Gram negative bacteria are not able to generate endospores. However, they can trigger a stress response with a highly-complex network of

Studies carried on bacterial stress response have started from bacterial physiology in natural

Subsequent studies have focused on the underlying molecular mechanisms, especially regarding a specific chemophysical stress and single-nutrients starvation (Eberl et al, 1996;

One aspect on which little is known is that, in natural environments, nutritional and chemophysical factors inducing bacterial stress response may change simultaneously, with one

Therefore, several mechanisms of bacterial stress responses should be activated. Unfortunately, few data are available about bacterial response to simultaneously-acting multiple stresses.

Bacterial responses to chemophysical stresses, such as cold- and heat-shock,

It has been observed that *E. coli*, when subjected to a temperature decrease, triggers the cold shock response, in which sets of proteins are synthesised, globally indicated as CIPs (Cold

specific factor influencing another and being, in turn, influenced by a third one.

oligotrophic environments (Kurath & Morita, 1983; Morita, 1982).

Matin, 1991; Nakashima et al., 1996; Rockabrand et al., 1995).

**5.1 The bacterial stress response to chemophysical stress** 

hyperosmolarity and acid pH, have been investigated.

**5.1.1 The cold shock response** 

and make traceability feasible.

**5. The bacterial stress response** 

others, differentiate into **endospore**.

restored and cell turns to vegetative life.

molecular mechanisms.

Many types of food may be contaminated by Group A *Streptococcus*, including raw milk, ice cream, ham, potato salad, shrimps salad, steamed lobster via manipulation by ill handlers. Moreover, they may contaminate food with low content of free water, rich in salt or with very acidic pH, which is generally difficult to be contaminated by other bacterial species.

Group A *Streptococcus* may cause several diseases, as bacteraemia, impetigo, erysipelas, pneumonia, osteomyelitis, septic arthritis, meningitis and toxic shock syndrome.

Among complications, acute rheumatic fever may follow respiratory infections as an autoimmune disease, in which antibodies raised against the streptococcal M-protein crossreact with autoantigens of pericardium and synovium.

Enterococci can cause food intoxication through production of biogenic amines, such as histamine and tyramine, mainly by the decarboxylation of amino acids in fish, meat and dairy products, wine, beer, vegetables, fruits, and nuts.

The genus *Enterococcus* is responsible for severe diseases, such as urinary tract infections, bacteraemia, bacterial endocarditis, diverticulitis, and meningitis.

#### *Vibrio cholerae*

Gram negative, slightly curved rod-shaped bacterium, which lives naturally in brackish waters, but can easily spread also in freshwater.

It can contaminate fish and seafood.

It is the aetiological agent of cholera, a serious illness that stroke Europe and North America during the XIX century with different outbreaks. Symptoms are nausea, vomiting, abdominal pain, watery diarrhoea which may induce severe dehydratation, that can be fatal if untreated.

Nowadays, cholera is endemic in India and rarely it causes outbreaks in Western countries. In 1991-1994, Latin America had an epidemic, in consequence of importation of contaminated seafood from Asia, with more than one million cases and 9600 deaths. In 2010, a new outbreak in Haiti has occurred.

#### *Vibrio parahaemolyticus*

Gram negative, slightly curved rod-shaped bacterium, which lives naturally in salt waters.

The related gastroenteritis follows the consumption of contaminated seafood. Symptoms include abdominal pain, vomiting, a burning sensation in the stomach and cholera-like watery faeces. Remission occurs within few days.

#### *Vibrio vulnificus*

As *V. parahemolyticus*, it is found in estuarine waters and contaminates seafood. It represents a serious threat for people with compromised immune system. In people with liver disease, it may cause septicaemia, with mortality rates often exceeding 50%.

#### *Yersinia enterocolitica* **and** *Y. pseudotuberculosis*

Psycrofile Gram-negative bacteria which usually colonise the bowel of domestic animals and are transmitted by milk and meat. Outside their natural habitat, they are able to grow at refrigeration temperature (4°C). The symptoms are diarrhoea, fever, headache and abdominal pain.

Many types of food may be contaminated by Group A *Streptococcus*, including raw milk, ice cream, ham, potato salad, shrimps salad, steamed lobster via manipulation by ill handlers. Moreover, they may contaminate food with low content of free water, rich in salt or with very acidic pH, which is generally difficult to be contaminated by other bacterial species.

Group A *Streptococcus* may cause several diseases, as bacteraemia, impetigo, erysipelas,

Among complications, acute rheumatic fever may follow respiratory infections as an autoimmune disease, in which antibodies raised against the streptococcal M-protein cross-

Enterococci can cause food intoxication through production of biogenic amines, such as histamine and tyramine, mainly by the decarboxylation of amino acids in fish, meat and

The genus *Enterococcus* is responsible for severe diseases, such as urinary tract infections,

Gram negative, slightly curved rod-shaped bacterium, which lives naturally in brackish

It is the aetiological agent of cholera, a serious illness that stroke Europe and North America during the XIX century with different outbreaks. Symptoms are nausea, vomiting, abdominal pain, watery diarrhoea which may induce severe dehydratation, that can be fatal if untreated. Nowadays, cholera is endemic in India and rarely it causes outbreaks in Western countries. In 1991-1994, Latin America had an epidemic, in consequence of importation of contaminated seafood from Asia, with more than one million cases and 9600 deaths. In 2010,

Gram negative, slightly curved rod-shaped bacterium, which lives naturally in salt waters. The related gastroenteritis follows the consumption of contaminated seafood. Symptoms include abdominal pain, vomiting, a burning sensation in the stomach and cholera-like

As *V. parahemolyticus*, it is found in estuarine waters and contaminates seafood. It represents a serious threat for people with compromised immune system. In people with liver disease,

Psycrofile Gram-negative bacteria which usually colonise the bowel of domestic animals and are transmitted by milk and meat. Outside their natural habitat, they are able to grow at refrigeration temperature (4°C). The symptoms are diarrhoea, fever, headache and

pneumonia, osteomyelitis, septic arthritis, meningitis and toxic shock syndrome.

react with autoantigens of pericardium and synovium.

dairy products, wine, beer, vegetables, fruits, and nuts.

waters, but can easily spread also in freshwater.

It can contaminate fish and seafood.

a new outbreak in Haiti has occurred.

watery faeces. Remission occurs within few days.

*Yersinia enterocolitica* **and** *Y. pseudotuberculosis* 

it may cause septicaemia, with mortality rates often exceeding 50%.

*Vibrio parahaemolyticus* 

*Vibrio vulnificus* 

abdominal pain.

*Vibrio cholerae* 

bacteraemia, bacterial endocarditis, diverticulitis, and meningitis.

For the most of the above described foodborne bacteria, the VBNC state has been proven by several studies. Therefore, the meaning of VBNC state as well as inducing and resuscitating factors have to be well understood, in order to plan how to face the difficulties of detection and make traceability feasible.

#### **5. The bacterial stress response**

In their natural environments, bacteria undergo fluctuating chemo-physical conditions, such as nutrient availability, temperature, osmolarity, and pH, which may interfere with their growth and survival. In such a situation, they modulate their gene expression, in order to survive, by activating the **bacterial stress response**, which consists in variations of cell morphology, dimensions, energetic levels, directly related to cell survival. The final effect is an increased global resistance of surviving cells against further stresses, such as exposure to antibiotics, hydrogen peroxide and high osmolarity (Matin, 1991; Nyström et al., 1992).

In such a situation, some genera of Gram positive bacteria, as *Bacillus*, *Clostridium* and few others, differentiate into **endospore**.

Endospore is a differentiated bacterial cell in which new structures, required for mechanical and physical resistance, are synthesised. In the endospore, metabolism has been abolished by a controlled process of dehydration. The lack of metabolism confers a global resistance extended for prolonged periods of time. When environmental conditions become favourable, endospore undergoes **germination**, a process by which the metabolic activity is restored and cell turns to vegetative life.

The most of Gram positive and all Gram negative bacteria are not able to generate endospores. However, they can trigger a stress response with a highly-complex network of molecular mechanisms.

Studies carried on bacterial stress response have started from bacterial physiology in natural oligotrophic environments (Kurath & Morita, 1983; Morita, 1982).

Subsequent studies have focused on the underlying molecular mechanisms, especially regarding a specific chemophysical stress and single-nutrients starvation (Eberl et al, 1996; Matin, 1991; Nakashima et al., 1996; Rockabrand et al., 1995).

One aspect on which little is known is that, in natural environments, nutritional and chemophysical factors inducing bacterial stress response may change simultaneously, with one specific factor influencing another and being, in turn, influenced by a third one.

Therefore, several mechanisms of bacterial stress responses should be activated. Unfortunately, few data are available about bacterial response to simultaneously-acting multiple stresses.

#### **5.1 The bacterial stress response to chemophysical stress**

Bacterial responses to chemophysical stresses, such as cold- and heat-shock, hyperosmolarity and acid pH, have been investigated.

#### **5.1.1 The cold shock response**

It has been observed that *E. coli*, when subjected to a temperature decrease, triggers the cold shock response, in which sets of proteins are synthesised, globally indicated as CIPs (Cold

Viable but Nonculturable Bacteria in Food 201

2002), osmotic stress (Asakura et al., 2008), oxygen availability (Kana et al., 2008), several food preservatives (Quirós et al., 2009), heavy metals (Ghezzi & Steck, 1999), exposure to white light (Gourmelon et al., 1994) and decontaminating processes, as pasteurization of

In such a state, bacteria lose the ability to grow on solid media and undergo reduction in size; moreover, several metabolic variations occur, such as reductions in nutrient transport across cytoplasmic membrane, respiration rates, and macromolecular synthesis (Oliver, 2000; Porter et al., 1995). Biosynthesis does not cease, in that starvation and cold shock proteins are synthesized (McGovern & Oliver, 1995; Morton & Oliver, 1994). ATP levels remain high in VBNC cells (Beumer et al., 1992; Federighi et al., 1998). Further, recent studies have demonstrated continued gene expression by cells in the VBNC state (Lleò et al., 2000, 2001; Yaron and Matthews, 2002). Other cellular characteristics, such as cell wall (Signoretto et al., 2000; Signoretto et al., 2002) and membrane composition (Day and Oliver,

When environmental conditions become permissive, **resuscitation** of VBNC bacteria occurs. Resuscitated bacteria are culturable on solid media and display the vegetative lifecycle.

