**Part 2**

**Genetic Modified Organisms in Today's Agriculture** 

52 Biochemical Testing

World Health Organization. (2007). The Global MDR-TB y XDR-TB Response Plan 2007-

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Yu M, chen H, Wu M, Huang L, Kuo Y, Yu F, and Jou R. (2011). Evaluation of the rapid

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### **Testing Methods for Agriculture and Food Safety**

Jose C. Jimenez-Lopez1\* and María C. Hernandez-Soriano2

*1Purdue University, Department of Biological Sciences, College of Science, West Lafayette, IN 2North Carolina State University, Department of Soil Science, College of Agriculture and Life Sciences, Raleigh, NC USA* 

#### **1. Introduction**

Sustainable agricultural production is a critical concern in response to global climate change and population increase (Brown and Funk, 2008; Turner et al., 2009). In addition, recent increased demand for Biofuel crops has created a new market for agricultural products. One potential solution is to increase plant yield by designing plants based on a molecular understanding of gene function and on the regulatory networks involved in stress tolerance, development and growth (Takeda & Matsuoka, 2008). Recent progress in plant genomics, as well as sequencing of the whole genome of new plant species have allowed to isolate important genes, discover new traits and to analyze functions that regulate yields and tolerance to environmental stress.

Recent remarkable innovations in platforms for omics-based research (genomic, proteomic, transcriptomic, metabolomic) and application development provide crucial resources to promote research in model and applied plant species. A combinatorial approach using multiple omics platforms and integration of their outcomes is now an effective strategy for clarifying molecular systems integral to improving plant productivity. Furthermore, support of comparative genomics and proteomis among model and applied plants allow to reveal the biological properties of each species and to accelerate gene discovery and functional analyses of genes. Bioinformatics platforms and their associated databases are also essential for the effective design of approaches making the best use of genomic resources, including resource integration.

Currently, many crop species can be considered important on a global scale for food security in which, have been developed large-scale genomic and genetic resources, i.e. array technology markers, expressed sequence tags or transcript reads, bacterial artificial chromosome libraries, genetic and physical maps, and germplasm stocks with rich genetic diversity, among others. These resources have the potential to accelerate gene discovery and initiate molecular breeding in these crops, thereby enhancing crop productivity to ensure food security in developing countries. In this line, and with all above molecular genetics

<sup>\*</sup> Corresponding Author

tools development, it has been possible that more and more plant crop species has been modified genetically obtaining desirable traits compared with wild plants.

Classical plant breeding uses deliberate interbreeding (crossing) of closely or distantly related individuals to produce new crop varieties or lines with desirable properties. Plants are crossbred to introduce traits/genes from one variety or line into a new genetic background. Progeny from the cross would then be crossed with the high-yielding parent to ensure that the progeny were most like the high-yielding parent, (backcrossing). The progeny from that cross would then be tested for desirable traits, i.e. resistance and highyielding resistant plants to be further developed. Plants may also be crossed with themselves to produce inbred varieties for breeding.

Classical breeding relies largely on homologous recombination between chromosomes to generate genetic diversity (Zhu et al., 2009). The classical plant breeder may also makes use of a number of in vitro techniques such as protoplast fusion, embryo rescue or mutagenesis to generate diversity and produce hybrid plants that would not exist in nature.

Traits that breeders have tried to incorporate into crop plants in the last 100 years include:

1) Increased quality and yield of the crop, 2) Increased tolerance of environmental pressures (salinity, extreme temperature, drought), 3) Resistance to viruses, fungi and bacteria, 4) Increased tolerance to insect pests, and 5) Increased tolerance of herbicides.

Modern plant breeding uses techniques of molecular biology to select, or in the case of genetic modification, to insert, desirable traits into plants (Bhatnagar-Mathur et al., 2008). Genetic modification of plants is achieved by adding a specific gene or genes to a plant, or by knocking out a gene with RNAi, to produce a desirable phenotype. Genetic modification can produce a plant with the desired trait or traits faster than classical breeding because the majority of the plant's genome is not altered. The use of tools such as molecular markers or DNA fingerprinting can map thousands of genes, allowing plant breeders to screen large populations of plants for those that possess the trait of interest. The screening is based on the presence or absence of a certain gene as determined by laboratory procedures, rather than on the visual identification of the expressed trait in the plant. The majority of commercially released transgenic plants are currently limited to plants that have introduced resistance to insect pests and herbicides. Insect resistance is achieved through incorporation of a gene from Bacillus thuringiensis (Bt) that encodes a protein that is toxic to some insects. For example, the cotton bollworm, a common cotton pest, feeds on Bt cotton it will ingest the toxin and die. Herbicides usually work by binding to certain plant enzymes and inhibiting their action. The enzymes that the herbicide inhibits are known as the herbicides target site. Herbicide resistance can be engineered into crops by expressing a version of target site protein that is not inhibited by the herbicide. This is the method used to produce glyphosate resistant crop plants (Pollegioni et al., 2011).

Testing for the presence of agricultural biotechnology products is being performed on many grain and food products. Currently, there is an absence of standardized tests to detect genetic modified (GM) crops, which can result in inaccurate claims and enforcement actions being taken without a means to challenge the results. Development of reliable, validated methods is necessary to avoid negative economic impacts due in invalid test results, as well as to ensure the safety of the consumer under arising of GM product in the markets. We also emphasize the need for such global compatibility of test results in order to facilitate international trade. A quality control of GM-crops product should be implemented through

tools development, it has been possible that more and more plant crop species has been

Classical plant breeding uses deliberate interbreeding (crossing) of closely or distantly related individuals to produce new crop varieties or lines with desirable properties. Plants are crossbred to introduce traits/genes from one variety or line into a new genetic background. Progeny from the cross would then be crossed with the high-yielding parent to ensure that the progeny were most like the high-yielding parent, (backcrossing). The progeny from that cross would then be tested for desirable traits, i.e. resistance and highyielding resistant plants to be further developed. Plants may also be crossed with

Classical breeding relies largely on homologous recombination between chromosomes to generate genetic diversity (Zhu et al., 2009). The classical plant breeder may also makes use of a number of in vitro techniques such as protoplast fusion, embryo rescue or mutagenesis

Traits that breeders have tried to incorporate into crop plants in the last 100 years include: 1) Increased quality and yield of the crop, 2) Increased tolerance of environmental pressures (salinity, extreme temperature, drought), 3) Resistance to viruses, fungi and bacteria, 4)

Modern plant breeding uses techniques of molecular biology to select, or in the case of genetic modification, to insert, desirable traits into plants (Bhatnagar-Mathur et al., 2008). Genetic modification of plants is achieved by adding a specific gene or genes to a plant, or by knocking out a gene with RNAi, to produce a desirable phenotype. Genetic modification can produce a plant with the desired trait or traits faster than classical breeding because the majority of the plant's genome is not altered. The use of tools such as molecular markers or DNA fingerprinting can map thousands of genes, allowing plant breeders to screen large populations of plants for those that possess the trait of interest. The screening is based on the presence or absence of a certain gene as determined by laboratory procedures, rather than on the visual identification of the expressed trait in the plant. The majority of commercially released transgenic plants are currently limited to plants that have introduced resistance to insect pests and herbicides. Insect resistance is achieved through incorporation of a gene from Bacillus thuringiensis (Bt) that encodes a protein that is toxic to some insects. For example, the cotton bollworm, a common cotton pest, feeds on Bt cotton it will ingest the toxin and die. Herbicides usually work by binding to certain plant enzymes and inhibiting their action. The enzymes that the herbicide inhibits are known as the herbicides target site. Herbicide resistance can be engineered into crops by expressing a version of target site protein that is not inhibited by the herbicide. This is the method used to produce

Testing for the presence of agricultural biotechnology products is being performed on many grain and food products. Currently, there is an absence of standardized tests to detect genetic modified (GM) crops, which can result in inaccurate claims and enforcement actions being taken without a means to challenge the results. Development of reliable, validated methods is necessary to avoid negative economic impacts due in invalid test results, as well as to ensure the safety of the consumer under arising of GM product in the markets. We also emphasize the need for such global compatibility of test results in order to facilitate international trade. A quality control of GM-crops product should be implemented through

to generate diversity and produce hybrid plants that would not exist in nature.

Increased tolerance to insect pests, and 5) Increased tolerance of herbicides.

modified genetically obtaining desirable traits compared with wild plants.

themselves to produce inbred varieties for breeding.

glyphosate resistant crop plants (Pollegioni et al., 2011).

self, supervisory, and peer review systems, which have to be organizationally independent of the testing staff or organization, performing internal test in accordance with the government requirements for.

Identification of GM product in the markets is another pending task, due that government from different countries are not in agreement about information that labelling product should contain. Authoritative, independent and public acceptable of green (eco-)/GM label scheme that identify products no-GM, in many case more environmentally desirable than other similar GM products with the same function in the market, is urged to be implemented with a general acceptance. Establishing compliance with GM food labeling laws is dependent on the availability of test methods capable of determining the presence and or concentration of GM ingredients in food or bulk consignments of agricultural commodities such as seed and grain.

Thus, current global regulatory requirements for labeling of products derived from plant biotechnology means that test methods, i.e. for the introduced trait(s) have to be developed and validated. Such methods require appropriate reference materials, controls and protocols in order to give accurate and precise identification and quantification of GM products.

#### **2. Genetic modified crops**

The term genetically-modified (GM) foods u organisms (GMOs) is most commonly used to refer to crop plants created for human or animal consumption, using molecular biology techniques. These plants have been modified in the laboratory to enhance desired traits such as increased resistance to herbicides or improved nutritional content. The enhancement of desired traits has traditionally been undertaken through breeding, but conventional plant breeding methods can be very time consuming and are often not very accurate.

During the past decade, a large number of genetically modified (GM) crops have been developed using methods of modern biotechnology. These GM or "biotech" crops exhibit unique agronomic traits such as herbicide tolerance or insect resistance, which offer significant benefits to farmers. The development of GM crops is accomplished by using molecular biology methods, essentially by the integration of novel DNA sequences into the plant genome. The new DNA encodes for the expression of the novel protein in the targeted tissue, resulting in the unique agronomic trait. The novel protein and DNA are present in many parts of the plant, in harvested grain, and often in the food fractions prepared from grain.

The production and global trade of genetically modified (GM) grain is increasing. At the same time, companies are required to provide validated diagnostic methods proving the inclusion of GM material, as a condition of the regulatory approval process in some jurisdictions. Numerous governmental agencies and industry organizations are attempting to develop standardization guidelines independently. Global harmonization of these efforts is necessary to ensure a consistent standard. An international coordination of detection methods for plant biotechnology products and the proper development of guidelines for their use are necessary and an awaiting mission.

#### **2.1 Detection methods for GM-Crops**

The detection of genetically modified organisms in food or feed is possible by using welldeveloped genetic and biochemical tools. It can either be qualitative, showing which genetically modified organism (GMO) is present, or quantitative, measuring in which amount a certain GMO is present. Being able to detect a GMO is an important part of food safety, as without detection methods the traceability of GMOs would rely solely on documentation.

#### **2.1.1 Methods based on DNA: polymerase chain reaction**

Methods for GMO should contain three analytical components of tests for detecting the presence of transgenic plant products: (1) detection, to screen for the presence of GM events in food and agricultural products; (2) identification, to reveal how many GM events are present and determine their molecular registers; and (3) quantification, to determine the amount of authorized GM product and compliance with threshold regulation. Analytical procedures for GM plants are directed to detect either the novel gene product or the gene construct itself.

Testing on GMOs in food and feed is routinely done by molecular techniques like DNA microarrays or qPCR, and the test can be based on screening elements (like p35S or terminator Nos) or specific markers for the official GMOs (like Bt11 or GT73).