Also resuscitation is a very complex phenomenon. A group of extracellular proteins, indicated as **resuscitation promoting factors** (Rpfs), play a key role in several other bacterial

Another class of resuscitation factors is a heat-stable **autoinducer of growth** (Reissbrodt et al., 2002), which has been identified as a novel quorum-sensing system, termed AI-3 (Sperandio et al., 2003) and secreted after incubation in media containing norepinephrine (Freestone et al., 1999). Norepinephrine is produced in large amounts in humans following severe tissue injury, and is thus considered to be a stress-related hormone. Both epinephrine and norepinephrine could replace AI-3 in activating enterohaemorrhagic *E. coli* virulence

These findings would support the hypothesis of resuscitation of VBNC enteropathogens in the human intestinal tract, at a time (e.g. tissue damage) when the host may be under

Interestingly, even several higher organisms may induce resuscitation from the VBNC state. Many conditions have been found to allow resuscitation of pathogens, as inoculation into yolk sacs of embryonated eggs (Cappelier et al., 1999b, 2007), into mice (Cappelier et al.,

The observations of the VBNC state for the most of foodborne pathogens have raised several questions about the retention of virulence in such a state as well as recovery of virulence together with resuscitation in the host. The matter is highly debated because contrasting results have been presented. In some cases, virulence of *L. monocytogenes* in the VBNC state has been shown to depend on the experimental conditions adopted for resuscitation (Cappelier et al., 2005, 2007). It seems that VBNC pathogens are not generally able to initiate disease, but virulence is retained and infection can be initiated following their resuscitation. In fact, VBNC cells of *Vibrio harveyi* were avirulent, but resuscitated cells were lethal, indicating that VBNC *V. harveyi* cells retained pathogenic potential (Sun et al., 2008). Similarly, Oliver & Bockian (1995) reported *V. vulnificus* to lose virulence for mice in

species (Hett et al., 2007; Mukamolova et al., 1998a, 1998b; Shleeva et al., 2004).

significant physiological stress, with consequent secretion of norepinephrine.

milk (Gunasekera et al., 2002) and chlorination of wastewater (Oliver et al., 2005).

2004), differ remarkably from culturable cells.

gene expression (Sperandio et al., 2003).

1999a) and into human volunteers (Colwell et al., 1996).

Induced Proteins), as transcription regulators, ribosomal proteins, elongation factors and β subunit of RNA-polymerase (Berger et al., 1996; Jones & Inouye, 1996; Nakashima et al., 1996), in order to allow mRNAs transcription and protein synthesis.

#### **5.1.2 The heat shock response**

When subjected to heat shock, bacteria have to counteract macromolecules denaturation, so the overexpression of chaperones is induced to stabilize cell macromolecules and to degrade denaturised polypeptides (Gage & Neidhardt, 1993; Spence, 1990).

#### **5.1.3 The osmotic shock response**

Osmotic shock causes considerable shrinkage of the cytoplasmic volume. In this process, known as **plasmolysis**, intracellular water tends to migrate towards the external environment. As a consequence, the concentrations of all the intracellular metabolites increase and thus reduce the intracellular water activity (wa). Bacteria may react by increasing the concentrations of solutes that have no effects on cell processes. Such solutes, called **compatible solutes**, are potassium ions, some amino acids (such as glutamate, glutamine, proline, γ-aminobutyrate, alanine), the quaternary amines glycinebetaine and some sugars (sucrose, threalose). Their accumulation inside the cells allows the cytoplasmic wa to be restored, with maintaining of metabolism (Csonka, 1989).

#### **5.1.4 The acid shock response**

When a sudden drop in pH occurs, bacteria generally use proton pumps, which literally pump protons out of the cell to keep the cytoplasmic pH. Another approach is to increase the concentration of alkaline compounds within the cell to counteract the acidification of the cytoplasm (Bore et al., 2007).

#### **5.2 The bacterial stress response to nutrient starvation**

Under nutrient starvation, variations at structural, metabolic and physiological levels are observed.

During carbon starvation, heterotrophic bacteria enter a quiescent state, with a decrease in ATP content and a general reduction in metabolic activity. Moreover, gram negative rod bacteria undergo a characteristic morphology transition to spherical shape. When an energetic source becomes newly available, carbon-starved bacteria rapidly resume their metabolic activities, simultaneously restoring the rod shape (Givskov et al., 1994).

On the other hand, under nitrogen or phosphate starvation, anabolism is strongly reduced and the cell undergoes an energetic surplus, which is dissipated by futile cycles reactions or accumulation of high-energy storage compounds, such as PHAs (Eberl et al., 1996).

#### **6. The viable but nonculturable (VBNC) state**

One of the most intriguing findings on stressed bacteria in natural environments was the socalled **viable but nonculturable** (VBNC) **state** (Xu et al., 1982).

The VBNC state is defined as a state of dormancy triggered by environmental harsh conditions, such as nutrient starvation (Cook & Bolster, 2007), temperature (Besnard et al.,

Induced Proteins), as transcription regulators, ribosomal proteins, elongation factors and β subunit of RNA-polymerase (Berger et al., 1996; Jones & Inouye, 1996; Nakashima et al.,

When subjected to heat shock, bacteria have to counteract macromolecules denaturation, so the overexpression of chaperones is induced to stabilize cell macromolecules and to degrade

Osmotic shock causes considerable shrinkage of the cytoplasmic volume. In this process, known as **plasmolysis**, intracellular water tends to migrate towards the external environment. As a consequence, the concentrations of all the intracellular metabolites increase and thus reduce the intracellular water activity (wa). Bacteria may react by increasing the concentrations of solutes that have no effects on cell processes. Such solutes, called **compatible solutes**, are potassium ions, some amino acids (such as glutamate, glutamine, proline, γ-aminobutyrate, alanine), the quaternary amines glycinebetaine and some sugars (sucrose, threalose). Their accumulation inside the cells allows the cytoplasmic

When a sudden drop in pH occurs, bacteria generally use proton pumps, which literally pump protons out of the cell to keep the cytoplasmic pH. Another approach is to increase the concentration of alkaline compounds within the cell to counteract the acidification of the

Under nutrient starvation, variations at structural, metabolic and physiological levels are

During carbon starvation, heterotrophic bacteria enter a quiescent state, with a decrease in ATP content and a general reduction in metabolic activity. Moreover, gram negative rod bacteria undergo a characteristic morphology transition to spherical shape. When an energetic source becomes newly available, carbon-starved bacteria rapidly resume their

On the other hand, under nitrogen or phosphate starvation, anabolism is strongly reduced and the cell undergoes an energetic surplus, which is dissipated by futile cycles reactions or

One of the most intriguing findings on stressed bacteria in natural environments was the so-

The VBNC state is defined as a state of dormancy triggered by environmental harsh conditions, such as nutrient starvation (Cook & Bolster, 2007), temperature (Besnard et al.,

metabolic activities, simultaneously restoring the rod shape (Givskov et al., 1994).

accumulation of high-energy storage compounds, such as PHAs (Eberl et al., 1996).

1996), in order to allow mRNAs transcription and protein synthesis.

denaturised polypeptides (Gage & Neidhardt, 1993; Spence, 1990).

wa to be restored, with maintaining of metabolism (Csonka, 1989).

**5.2 The bacterial stress response to nutrient starvation** 

**6. The viable but nonculturable (VBNC) state** 

called **viable but nonculturable** (VBNC) **state** (Xu et al., 1982).

**5.1.2 The heat shock response** 

**5.1.3 The osmotic shock response** 

**5.1.4 The acid shock response** 

cytoplasm (Bore et al., 2007).

observed.

2002), osmotic stress (Asakura et al., 2008), oxygen availability (Kana et al., 2008), several food preservatives (Quirós et al., 2009), heavy metals (Ghezzi & Steck, 1999), exposure to white light (Gourmelon et al., 1994) and decontaminating processes, as pasteurization of milk (Gunasekera et al., 2002) and chlorination of wastewater (Oliver et al., 2005).

In such a state, bacteria lose the ability to grow on solid media and undergo reduction in size; moreover, several metabolic variations occur, such as reductions in nutrient transport across cytoplasmic membrane, respiration rates, and macromolecular synthesis (Oliver, 2000; Porter et al., 1995). Biosynthesis does not cease, in that starvation and cold shock proteins are synthesized (McGovern & Oliver, 1995; Morton & Oliver, 1994). ATP levels remain high in VBNC cells (Beumer et al., 1992; Federighi et al., 1998). Further, recent studies have demonstrated continued gene expression by cells in the VBNC state (Lleò et al., 2000, 2001; Yaron and Matthews, 2002). Other cellular characteristics, such as cell wall (Signoretto et al., 2000; Signoretto et al., 2002) and membrane composition (Day and Oliver, 2004), differ remarkably from culturable cells.

When environmental conditions become permissive, **resuscitation** of VBNC bacteria occurs. Resuscitated bacteria are culturable on solid media and display the vegetative lifecycle.

Also resuscitation is a very complex phenomenon. A group of extracellular proteins, indicated as **resuscitation promoting factors** (Rpfs), play a key role in several other bacterial species (Hett et al., 2007; Mukamolova et al., 1998a, 1998b; Shleeva et al., 2004).

Another class of resuscitation factors is a heat-stable **autoinducer of growth** (Reissbrodt et al., 2002), which has been identified as a novel quorum-sensing system, termed AI-3 (Sperandio et al., 2003) and secreted after incubation in media containing norepinephrine (Freestone et al., 1999). Norepinephrine is produced in large amounts in humans following severe tissue injury, and is thus considered to be a stress-related hormone. Both epinephrine and norepinephrine could replace AI-3 in activating enterohaemorrhagic *E. coli* virulence gene expression (Sperandio et al., 2003).

These findings would support the hypothesis of resuscitation of VBNC enteropathogens in the human intestinal tract, at a time (e.g. tissue damage) when the host may be under significant physiological stress, with consequent secretion of norepinephrine.

Interestingly, even several higher organisms may induce resuscitation from the VBNC state.

Many conditions have been found to allow resuscitation of pathogens, as inoculation into yolk sacs of embryonated eggs (Cappelier et al., 1999b, 2007), into mice (Cappelier et al., 1999a) and into human volunteers (Colwell et al., 1996).

The observations of the VBNC state for the most of foodborne pathogens have raised several questions about the retention of virulence in such a state as well as recovery of virulence together with resuscitation in the host. The matter is highly debated because contrasting results have been presented. In some cases, virulence of *L. monocytogenes* in the VBNC state has been shown to depend on the experimental conditions adopted for resuscitation (Cappelier et al., 2005, 2007). It seems that VBNC pathogens are not generally able to initiate disease, but virulence is retained and infection can be initiated following their resuscitation. In fact, VBNC cells of *Vibrio harveyi* were avirulent, but resuscitated cells were lethal, indicating that VBNC *V. harveyi* cells retained pathogenic potential (Sun et al., 2008). Similarly, Oliver & Bockian (1995) reported *V. vulnificus* to lose virulence for mice in

Viable but Nonculturable Bacteria in Food 203

conservation technique for meat and vegetables consists, ultimately, in bringing down aw to

As already stated, bacteria may face osmotic shock; moreover, high osmolarity may induce

Biological macromolecules are used as substrates for growth, but bacteria may use only a limited part of the macromolecular content. Moreover, several compounds, such as polyphenols of fruit and vegetables and organic acids, display bacteriostatic and bactericide

Decontamination procedures, such as pasteurisation, salting and acidification, can induce VBNC state (Gunasekera et al., 2002; Makino et al., 2000; Quirós et al., 2009). Moreover,

Before consumption, food is stored during packaging, distribution and commercialization. Time and temperature are critical factors which are related to bacterial stress response and

Feedstock and untreated food possess a resident microflora, not necessarily hazardous for human health. Such microflora may interact with contaminating bacterial pathogens by intercellular exchange of signalling molecules, as the **autoinducers**, which may activate secretion of virulence factors or resuscitation in VBNC bacteria (Reissbrodt et al., 2002).