Technologies involving amplification of gene fragments by the polymerase chain reaction (PCR) have the greatest potential for detecting transgenic plants and foodstuffs derived from them (Ahmed, 2002; Schmidt et al., 2008). PCR is a biochemistry and molecular biology technique for isolating and exponentially amplifying a fragment of DNA, via enzymatic replication, without using a living organism. It enables the detection of specific strands of DNA by making millions of copies of a target genetic sequence. Target sequences may be those of the marker gene, the promoter, the terminator, or the transgenes themselves. Confirmatory tests are essential to ensure authenticity of PCR product. The use of genomic fragments that include the border sequence at the insertion site and the inserted genes (edge or junction fragments) may be a better target for unequivocal identification of GM plant sources (Windels et al., 2001), particularly when detection is based on regulatory sequences in promoters and terminators that could occur in microbial contaminants.

Improving PCR based detection of GMOs is a further goal of different governmental research programme. Research is now underway to develop multiplex PCR methods that can simultaneously detect many different transgenic lines. Another major challenge is the increasing prevalence of transgenic crops with stacked traits. This refers to transgenic cultivars derived from crosses between transgenic parent lines, combining the transgenic traits of both parents.

Whether or not a GMO is present in a sample can be tested by qPCR, but also by multiplex PCR. Multiplex PCR uses multiple, unique primer sets within a single PCR reaction to produce amplicons of varying sizes specific to different DNA sequences. By targeting multiple genes at once, additional information may be gained from a single test run that otherwise would require several times the reagents and more time to perform. Furthermore, DNA array technology using chip platforms may also be a useful tool for determining the presence of insertional sequences. The possibility of testing against a large number of oligonucleotides representing various gene sequences will be particularly useful when the specific construct is unknown. To avoid any kind of false positive or false negative testing

genetically modified organism (GMO) is present, or quantitative, measuring in which amount a certain GMO is present. Being able to detect a GMO is an important part of food safety, as without detection methods the traceability of GMOs would rely solely on

Methods for GMO should contain three analytical components of tests for detecting the presence of transgenic plant products: (1) detection, to screen for the presence of GM events in food and agricultural products; (2) identification, to reveal how many GM events are present and determine their molecular registers; and (3) quantification, to determine the amount of authorized GM product and compliance with threshold regulation. Analytical procedures for GM plants are directed to detect either the novel gene product or the gene

Testing on GMOs in food and feed is routinely done by molecular techniques like DNA microarrays or qPCR, and the test can be based on screening elements (like p35S or

Technologies involving amplification of gene fragments by the polymerase chain reaction (PCR) have the greatest potential for detecting transgenic plants and foodstuffs derived from them (Ahmed, 2002; Schmidt et al., 2008). PCR is a biochemistry and molecular biology technique for isolating and exponentially amplifying a fragment of DNA, via enzymatic replication, without using a living organism. It enables the detection of specific strands of DNA by making millions of copies of a target genetic sequence. Target sequences may be those of the marker gene, the promoter, the terminator, or the transgenes themselves. Confirmatory tests are essential to ensure authenticity of PCR product. The use of genomic fragments that include the border sequence at the insertion site and the inserted genes (edge or junction fragments) may be a better target for unequivocal identification of GM plant sources (Windels et al., 2001), particularly when detection is based on regulatory sequences

Improving PCR based detection of GMOs is a further goal of different governmental research programme. Research is now underway to develop multiplex PCR methods that can simultaneously detect many different transgenic lines. Another major challenge is the increasing prevalence of transgenic crops with stacked traits. This refers to transgenic cultivars derived from crosses between transgenic parent lines, combining the transgenic

Whether or not a GMO is present in a sample can be tested by qPCR, but also by multiplex PCR. Multiplex PCR uses multiple, unique primer sets within a single PCR reaction to produce amplicons of varying sizes specific to different DNA sequences. By targeting multiple genes at once, additional information may be gained from a single test run that otherwise would require several times the reagents and more time to perform. Furthermore, DNA array technology using chip platforms may also be a useful tool for determining the presence of insertional sequences. The possibility of testing against a large number of oligonucleotides representing various gene sequences will be particularly useful when the specific construct is unknown. To avoid any kind of false positive or false negative testing

terminator Nos) or specific markers for the official GMOs (like Bt11 or GT73).

in promoters and terminators that could occur in microbial contaminants.

**2.1.1 Methods based on DNA: polymerase chain reaction** 

documentation.

construct itself.

traits of both parents.

outcome, comprehensive controls for every step of the process is mandatory. A CaMV check is important to avoid false positive outcomes based on virus contamination of the sample.

Sometime it is required to quantify GM product that contain food or directly in plants. For that purpose it is frequently used quantitative PCR (qPCR), to measure amounts of transgene DNA or PCR product in a food or feed sample, preferably real-time qRT-PCR (Ref.), with currently the highest level of accuracy. If the targeted genetic sequence is unique to a certain GMO, a positive PCR test proves that the GMO is present in the sample.

In addition, the array-based methods combine multiplex PCR and array technology to screen samples for different potential GMOs (Querci et al., 2009; Dorries et al., 2010) combining different approaches (screening elements, plant-specific markers, and eventspecific markers). Advanced PCR technologies, including competitive multiplex PCR and real-time PCR, are useful for quantifying the level of GM plant material in foodstuffs (Matsuoka et al., 2000).

Alternatively, there are methods to detect specific genetic construct, instead the specific genetic product. Since different GMOs may produce the same protein, construct-specific detection can test a sample for several GMOs in one step, but is unable to tell precisely, which of the similar GMOs are present.

Almost all transgenic plants contain a few common building blocks that make unknown GMOs easier to find. Even though detecting a novel gene in a GMO can be like finding a needle in a haystack, the fact that the needles are usually similar makes it much easier. Researchers now compile a set of genetic sequences characteristic of GMOs. After genetic elements characteristic of GMOs are selected, methods and tools are developed for detecting them in test samples. Approaches being considered include microarrays and anchor PCR profiling.

#### **2.1.2 Methods based on protein: immunoassay technology**

GM content can be determined by methods that detect either the novel protein or the inserted DNA. Detection of the novel proteins produced by GM crops relies almost exclusively on the application of immunoassay technology (Fantozzi et al., 2007). Commercial immunoassays are available for most of the GM crops on the market today and have been used in a variety of large-scale applications, determining GM content (%GM) ensuring compliance with non-GM labeling requirements, and confirming the presence of high-value commodities.

Immunoassays are based on the reaction of an antigen (Ag), e.g., transgenic protein, with a specific antibody (Ab) to give a product (Ag-Ab complex) that can be measured. There are many different immunoassay formats, and the choice of format is dependent on the target molecule and application. For macromolecules, the most commonly used test formats are enzyme-linked immunosorbent assay (ELISA) that can be used as either a qualitative or a quantitative assay, and lateral flow device (LFD) designed for qualitative yes/no testing.

ELISA based commercial kits are available for serological detection of selected GM gene products. ELISA is a comparatively easy and cost-effective procedure to apply to large numbers of samples, but specificity of antibodies is critical for an accurate test.

Two other test formats used for seed quality testing are Western blot and immunohistochemical staining.

LFDs are used for qualitative or semiquantitative detection of antigens. LFDs for the detection of GM proteins use antibodies in the same sandwich immunoassay format used in ELISA, except that the secondary antibody is labeled with a colored particle such as colloidal gold rather than an enzyme as a means of generating a visible signal.

Furthermore, the Western blot is primarily a qualitative analytical method and is particularly useful in protein characterization because it provides additional information regarding molecular weight. Immunohistochemical staining is used to determine the location of the expressed proteins in the plant.

The key component of immunoassay, antibody, have the atribute that makes it useful as a reagent in a diagnostic kit, being its capacity to bind specifically and with high affinity to the antigen that elicited its production. Polyclonal antibodies are relatively easy and inexpensive to prepare in a relatively short time frame (e.g., 3–4 months); however, the quality of the antibody reagent varies from animal to animal, and it is necessary to prepare large pools of qualified reagent to support long-term commercial production of uniform product. Monoclonal antibodies require greater time (e.g., 6 months) and skill to produce and are more expensive to develop than polyclonal antibodies. In applications where discrimination between very closely related molecules is required, it may be more advantageous to use a highly specific monoclonal antibody reagent. Conversely, in an application designed to detect all the members of a family of closely related molecules it may be more advantageous to use a polyclonal antibody reagent. The selection of one reagent type over another is dependent on the desired performance characteristics of the test method.

Another key component of an immunoassay is the antigen that can be defined as substances that induce a specific immune response resulting in production of antibodies. The interaction between antibody and antigen involves binding of the antigenic epitope to the complementarily determining region (CDR) of the antibody. The strength of binding between the 2 is referred to as the affinity of the bond. In general, the greater the affinity of the bond, the greater the sensitivity (lower limit of detection; LOD) of the test method. Sensitivity of a test method is determined not only by the affinity of the antibody for the antigen, but by factors such as protein expression level, extraction efficiency, and the size of the sample taken for analysis. In addition, an antibody binds only to the antigenic determinant that elicited its production. This specificity enables the development of test methods that require minimal sample preparation. Cross-reactivity can result in falsepositive responses over-estimation of antigen concentrations. Cross-reactivity of an antibody to a component of the sample or other GM crop is highly unlikely and almost never a significant issue.

The ideal antigen for immunization would be the actual GM protein as it is expressed in the plant. However, purification of the novel protein from plant tissue can be difficult and may result in undesirable modifications to the target protein. In addition, purification rarely results in 100% pure protein and immunization of animals with such preparations results not only in the production of antibodies to the target protein but to the contaminants as well. Polyclonal antibodies made from these preparations typically exhibit high background and poor sensitivity. A more common approach to making antibodies to GM proteins is to express and purify the protein of interest from an alternate host such as *E. coli* using genetic engineering techniques. Although the amino acid sequence of these recombinant proteins may be the same as the plant-produced protein, post-translational modification may be subtly different, and purification may result in modifications to the secondary and tertiary structure (e.g., denaturation). As long as antibodies that bind to the plant-produced protein with sufficient sensitivity and specificity can be isolated, then differences in structure between plant-produced and microbial-derived proteins are not an issue.

In certain instances where purified or recombinant antigens are not available or are exceedingly difficult to obtain, or where antibodies to very specific amino acids are desired, short peptides conjugated to carrier proteins may be used to develop antibodies. However, peptide antibodies may be more reactive to denatured forms of the protein and therefore often find better utility in Western blot (De Boer, 2003; Grothaus et al., 2006).