Despite the few studies, strong evidences of VBNC bacteria in food have been reported. In stored wine, for example, acetic acid and lactic acid bacteria entered VBNC state as consequence of lack of oxygen and presence of sulphites, respectively (Millet and Lonvaud-

The role played by chemo-physical characteristics of food in triggering VBNC state in foodborne pathogen bacteria is poorly undestood. The most of the studies consists in analysing the effects provoked by a single stress on a homogeneous population of

However, it has to be considered that, in their habitat, bacteria are continuously subjected to the simultaneous action of factors, as nutrient availability, pH, osmolarity, temperature, presence of toxic compounds, ecological competition with other organisms, which are continuously changing. The time and the entity of the change of a single factor influences, and in turn is influenced by, changes and entities of other factors, according to chaos

**8. VBNC state of foodborne bacteria – The new challenge in food safety** 

untreated food is more susceptible to microbial contamination (Erickson et al., 2010).

reduce microbial development.

VBNC state (Asakura et al., 2008).

activities, which are further stress-inducing factors.

induction of VBNC state (Besnard et al., 2002).

dynamics observed in nature, in an unpredictable way.

**7.7 Presence of endogenous microflora** 

**7.4 Chemical composition** 

**7.5 Feedstock treatment** 

**7.6 Food storage** 

Funel, 2000).

bacteria.

proportion to the length of time that the cells were in the VBNC state. The cells retained virulence, however, and even when fully nonculturable, were able to cause fatal infections, with resuscitation occurring within the mouse. Continued virulence for a variety of pathogenic vibrios has also been demonstrated (Baffone et al., 2003).

Then, it can be concluded that VBNC forms of foodborne pathogen bacteria into a human or animal host may be a realistic threat for public health, because it has been demonstrated that virulence can be maintained or recovered after resuscitation.

#### **7. Role of food in induction of VBNC state**

Chemophysical characteristics of food select the bacteria able to colonize and survive.

Such characteristics, defined as **intrinsic factors**, are pH, redox potential (or oxygen concentration) and aw. However, other factors, that have to be considered, will be also discussed.

#### **7.1 pH**

Although optimal bacterial growth requires a pH near neutrality, it has been demonstrated that foodborne pathogens can trigger the acid shock response for survival in acidic environments.

Then, acidic food may be an environment inducing the VBNC state and risk becomes higher for food, as fresh juices and salads, not subjected to antimicrobial treatments.

#### **7.2 Redox potential (or oxygen concentration)**

Food redox potential is a factor which influences the development of bacteria. Vegetable food has a redox potential ranging from +300 and -400 mV, which favours aerobes and eukaryotic microrganisms (yeasts and moulds). Meat has a value of -200 mV, which allows persistence of microaerobes and anaerobes bacteria.

Oxygen influences food redox potential, so its concentration is critical for bacterial growth. Aerobe bacteria need oxygen for growth, whereas anaerobes use compounds other than oxygen, such as nitrate, nitrite, sulphate, sulphite, etc. Some anaerobes, defined **obligate**, have been considered as totally inhibited by presence of oxygen for a long time, in that were believed to be unable to face the oxidative damage caused by Reactive Oxygen Species (ROS). Nowadays, it has been demonstrated that *Clostridium* may withstand oxidative stress, in that genome sequencing revealed the presence of genes related to oxygen metabolism (Kawasaki et al., 2005).

Also oxidative stress may induce VBNC state, as demonstrated in *Vibrio* sp. (McDougald & Kjelleberg, 2006).

#### **7.3 Water activity (aw)**

The aw is a measure of free water available for microbial growth and its value ranges from 1.00 of mineral water to 0.60 of bakery products, candies and dried fruit. A range of aw around 0.995 to 0.998 allows the growth of most of bacteria. The use of salting as ancient conservation technique for meat and vegetables consists, ultimately, in bringing down aw to reduce microbial development.

As already stated, bacteria may face osmotic shock; moreover, high osmolarity may induce VBNC state (Asakura et al., 2008).

#### **7.4 Chemical composition**

202 Public Health – Methodology, Environmental and Systems Issues

proportion to the length of time that the cells were in the VBNC state. The cells retained virulence, however, and even when fully nonculturable, were able to cause fatal infections, with resuscitation occurring within the mouse. Continued virulence for a variety of

Then, it can be concluded that VBNC forms of foodborne pathogen bacteria into a human or animal host may be a realistic threat for public health, because it has been demonstrated that

Such characteristics, defined as **intrinsic factors**, are pH, redox potential (or oxygen concentration) and aw. However, other factors, that have to be considered, will be also

Although optimal bacterial growth requires a pH near neutrality, it has been demonstrated that foodborne pathogens can trigger the acid shock response for survival in acidic

Then, acidic food may be an environment inducing the VBNC state and risk becomes higher

Food redox potential is a factor which influences the development of bacteria. Vegetable food has a redox potential ranging from +300 and -400 mV, which favours aerobes and eukaryotic microrganisms (yeasts and moulds). Meat has a value of -200 mV, which allows

Oxygen influences food redox potential, so its concentration is critical for bacterial growth. Aerobe bacteria need oxygen for growth, whereas anaerobes use compounds other than oxygen, such as nitrate, nitrite, sulphate, sulphite, etc. Some anaerobes, defined **obligate**, have been considered as totally inhibited by presence of oxygen for a long time, in that were believed to be unable to face the oxidative damage caused by Reactive Oxygen Species (ROS). Nowadays, it has been demonstrated that *Clostridium* may withstand oxidative stress, in that genome sequencing revealed the presence of genes related to oxygen

Also oxidative stress may induce VBNC state, as demonstrated in *Vibrio* sp. (McDougald &

The aw is a measure of free water available for microbial growth and its value ranges from 1.00 of mineral water to 0.60 of bakery products, candies and dried fruit. A range of aw around 0.995 to 0.998 allows the growth of most of bacteria. The use of salting as ancient

for food, as fresh juices and salads, not subjected to antimicrobial treatments.

Chemophysical characteristics of food select the bacteria able to colonize and survive.

pathogenic vibrios has also been demonstrated (Baffone et al., 2003).

virulence can be maintained or recovered after resuscitation.

**7. Role of food in induction of VBNC state** 

**7.2 Redox potential (or oxygen concentration)** 

persistence of microaerobes and anaerobes bacteria.

metabolism (Kawasaki et al., 2005).

Kjelleberg, 2006).

**7.3 Water activity (aw)** 

discussed.

environments.

**7.1 pH** 

Biological macromolecules are used as substrates for growth, but bacteria may use only a limited part of the macromolecular content. Moreover, several compounds, such as polyphenols of fruit and vegetables and organic acids, display bacteriostatic and bactericide activities, which are further stress-inducing factors.

#### **7.5 Feedstock treatment**

Decontamination procedures, such as pasteurisation, salting and acidification, can induce VBNC state (Gunasekera et al., 2002; Makino et al., 2000; Quirós et al., 2009). Moreover, untreated food is more susceptible to microbial contamination (Erickson et al., 2010).

#### **7.6 Food storage**

Before consumption, food is stored during packaging, distribution and commercialization. Time and temperature are critical factors which are related to bacterial stress response and induction of VBNC state (Besnard et al., 2002).

#### **7.7 Presence of endogenous microflora**

Feedstock and untreated food possess a resident microflora, not necessarily hazardous for human health. Such microflora may interact with contaminating bacterial pathogens by intercellular exchange of signalling molecules, as the **autoinducers**, which may activate secretion of virulence factors or resuscitation in VBNC bacteria (Reissbrodt et al., 2002).

#### **8. VBNC state of foodborne bacteria – The new challenge in food safety**

Despite the few studies, strong evidences of VBNC bacteria in food have been reported. In stored wine, for example, acetic acid and lactic acid bacteria entered VBNC state as consequence of lack of oxygen and presence of sulphites, respectively (Millet and Lonvaud-Funel, 2000).

The role played by chemo-physical characteristics of food in triggering VBNC state in foodborne pathogen bacteria is poorly undestood. The most of the studies consists in analysing the effects provoked by a single stress on a homogeneous population of bacteria.

However, it has to be considered that, in their habitat, bacteria are continuously subjected to the simultaneous action of factors, as nutrient availability, pH, osmolarity, temperature, presence of toxic compounds, ecological competition with other organisms, which are continuously changing. The time and the entity of the change of a single factor influences, and in turn is influenced by, changes and entities of other factors, according to chaos dynamics observed in nature, in an unpredictable way.

Viable but Nonculturable Bacteria in Food 205

known as **tetrazolium salts**, as 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and 2-(4-

In recent years, a new differential staining assay, the BacLight® Live/Dead assay, has been developed. The assay allows to simultaneously count total and viable (metabolically active) cells, by using two nucleic acid stains, that is green-fluorescent SYTO® 9 stain and redfluorescent propidium iodide stain. These stains differ in their ability to penetrate intact cell membranes. When used alone, SYTO® 9 stain labels both live and dead bacteria. In contrast, propidium iodide penetrates only bacteria with damaged membranes, reducing SYTO® 9 fluorescence when both dyes are present. Thus, live bacteria with intact membranes

CTC, INT and BacLight® Live/Dead assay are commonly used for the Direct Viable Count (DVC), by visualization of VBNC bacteria under fluorescence microscopy, one of the most

When DVC values are higher than culturable cells, obtained by CFU assay (that is when cell viability is higher than culturability), then it is assumed that bacteria have entered VBNC

Fluorescent antibodies have also been employed in DVC for VBNC detection of *E. coli* in recreational water (Zimmerman et al., 2009). In such technique, the sample is incubated in presence of yeast extract and nalidixic acid, which inhibits bacterial replication. Then, living cells increase their biomass, but cannot duplicate, so they elongate or enlarge. After addition of fluorescent antibody, fluorescing cells with altered morphology can be counted with epifluorescence microscopy. The difference between fluorescing cells, obtained by DVC, and

DNA hybridization is based on the identification of nucleic acid sequences that are specific for a given species, such as those present in ribosomes (16S and 23S rRNAs), by using a

In **fluorescence in situ hybridization** (FISH), the hybridization between probe and nucleic acid sequence can be visualized in epifluorescence microscopy and used for DVC after incubation of the sample in a medium containing an antibiotic which prevents bacterial division. Comparison between DVC and culture count by miniaturized most-probable number (MPN) gives information about the presence of VBNC cells (Garcia-Armisen, 2004;

**Flow cytometry** has been successfully employed to gather information about cell viability, antigenic surface components, and the quantification of morphological variations of *V.* 

**mRNA quantization** is performed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Such technique is derived from the classic polymerase chain reaction

Dunaev et al. (2008) recently reported on the rapid and accurate quantification of VBNC pathogens in biosolids via monitoring and quantifying stress-related genes in *Salmonella* spp. using cDNA microarrays combined with qRT-PCR. Quantification of mRNA was

*parahaemolyticus* during entry into the VBNC state (Falcioni et al., 2008).