#### **2.1.3 Others methods**

60 Biochemical Testing

Two other test formats used for seed quality testing are Western blot and

LFDs are used for qualitative or semiquantitative detection of antigens. LFDs for the detection of GM proteins use antibodies in the same sandwich immunoassay format used in ELISA, except that the secondary antibody is labeled with a colored particle such as

Furthermore, the Western blot is primarily a qualitative analytical method and is particularly useful in protein characterization because it provides additional information regarding molecular weight. Immunohistochemical staining is used to determine the

The key component of immunoassay, antibody, have the atribute that makes it useful as a reagent in a diagnostic kit, being its capacity to bind specifically and with high affinity to the antigen that elicited its production. Polyclonal antibodies are relatively easy and inexpensive to prepare in a relatively short time frame (e.g., 3–4 months); however, the quality of the antibody reagent varies from animal to animal, and it is necessary to prepare large pools of qualified reagent to support long-term commercial production of uniform product. Monoclonal antibodies require greater time (e.g., 6 months) and skill to produce and are more expensive to develop than polyclonal antibodies. In applications where discrimination between very closely related molecules is required, it may be more advantageous to use a highly specific monoclonal antibody reagent. Conversely, in an application designed to detect all the members of a family of closely related molecules it may be more advantageous to use a polyclonal antibody reagent. The selection of one reagent type over another is dependent on the desired performance characteristics of the test

Another key component of an immunoassay is the antigen that can be defined as substances that induce a specific immune response resulting in production of antibodies. The interaction between antibody and antigen involves binding of the antigenic epitope to the complementarily determining region (CDR) of the antibody. The strength of binding between the 2 is referred to as the affinity of the bond. In general, the greater the affinity of the bond, the greater the sensitivity (lower limit of detection; LOD) of the test method. Sensitivity of a test method is determined not only by the affinity of the antibody for the antigen, but by factors such as protein expression level, extraction efficiency, and the size of the sample taken for analysis. In addition, an antibody binds only to the antigenic determinant that elicited its production. This specificity enables the development of test methods that require minimal sample preparation. Cross-reactivity can result in falsepositive responses over-estimation of antigen concentrations. Cross-reactivity of an antibody to a component of the sample or other GM crop is highly unlikely and almost never a

The ideal antigen for immunization would be the actual GM protein as it is expressed in the plant. However, purification of the novel protein from plant tissue can be difficult and may result in undesirable modifications to the target protein. In addition, purification rarely results in 100% pure protein and immunization of animals with such preparations results not only in the production of antibodies to the target protein but to the contaminants as well. Polyclonal antibodies made from these preparations typically exhibit high background

colloidal gold rather than an enzyme as a means of generating a visible signal.

immunohistochemical staining.

method.

significant issue.

location of the expressed proteins in the plant.

To date there is no molecular approach to testing that can distinguish between the presence of a low (or high) percentage of GM events in a bulk sample, and the presence of a mixture of the two or more individual events that comprise the stack. In addition, there are added problems because gene products can become degraded during preparation and cooking. Thus, serology may not be suitable for their detection in some processed food products.

These reasons have lead to use alternative methods, por example to monitor changes in the chemical profile of oils derived from GM Plants by using chromatophaphic methods, i.e. HPLC (Lopez et al., 2009), or near infrared spectroscopy (NIR) for detection of changes in fibre structure (Michelini et al., 2008).

NIR detection is a method that can reveal what kinds of chemicals are present in a sample based on their physical properties. It is not yet known if the differences between GMOs and conventional plants are large enough to detect with NIR imaging. Although the technique would require advanced machinery and data processing tools, a non-chemical approach could have some advantages such as lower costs and enhanced speed and mobility.

Another alternative method to differentiate and quantify GM from non GM seed contained in a seed lot is a statistical approach. The approach is a pooled testing approach and involves the examination of as many as 10-20 pools. However, if the percentage of positive seeds in the sample is higher than a few percent of the seeds, the model may not give clear results.

#### **2.1.4 Reference materials, standards and control for validation and standardization of detection and analysis methods**

The request for powerful analytical methods for routine detection of GMOs by accredited laboratories has called attention to international validation and preparation of official and non-commercial guidelines. Among these guidelines are preparations of certified reference material (CRM), sampling, treatment of samples, production of stringent analytical protocols, and extensive ring-trials for determination of the efficacy of selected GMO detection procedures. Any detection method to be implemented in the identification of GM crop plants and its derived products used in food needs materials to be used for calibration and validation of such detection methods as well as proficiency testing of laboratories. The reference materials should be controlled and regulated by government agencies as general use, in order to ensure a globally harmonized approach and provide them under principles for transfer in order to control the distribution and use of intellectual property (Trapmann et al., 2010).

Reference material is material with sufficiently stable and homogeneous properties and well established to be used for calibration, the assessment of a measurement method or for assigning values to materials. Certified reference material (CRM) is reference material accompanied by a certificate issued by a recognised body indicating the value of one or more properties and their uncertainty. The certified values of these materials have been established during the course of a certification campaign including inter-laboratory studies (which should be available upon request). In the absence of CRM, standards validated by a laboratory can be used.

Reference Materials are required as reference standards in method calibration and must be produced according to international standards and guidelines and may be certified. Reference materials will be made available for all products which are commercially available. These reference materials will be made available globally and on a single GM event detection, and designated by a third-party source. This source will be selected by each company based upon factors such as global presence, operational independence or experience in working with such materials under ISO standards. For compliance with the 1% threshold level of cross-contamination of unmodified foods with GM food products, certified reference materials for precise quantification and method validation are needed. According to commonly accepted rules, the production of reference materials should preferably follow metrological principles and should be traceable to the SI system. Arbitrary definition of measurement units could lead, as a consequence, to difficulties with nonconsistent standards and a lack of long-term reproducibility. In the future, efforts should be concentrated on establishing reliable quantification methods accompanied by the production of reference materials with high DNA quality and DNA degraded under controlled conditions (simulating real samples in food production) using very well characterized base materials.

European Union's Joint Research Centre, Institute for Reference Materials and Measurements, Belgium, is currently developing a system for distribution of GMO reference material (http://www.irmm.jrc.be/).

Identification and quantification of gene products in a GM plant must be done with standards that correlate to known concentrations of the antigen (protein) that it is used to produce a dose-response curve. The standard curve and the assay response from the samples are used to determine the antigen concentration. The material used to make the standards should yield a response that correlates to the actual concentration of antigen in the sample type and assay conditions specified by the test procedure. Recombinant proteins, which contain a similar or identical amino acid sequence and immunoreactivity as the GM plant-expressed protein, are often used as ELISA standards. Uniform preparations of actual samples (such as ground corn) having known concentrations of GM proteins may also be used as standards (Trapmann et al., 2002). Protein reference materials are critical for the

crop plants and its derived products used in food needs materials to be used for calibration and validation of such detection methods as well as proficiency testing of laboratories. The reference materials should be controlled and regulated by government agencies as general use, in order to ensure a globally harmonized approach and provide them under principles for transfer in order to control the distribution and use of intellectual property (Trapmann et

Reference material is material with sufficiently stable and homogeneous properties and well established to be used for calibration, the assessment of a measurement method or for assigning values to materials. Certified reference material (CRM) is reference material accompanied by a certificate issued by a recognised body indicating the value of one or more properties and their uncertainty. The certified values of these materials have been established during the course of a certification campaign including inter-laboratory studies (which should be available upon request). In the absence of CRM, standards validated by a

Reference Materials are required as reference standards in method calibration and must be produced according to international standards and guidelines and may be certified. Reference materials will be made available for all products which are commercially available. These reference materials will be made available globally and on a single GM event detection, and designated by a third-party source. This source will be selected by each company based upon factors such as global presence, operational independence or experience in working with such materials under ISO standards. For compliance with the 1% threshold level of cross-contamination of unmodified foods with GM food products, certified reference materials for precise quantification and method validation are needed. According to commonly accepted rules, the production of reference materials should preferably follow metrological principles and should be traceable to the SI system. Arbitrary definition of measurement units could lead, as a consequence, to difficulties with nonconsistent standards and a lack of long-term reproducibility. In the future, efforts should be concentrated on establishing reliable quantification methods accompanied by the production of reference materials with high DNA quality and DNA degraded under controlled conditions (simulating real samples in food production) using very well

European Union's Joint Research Centre, Institute for Reference Materials and Measurements, Belgium, is currently developing a system for distribution of GMO reference

Identification and quantification of gene products in a GM plant must be done with standards that correlate to known concentrations of the antigen (protein) that it is used to produce a dose-response curve. The standard curve and the assay response from the samples are used to determine the antigen concentration. The material used to make the standards should yield a response that correlates to the actual concentration of antigen in the sample type and assay conditions specified by the test procedure. Recombinant proteins, which contain a similar or identical amino acid sequence and immunoreactivity as the GM plant-expressed protein, are often used as ELISA standards. Uniform preparations of actual samples (such as ground corn) having known concentrations of GM proteins may also be used as standards (Trapmann et al., 2002). Protein reference materials are critical for the

al., 2010).

laboratory can be used.

characterized base materials.

material (http://www.irmm.jrc.be/).

validation of externally operated immunochemistry processes. Reference materials can be derived from a number of production sources, and can take on a variety of final forms (stabilized plant extracts to highly pure protein). Three types of certified GMO reference samples for GMO testing are especially needed: 1) DNA-CRM, 2) matrix-CRM for events of major importance, and 3) protein-CRM. An important issue to consider is that the CRMs are stable and non-degraded. Often problems with degradation of CRMs are encountered.

The European Network of GMO Laboratories has prepared a list of wishes concerning CRMs for GMO inspection as follows:


**Controls** are reagents and specifications that validate each method run. Reagent controls may be different from standards. Per example, every ELISA test, qualitative or quantitative, should include known positive and negative controls to ensure assay validity. Typical controls specify limits for background, assay response to a known concentration, quantitative range, and variability between replicates.

**Validation of methods** is the process of showing that the combined procedures of sample extraction, preparation, and analysis will yield acceptably accurate and reproducible results for a given analysis in a specified matrix. For validation of an analytical method, the testing objective must be defined and performance characteristics must be demonstrated. Performance characteristics include accuracy, extraction efficiency, precision, reproducibility, sensitivity, specificity, and robustness. The use of validated methods is important to assure acceptance of results produced by analytical laboratories.

Each new method should be tested in trials using numerous laboratories in order to demonstrate reproducible, sensitive and specific results. In these trials the same measurements should be assessed on identical materials. The experimental designs of each trial are crucial and several questions should be considered when planning such experiments. Examples of important issues to consider include availability of satisfactory standards, number of laboratories and how they should be recruited. It is also necessary to specify the manner of calculating and expressing test result.

Unfortunally, at this moment, no single validated method has yet been developed which is capable of accurately determining all GM products in a timely and cost effective manner. Testing programs will need to incorporate the best qualities of each technology in developing testing programs. The collaborative efforts of many organizations will be required to facilitate the development of reliable, validated diagnostic tests with broad global acceptance among users and regulators.

According to European Union legislation state laboratories participating in inspection should, whenever possible, use validated analytical methods. This is also the case for all laboratories aiming at accreditation. There are some examples of methods that have been validated or accredited recently are given below:


However, these methods based on a relatively expensive instrumentation, requiring substantial efforts in training and available only to a limited number of participants as e.g. Real-time PCR or Microarrays for validation studies may not be useful at the moment, as methods to be implemented in routine laboratories on European scale. Furthermore, GMO testing laboratories should participate in an internationally recognised external quality control assessment and accreditation scheme. In accordance with this, authorised laboratories (approved for official inspection purposes) must participate regularly in appropriate proficiency testing schemes.

#### **2.2 Uses and concerns about genetically modified organisms**

GMOs are used in biological and medical research, production of pharmaceutical drugs, experimental medicine (e.g. gene therapy), and agriculture (e.g. golden rice). The term "genetically modified organism" does not always imply, but can include, targeted insertions of genes from one species into another.

To date the most controversial but also the most widely adopted application of GMO technology is patent-protected food crops which are resistant to commercial herbicides or are able to produce pesticidal proteins from within the plant, or stacked trait seeds, which do both. Transgenic animals are also becoming useful commercially. On February 6, 2009 the U.S. Food and Drug Administration approved the first human biological drug produced from such an animal, a goat. The drug, ATryn, is an anticoagulant which reduces the probability of blood clots during surgery or childbirth. It is extracted from the goat's milk (Niemann & Kues, 2007). Furthermore, transgenic plants have been engineered to possess several desirable traits, such as resistance to pests, herbicides, or harsh environmental conditions, improved product shelf life, and increased nutritional value. Since the first commercial cultivation of genetically modified plants in 1996, they have been modified to be tolerant to the herbicides glufosinate and glyphosate, to be resistant to virus damage as in Ring-spot virus-resistant GM papaya, grown in Hawaii, and to produce the Bt toxin, an insecticide that is non-toxic to mammals (Nasiruddin & Nasim, 2007).