(PCR), which allows the synthesis of huge quantities of a DNA sequence.

culturable cells, obtained by CFU assay, indicates the presence of VBNC bacteria.

fluorescent known DNA sequence, called **probe**.

correlated to cell viability and their ability to grow.

iodophenyl)-3-(4-nitrophenyl)-5-phenyl tetrazolium chloride (INT).

fluoresce green, while dead bacteria with damaged membranes fluoresce red.

widespread techniques for assessing bacterial viability (Rowan, 2011).

state.

Servais et al., 2009).

In the same way, food and its surrounding environment have to be considered as a complex system, in which the chemo-physical characteristics (pH, aw, chemical composition) and environmental factors (storage temperature and time, decontamination treatments, packaging under modified atmosphere) act simultaneously on contaminating bacteria.

It has been demonstrated (Nicolò et al., 2011) that refrigerated pasteurised grapefruit juice induces VBNC state in *E. coli* O157:H7 and *S.* Typhimurium within 24 hours of incubation.

Grapefruit has a very acidic pH, low content in carbohydrates and several antimicrobial compounds. Such characteristics, generally described as factors inducing VBNC state in laboratory, together with the refrigeration used for storage, suggested the hypothesis that grapefruit juice could induce VBNC state in foodborne pathogens.

On the contrary, grape juice, which differs from grapefruit juice for the higher content in carbohydrates, did not induce VBNC state (Nicolò et al., unpublished data), despite the acidic pH and refrigeration temperature.

Therefore, the role of food in induction of VBNC state has to be elucidated. Predictive models offered by biomathematics and bioinformatics would be very helpful tools, in order to evaluate the possibility that, under certain conditions, pathogen bacteria contaminating a tipology of food may enter the VBNC state.

In pasteurised milk, de novo expression of a *gfp* reporter gene has been demonstrated to be higher than culturable cells for both *E. coli* and *Pseudomonas putida*, so showing that, after thermal treatment, contaminant bacteria had lost the ability to form colonies, but retained transcription and translation machineries (Gunasekera et al., 2002).

A study on dried salted squid contaminated by *Salmonella enterica* subsp. *enterica* Oranienburg, responsible for an outbreak in Japan in 1999, has showed the inefficacy of cultural methods for detection of VBNC bacteria. In such a study, less than 20 culturable cells were recovered by plating a sample of salted squid, a value that cannot support the septicaemia observed in patients, but BacLight assay showed that more than 90% of the population was viable. In such a way, the hypothesis that an outbreak of foodborne bacteria in VBNC state could be underestimated on the basis of culturable cells has been demonstrated (Asakura et al., 2002).

In another outbreak in Japan, due to contamination of salted salmon roe by *E. coli* O157 in VBNC state, the authors demonstrated the resuscitation *in vivo* of VBNC bacteria and the maintaining of virulence in mice (Makino et al., 2000).

Such studies demonstrate that also treatments for food preservation have to be considered as a possible factor inducing pathogen bacteria into VBNC state. On the basis of such observations, the role of preservation treatments has to be investigated, in order to identify the critical points of a given procedure and make the adequate corrections.

#### **9. Detection of VBNC bacteria**

Actually, several systems for VBNC detection in water environments have been set up. Such systems are based on fluorescent staining, DNA hybridization and mRNA quantization.

Many fluorescent stains are used as indicators of metabolic activity (or viability), because they can accept the electrons flowing through cell respiratory chains. The most common are

In the same way, food and its surrounding environment have to be considered as a complex system, in which the chemo-physical characteristics (pH, aw, chemical composition) and environmental factors (storage temperature and time, decontamination treatments, packaging under modified atmosphere) act simultaneously on contaminating bacteria.

It has been demonstrated (Nicolò et al., 2011) that refrigerated pasteurised grapefruit juice induces VBNC state in *E. coli* O157:H7 and *S.* Typhimurium within 24 hours of incubation. Grapefruit has a very acidic pH, low content in carbohydrates and several antimicrobial compounds. Such characteristics, generally described as factors inducing VBNC state in laboratory, together with the refrigeration used for storage, suggested the hypothesis that

On the contrary, grape juice, which differs from grapefruit juice for the higher content in carbohydrates, did not induce VBNC state (Nicolò et al., unpublished data), despite the

Therefore, the role of food in induction of VBNC state has to be elucidated. Predictive models offered by biomathematics and bioinformatics would be very helpful tools, in order to evaluate the possibility that, under certain conditions, pathogen bacteria contaminating a

In pasteurised milk, de novo expression of a *gfp* reporter gene has been demonstrated to be higher than culturable cells for both *E. coli* and *Pseudomonas putida*, so showing that, after thermal treatment, contaminant bacteria had lost the ability to form colonies, but retained

A study on dried salted squid contaminated by *Salmonella enterica* subsp. *enterica* Oranienburg, responsible for an outbreak in Japan in 1999, has showed the inefficacy of cultural methods for detection of VBNC bacteria. In such a study, less than 20 culturable cells were recovered by plating a sample of salted squid, a value that cannot support the septicaemia observed in patients, but BacLight assay showed that more than 90% of the population was viable. In such a way, the hypothesis that an outbreak of foodborne bacteria in VBNC state could be underestimated on the basis of culturable cells has been demonstrated (Asakura et al., 2002). In another outbreak in Japan, due to contamination of salted salmon roe by *E. coli* O157 in VBNC state, the authors demonstrated the resuscitation *in vivo* of VBNC bacteria and the

Such studies demonstrate that also treatments for food preservation have to be considered as a possible factor inducing pathogen bacteria into VBNC state. On the basis of such observations, the role of preservation treatments has to be investigated, in order to identify

Actually, several systems for VBNC detection in water environments have been set up. Such systems are based on fluorescent staining, DNA hybridization and mRNA quantization.

Many fluorescent stains are used as indicators of metabolic activity (or viability), because they can accept the electrons flowing through cell respiratory chains. The most common are

the critical points of a given procedure and make the adequate corrections.

grapefruit juice could induce VBNC state in foodborne pathogens.

transcription and translation machineries (Gunasekera et al., 2002).

maintaining of virulence in mice (Makino et al., 2000).

**9. Detection of VBNC bacteria** 

acidic pH and refrigeration temperature.

tipology of food may enter the VBNC state.

known as **tetrazolium salts**, as 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and 2-(4 iodophenyl)-3-(4-nitrophenyl)-5-phenyl tetrazolium chloride (INT).

In recent years, a new differential staining assay, the BacLight® Live/Dead assay, has been developed. The assay allows to simultaneously count total and viable (metabolically active) cells, by using two nucleic acid stains, that is green-fluorescent SYTO® 9 stain and redfluorescent propidium iodide stain. These stains differ in their ability to penetrate intact cell membranes. When used alone, SYTO® 9 stain labels both live and dead bacteria. In contrast, propidium iodide penetrates only bacteria with damaged membranes, reducing SYTO® 9 fluorescence when both dyes are present. Thus, live bacteria with intact membranes fluoresce green, while dead bacteria with damaged membranes fluoresce red.

CTC, INT and BacLight® Live/Dead assay are commonly used for the Direct Viable Count (DVC), by visualization of VBNC bacteria under fluorescence microscopy, one of the most widespread techniques for assessing bacterial viability (Rowan, 2011).

When DVC values are higher than culturable cells, obtained by CFU assay (that is when cell viability is higher than culturability), then it is assumed that bacteria have entered VBNC state.

Fluorescent antibodies have also been employed in DVC for VBNC detection of *E. coli* in recreational water (Zimmerman et al., 2009). In such technique, the sample is incubated in presence of yeast extract and nalidixic acid, which inhibits bacterial replication. Then, living cells increase their biomass, but cannot duplicate, so they elongate or enlarge. After addition of fluorescent antibody, fluorescing cells with altered morphology can be counted with epifluorescence microscopy. The difference between fluorescing cells, obtained by DVC, and culturable cells, obtained by CFU assay, indicates the presence of VBNC bacteria.

DNA hybridization is based on the identification of nucleic acid sequences that are specific for a given species, such as those present in ribosomes (16S and 23S rRNAs), by using a fluorescent known DNA sequence, called **probe**.

In **fluorescence in situ hybridization** (FISH), the hybridization between probe and nucleic acid sequence can be visualized in epifluorescence microscopy and used for DVC after incubation of the sample in a medium containing an antibiotic which prevents bacterial division. Comparison between DVC and culture count by miniaturized most-probable number (MPN) gives information about the presence of VBNC cells (Garcia-Armisen, 2004; Servais et al., 2009).

**Flow cytometry** has been successfully employed to gather information about cell viability, antigenic surface components, and the quantification of morphological variations of *V. parahaemolyticus* during entry into the VBNC state (Falcioni et al., 2008).

**mRNA quantization** is performed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Such technique is derived from the classic polymerase chain reaction (PCR), which allows the synthesis of huge quantities of a DNA sequence.

Dunaev et al. (2008) recently reported on the rapid and accurate quantification of VBNC pathogens in biosolids via monitoring and quantifying stress-related genes in *Salmonella* spp. using cDNA microarrays combined with qRT-PCR. Quantification of mRNA was correlated to cell viability and their ability to grow.

Viable but Nonculturable Bacteria in Food 207

As consequence of the false information, Spanish exporters lost more than 200 millions of Euro per week. Political tension between Spain and Germany exploded on behalf of the European summit in Debrecen, Hungary, where the Spanish Minister of Agriculture publicly accused the German government of irresponsible behaviour. Spain, in fact, is the first world producer of cucumbers and more than 90% of its agricultural produce is exported in Europe. The destruction of vegetables and fruit had dramatic economic losses,

Moreover, Russia banned the import of fresh vegetable from Europe, with economic

Another cause of diplomatic tension was a joint risk-assessment by European Food Safety Agency (EFSA)/European Centre for Disease Prevention and Control (ECDC), which identified a link between the German outbreak and a simultaneous haemolytic urea syndrome outbreak in France, caused by the same *E. coli* strain. The assessment indicated fenugreek seeds imported in Europe from Egypt as a possible source of contamination, even if in the same document it was stated that such hypothesis had to be truly demonstrated. Such behaviour induced the Egyptian Minister of Agriculture to comment such opinions as

Several critical points about the German outbreak management emerge. First of all, standard procedures for the fast and unambiguous pathogen(s) identification are required, in order to alert health care points and allow them to plan the best response for

Then, a real-time communication between health care points and national health authorities

Health authorities have to communicate with politics, to plan the best way to inform people

Further, communication among health authorities of the countries involved in the outbreak

Communication, then, is the keystone for the best management of an outbreak. Appropriate procedures have to be established, with the creation of multilevel joint committees, formed by health and political authorities, which have to control quality and completeness of

The importance of communication has been matter of the WHO Expert Consultation on Outbreak Communications held in Singapore on 2004. As result, a manual containing the

Surveillance of foodborne disease is a fundamental component of food safety systems and data are used for planning, implementing and evaluating public health policies. There is therefore a strong need to strengthen such systems, particularly for establishing whether

has to be promoted, to efficiently identify the causes of the outbreak and track it.

available information and manage subsequent communication to public opinion.

guidelines to be followed has been published and is freely available (WHO, 2005).

**11. International programs devoted to monitoring and tracking of foodborne** 

because at that time Spain was already heavily hit by international financial crisis.

consequences which impacted on all the countries of European Union.

is needed, for continuous monitoring of the outbreak.

about the situation, giving appropriate information and warnings.