Most GM crops grown today have been modified with "input traits", which provide benefits mainly to farmers. The GM oilseed crops on the market today offer improved oil profiles for

According to European Union legislation state laboratories participating in inspection should, whenever possible, use validated analytical methods. This is also the case for all laboratories aiming at accreditation. There are some examples of methods that have been

1. Bt176, Bt11, T25 and MON810 maize using real time quantitative PCR have been accomplished by the BgVV, Federal Institute for Health protection of Consumers and

2. A PCR and an ELISA method for Roundup ReadyTM soybean and a PCR for Maximizer maize (Bt176) have been validated for commercial testing of grain by the European

3. An ELISA for MON810 maize has also been validated by AACC (American Association

4. The Varietal ID PCR methods (based on primers that span unique sequence junctions) have been accredited through the United Kingdom Accreditation System (UKAS). However, these methods based on a relatively expensive instrumentation, requiring substantial efforts in training and available only to a limited number of participants as e.g. Real-time PCR or Microarrays for validation studies may not be useful at the moment, as methods to be implemented in routine laboratories on European scale. Furthermore, GMO testing laboratories should participate in an internationally recognised external quality control assessment and accreditation scheme. In accordance with this, authorised laboratories (approved for official inspection purposes) must participate regularly in

GMOs are used in biological and medical research, production of pharmaceutical drugs, experimental medicine (e.g. gene therapy), and agriculture (e.g. golden rice). The term "genetically modified organism" does not always imply, but can include, targeted insertions

To date the most controversial but also the most widely adopted application of GMO technology is patent-protected food crops which are resistant to commercial herbicides or are able to produce pesticidal proteins from within the plant, or stacked trait seeds, which do both. Transgenic animals are also becoming useful commercially. On February 6, 2009 the U.S. Food and Drug Administration approved the first human biological drug produced from such an animal, a goat. The drug, ATryn, is an anticoagulant which reduces the probability of blood clots during surgery or childbirth. It is extracted from the goat's milk (Niemann & Kues, 2007). Furthermore, transgenic plants have been engineered to possess several desirable traits, such as resistance to pests, herbicides, or harsh environmental conditions, improved product shelf life, and increased nutritional value. Since the first commercial cultivation of genetically modified plants in 1996, they have been modified to be tolerant to the herbicides glufosinate and glyphosate, to be resistant to virus damage as in Ring-spot virus-resistant GM papaya, grown in Hawaii, and to produce the Bt toxin, an

Most GM crops grown today have been modified with "input traits", which provide benefits mainly to farmers. The GM oilseed crops on the market today offer improved oil profiles for

validated or accredited recently are given below:

Veterinary Medicine in Germany).

Union's Joint Research Centre, JRC.

appropriate proficiency testing schemes.

of genes from one species into another.

**2.2 Uses and concerns about genetically modified organisms** 

insecticide that is non-toxic to mammals (Nasiruddin & Nasim, 2007).

of Cereal Chemists).

processing or healthier edible oils (Sayanova & Napier, 2011). The GM crops in development offer a wider array of environmental and consumer benefits such as nutritional enhancement, drought and stress tolerance. Other examples include a genetically modified sweet potato, enhanced with protein and other nutrients, while golden rice, developed by the International Rice Research Institute (IRRI), has been discussed as a possible cure for Vitamin A deficiency.

The most common genetically engineered (GE) crops now being grown are transgenic varieties of soybean, canola, cotton, and corn. Varieties of each of these crops have been engineered to have either herbicide tolerance or insect resistance (or in a few cases, both). All of the genetically engineered insect-resistant crop varieties produced so far use specific genes taken from *Bacillus thuringiensis*, a common soil bacterium, to produce proteins that are toxic to certain groups of insects that feed on them. Currently, only Bt corn and Bt cotton varieties are being grown in the U.S., but Bt potatoes were on the market for several years until being discontinued in 2001. In addition, several different genetic modifications have been used to engineer tolerance to herbicides, the most widely adopted GE trait overall. Genetically engineered herbicide tolerant varieties of each of the four major crops listed above have been developed for use with glyphosate or glufosinate herbicides, and some cotton varieties grown in the U.S. have genetically engineered tolerance to bromoxynil or sulfonylurea herbicides. About half of the papaya crop produced in Hawaii is now from genetically engineered virus-resistant varieties, but most of the world-wide papaya crop is not genetically engineered. There is currently some limited production of squash genetically engineered for virus resistance in the U.S.

All together, about 50 different kinds of genetically engineered plants (each developed from a unique "transformation event") have been approved for commercial production in the U.S. These include 12 different crops modified to have six general kinds of traits:


Table 1. Genetically modified crops.

Not all of the genetically engineered varieties that have received regulatory approval are currently being grown. Some have not yet been marketed (herbicide tolerant sugarbeets and most kinds of GE tomatoes, for example), and some have been commercially grown but were later withdrawn from the market. More details on the transgenic crops listed in the table above and short descriptions of how each of the transgenic traits works are available at http://www.comm.cornell.edu/gmo/traits/traits.html.

There are many perceived risks and benefits associated with the use of transgenic crop plants for agricultural food production (Wolfenbarger & Phifer, 2000). Some of the risks relate to the use of specific transgenes while others emphasize a broader concern that addresses the entire approach of engineering heterologous genes into plants.

Most concerns about GM foods fall into three categories: 1) *environmental hazards*, including an unintended harm to other organisms, i.e. B.t. corn caused high mortality rates in monarch butterfly caterpillars; reduction of the effectiveness of pesticides; and gene transfer to nontarget species, i.e. transfer of the herbicide resistance genes from the crops into the weeds.

2) *Human health risks*, including Allergenicity and toxic effects and 3) *Economic concerns*, since GM food production is a lengthy and costly process, being many new plants GM technologies patented raising the price of seeds.

One of the primary concerns about genetically engineered crop plants is that they will hybridize with wild relatives, permitting the transgene to escape and spread into the environment, which depends on its potential fitness impact. Depending on the nature of the plant and its propensity to cross-pollinate, the flow of transgenes in the field may be an important consideration. Perhaps in some instances, transgenic plants should not be grown in geographic areas where close relatives may be pollinated with transgene containing pollen. It is feared that gene flow to non target plant populations may diminish diversity within plant species. Whether or not genetic diversity is threatened by gene flow when transgenic plants are grown in the vicinity of a native gene pool, the susceptibility of native flora to contamination by transgenes ought to be taken into account.

There are several possible solutions to the problems mentioned above. Genes are exchanged between plants via pollen. Two ways to ensure that non target species will not receive introduced genes from GM plants are to create GM plants that are male sterile (do not produce pollen) or to modify the GM plant so that the pollen does not contain the introduced gene (Warwick et al., 2009). Cross-pollination would not occur, and if harmless insects such as Monarch caterpillars were to eat pollen from GM plants, the caterpillars would survive.

Another possible solution is to create buffer zones around fields of GM crops. Beneficial or harmless insects would have a refuge in the non GM corn, and insect pests could be allowed to destroy the non GM corn and would not develop resistance to Bt pesticides. Gene transfer to weeds and other crops would not occur because the wind blown pollen would not travel beyond the buffer zone (Hüsken & Dietz-Pfeilstetter, 2007).

Potential unanticipated events relating to the safety and acceptability of transgenic plants include the transfer of antibiotic-resistant genes, up regulation of non-target genes by foreign promoter sequences, production of allergenic compounds and proteins, including cross-reactivity between plant-derived food and/or pollen (Jimenez-Lopez et al. 2011) , or gene products with mammalian toxicity. Different strategies have been developed for reducing the probability and impact of gene flow, including physical separation from wild relatives and genetic engineering. Mathematical models and empirical experimental evidence suggest that genetic approaches have the potential to effectively prevent transgenes from incorporating into wild relatives and becoming established in wild populations that are not reproductively isolated from genetically engineered crops. In addition, transgene strategies for controlling plant disease do not raise some of the same concerns that relate to the release of herbicide-tolerant cultivars or insect-protected varieties. The environmental and food safety aspect of each gene construct, however, must be

relate to the use of specific transgenes while others emphasize a broader concern that

Most concerns about GM foods fall into three categories: 1) *environmental hazards*, including an unintended harm to other organisms, i.e. B.t. corn caused high mortality rates in monarch butterfly caterpillars; reduction of the effectiveness of pesticides; and gene transfer to nontarget species, i.e. transfer of the herbicide resistance genes from the crops into the weeds. 2) *Human health risks*, including Allergenicity and toxic effects and 3) *Economic concerns*, since GM food production is a lengthy and costly process, being many new plants GM

One of the primary concerns about genetically engineered crop plants is that they will hybridize with wild relatives, permitting the transgene to escape and spread into the environment, which depends on its potential fitness impact. Depending on the nature of the plant and its propensity to cross-pollinate, the flow of transgenes in the field may be an important consideration. Perhaps in some instances, transgenic plants should not be grown in geographic areas where close relatives may be pollinated with transgene containing pollen. It is feared that gene flow to non target plant populations may diminish diversity within plant species. Whether or not genetic diversity is threatened by gene flow when transgenic plants are grown in the vicinity of a native gene pool, the susceptibility of native

There are several possible solutions to the problems mentioned above. Genes are exchanged between plants via pollen. Two ways to ensure that non target species will not receive introduced genes from GM plants are to create GM plants that are male sterile (do not produce pollen) or to modify the GM plant so that the pollen does not contain the introduced gene (Warwick et al., 2009). Cross-pollination would not occur, and if harmless insects such as Monarch caterpillars were to eat pollen from GM plants, the caterpillars

Another possible solution is to create buffer zones around fields of GM crops. Beneficial or harmless insects would have a refuge in the non GM corn, and insect pests could be allowed to destroy the non GM corn and would not develop resistance to Bt pesticides. Gene transfer to weeds and other crops would not occur because the wind blown pollen would not travel

Potential unanticipated events relating to the safety and acceptability of transgenic plants include the transfer of antibiotic-resistant genes, up regulation of non-target genes by foreign promoter sequences, production of allergenic compounds and proteins, including cross-reactivity between plant-derived food and/or pollen (Jimenez-Lopez et al. 2011) , or gene products with mammalian toxicity. Different strategies have been developed for reducing the probability and impact of gene flow, including physical separation from wild relatives and genetic engineering. Mathematical models and empirical experimental evidence suggest that genetic approaches have the potential to effectively prevent transgenes from incorporating into wild relatives and becoming established in wild populations that are not reproductively isolated from genetically engineered crops. In addition, transgene strategies for controlling plant disease do not raise some of the same concerns that relate to the release of herbicide-tolerant cultivars or insect-protected varieties. The environmental and food safety aspect of each gene construct, however, must be

addresses the entire approach of engineering heterologous genes into plants.

flora to contamination by transgenes ought to be taken into account.

beyond the buffer zone (Hüsken & Dietz-Pfeilstetter, 2007).

technologies patented raising the price of seeds.

would survive.

evaluated on the basis of its genetic background, specific gene product, and the environmental context of the host crop.

Resistance to disease based on a product from a single gene, like resistance to insects, is often overcome by development of new pest strains. Much concern has surrounded the development of plants engineered to produce their own pesticide and the subsequent development of resistant insects. Engineered resistance to insects has focused on the use of the gene for Cry (Bt) toxins, for controlling lepidopterous. Carefully management strategies will be further required to prevent development of resistance breaking pathogens, when disease-protected GM plants are grown commercially. Certainly, the breakdown of resistance as a result of pathogen adaptation, occurs in cultivars developed by classical breeding and can be anticipated if single genes are used for engineering disease-protected GM plants (Wally & Punja, 2010). Moreover, evolution of recombinant pathogens has been raised as a particular concern in strategies using structural viral genes that could recombine with naturally infecting viruses to form new viral forms (Regev et al., 2006).

Finally, the possibility of increased weediness of plants is particularly relevant to the design of plants with herbicide tolerance. Genetically modified plants themselves could become weedy and difficult to control on account of their resistance to a particular herbicide of equal concern is movement of the herbicide-tolerance trait to weedy relatives by pollen transfer. The invasiveness of any species and long-term environmental effects are difficult to project for any modified plant released into the ecosystem. However, it is not well established whether genetic modifications created by molecular techniques pose a greater risk of invasiveness than those created by classical breeding techniques (Jacobsen & Schouter, 2007).

#### **2.2.1 Food safety and side effects of GM-crops**

The safety of GM foods has been a controversial issue over the past decade. Despite major concerns, very little independent research has been carried out to establish their long-term safety.

In general terms, the safety assessment of GM foods should investigate: a) toxicity, b) allergenicity, c) specific components thought to have nutritional or toxic properties, d) stability of the inserted gene, e) nutritional effects associated with genetic modification, and f) any unintended effects which could result from the gene insertion (WHO, 2002).

Construction of transgenic crop plants must take into consideration any possible impact on food safety, concretely in the two areas of concern which are allergenicity and the production of gene products that are toxic to mammalian metabolism (Uzogara, 2000; Malarkey, 2003; Dona & Arvanitoyannis, 2009).

Biotechnology companies argue that many of the individual proteins used in GM crops have been consumed over a long period in their natural host with no health effects seen, so simply creating the proteins in a new plant will surely be the same. This assumes both that the new protein in the GM plant is identical to the naturally produced protein, and that no unintended effects have occurred during the genetic modification process that could produce other proteins. This assumption can be seen in action in the USA, where after ten years of commercialisation of GM crops there is still no post-market surveillance for allergic reactions (Davies, 2005). New research now challenges this assumption, and brings into question the safety of both new and previously approved GM foods.

About 1–2% of adults population in the world and about 5% of children display food allergies. Around 90% of food allergies are induced by peanuts, soybeans, vegetables, fruits, milk, eggs, cereals, nuts, some fish and shellfish. Generally speaking, the allergic reaction is caused not by whole food items, but only by certain components called allergens, which most commonly are proteins, or in fact only segments of proteins (peptides) called allergenic epitopes. Biotechnology allows crop breeders to add new genes to a plant, but also to remove or inactivate a specific gene. This opens the possibility of removing specific allergens so that those people who suffer from a specific food allergy can again eat that GM food. Such "allergen-free" foods have not yet come on the market, but they are being developed in various laboratories. One group in Japan reported several years ago that they had removed the major allergen from a variety of rice. In the US research is being done to remove the main allergen from peanuts and shrimps (Randhawa et al., 2011).

Allergenicity Many children in the US and Europe have developed life-threatening allergies to peanuts and other foods. There is a possibility that introducing a gene into a plant may create a new allergen or cause an allergic reaction in susceptible individuals.

A proposal to incorporate a gene from Brazil nuts into soybeans (a methionine-rich protein) was abandoned because of the fear of causing unexpected allergic reactions (Nordlee et al., 1996). Some people are allergic to proteins that occur naturally in soybeans, and they could have a reaction if they are exposed to either conventional or transgenic soybeans or soy products. Soybeans are one of the eight most common sources of food allergies. Although less common, some people have food allergies associated with corn and they could be affected by either conventional or transgenic corn. No allergic reactions attributable to the proteins present as a result of genetic engineering have been reported in the transgenic soybeans being grown commercially at this time. Reports of an allergenic protein made as a result of genetic engineering in one particular type of transgenic corn could not be confirmed by subsequent testing.

While there isn't any evidence that allergens have been introduced into food crops by genetic engineering, two incidents have received quite a bit of publicity and caused public concern about food allergies resulting from transgenic crops:

The first incident involved soybean plants, and a gene from Brazil nuts to make soybeans that contained higher levels of the amino acid methionine, to improve nutritious chicken feed that would eliminate the need for expensive feed supplements.

The second incident involved reports of allergic reactions in people who may have eaten food containing the insecticidal protein called Cry9C, one of several forms of the Bt insecticide. When food from grocery shelves tested positive for Cry9C, demonstrating an accidental way into the food supply. During this time, the reports surfaced of allergic reactions in people who had eaten corn products that may have been contaminated by Cry9C, but special test developed by the FDA (an enzyme-linked immunosorbent assay, or ELISA test, to detect people's antibodies to the Cry9C protein) did not find any evidence that the reactions in the affected people were associated with hypersensitivity to the Cry9C protein. The test isn't 100% conclusive, though, partly because food allergies may sometimes

reactions (Davies, 2005). New research now challenges this assumption, and brings into

About 1–2% of adults population in the world and about 5% of children display food allergies. Around 90% of food allergies are induced by peanuts, soybeans, vegetables, fruits, milk, eggs, cereals, nuts, some fish and shellfish. Generally speaking, the allergic reaction is caused not by whole food items, but only by certain components called allergens, which most commonly are proteins, or in fact only segments of proteins (peptides) called allergenic epitopes. Biotechnology allows crop breeders to add new genes to a plant, but also to remove or inactivate a specific gene. This opens the possibility of removing specific allergens so that those people who suffer from a specific food allergy can again eat that GM food. Such "allergen-free" foods have not yet come on the market, but they are being developed in various laboratories. One group in Japan reported several years ago that they had removed the major allergen from a variety of rice. In the US research is being done to

Allergenicity Many children in the US and Europe have developed life-threatening allergies to peanuts and other foods. There is a possibility that introducing a gene into a plant may

A proposal to incorporate a gene from Brazil nuts into soybeans (a methionine-rich protein) was abandoned because of the fear of causing unexpected allergic reactions (Nordlee et al., 1996). Some people are allergic to proteins that occur naturally in soybeans, and they could have a reaction if they are exposed to either conventional or transgenic soybeans or soy products. Soybeans are one of the eight most common sources of food allergies. Although less common, some people have food allergies associated with corn and they could be affected by either conventional or transgenic corn. No allergic reactions attributable to the proteins present as a result of genetic engineering have been reported in the transgenic soybeans being grown commercially at this time. Reports of an allergenic protein made as a result of genetic engineering in one particular type of transgenic corn could not be

While there isn't any evidence that allergens have been introduced into food crops by genetic engineering, two incidents have received quite a bit of publicity and caused public

The first incident involved soybean plants, and a gene from Brazil nuts to make soybeans that contained higher levels of the amino acid methionine, to improve nutritious chicken

The second incident involved reports of allergic reactions in people who may have eaten food containing the insecticidal protein called Cry9C, one of several forms of the Bt insecticide. When food from grocery shelves tested positive for Cry9C, demonstrating an accidental way into the food supply. During this time, the reports surfaced of allergic reactions in people who had eaten corn products that may have been contaminated by Cry9C, but special test developed by the FDA (an enzyme-linked immunosorbent assay, or ELISA test, to detect people's antibodies to the Cry9C protein) did not find any evidence that the reactions in the affected people were associated with hypersensitivity to the Cry9C protein. The test isn't 100% conclusive, though, partly because food allergies may sometimes

question the safety of both new and previously approved GM foods.

remove the main allergen from peanuts and shrimps (Randhawa et al., 2011).

create a new allergen or cause an allergic reaction in susceptible individuals.

confirmed by subsequent testing.

concern about food allergies resulting from transgenic crops:

feed that would eliminate the need for expensive feed supplements.

occur without detectable levels of antibodies to allergens. Extensive testing of GM foods may be required to avoid the possibility of harm to consumers with food allergies, and labeling of GM foods and food products will acquire new importance.

Before any GMO or derived product can be marketed in the EU, it must pass through an approval system which is intended to assess its safety for humans, animals and the environment. The GMO Panel of the European Food Safety Authority (EFSA), which provides scientific advice and technical support for GM food safety issues, published guidance for applicants seeking authorisation of GM food and/or feed, and a section of the guidance covers current requirements for assessment of allergenicity. The guidelines are based on the recommendations of the Codex Alimentarius Commission's ad hoc Intergovernmental Task Force on Foods Derived from Biotechnology. Codex is an organisation that develops international standards for food standards (Codex Procedural Manual 20th *Ed*. 2011, WHO/FAO).

Toxicity of transgene products in new and another major concern about GMOs, regardless of the source of the gene sequence. There is a growing concern that introducing foreign genes into food plants may have an unexpected and negative impact on human health. Various food plants produce compounds that would be toxic at high levels and enhancement of their production above normal levels in transgenic plants could be detrimental to human health. Up regulation of glycoalkaloids in potato by genetic manipulation, for example, would be of concern whether the increased levels resulted from classical breeding or genetic engineering (Friedman & McDonald, 1997). The gene introduced into the potatoes was a snowdrop flower lectin, a substance known to be toxic to mammals. The scientists who created this variety of potato chose to use the lectin gene simply to test the methodology, and these potatoes were never intended for human or animal consumption.

Side effects of gene products expressed in transgenic plants are also not to be ignored. The possible effect of Bt endotoxin in pollen ingested by monarch butterflies has received public attention, although further field research demonstrated low impact on lepidopterans that could be at high potential risk (Losey et al., 1999). In additinon, concerns about secondary effects on non-target insects must be balanced by the impact of traditional pesticides that would have been used if Bt transgenics were not grown.

Other example of possible secondary effects can be envisioned, such as the effect of antifungal or antibacterial gene products on degradation of crop residues in the field. Decrease in plant decomposition could affect soil fertility and, in some cases, antimicrobial gene products could lower the diversity of soil microorganism communities (Wolfenbarger & Phifer, 2000).

Whether or not these effects are important for the environment or ecology of particular macro- or micro-organisms is something that needs to be evaluated within the scope of establishing the safety of any particular transgenic plant species.

#### **2.2.2 Controversy about the safety to eat GM crops**

The primary concern many people have about genetically engineered (GE) crops is the safety of food made from them. It is unlikely that eating DNA poses any significant risk to human or animal health, and there is no evidence to suggest that there is any additional risk from the transgenes present in genetically engineered plants. Although there continues to be quite a bit of controversy over this issue, no evidence has been found that foods made with the genetically engineered crops now on the market are any less safe to eat than foods made with the same kinds of conventional crops.

The overall goal about GE crops is not to establish an absolute level of safety, but rather the relative safety of the new product so that there is a reasonable certainty that no harm will result from intended uses under the anticipated conditions of production, processing and consumption. Most of the DNA we eat is degraded in the digestive system, but some experiments have shown that small amounts of it can be found in some cells in the body. It is thought to be unlikely that this DNA would be incorporated into the DNA of those cells, but even if it was, the chance of any undesirable effect on the whole organism is thought to be very low. Normal diets for humans and other animals contain large amounts of DNA. This DNA comes not only from the cells of the various kinds of plants or animals constituting the food, but also from any contaminating microorganisms or viruses that may be present in or on the food. We have been exposed to this variety of DNA throughout our entire history. It seems that we are well adapted to handling exposure to DNA, and there is no obvious reason that the DNA from other organisms introduced into crops by genetic engineering would have any additional effect.

Some critics of GE crops point out that a lack of evidence for harmful effects does not mean they do not exist, but just as likely could mean that we have not done the proper studies to document them. Some reject the idea that we face the same kinds of risks from GE crops as from conventionally developed crops, believing the genetic engineering process itself introduces unique risks. Genetically engineered crop varieties are being subjected to far greater scientific scrutiny than that ordinarily given to conventional varieties, even though many scientists have argued that there is no strict distinction between the food safety risks posed by genetically engineered plants and those developed using conventional breeding practices.