VBNC foodborne pathogens may be responsible for an outbreak.

"sheer lies".

**pathogens** 

people assistance.

Other techniques, such as microradiography, have been proposed for VBNC identification, but they resulted to be time-consuming and quite expensive.

The above described detection methods, used in water environment, are not part of routinary food safety procedures, in that they are not simple enough, the equipment is too expensive and specialised technical personnel is necessary.

Moreover, it has to be considered that food could be quite difficult to investigate, because of the presence of heterogeneous compounds that could interfere with the molecular reaction required by a single detection technique.

However, new methods for VBNC detection should be designed, in terms of rapidity, sensitivity and ease of use.

#### **10. Social and economical consequences of an outbreak**

In any outbreak, beyond the health aspect, several social and economic implications are strictly associated to the emergence of a foodborne disease.

20% of illnesses are referable to known pathogens, but the remaining 80% is due to unspecified agents, intended as known agents not yet identified as cause of foodborne illness, agents known to be in food whose pathogenicity is not proven and unidentified agents.

As consequence, their traceability by traditional food safety methods is difficult.

In this regard, VBNC role in foodborne outbreaks has still to be defined.

In fact, the appearance of an outbreak has a critical effect on public opinion, generating fears that often become panic. The difficulty of a correct communication among health authorities, politics and people has been matter of study by World Health Organization, which published a manual containing the guidelines to overcome such problem.

Moreover, the difficulty to identify in a timely fashion the primary causes of the outbreak has a negative impact at economic level, because any foodstuff suspected to be infected is removed from the market and destroyed, with consequent economic losses.

The management of the recent outbreak of *E. coli* O104:H4 in Germany can be considered as a good source for several considerations.

At the onset of the outbreak, the identification of the aetiological agent was erroneous and only later it was demonstrated that the true cause was an enteroaggregative *E. coli strain,*  able to synthesize *Shiga* toxins.

The source of infection was sprouted food produced by an organic farm in Bienenbüttel, Lower Saxony, Germany; local laboratories failed in finding the pathogen and correct identification was achieved later by a laboratory in North Rine-Westphalia.

Before the test results, German health authorities erroneously declared that the pathogen was present in cucumbers imported from Spain. Later, they admitted that the *E. coli* strain responsible of the outbreak was not found in cucumber samples they had analysed.

Other techniques, such as microradiography, have been proposed for VBNC identification,

The above described detection methods, used in water environment, are not part of routinary food safety procedures, in that they are not simple enough, the equipment is too

Moreover, it has to be considered that food could be quite difficult to investigate, because of the presence of heterogeneous compounds that could interfere with the molecular reaction

However, new methods for VBNC detection should be designed, in terms of rapidity,

In any outbreak, beyond the health aspect, several social and economic implications are

20% of illnesses are referable to known pathogens, but the remaining 80% is due to unspecified agents, intended as known agents not yet identified as cause of foodborne illness, agents known to be in food whose pathogenicity is not proven and unidentified

In fact, the appearance of an outbreak has a critical effect on public opinion, generating fears that often become panic. The difficulty of a correct communication among health authorities, politics and people has been matter of study by World Health Organization,

Moreover, the difficulty to identify in a timely fashion the primary causes of the outbreak has a negative impact at economic level, because any foodstuff suspected to be infected is

The management of the recent outbreak of *E. coli* O104:H4 in Germany can be considered as

At the onset of the outbreak, the identification of the aetiological agent was erroneous and only later it was demonstrated that the true cause was an enteroaggregative *E. coli strain,* 

The source of infection was sprouted food produced by an organic farm in Bienenbüttel, Lower Saxony, Germany; local laboratories failed in finding the pathogen and correct

Before the test results, German health authorities erroneously declared that the pathogen was present in cucumbers imported from Spain. Later, they admitted that the *E. coli* strain

responsible of the outbreak was not found in cucumber samples they had analysed.

As consequence, their traceability by traditional food safety methods is difficult.

which published a manual containing the guidelines to overcome such problem.

removed from the market and destroyed, with consequent economic losses.

identification was achieved later by a laboratory in North Rine-Westphalia.

In this regard, VBNC role in foodborne outbreaks has still to be defined.

but they resulted to be time-consuming and quite expensive.

expensive and specialised technical personnel is necessary.

**10. Social and economical consequences of an outbreak** 

strictly associated to the emergence of a foodborne disease.

required by a single detection technique.

a good source for several considerations.

able to synthesize *Shiga* toxins.

sensitivity and ease of use.

agents.

As consequence of the false information, Spanish exporters lost more than 200 millions of Euro per week. Political tension between Spain and Germany exploded on behalf of the European summit in Debrecen, Hungary, where the Spanish Minister of Agriculture publicly accused the German government of irresponsible behaviour. Spain, in fact, is the first world producer of cucumbers and more than 90% of its agricultural produce is exported in Europe. The destruction of vegetables and fruit had dramatic economic losses, because at that time Spain was already heavily hit by international financial crisis.

Moreover, Russia banned the import of fresh vegetable from Europe, with economic consequences which impacted on all the countries of European Union.

Another cause of diplomatic tension was a joint risk-assessment by European Food Safety Agency (EFSA)/European Centre for Disease Prevention and Control (ECDC), which identified a link between the German outbreak and a simultaneous haemolytic urea syndrome outbreak in France, caused by the same *E. coli* strain. The assessment indicated fenugreek seeds imported in Europe from Egypt as a possible source of contamination, even if in the same document it was stated that such hypothesis had to be truly demonstrated. Such behaviour induced the Egyptian Minister of Agriculture to comment such opinions as "sheer lies".

Several critical points about the German outbreak management emerge. First of all, standard procedures for the fast and unambiguous pathogen(s) identification are required, in order to alert health care points and allow them to plan the best response for people assistance.

Then, a real-time communication between health care points and national health authorities is needed, for continuous monitoring of the outbreak.

Health authorities have to communicate with politics, to plan the best way to inform people about the situation, giving appropriate information and warnings.

Further, communication among health authorities of the countries involved in the outbreak has to be promoted, to efficiently identify the causes of the outbreak and track it.

Communication, then, is the keystone for the best management of an outbreak. Appropriate procedures have to be established, with the creation of multilevel joint committees, formed by health and political authorities, which have to control quality and completeness of available information and manage subsequent communication to public opinion.

The importance of communication has been matter of the WHO Expert Consultation on Outbreak Communications held in Singapore on 2004. As result, a manual containing the guidelines to be followed has been published and is freely available (WHO, 2005).

#### **11. International programs devoted to monitoring and tracking of foodborne pathogens**

Surveillance of foodborne disease is a fundamental component of food safety systems and data are used for planning, implementing and evaluating public health policies. There is therefore a strong need to strengthen such systems, particularly for establishing whether VBNC foodborne pathogens may be responsible for an outbreak.

Viable but Nonculturable Bacteria in Food 209

One program will track processed foods, and the other will trace raw fruits and vegetables. The program will focus on keeping more detailed records of food and the path it makes as it

A new project has been established at Centers for Disease Control and Prevention (CDC), in collaboration with the Food and Drug Administration (FDA), which is called the Food-

The project consists of active surveillance for foodborne diseases and related epidemiologic studies designed to help public health officials better understand the epidemiology of

In the United States, using FoodNet data from 2000–2007, the Centers for Disease Control and Prevention estimated that 48 million people get sick, 127,839 were hospitalized and

Similarly, the European Food Safety Authority (EFSA) is the keystone of European Union (EU) risk assessment regarding food and feed safety and emerging risks, in close

Particularly, European food safety policy is to ensure a high level of protection of human health and consumers' interests in relation to food, taking into account diversity, including

Since 2000, the European Commission's guiding principle, primarily set out in its White Paper on Food Safety, is to apply an integrated approach from farm to table covering all

Moreover, the regional office for Europe of WHO supports countries in building capacity to manage food safety challenges in accordance with the WHO European Action Plan for Food and Nutrition Policy 2007–2012 and the WHO global strategy for food safety. The Action Plan is an important guide for policy-makers and health professionals that includes a wide

Despite the care by public health entities to track efficiently many known foodborne pathogens and identify the possibility of outbreaks, it is known that a consistent number of outbreaks is due to unknown pathogens. Such data may be due to microbes that are not proven to cause diseases as well as to VBNC state of known foodborne pathogen bacteria

Foodborne pathogens are the greatest threat to food safety. Despite the several efforts, much

The first action to be taken is the enhancement of measures to reduce or prevent contamination in the food and to educate stakeholders more effectively about risks and

Whereas in developed countries food safety procedures and surveillance network monitor continuously the insurgence of possible emergencies, the situation is drastically different in

traditional products, whilst ensuring the effective functioning of the internal market.

sectors of the food chain, from feed production to transport and retail sale.

travels across the country.

3,037 people died.

borne Disease Active Surveillance Network or FoodNet.

foodborne diseases in the United States.

collaboration with national authorities.

range of actions in the area of food safety.

remains to be done to reach the national health objectives.

developing countries, where many difficulties have to be considered.

that, up to date, are undetectable.

**12. Conclusions** 

prevention measures.

In this regard, the New Zealand Food Safety Authority has charged the Institute of Environmental Science and Research Limited to investigate the resuscitation of putative VBNC foodborne bacteria of significance to New Zealand. The final report concluded that some foodborne pathogen bacteria may become VBNC under certain conditions, that there may be no universal system for resuscitation of VBNC cells and that the phenomenon may be highly variable and bacterial species specific.

The difficulty in investigating the VBNC state and the related potential risk did not allow health authorities to establish guidelines for their detection.

Anyway, several efforts are carried out for surveillance of emerging foodborne diseases, creating new and interdisciplinary teams of research for data generation, collection and analysis.

In 2000, the Food and Agriculture Organization (FAO) of the United Nations and World Health Organization (WHO) started to expand their activities in the area of microbiological risk assessment to meet the increasing need for risk-based scientific advice and information and tools to undertake microbiological risk assessment. FAO and WHO coordinate their work in this area through the implementation of joint FAO/WHO meetings on microbiological risk assessment (JEMRA).

The activities of JEMRA can be categorised as follows:


Moreover, WHO is promoting many programs and projects, by the creation of several worldwide collaborations involving technical existing structures, to give support, information and instructions on how to face an incoming outbreak.

The Global Alert and Response (GAR) is an integrated system for epidemics and other public health emergencies based on strong national public health systems and capacity and an effective international system for coordinated response.

The Global Outbreak Alert and Response Network (GOARN) is a technical collaboration of existing institutions and networks that pool human and technical resources for the rapid identification, confirmation and response to outbreaks of international importance. The Network provides an operational framework to link this expertise and skill to keep the international community constantly alert to the threat of outbreaks and ready to respond.

In 2011, United States President Obama signed into law the FDA Food Safety Modernization Act (FSMA). It aims to ensure the U.S. food supply is safe by shifting the focus of federal regulators from responding to contamination to preventing it. As a mandate, the FDA will launch a test of two different programs that they hope will help with locating the source of food contamination more quickly.