Safety assessments of foods developed using genetic engineering include the following considerations:


A major concern often expressed about GE food safety is the risk for unintentional, potentially harmful changes that may escape detection in the evaluation process. It is true that the number of factors that are examined for change is small compared to the total number of components produced by plants. Also, more extensive comparisons of plant chemical compositions would be difficult because complete data describing the composition of conventional crop plants, including knowledge of variability among different cultivars or

human or animal health, and there is no evidence to suggest that there is any additional risk from the transgenes present in genetically engineered plants. Although there continues to be quite a bit of controversy over this issue, no evidence has been found that foods made with the genetically engineered crops now on the market are any less safe to eat than foods made

The overall goal about GE crops is not to establish an absolute level of safety, but rather the relative safety of the new product so that there is a reasonable certainty that no harm will result from intended uses under the anticipated conditions of production, processing and consumption. Most of the DNA we eat is degraded in the digestive system, but some experiments have shown that small amounts of it can be found in some cells in the body. It is thought to be unlikely that this DNA would be incorporated into the DNA of those cells, but even if it was, the chance of any undesirable effect on the whole organism is thought to be very low. Normal diets for humans and other animals contain large amounts of DNA. This DNA comes not only from the cells of the various kinds of plants or animals constituting the food, but also from any contaminating microorganisms or viruses that may be present in or on the food. We have been exposed to this variety of DNA throughout our entire history. It seems that we are well adapted to handling exposure to DNA, and there is no obvious reason that the DNA from other organisms introduced into crops by genetic

Some critics of GE crops point out that a lack of evidence for harmful effects does not mean they do not exist, but just as likely could mean that we have not done the proper studies to document them. Some reject the idea that we face the same kinds of risks from GE crops as from conventionally developed crops, believing the genetic engineering process itself introduces unique risks. Genetically engineered crop varieties are being subjected to far greater scientific scrutiny than that ordinarily given to conventional varieties, even though many scientists have argued that there is no strict distinction between the food safety risks posed by genetically engineered plants and those developed using conventional breeding

Safety assessments of foods developed using genetic engineering include the following

1. Evaluation of the methods used to develop the crop, including the molecular biological

3. The general chemical composition of the novel food compared to conventional

A major concern often expressed about GE food safety is the risk for unintentional, potentially harmful changes that may escape detection in the evaluation process. It is true that the number of factors that are examined for change is small compared to the total number of components produced by plants. Also, more extensive comparisons of plant chemical compositions would be difficult because complete data describing the composition of conventional crop plants, including knowledge of variability among different cultivars or

with the same kinds of conventional crops.

engineering would have any additional effect.

data which characterizes the genetic change, 2. The evaluation for the expected phenotype,

5. The potential for introducing new toxins, and 6. The potential for causing allergic reactions.

4. The nutritional content compared to conventional counterparts,

practices.

considerations:

counterparts,

that due to environmental influences, is lacking. The random nature of transgene insertion when making GE plants, it is argued, may cause disruption of important genes, causing significant effects but little obvious change to the plant's phenotype.

Antibiotic resistance genes are frequently used at several stages in the creation of GE plants as convenient "selectable markers". Bacteria or plant cells without a gene for resistance to the antibiotics used can be killed when the antibiotic is applied to them. So when scientists link the gene for the desired trait being introduced into a plant with an antibiotic resistance gene, they can separate cells carrying the desired gene from those that don't by exposing them to the antibiotic. The antibiotic resistance genes end up in the genetically engineered plants as excess baggage whose function is no longer required after the process of making them is complete. Concern has been raised about the possibility that antibiotic resistance genes used to make transgenic plants could be transferred to microorganisms that inhabit the digestive tracts of humans or other animals that eat them, and therefore might contribute to the already serious problem of antibiotic resistant pathogens. Transfer of DNA from one microbe to another (horizontal gene transfer) is known to occur in nature and has been observed in some laboratory experiments under specific conditions, but the likelihood of DNA being transferred from plant material in the digestive system to microbes has not yet been experimentally determined. It is thought that for such a transfer to be possible, it would have to come from consumption of fresh food since most processing would degrade the plant's DNA. Also, there is evidence that most DNA is rapidly degraded by the digestive system. Overall, the risk of antibiotic resistance genes from transgenic plants ending up in microorganisms appears to be low.

A second concern about the use of some antibiotic resistance genes is that they could reduce the effectiveness of antibiotics taken at the same time transgenic food carrying the resistance gene for that antibiotic was consumed. In cases where this has been identified as a risk based on the mechanism of resistance, studies have suggested the chance of this happening was probably very low due to rapid digestion of the inactivating enzymes produced by the transgenic resistance gene. Most transgenic plants do not carry resistance genes for antibiotics commonly used to treat infections in humans. Scientists are developing and using different selectable markers, and are also experimenting with methods for removing the antibiotic resistance genes before the plants are released for commercial use.

#### **2.2.3 Advantages of transgenic plants**

Despite the many concerns transgenic plants raise, they do have immense potential for benefit to society (Peterson et al., 2000).

Positive effects may include soil conservation, as new cultural practices permit low till methods and consequential maintenance of soil structure and decreased erosion.

Transgenic plants with stable resistance to disease will restrict crop losses and permit increased yield. Losses of food during postharvest storage can be decreased. There are also direct benefices to decreased use of pesticides and savings in resources and energy to manufacture and apply chemicals. Genetically modified plants may one day allow us to grow profitable crops without the need for environmentally unfriendly disease control plans. Globalization of the agricultural industry inevitably results in globalization of plant diseases. Various diseases, such as blight on potato, appear to be spreading worldwide, karnal bunt on wheat is on the increase in Asia and parts of North America, mosaic is increasing on cassava in Africa, and leaf blight continues to spread on rice in Japan and India (Moffat, 2001). Disease protected transgenic plants may yet demonstrate to be an important alternative against plant pathogens. Molecular biology has the potential to contribute significantly to a better society in which the environment is respected and an adequate food supply is provided.

Furthermore, the world population has topped 6 billion people and is predicted to double in the next 50 years. Ensuring an adequate food supply for this booming population is going to be a major challenge in the years to come. GM foods promise to meet this need in a number of ways: 1) Pest resistance Crop losses from insect pests can be staggering, resulting in devastating financial loss for farmers and starvation in developing countries, beside health hazards risks of chemical treatments, and contaminations of water and the environment; 2) Herbicide tolerance, avoiding utilization of chemicals to kill weed. Crop plants geneticallyengineered to be resistant to one very powerful herbicide could help prevent environmental damage by reducing the amount of herbicides needed; 3) Disease resistance caused by viruses, fungi and bacteria; 4) Cold tolerance, avoiding destruction of sensitive seedlings; 5) Drought tolerance/salinity tolerance; 6) Nutrition. Some crops do not contain necessary nutrients to prevent malnutrition. GM crops are directed toward increase minerals and vitamine, i.e. -carotene; 7) Pharmaceuticals production, such as edible vaccines in tomatoes and potatoes, which will be much easier to ship, store and administer than traditional injectable vaccines; 8) Phytoremediation. Soil and groundwater pollution continues to be a problem in all parts of the world. Plants have been genetically engineered to clean up heavy metal pollution from contaminated soil.

#### **3. Unification, development and implementation of official standard technologies**

The coexistence of GM plants with conventional and organic crops has raised significant concern in many European countries. Due to relatively high demand from European consumers for the freedom of choice between GM and non-GM foods, EU regulations require measures to avoid mixing of foods and feed produced from GM crops and conventional or organic crops. European research programs are investigating appropriate methods and tools to keep both GM and non GM crops isolated, i.e. isolation distance and pollen barriers, which are usually not used in North America because they are very costly and there are no safety-related reasons to employ them (Ramessar et al., 2010).

Certain global regulatory bodies require development of DNA detection methods that allow for unique identification of commercial transgenics, harmonised guidelines for the validation and use of these methods are not yet in place. As a result, numerous governmental agencies, global standards organizations, and industry organisations are attempting to develop their own independent standardisation guidelines for testing methodologies.

In the European Union, the (JRC) is playing a leading role in ensuring a harmonised approach between EU Member States, industry and stakeholders. It now hosts six European Union Reference Laboratories (EU-RLs) on food and feed safety in support of EU Member States' National Reference Laboratories (NRLs) in the respective fields. It is the National

karnal bunt on wheat is on the increase in Asia and parts of North America, mosaic is increasing on cassava in Africa, and leaf blight continues to spread on rice in Japan and India (Moffat, 2001). Disease protected transgenic plants may yet demonstrate to be an important alternative against plant pathogens. Molecular biology has the potential to contribute significantly to a better society in which the environment is respected and an

Furthermore, the world population has topped 6 billion people and is predicted to double in the next 50 years. Ensuring an adequate food supply for this booming population is going to be a major challenge in the years to come. GM foods promise to meet this need in a number of ways: 1) Pest resistance Crop losses from insect pests can be staggering, resulting in devastating financial loss for farmers and starvation in developing countries, beside health hazards risks of chemical treatments, and contaminations of water and the environment; 2) Herbicide tolerance, avoiding utilization of chemicals to kill weed. Crop plants geneticallyengineered to be resistant to one very powerful herbicide could help prevent environmental damage by reducing the amount of herbicides needed; 3) Disease resistance caused by viruses, fungi and bacteria; 4) Cold tolerance, avoiding destruction of sensitive seedlings; 5) Drought tolerance/salinity tolerance; 6) Nutrition. Some crops do not contain necessary nutrients to prevent malnutrition. GM crops are directed toward increase minerals and vitamine, i.e. -carotene; 7) Pharmaceuticals production, such as edible vaccines in tomatoes and potatoes, which will be much easier to ship, store and administer than traditional injectable vaccines; 8) Phytoremediation. Soil and groundwater pollution continues to be a problem in all parts of the world. Plants have been genetically engineered to clean up heavy

**3. Unification, development and implementation of official standard** 

and there are no safety-related reasons to employ them (Ramessar et al., 2010).

The coexistence of GM plants with conventional and organic crops has raised significant concern in many European countries. Due to relatively high demand from European consumers for the freedom of choice between GM and non-GM foods, EU regulations require measures to avoid mixing of foods and feed produced from GM crops and conventional or organic crops. European research programs are investigating appropriate methods and tools to keep both GM and non GM crops isolated, i.e. isolation distance and pollen barriers, which are usually not used in North America because they are very costly

Certain global regulatory bodies require development of DNA detection methods that allow for unique identification of commercial transgenics, harmonised guidelines for the validation and use of these methods are not yet in place. As a result, numerous governmental agencies, global standards organizations, and industry organisations are attempting to develop their own independent standardisation guidelines for testing

In the European Union, the (JRC) is playing a leading role in ensuring a harmonised approach between EU Member States, industry and stakeholders. It now hosts six European Union Reference Laboratories (EU-RLs) on food and feed safety in support of EU Member States' National Reference Laboratories (NRLs) in the respective fields. It is the National

adequate food supply is provided.

metal pollution from contaminated soil.

**technologies** 

methodologies.

Food Authorities who are responsible for the appropriate implementation of legislation. The latter is in place both to ensure the safety and quality of food products including animal feed and to ensure public health.

In order to ensure public health, potentially hazardous residues and contaminants are put under vigorous scrutiny and strict authorisation procedures for new additives and crops for feed and food production are in place. The aim of EU-RLs is to guarantee uniform detection, quantification and authorisation procedures. The activities of EU-RLs cover all the areas of feed and food law and animal health. In particular, those areas where there is a need for precise analytical and diagnostic results. The main objective of the EU-RLs is to contribute to a high quality and uniformity of results obtained in the various official food and feed control laboratories throughout the European Union.

Two JRC EU-RLs support authorisation for additives for feed production and of crops to be used in food and feed that have been genetically modified i.e. containing genetically modified organisms (GMOs). This work is carried out in close collaboration with the European Food Safety Authority (EFSA), the latter being responsible for risk assessment of such new substances and crops.

The main responsibilities of the EU-RLs for feed and food are to: 1) provide National Reference Laboratories (NRLs) with details of analytical methods, including reference methods, 2) organise comparative (proficiency) testing amongst the NRLs, 3) conduct training courses for the benefit of staff from the NRLs and of experts from developing countries, and 4) provide scientific and technical assistance to the European Commission, especially in cases when Member States contest the results of analyses.

The work of the EU-RLs contributes to increasing European and worldwide standardisation of analytical methods. This helps to ensure that the quality of analytical data obtained in various laboratories are increasingly comparable. Methods are developed by EU-RLs and then validated through collaborative trial testing in collaboration with the NRLs and other expert laboratories in the respective field. Proficiency tests are also organised by the EU-RLs (for NRLs) and by the NRLs (for national official laboratories) to ensure the quality of data obtained in the various laboratories that are also required for European and other international monitoring databases for exposure and risk assessment.

In this way, EU-RLs are working towards the best interests of the consumer. They are helping to build confidence in the results obtained by food control laboratories and to ensure that products purchased are in compliance with legislation and have the highest food hygiene standards.

EU-RLs also represent a unique platform for information exchange on analytical methodology and quality assurance tools for control laboratories. Together with the network of NRLs, they provide a pool of knowledge and facilities that makes them best placed to handle emerging issues.

The JRC is currently managing six EU-RLs. These are located in the Institute for Reference Materials and Measurements (IRMM) in Belgium and the Institute for Health and Consumer Protection (IHCP) in Italy. It is worth noting that the JRC-IRMM also chairs a board of expert laboratories which acts as EU-RL on behalf of the European Commission's Directorate General Agriculture. Its purpose is to harmonise analytical methodologies for the determination of water content in poultry to ensure the quality and to prevent fraud: 1) EU-RL for GMOs in food and feed, 2) EU-RL for feed additives, 3) EU-RL for food contact materials, 4) EU-RL for heavy metals in feed and food, 5) EU-RL for mycotoxins in food and feed, and 6) EU-RL for polycyclic aromatic hydrocarbons.

Regulation of genetically engineered crops by US government is made because guidance of the first the federal government adopted a "Coordinated Framework for Regulation of Biotechnology". Under this system, three federal agencies have regulatory authority over genetically engineered (GE) crops. Each agency has a different role to ensure safety under specific legislation. These agencies and their regulatory responsibilities are:

1) *The U.S. Department of Agriculture* (USDA), through the Animal and Plant Health Inspection Service (APHIS), is responsible for assuring that any organism, including genetically engineered organisms, will not become pests that can cause harm if they are released into the environment. APHIS has used their authority to grant permission and set the rules for field testing of genetically engineered crops. These crops cannot be commercialized until they are granted "non regulated" status by APHIS upon satisfactory review of the field testing data.

2) *The Food and Drug Administration* (FDA) is responsible for ensuring the safety of most food (except for meat, poultry and some egg products, which are regulated by the U.S. Department of Agriculture), including food from genetically engineered crops. If the allergen, nutrient and toxin content of new GE foods fall within the normal range found in the same kind of conventional food, the FDA does not regulate the GE food any differently. So far, all genetically modified foods in the U.S. marketplace have gone through a voluntary review process where the FDA determines whether they are "not substantially different" from the same conventional foods by consulting with developers of new GE foods to identify potential sources of differences, then reviewing a formal summary of data provided by the developer. Recently, the FDA has announced a new rule that would make pre-market consultation mandatory. The FDA has the authority to order foods to be pulled from the market at any time if are found to be unsafe, or to require labeling of any food that has different amounts of allergens, nutrients, or toxins than a consumer would expect to find in that kind of food.

3) *The Environmental Protection Agency* (EPA) evaluates the safety of any pesticides that are produced by genetically engineered plants. The EPA calls novel DNA and proteins genetically engineered into plants to protect them against pests "plant incorporated protectants" (PIPs) and regulates them the same way they regulate other pesticides.

Under the Coordinated Framework, some kinds of genetically engineered crops might not be subject to the oversight of all three agencies. For example, an ornamental flower like petunias engineered to have longer lasting blooms may only have to meet the requirements of APHIS, but a food crop like soybeans engineered to produce an insecticidal compound would be subject to the rules of all three agencies. Additional regulations are imposed by some states. Also, the National Institutes of Health has developed safety procedures for research with recombinant DNA. Most institutions developing genetically engineered crops follow the NIH guidelines, and they are required for federally funded research.

the determination of water content in poultry to ensure the quality and to prevent fraud: 1) EU-RL for GMOs in food and feed, 2) EU-RL for feed additives, 3) EU-RL for food contact materials, 4) EU-RL for heavy metals in feed and food, 5) EU-RL for mycotoxins in food and

Regulation of genetically engineered crops by US government is made because guidance of the first the federal government adopted a "Coordinated Framework for Regulation of Biotechnology". Under this system, three federal agencies have regulatory authority over genetically engineered (GE) crops. Each agency has a different role to ensure safety under

1) *The U.S. Department of Agriculture* (USDA), through the Animal and Plant Health Inspection Service (APHIS), is responsible for assuring that any organism, including genetically engineered organisms, will not become pests that can cause harm if they are released into the environment. APHIS has used their authority to grant permission and set the rules for field testing of genetically engineered crops. These crops cannot be commercialized until they are granted "non regulated" status by APHIS upon satisfactory

2) *The Food and Drug Administration* (FDA) is responsible for ensuring the safety of most food (except for meat, poultry and some egg products, which are regulated by the U.S. Department of Agriculture), including food from genetically engineered crops. If the allergen, nutrient and toxin content of new GE foods fall within the normal range found in the same kind of conventional food, the FDA does not regulate the GE food any differently. So far, all genetically modified foods in the U.S. marketplace have gone through a voluntary review process where the FDA determines whether they are "not substantially different" from the same conventional foods by consulting with developers of new GE foods to identify potential sources of differences, then reviewing a formal summary of data provided by the developer. Recently, the FDA has announced a new rule that would make pre-market consultation mandatory. The FDA has the authority to order foods to be pulled from the market at any time if are found to be unsafe, or to require labeling of any food that has different amounts of allergens, nutrients, or toxins than a consumer would expect to find in

3) *The Environmental Protection Agency* (EPA) evaluates the safety of any pesticides that are produced by genetically engineered plants. The EPA calls novel DNA and proteins genetically engineered into plants to protect them against pests "plant incorporated

Under the Coordinated Framework, some kinds of genetically engineered crops might not be subject to the oversight of all three agencies. For example, an ornamental flower like petunias engineered to have longer lasting blooms may only have to meet the requirements of APHIS, but a food crop like soybeans engineered to produce an insecticidal compound would be subject to the rules of all three agencies. Additional regulations are imposed by some states. Also, the National Institutes of Health has developed safety procedures for research with recombinant DNA. Most institutions developing genetically engineered crops

protectants" (PIPs) and regulates them the same way they regulate other pesticides.

follow the NIH guidelines, and they are required for federally funded research.

specific legislation. These agencies and their regulatory responsibilities are:

feed, and 6) EU-RL for polycyclic aromatic hydrocarbons.

review of the field testing data.

that kind of food.

#### **4. Labeling of genetically engineered foods**

GMO labelling was introduced to give consumers the freedom to choose between GMOs and conventional products. Essentially, if a foodstuff is produced using genetic engineering, this must be indicated on its label. The target of most labeling efforts is food products that were genetically engineered, that is, they contain genes artificially inserted from another organism.

Whether or not to require mandatory labeling of genetically engineered (GE) foods is a major issue in the debate over the risks and benefits of food crops produced using biotechnology. The issue is complex because 1) many arguments put forth in the debate are based on disagreements about the adequacy of our scientific understanding of the consequences of genetic engineering; and (2) significant changes to our current food marketing and manufacturing system, with potentially large economic impacts, would be required to implement mandatory labeling.

Actual labelling practice, however, is far more complicated, and must be planned and regulated with issues such as feasibility, legal responsibilities, coherence and standardisation in mind.

While some groups advocate the complete prohibition of GMOs, others call for mandatory labeling of genetically modified food or other products. Other controversies include the definition of patent and property pertaining to products of genetic engineering. According to the documentary Food, Inc. efforts to introduce labeling of GMOs has repeatedly met resistance from lobbyists and politicians affiliated with companies developing GM crops.

Governments around the world are hard at work to establish a regulatory process to monitor the effects of and approve new varieties of GM plants. Yet depending on the political, social and economic climate within a region or country, different governments are responding in different ways.

Agribusiness industries believe that labeling should be voluntary and influenced by the demands of the free market. If consumers show preference for labeled foods over nonlabeled foods, then industry will have the incentive to regulate itself or risk alienating the customer. Consumer interest groups, on the other hand, are demanding mandatory labeling.

Central to the arguments for mandatory labeling is that consumers have the right to know what they are eating. This is especially true for some products made with biotechnology where health and environmental concerns have not been satisfactorily resolved. Historically industry has proven itself to be unreliable at self-compliance with existing safety regulations. Some people do not wish to use genetically engineered products for religious or ethical reasons. Labeling is the only way consumers can make informed choices, whatever their reasons may be.

Major arguments against mandatory labeling have addressed the practical concerns about the expense and complex logistics that would be required to ensure GE and conventional foods are kept separate or to test all foods for GE content. It is argued that such measures are unnecessary since no significant differences have been found between today's GE foods and conventional foods. Enacting mandatory labeling will also require resolving certain other questions. Major issues include defining exactly what kinds of technologies would be covered, deciding on tolerance levels for genetically engineered content or ingredients before labeling would be required, and choosing a method for verifying that products are properly labeled.

In Europe, anti-GM food protestors have been especially active. In the last few years Europe has experienced two major foods scares: bovine spongiform encephalopathy (mad cow disease) in Great Britain and dioxin-tainted foods originating from Belgium. These food scares have undermined consumer confidence about the European food supply, and citizens are disinclined to trust government information about GM foods, establishing a mandatory food labeling of GM foods in stores, with a 1% threshold for contamination of unmodified foods with GM food products, a commonly proposed threshold. In other words, if any ingredient of a product exceeds one percent GM content, the product needs labeling. One percent is the labeling threshold decided upon by Australia and New Zealand. The European Union has decided on a level of 0.9 percent, while Japan has specified a five percent threshold. Thresholds as low as 0.01 percent (the approximate limit of detection) have been recommended (Davison, 2010).

The shift of global agriculture towards biotech varieties, however, has not been supported by all elements of society. In response to these differing levels of acceptance of the use of this technology, several countries have adopted regulations requiring that foods prepared from GM ingredients be labeled as such. However, labeling of foods is necessary only when the concentration of GM material in a food ingredient measures above a specified threshold concentration (%GM). The adoption and implementation of such laws can have significant consequences to global commerce in agriculture, food, and feed. Meeting these global market requirements for GM compliances is further complicated by the fact that each country has different regulations, including different GM ingredient thresholds for labeling and different methods of testing.

In the United States, the regulatory process is confused because there are three different government agencies that have jurisdiction over GM foods. The EPA evaluates GM plants for environmental safety, the USDA evaluates whether the plant is safe to grow, and the FDA evaluates whether the plant is safe to eat. The EPA is responsible for regulating substances such as pesticides or toxins that may cause harm to the environment. GM crops such as B.t. pesticide-laced corn or herbicide-tolerant crops but not foods modified for their nutritional value fall under the purview of the EPA. The USDA is responsible for GM crops that do not fall under the umbrella of the EPA such as drought-tolerant or disease-tolerant crops, crops grown for animal feeds, or whole fruits, vegetables and grains for human consumption. The FDA historically has been concerned with pharmaceuticals, cosmetics and food products and additives, not whole foods. Under current guidelines, a geneticallymodified ear of corn sold at a produce stand is not regulated by the FDA because it is a whole food, but a box of cornflakes is regulated because it is a food product. The FDA's stance is that GM foods are substantially equivalent to unmodified, "natural" foods, and therefore not subject to FDA regulation.