In this regard, the New Zealand Food Safety Authority has charged the Institute of Environmental Science and Research Limited to investigate the resuscitation of putative VBNC foodborne bacteria of significance to New Zealand. The final report concluded that some foodborne pathogen bacteria may become VBNC under certain conditions, that there may be no universal system for resuscitation of VBNC cells and that the phenomenon may

The difficulty in investigating the VBNC state and the related potential risk did not allow

Anyway, several efforts are carried out for surveillance of emerging foodborne diseases, creating new and interdisciplinary teams of research for data generation, collection and

In 2000, the Food and Agriculture Organization (FAO) of the United Nations and World Health Organization (WHO) started to expand their activities in the area of microbiological risk assessment to meet the increasing need for risk-based scientific advice and information and tools to undertake microbiological risk assessment. FAO and WHO coordinate their work in this area through the implementation of joint FAO/WHO meetings on

Moreover, WHO is promoting many programs and projects, by the creation of several worldwide collaborations involving technical existing structures, to give support,

The Global Alert and Response (GAR) is an integrated system for epidemics and other public health emergencies based on strong national public health systems and capacity and

The Global Outbreak Alert and Response Network (GOARN) is a technical collaboration of existing institutions and networks that pool human and technical resources for the rapid identification, confirmation and response to outbreaks of international importance. The Network provides an operational framework to link this expertise and skill to keep the international community constantly alert to the threat of outbreaks and ready to

In 2011, United States President Obama signed into law the FDA Food Safety Modernization Act (FSMA). It aims to ensure the U.S. food supply is safe by shifting the focus of federal regulators from responding to contamination to preventing it. As a mandate, the FDA will launch a test of two different programs that they hope will help with locating the source of

be highly variable and bacterial species specific.

microbiological risk assessment (JEMRA).

• Elaboration of guideline documents • Data collection and generation

• Information and technology transfer

food contamination more quickly.

The activities of JEMRA can be categorised as follows:

• Generation of scientific information - risk assessments

• Use of risk assessment within a risk management framework

information and instructions on how to face an incoming outbreak.

an effective international system for coordinated response.

analysis.

respond.

health authorities to establish guidelines for their detection.

One program will track processed foods, and the other will trace raw fruits and vegetables. The program will focus on keeping more detailed records of food and the path it makes as it travels across the country.

A new project has been established at Centers for Disease Control and Prevention (CDC), in collaboration with the Food and Drug Administration (FDA), which is called the Foodborne Disease Active Surveillance Network or FoodNet.

The project consists of active surveillance for foodborne diseases and related epidemiologic studies designed to help public health officials better understand the epidemiology of foodborne diseases in the United States.

In the United States, using FoodNet data from 2000–2007, the Centers for Disease Control and Prevention estimated that 48 million people get sick, 127,839 were hospitalized and 3,037 people died.

Similarly, the European Food Safety Authority (EFSA) is the keystone of European Union (EU) risk assessment regarding food and feed safety and emerging risks, in close collaboration with national authorities.

Particularly, European food safety policy is to ensure a high level of protection of human health and consumers' interests in relation to food, taking into account diversity, including traditional products, whilst ensuring the effective functioning of the internal market.

Since 2000, the European Commission's guiding principle, primarily set out in its White Paper on Food Safety, is to apply an integrated approach from farm to table covering all sectors of the food chain, from feed production to transport and retail sale.

Moreover, the regional office for Europe of WHO supports countries in building capacity to manage food safety challenges in accordance with the WHO European Action Plan for Food and Nutrition Policy 2007–2012 and the WHO global strategy for food safety. The Action Plan is an important guide for policy-makers and health professionals that includes a wide range of actions in the area of food safety.

Despite the care by public health entities to track efficiently many known foodborne pathogens and identify the possibility of outbreaks, it is known that a consistent number of outbreaks is due to unknown pathogens. Such data may be due to microbes that are not proven to cause diseases as well as to VBNC state of known foodborne pathogen bacteria that, up to date, are undetectable.

#### **12. Conclusions**

Foodborne pathogens are the greatest threat to food safety. Despite the several efforts, much remains to be done to reach the national health objectives.

The first action to be taken is the enhancement of measures to reduce or prevent contamination in the food and to educate stakeholders more effectively about risks and prevention measures.

Whereas in developed countries food safety procedures and surveillance network monitor continuously the insurgence of possible emergencies, the situation is drastically different in developing countries, where many difficulties have to be considered.

Viable but Nonculturable Bacteria in Food 211

Asakura, H., Kawamoto, K., Haishima, Y., Igimi, S., Yamamoto, S. & Makino, S.I. (2008).

Baffone, W., Citterio, B., Vittoria, E., Casaroli, A., Campana, R., Falzano, L. & Donelli, G.

Berger, F., Morellet, N., Menu, F. and Potier, P. (1996). Cold Shock and Cold Acclimation

Bore, E., Langsrud, S., Langsrud, Ø., Rode, T.M. & Holck, A. (2007). Acid-shock Responses

Cappelier, J.M., Minet, J., Magras, C., Colwell, R.R. & Federichi, M. (1999b). Recovery in

Cappelier, J.M., Besnard, V., Roche, S., Garrec, N., Zundel, E., Velge, P. & Federighi, M.

Cappelier, J.M., Besnard, V., Roche, S.M., Velge, P. & Federighi, M. (2007). Avirulent Viable

Cook, K.L. & Bolster, C.H. (2007). Survival of *Campylobacter jejuni* and *Escherichia coli* in

*Microbiology*, Vol.103, No.3, (September 2007), pp. 573-583, ISSN 1364-5072 Csonka, L.N. (1989). Physiological and Genetic Responses of Bacteria to Osmotic Stress. *Microbiological Reviews*, Vol.53, No.1, (March 1989), pp. 121-147, ISSN 0146-0749

*Research*, Vol.38, No.4, (July-August 2007), pp. 573-583, ISSN 0928-4249 Colwell, R.R., Brayton, P.R., Herrington, D., Tall, B.D., Huq, A. & Levine M.M. (1996). Viable

*Microbiology*, Vol.153, No.7, (July 2007), pp. 2289-2303, ISSN 1350-0872 Cappelier, J.M., Magras, C., Jouve, J.L. & Federighi, M. (1999a). Recovery of Viable but

Vol.16, No.4, (August 1999), pp. 375-383, ISSN 0740-0020

*Bacteriology*, Vol.178, No.11, (June 1996), pp. 2999-3007, ISSN 0021-9193 Besnard, V., Federighi, M., Declerq, E., Jugiau, F. & Cappelier, J.M. (2002). Environmental

(November-December 2008), pp. 709-717, ISSN 0923-2508

February 1992), pp. 153-163, ISSN 0168-1605

589-599, (July-August 2005), ISSN 0928-4249

1996), pp. 28-31, ISSN 0959-3993

ISSN 0168-1605

0099-2240

Differential Expression of the Outer Membrane Protein W (OmpW) Stress Response in Enterohaemorrhagic *Escherichia coli* O157:H7 Corresponds to the Viable but Non-culturable State. *Research in Microbiology*, Vol.159, No.9-10,

(2003). Retention of Virulence in Viable but Nonculturable Halophilic *Vibrio* spp. *International Journal of Food Microbiology*, Vol.89, No.1, (December 2003), pp. 31-39,

Proteins in the Psychrotrophic Bacterium *Arthrobacter globiformis* SI55. *Journal of* 

and Physico-chemical Factors Induce VBNC State in *Listeria monocytogenes*. *Veterinary Research*, Vol.33, No.4, (July-August 2002), pp. 359-370, ISSN 0928-4249 Beumer, R.R., de Vries, J. & Rombouts, F.M. (1992). *Campylobacter jejuni* Non-culturable

Coccoid Cells. *International Journal of Food Microbiology*, Vol.15, No.1-2, (January-

in *Staphylococcus aureus* Investigated by Global Gene Expression Analysis.

Nonculturable *Campylobacter jejuni* Cells in Two Animal Models. *Food Microbiology,*

Embryonated Eggs of Viable but Nonculturable *Campylobacter jejuni* Cells and Maintenance of Ability to Adhere to HeLa Cells after Resuscitation. *Applied and Environmental Microbiology*, Vol.65, No.11, (November 1999), pp. 5154-5157, ISSN

(2005). Avirulence of Viable but Non-culturable *Listeria monocytogenes* Cells Demonstrated by in Vitro and in Vivo Models. *Veterinary Research*, Vol.36, No.4, pp.

but Non-culturable Cells of *Listeria monocytogenes* Need the Presence of an Embryo to be Recovered in Egg Yolk and Regain Virulence after Recovery. *Veterinary* 

but Nonculturable *Vibrio cholerae* O1 Revert to a Culturable State in Human Intestine. *World Journal of Microbiology and Biotechnology*, Vol.12, No.1, (January

Groundwater During Prolonged Starvation at Low Temperatures. *Journal of Applied* 

In general, the lack of people education is the first problem, in that social habits as well as cultural factors may favour the spread of a foodborne infection.

Then, people education is the keystone needed to strongly limit the raise and diffusion of an outbreak within a community.

Moreover, a continuous support, in terms of updating of political authorities and retraining of health personnel, could contribute to an efficient action by the local governments.

Another step for developing countries is the creation of a permanent educational system that allows people, since infancy to adult age, to be informed about foodborne infections, their causes and ways of spreading. In this way, such information will become part of the community culture and will be transmitted to the future generations.

Overall, it is important to encourage the local formation of specialised personnel in healthcare sector for medical care, research, monitoring and communication.

Furthermore, it would be also desirable that international health authorities could take into account the focusing of VBNC state of foodborne bacteria. In fact, despite the studies performed up to date, several topics have to be clarified, that is *i*) the risk that food may induce VBNC state in contaminating foodborne pathogens; *ii*) wheter VBNC state can be considered as a resistance strategy against stress or a transitory condition which precedes cell death; *iii*) the factors triggering the VBNC state; *iv*) the factors triggering the resuscitation and *v*) the maintaining of the virulence.

In fact, in food field it is very important to fit into a global frame the effects of the various food parameters, as chemophysical characteristics, decontamination procedures and storage conditions and time. All these factors, in fact, may be source of multiple and subsequent stresses for contaminating pathogen bacteria and evaluating the possibility of onset of VBNC state is a primary need. In such sense, bioinformatics and predictive mathematical models could be a powerful tool to identify whether a specific association of different stresses acting on a bacterial population may induce the entry into VBNC state.

Unfortunately, up to date detection of VBNC foodborne pathogen bacteria in food is problematic.

New methods have been proposed for VBNC detection, but they are not satisfying in that difficult to use as routinary procedures.

Therefore, innovative detection procedures, in terms of ease of use and rapidity of analysis, are urgently needed to allow an efficient monitoring and tracking of foodborne VBNC to prevent outbreaks in today's and future global market.

#### **13. References**

Asakura, H., Makino, S.I., Takagi, T., Kuri, A., Kurazano, T., Watarai M. & Shirahata, T. (2002). Passage in Mice Causes a Change in the Ability *of Salmonella enterica* Serovar Oranienburg to Survive NaCl Osmotic Stress: Resuscitation from the Viable but Non-culturable State. *FEMS Microbiology Letters*, Vol.212, No.1, (June 2002), pp. 87- 93, ISSN 0378-1097

In general, the lack of people education is the first problem, in that social habits as well as

Then, people education is the keystone needed to strongly limit the raise and diffusion of an

Moreover, a continuous support, in terms of updating of political authorities and retraining

Another step for developing countries is the creation of a permanent educational system that allows people, since infancy to adult age, to be informed about foodborne infections, their causes and ways of spreading. In this way, such information will become part of the

Overall, it is important to encourage the local formation of specialised personnel in

Furthermore, it would be also desirable that international health authorities could take into account the focusing of VBNC state of foodborne bacteria. In fact, despite the studies performed up to date, several topics have to be clarified, that is *i*) the risk that food may induce VBNC state in contaminating foodborne pathogens; *ii*) wheter VBNC state can be considered as a resistance strategy against stress or a transitory condition which precedes cell death; *iii*) the factors triggering the VBNC state; *iv*) the factors triggering the

In fact, in food field it is very important to fit into a global frame the effects of the various food parameters, as chemophysical characteristics, decontamination procedures and storage conditions and time. All these factors, in fact, may be source of multiple and subsequent stresses for contaminating pathogen bacteria and evaluating the possibility of onset of VBNC state is a primary need. In such sense, bioinformatics and predictive mathematical models could be a powerful tool to identify whether a specific association of different

Unfortunately, up to date detection of VBNC foodborne pathogen bacteria in food is

New methods have been proposed for VBNC detection, but they are not satisfying in that

Therefore, innovative detection procedures, in terms of ease of use and rapidity of analysis, are urgently needed to allow an efficient monitoring and tracking of foodborne VBNC to

Asakura, H., Makino, S.I., Takagi, T., Kuri, A., Kurazano, T., Watarai M. & Shirahata, T.

(2002). Passage in Mice Causes a Change in the Ability *of Salmonella enterica* Serovar Oranienburg to Survive NaCl Osmotic Stress: Resuscitation from the Viable but Non-culturable State. *FEMS Microbiology Letters*, Vol.212, No.1, (June 2002), pp. 87-

of health personnel, could contribute to an efficient action by the local governments.

cultural factors may favour the spread of a foodborne infection.

community culture and will be transmitted to the future generations.

resuscitation and *v*) the maintaining of the virulence.

difficult to use as routinary procedures.

93, ISSN 0378-1097

prevent outbreaks in today's and future global market.

healthcare sector for medical care, research, monitoring and communication.

stresses acting on a bacterial population may induce the entry into VBNC state.

outbreak within a community.

problematic.

**13. References** 


Viable but Nonculturable Bacteria in Food 213

Hett, E.C., Chao, M.C., Steyn, A.J., Fortune, S.M., Deng, L.L. & Rubin, E.J. (2007). A Partner

*Microbiology*, Vol.66, No.3, (November 2007), pp. 658-668, ISSN 0950-382X Jones, P.G. & Inouye, M. (1996). RbfA, a 30 S Ribosomal Binding Factor, is a Cold-Shock

Kana, B.D., Gordhan, B.G., Downing, K.J., Sung, N., Vostroktunova, G., Machowski, E.E.,

Kawasaki, S., Watamura, Y., Ono, M., Watanabe, T., Takeda, K. & Niimura, Y. (2005).

*Microbiology*, Vol.71, No.12, (December 2005), pp. 8442-8450, ISSN 0099-2240 Kelly, S.M., Pitcher, M.C.L., Farmery, S.M. & Gibson, G.R. (1994). Isolation of *Helicobacter* 

Lleò, M.M., Pierobon, S., Tafi, M.C., Signoretto, C. & Canepari, P. (2000). mRNA Detection

Lleò, M.M.; Bonato, B.; Tafi, M.C.; Signoretto, C.; Boaretti, M. & Canepari, P. (2001).

Loftis, A.D.; Priestley, R.A. & Massung, R.F. (2010). Detection of *Coxiella burnetii* in

Matin, A. (1991). The Molecular Basis of Carbon-Starvation-Induced General Resistance in

McDougald, D. & Kjelleberg, S. (2006). Adaptive Responses of Vibrios. In: *The biology of* 

*Disease*, Vol.7, No.12, (December 2010), pp. 1453-1456, ISSN 1535-3141 Makino, S.I.; Kii, T.; Asakura, H.; Shirahata, T.; Ikeda, T.; Takeshi, K. & Itoh, K. (2000). Does

Vol.21, No.6, (September 1996), pp. 1207-1218, ISSN 0950-382X

0099-2240

ISSN 0950-382X

pp. 1206-1211, ISSN 0099-2240

1095-1102, ISSN 1364-5072,

0950-382X

(October 2000), pp. 4564-4567, ISSN 0099-2240

(December 2001), pp. 5536-5539, ISSN 0099-2240

1555813658, Herndon, Virginia, United States

*and Environmental Microbiology*, Vol.68, No.4, (April 2002), pp. 1988-1993, ISSN

for the Resuscitation-promoting Factors of *Mycobacterium tuberculosis*. *Molecular* 

Protein Whose Absence Triggers the Cold-Shock Response. *Molecular Microbiology*,

Tsenova, L., Young, M., Kaprelyants, A., Kaplan, G. & Mizrahi, V.l. (2008). The Resuscitation-promoting Factors of *Mycobacterium tuberculosis* are Required for Virulence and Resuscitation from Dormancy but are Collectively Dispensable for Growth in Vitro. *Molecular Microbiology*, Vol.67, No.3, (February 2008), pp. 672-684,

Adaptive Responses to Oxygen Stress in Obligatory Anaerobes *Clostridium acetobutylicum* and *Clostridium aminovalericum*. *Applied and Environmental* 

*pylori* from Feces of Patients with Dyspepsia in the United Kingdom. *Gastroenterology*, Vol.107, No.6, (December 1994), pp. 1671-1674, ISSN 0016-5085 Kurath, G. & Morita, R.Y. (1983). Starvation-Survival Physiological Studies of a Marine

*Pseudomonas* sp. *Applied and Environmental Microbiology*, Vol.45, No.4, (April 1983),

by Reverse Transcription-PCR for Monitoring Viability over Time in an *Enterococcus faecalis* Viable but Nonculturable Population Maintained in a Laboratory Microcosm. *Applied and Environmental Microbiology*, Vol.66, No.10,

Resuscitation Rate in Different Enterococcal Species in the Viable but Nonculturable State. *Journal of Applied Microbiology*, Vol.91, No.6, (December 2001), pp.

Commercially Available Raw Milk from the United States. *Foodborne Pathogens and* 

Enterohaemorrhagic *Escherichia coli* O157:H7 Enter the Viable but Nonculturable State in Salted Salmon Roe? *Applied and Environmental Microbiology*, Vol.66, No.12,

*Escherichia coli*. *Molecular Microbiology*, Vol.5, No.1, (January 1991), pp. 3-10, ISSN

*vibrios*, F. L Thompson,. B. Austin & J. G. Swings (Eds.), 133-155, ASM Press, ISBN


Day, A.P. & Oliver, J.D. (2004). Changes in Membrane Fatty Acid Composition during Entry

Dunaev, T., Alanya, S. & Duran, M. (2008). Use of RNA-based Genotypic Approaches for

Eberl, L., Givskov, M., Sternberg, C., Møller, S., Christiansen, G. & Molin, S. (1996).

Falcioni, T., Papa, S., Campana, R., Manti, A., Battistelli, M. & Baffone, W. (2008). State

Federighi, M., Tholozan, J.L., Cappelier, J.M., Tissier, J.P. & Jouve, J.L. (1998). Evidence of

Freestone, P.P.E., Haigh, R.D., Williams, P.H. & Lyte, M. (1999). Stimulation of Bacterial

Gage, J. & Neidhardt, F.C. (1993). Modulation of the Heat Shock Response by One-carbon

Garcia-Armisen, T. & Servais, P. (2004). Combining Direct Viable Counts (DVC) and

Ghezzi, J.I. & Steck, T.R. (1999). Induction of the Viable but Nonculturable Conditions in

Gourmelon, M., Cillard, J. & Pommepuy, M. (1994) Visible Light Damage to *Escherichia coli*

Gunasekera, T.S., Sørensen, A., Attfield, P.V., Sørensen, S.J. & Veal, D.A. (2002). Inducible

*Letters*, Vol.172, No.1, (March 1999), pp. 53-60, ISSN 0378-1097

No.1, (January 1994), pp. 7-14, ISSN 0021-9193

No.1, (July 1994), pp. 105-112, ISSN 1364-5072

*Microbiology*, Vol.142, No.1, (January 1996), pp. 155-163, ISSN 1350-0872. Erickson, M.C., Webb, C.C., Diaz-Perez, J.C., Phatak, S.C., Silvoy, J.J., Davey, L., Payton,

*Microbiology*, Vol.42, No.2, (June 2004), pp. 69-73, ISSN 1225-8873

*and Technology*, Vol.58, No.9, (n.d.), pp. 1823-1828, ISSN 0273-1223

ISSN 0362-028X

ISSN 1552-4949

1961-1970 ISSN 0021-9193

275, ISSN 0273-1223

0020

of *Vibrio vulnificus* into the Viable but Nonculturable State. *The Journal of* 

Quantification of Viable but Nonculturable *Salmonella* sp. in Biosolids. *Water Science* 

Physiological Responses of *Pseudomonas putida* KT2442 to Phosphate Starvation.

A.S., Liao, J., Ma, L. & Doyle M.P. (2010). Surface and Internalized *Escherichia coli* O157:H7 on Field-Grown Spinach and Lettuce Treated with Spray-Contaminated Irrigation Water. *Journal of Food Protection*, Vol.73, No.6, (June 2010), pp. 1023-1029,

Transitions of *Vibrio parahaemolyticus* VBNC Cells Evaluated by Flow Cytometry*. Cytometry Part B (Clinical Cytometry)*, Vol.74, No.5, (September 2008), pp. 272-281,

Non-coccoid Viable but Non-culturable *Campylobacter jejuni* Cells in Microcosm Water by Direct Viable Count, CTC-DAPI Double Staining, and Scanning Electron Microscopy. *Food Microbiology*, Vol.15, No.5, (October 1998), pp. 539-550, ISSN 0740-

Growth by Heat-stable, Norepinephrine-induced Autoinducers. *FEMS Microbiology* 

Metabolism in *Escherichia coli*. *Journal of Bacteriology*, Vol.175, No.7, (April 1993), pp.

Fluorescent in Situ Hybridization (FISH) to Enumerate Viable *Escherichia coli* in Rivers and Wastewaters. *Water Science and Technology*, Vol.50, No.1, (n.d.), pp. 271-

*Xanthomonas campestris* pv. *campestris* in Liquid Microcosms and Sterile Soil. *FEMS Microbiology Ecology*, Vol.30, No.3, (November 1999), pp. 203-208, ISSN 0168-6496 Givskov, M., Eberl, L., Møller, S., Poulsen, L.K., Molin, S. (1994). Responses to Nutrient

Starvation in *Pseudomonas putida* KT2442: Analisys of General Cross-protection, Cell Shape, and Macromolecular Content. *Journal of Bacteriology*,. 1994 Vol.176,

in Seawater: Oxidative Stress Hypothesis. *Journal of Applied Microbiology*, Vol.77,

Gene Expression by Nonculturable Bacteria in Milk after Pasteurization. *Applied* 

*and Environmental Microbiology*, Vol.68, No.4, (April 2002), pp. 1988-1993, ISSN 0099-2240


Viable but Nonculturable Bacteria in Food 215

Reissbrodt, R., Rienaecker, I., Romanova, J.M., Freestone, P.P.E., Haigh, R.D., Lyte, M., Tschäpe,

Rockabrand, T.A., Arthur, T., Korinek, G., Livers, K. & Blum, P. (1995). An Essential Role for

Rowan, N.J. (2011). Defining Established and Emerging Microbial Risks in the Aquatic

Servais, P., Prats, J., Passerat, J. & Garcia-Armisen, T. (2009). Abundance of Culturable

Shleeva, M., Mukamolova, G.V., Young, M., Williams, H.D. & Kaprelyants, A.S. (2004).

Signoretto, C., Lleò, M.M., Tafi, M.C. & Canepari, P. (2000). Cell Wall Chemical Composition

Signoretto, C., Lleò, M.M. & Canepari, P. (2002). Modification of the Peptidoglycan of

Spence, J., Cegielska, A. & Georgopoulos, C. (1990). Role of *Escherichia coli* Heat Shock

Sun, F., Chen, J., Zhong, L., Zhang, X.-H., Wang, R., Guo, Q. & Dong, Y. (2008).

Terpeluk, C., Goldmann, A., Bartmann, P. & Pohlandt, F. (1992). *Plesiomonas shigelloides*

Thomas, J.E., Gibson, G., Darboe, M., Dale, A. & Weaver L.T. (1992). Isolation of *Helicobacter* 

*Sciences*, Vol.100, No.15, (July 2003), pp. 8951–8956, ISSN 0027-8424

No.9, (May 2009), pp. 2977-2981, ISSN 0099-2240

No.10, (October 2002), pp. 4788-4794. ISSN 0099-2240

*of Microbiology*, Vol.2011, (n.d.), pp. 1-15, ISSN 1687-918X

No.7, (July 2009), pp. 905-909, ISSN 0008-4166

No.2, (February 2002), pp. 125-131, ISSN 0343-8651

Vol.151, No.7, (July 1992), pp. 499-501, ISSN 0340-6199

WHO. (2005). WHO Outbreak Communication Guidelines. Available at:

news/IDdocs/whocds200528/whocds200528en.pdf.

0168-6496

1095, ISSN 0140-6736

www.who.int/infectious-disease-

1995), pp. 3695-3703, ISSN 0021-9193

Malolactic Fermentation Processes. *Applied and Environmental Microbiology* Vol.75,

H. & Williams, P.H. (2002). Resuscitation of *Salmonella enterica* Serovar Typhimurium and Enterohemorrhagic *Escherichia coli* from the Viable but Nonculturable State by Heat-stable Enterobacterial Autoinducer. *Applied and Environmental Microbiology*, Vol.68,

the *Escherichia coli* DnaK Protein in Starvation-induced Thermotolerance, H2O2 Resistance, and Reductive Division. *Journal of Bacteriology*, Vol.177, No.13, (July

Environment: Current Knowledge, Implications, and Outlooks. *International Journal* 

versus Viable *Escherichia coli* in Freshwater. *Canadian Journal of Microbiology*, Vol.55,

Formation of 'Non-culturable' Cells of *Mycobacterium smegmatis* in Stationary Phase in Response to Growth under Suboptimal Conditions and Their Rpf-mediated Resuscitation. *Microbiology*, Vol.150, No.6, (June 2004), pp. 1687-1697, ISSN 1350-0872

of *Enterococcus faecalis* in the Viable but Nonculturable State. *Applied and Environmental Microbiology*, Vol.66, No.5, (May 2000), pp. 1953-1959, ISSN 0099-2240

*Escherichia coli* in the Viable but Nonculturable State. *Current Microbiology*, Vol.44,

Proteins DnaK and HtpG (C62.5) in Response to Nutritional Deprivation. *Journal of Bacteriology*, Vol.172, No.12, (December 1990), pp. 7157-7166, ISSN 0021-9193 Sperandio, V., Torres, A.G., Jarvis, B., Nataro, J.P. & Kaper, J.B. (2003). Bacteria–host

Communication: the Language of Hormones. *Proceedings of the National Academy of* 

Characterization and Virulence Retention of Viable but Nonculturable *Vibrio harveyi*. FEMS Microbiology Ecology , Vol.64, No.1, (April 2008), pp. 37–44, ISSN

Sepsis and Meningoencephalitis in a Neonate. *European Journal of Pediatrics*.

*pylori* from Human Faeces. *Lancet*, Vol.340, No.8829, (November 1992), pp. 1094-


McGovern, V.P. & Oliver, J.D. (1995). Induction of Cold Responsive Proteins in *Vibrio* 

Millet, V., & Lonvaud-Funel, A. (2000). The viable but non-culturable state of wine

Morita, R.Y. (1982). Starvation-Survival of Heterotophs in the Marine Environment. *Advances in Microbial Ecology*, Vol.6, (n.d.), pp. 117-198, ISSN 0147-4863 Morton, D. & Oliver, J.D. (1994). Induction of Carbon Starvation Proteins in *Vibrio vulnificus*.

Mukamolova, G.V., Kaprelyants, A.S., Young D.I., Young, M. & Kell, D.B. (1998a). A

Mukamolova, G.V.; Yanopolskaya, N.D.; Kell, D.B. & Kaprelyants, A.S. (1998b). On

Leeuwenhoek, Vol.73, No.3, (Apr 1998), pp. 237-243, ISSN 0003-6012 Nakashima, K., Kanamaru, K., Mizuno, T. & Horikoshi, K. (1996). A Novel Member of the

*Bacteriology*, Vol.178, No.10, (May 1996), pp. 2994-2998, ISSN 0021-9193 Nesbakken, T. (2007). Pig Herds Free from Human Pathogenic *Yersinia enterocolitica*.

Nicolò, M.S., Gioffre`, A., Carnazza, S., Platania, G., Di Silvestro, I. and Guglielmino, S.P.P.

Nyström, T., Olsson, R.M. & Kjelleberg, S. (1992). Survival, Stress Resistance, and

Oliver, J.D. (2005). Viable but Nonculturable Bacteria in Food Environments. In: *Food Borne* 

Oliver, J.D. & Bockian, R. (1995). In Vivo Resuscitation, and Virulence Towards Mice, of

Quirós, C., Herrero, M., Garcia, L.A. & Diaz, M. (2009). Quantitative Approach to

*Microbiology*, Vol.61, No.7, (July 1995), pp. 2620-2623, ISSN 0099-2240 Porter, J., Edwards, C. & Pickup, R.W. (1995). Rapid Assessment of Physiological Sstatus in

*Microbiology*, Vol.58, No.1, (January 1992), pp. 55-65, ISSN 0099-2240 Oliver, J.D. (2000). Problems in Detecting Dormant (VBNC) Cells and the Role of DNA

(February 2000), pp.136-141, ISSN 0266-8254

1998), pp. 8916-8921, ISSN 0027-8424

0021-9193

1080-6059

pp. 11-17, ISSN 1535-3141

58706-009-0, Georgetown, Texas, USA.

No.4, (October 1995), pp. 399-408, ISSN 1364-5072

3659, ISSN 0099-2240

*vulnificus*. *Journal of Bacteriology*, Vol.177, No.14, (July 1995), pp. 4131-4133, ISSN

microorganisms during storage. *Letters in Applied Microbiology*, Vol.30, No.2,

*Applied and Environmental Microbiology*, Vol.60, No.10, (October 1994), pp. 3653-

bacterial cytokine. *Proceedings of National Academy of Sciences*, Vol.95, No.15, (July

resuscitation from the dormant state of *Micrococcus luteus*. Antonie Van

*cspA* Family of Genes that is Induced by Cold Shock in *Escherichia coli*. *Journal of* 

*Emerging Infectious Diseases*, Vol.13, No.12, (December 2007), pp. 1860-1864, ISSN

(2011) Viable but Nonculturable State of Foodborne Pathogens in Grapefruit Juice: a Study of Laboratory. *Foodborne Pathogens and Disease*, Vol.8, No.1, (January 2011),

Alterations in Protein Expression in the Marine *Vibrio* sp. Strain S14 during Starvation for Different Individual Nutrients. *Applied and Environmental* 

Elements in This Response, In: *Tracking Genetically-engineered Microorganisms*, J.K. Jansson, J.D. van Elsas, and M.J. Bailey (eds.), 1-15, Landes Biosciences, ISBN 978-1-

*Pathogens: Microbiology and Molecular Biology*, P.M. Fratamico & A.K. Bhunia (Eds.), 99-112, Horizon Scientific Press, ISBN: 978-1-898486-52-7, Norfolk, United Kingdom

Viable but Nonculturable Cells of *Vibrio vulnificus*. *Applied and Environmental* 

*Escherichia coli* Using Fluorescent Probes. *Journal of Applied Bacteriology*, Vol.79,

Determining the Contribution of Viable-butnonculturable Subpopulations of

Malolactic Fermentation Processes. *Applied and Environmental Microbiology* Vol.75, No.9, (May 2009), pp. 2977-2981, ISSN 0099-2240


**10** 

**Waste Minimization for the Safe Use of** 

Winslow (1920) has defined the meaning of public health as "the science and art of preventing disease, prolonging life and promoting health through the organized efforts and informed choices of society, organizations, public and private, communities and individuals". Thus, the focus of public health intervention is the improvement of health and quality of life through the prevention and treatment of disease, and promotion of healthy behaviors. Promotion of hand washing is a typical common public health practice to prevent

With the rapid increase on applications for nanoparticle silver, its potential impact on public health has become a critical issue. Nanosized silver can be made with different shapes such as particles, wires, and rods. Silver nanoparticles (AgNPs; many other names such as nanosilver (nAg) and colloidal silver) have already been used in everyday consumer

\* S-H. Hsu2, D. S. Lee3, Z. Iqbal4, S. Sund5, S. Curran6, C. Brumlik7, A. Choudhury7,

*8 Dept of Mat Sci and Eng, National Taiwan University Science and Technology, Taipei, Taiwan,* 

*11 Department of Materials Chemistry and Engineering, Konkuk University, Seoul, Korea.* 

 *Department of Physics, MTSE Program, New Jersey Institute of Technology, NJ, USA, Institute of Polymer Science and Engineering, National Taiwan University, Taipei, Taiwan, Department of Chemical Engineering, Chonbuk National University, Chonju, Korea, 4 Department of Chemistry and Environ Science, New Jersey Institute of Technology, NJ, USA,* 

*10 Ernest Mario School of Pharmacy, Rutgers University, Piscataway, NJ, USA,* 

the spread of "unwanted" diseases (Samuel et al., 2005).

D. S-G. Hu8, N. Chiu9, R. C. Lem10 and J. R. Haw11,#

*9 UMDNJ-New Jersey Medical School, Newark, NJ, USA,* 

*5 Nygard Consulting, LLC, NJ, USA, 6 Boston Scientific, MA, USA, 7 Nanobiz, LLC, NJ, USA,* 