Independently of the government organism, there should be regulated the verification claims to know if a food is or is not genetically engineered. There are two ways this can be done: 1) Content-based verification requires testing foods for the physical presence of foreign DNA or protein. A current application of this type of procedure is the analysis and

covered, deciding on tolerance levels for genetically engineered content or ingredients before labeling would be required, and choosing a method for verifying that products are

In Europe, anti-GM food protestors have been especially active. In the last few years Europe has experienced two major foods scares: bovine spongiform encephalopathy (mad cow disease) in Great Britain and dioxin-tainted foods originating from Belgium. These food scares have undermined consumer confidence about the European food supply, and citizens are disinclined to trust government information about GM foods, establishing a mandatory food labeling of GM foods in stores, with a 1% threshold for contamination of unmodified foods with GM food products, a commonly proposed threshold. In other words, if any ingredient of a product exceeds one percent GM content, the product needs labeling. One percent is the labeling threshold decided upon by Australia and New Zealand. The European Union has decided on a level of 0.9 percent, while Japan has specified a five percent threshold. Thresholds as low as 0.01 percent (the approximate limit of detection)

The shift of global agriculture towards biotech varieties, however, has not been supported by all elements of society. In response to these differing levels of acceptance of the use of this technology, several countries have adopted regulations requiring that foods prepared from GM ingredients be labeled as such. However, labeling of foods is necessary only when the concentration of GM material in a food ingredient measures above a specified threshold concentration (%GM). The adoption and implementation of such laws can have significant consequences to global commerce in agriculture, food, and feed. Meeting these global market requirements for GM compliances is further complicated by the fact that each country has different regulations, including different GM ingredient thresholds for labeling

In the United States, the regulatory process is confused because there are three different government agencies that have jurisdiction over GM foods. The EPA evaluates GM plants for environmental safety, the USDA evaluates whether the plant is safe to grow, and the FDA evaluates whether the plant is safe to eat. The EPA is responsible for regulating substances such as pesticides or toxins that may cause harm to the environment. GM crops such as B.t. pesticide-laced corn or herbicide-tolerant crops but not foods modified for their nutritional value fall under the purview of the EPA. The USDA is responsible for GM crops that do not fall under the umbrella of the EPA such as drought-tolerant or disease-tolerant crops, crops grown for animal feeds, or whole fruits, vegetables and grains for human consumption. The FDA historically has been concerned with pharmaceuticals, cosmetics and food products and additives, not whole foods. Under current guidelines, a geneticallymodified ear of corn sold at a produce stand is not regulated by the FDA because it is a whole food, but a box of cornflakes is regulated because it is a food product. The FDA's stance is that GM foods are substantially equivalent to unmodified, "natural" foods, and

Independently of the government organism, there should be regulated the verification claims to know if a food is or is not genetically engineered. There are two ways this can be done: 1) Content-based verification requires testing foods for the physical presence of foreign DNA or protein. A current application of this type of procedure is the analysis and

properly labeled.

have been recommended (Davison, 2010).

and different methods of testing.

therefore not subject to FDA regulation.

labeling of vitamin content of foods. As the number of transgenes in commercialized crops increases, the techniques for detecting an array of different transgenes have become more sophisticated (Shrestha et al., 2008). 2) Process-based verification entails detailed recordkeeping of seed source, field location, harvest, transport, and storage (Sundstrom et al., 2002).

There are many questions that must be answered whether labeling of GM foods becomes mandatory:

First, it is concern about whether consumers are willing to absorb the cost of labeling. Accurate labeling requires an extensive identity preservation system from farmer to elevator to grain processor to food manufacturer to retailer (Maltsbarger & Kalaitzandonakes, 2000). If the food production industry is required to label GM foods, factories will need to construct two separate processing streams and monitor the production lines accordingly. Farmers must be able to keep GM crops and non GM crops from mixing during planting, harvesting and shipping. It is almost assured that industry will pass along these additional costs to consumers in the form of higher prices. Either testing or detailed recordkeeping needs to be done at various steps along the food supply chain. Estimates of the costs of mandatory labeling vary from a few dollars per person per year to 10 percent of a consumer's food bill (Gruere & Rao, 2007). Consumer willingness to pay for GE labeling information varies widely according to a number of surveys, but it is generally low in North America. Another potential economic impact for certain food manufacturers is that some consumers may avoid foods labelled as containing GE ingredients.

Secondly, the acceptable limits of GM contamination in non GM products. The EC has determined that 1% is an acceptable limit of cross-contamination, yet many consumer interest groups argue that only 0% is acceptable. In addition, it is necessary to know who is going to monitor these companies for compliance and what could be the penalty if they fail.

Third, concerns the level of traceability of GM food cross-contamination. Scientists agree that current technology is unable to detect minute quantities of contamination, so ensuring 0% contamination using existing methodologies is not guaranteed. Yet researchers disagree on what level of contamination really is detectable, especially in highly processed food products such as vegetable oils or breakfast cereals where the vegetables used to make these products have been pooled from many different sources. A 1% threshold may already be below current levels of traceability.

Finally, who should be responsible for educating the public about GM food labels. Food labels must be designed to contain clearly and accurate information about the product in simple language that everyone can understand. This may be the greatest challenge faced be a new food labeling policy: how to educate and inform the public without damaging the public trust and causing alarm or fear of GM food products.

Under current policy, the U.S. Food and Drug Administration do not automatically require all genetically engineered food to be labeled. Conventional and genetically engineered (GE) foods are all subject to the same labeling requirements, and both may require special labeling if particular food products have some property that is significantly different than what consumers might reasonably expect to find in that kind of food. Therefore, particular genetically engineered foods are subject to special labeling requirements if the FDA concludes they have significantly different properties including:


Examples of genetically engineered foods that require special labeling are those that contain vegetable oil made from varieties of GE soybeans and canola where the fatty acid composition of the oils extracted from the seeds of these crops was altered. Since the oils from these varieties have different nutritional properties than conventional soy and canola oils, foods made with them must be labeled to clearly indicate how they are different. You might see "high laurate canola" or "high oleic soybean" on food labels if these products were used. The FDA does not require them to be labeled as "genetically engineered", but that information could also be included on the label.

So far, no approved, commercially grown genetically engineered food crops have known properties that would require foods made from them to be labeled because they contain a new allergen or excess levels of toxic substances.

Federal legislation has been proposed that would require mandatory labeling of genetically engineered foods and similar initiatives at the state or local level have been considered or are currently pending.

#### **5. Conclusions**

The present atmosphere surrounding genetically engineered crops has led to a situation where food safety assessment is not just about science, but also about concerns, and standards about how to assure "safety." The detection, identification and quantification of the GMO content in food or feed products are a great challenge. The existing analytical methods for GMO testing leave the inspection authorities with many choices and compromises.

Methods, which can guarantee absence of non approved GMOs in seed samples even at the suggested 0.1% level of GM contamination does not exist at present. However, PCR and immunoassay based technologies are often used for the detection of products of agricultural biotechnology. They are valuable and reliable tools for the detection of GM products in seed production and very early in the food and feed supply chain. Concretely, when operated within specifications, immunoassays have been proven, in most cases, to be fast, reliable, and economic test methods.

It is critical that such methods are reliable and give the consistent results in laboratories across the world. This includes the need for a proper validation of the methods. The choice of the appropriate reference material will impact the reliability and accuracy of the analytical results, and numerous biological and analytical factors need to be taken into account when reporting results. Furthermore, as scientific opportunities advance, agreement on reasonable standards of safety for developing countries will be critical, and exchange of data as well, which will help ensure that data requirements are manageable across the developing world.

We don't know yet all the potential risks that the GMO could have, by long term accumulation, upon the environment. New strategies, therefore, are required to face the

2. A new allergen consumers would not expect to be in that kind of food (a hypothetical example would be an allergenic peanut protein in GE corn or some other crop),

Examples of genetically engineered foods that require special labeling are those that contain vegetable oil made from varieties of GE soybeans and canola where the fatty acid composition of the oils extracted from the seeds of these crops was altered. Since the oils from these varieties have different nutritional properties than conventional soy and canola oils, foods made with them must be labeled to clearly indicate how they are different. You might see "high laurate canola" or "high oleic soybean" on food labels if these products were used. The FDA does not require them to be labeled as "genetically engineered", but that

So far, no approved, commercially grown genetically engineered food crops have known properties that would require foods made from them to be labeled because they contain a

Federal legislation has been proposed that would require mandatory labeling of genetically engineered foods and similar initiatives at the state or local level have been considered or

The present atmosphere surrounding genetically engineered crops has led to a situation where food safety assessment is not just about science, but also about concerns, and standards about how to assure "safety." The detection, identification and quantification of the GMO content in food or feed products are a great challenge. The existing analytical methods for GMO testing leave the inspection authorities with many choices and

Methods, which can guarantee absence of non approved GMOs in seed samples even at the suggested 0.1% level of GM contamination does not exist at present. However, PCR and immunoassay based technologies are often used for the detection of products of agricultural biotechnology. They are valuable and reliable tools for the detection of GM products in seed production and very early in the food and feed supply chain. Concretely, when operated within specifications, immunoassays have been proven, in most cases, to be fast, reliable,

It is critical that such methods are reliable and give the consistent results in laboratories across the world. This includes the need for a proper validation of the methods. The choice of the appropriate reference material will impact the reliability and accuracy of the analytical results, and numerous biological and analytical factors need to be taken into account when reporting results. Furthermore, as scientific opportunities advance, agreement on reasonable standards of safety for developing countries will be critical, and exchange of data as well, which will help ensure that data requirements are manageable across the

We don't know yet all the potential risks that the GMO could have, by long term accumulation, upon the environment. New strategies, therefore, are required to face the

1. A different nutritional property from the same kind of conventional food,

3. a toxicant in excess of acceptable limits.

information could also be included on the label.

new allergen or excess levels of toxic substances.

are currently pending.

**5. Conclusions** 

compromises.

and economic test methods.

developing world.

continuing challenge of disease spread to new environments and emergence of resistancebreaking strains of microbial plant pathogens. Disease-protected transgenic plants may yet prove to be an important arsenal in the battle against plant pathogens. With a judicious approach and careful development of new innovations, molecular biology has the potential to contribute significantly to a better society in which the environment is respected and an adequate food supply is provided. Genetically-modified foods have the potential to solve many of the world's hunger and malnutrition problems, and to help protect and preserve the environment by increasing yield and reducing reliance upon chemical pesticides and herbicides. Yet there are many challenges ahead for governments, especially in the areas of safety testing, regulation, international policy and food labeling.

The achievements of the genetic engineering have nowadays considerable benefits, but now we don't know the price we, or the future generations, will have to pay for these benefits. The long term risks of the GMO are not entirely known today, and long-term studies are clearly necessary. We must proceed with caution to avoid causing unintended harm to human health and the environment as a result of our enthusiasm for this powerful technology.

Globalization of the agricultural industry inevitably results in globalization of markets, so competency in assuring food safety for GM crops is essential. This competency will enable countries to conduct independent research when necessary. Building such capacity also creates sufficient infrastructure to allow scientifically defensible decisions in the face of food safety questions colored by each country's perceptions and circumstances. It is obvious that international collaboration is needed to ensure that the methods offered by the different companies hold promises, which can be done by elaboration of:


In the future, the number of different GMOs is expected to grow, and research is going in the direction to develop GM plants with inducible promoters that activate specific traits when needed. It can be expected that detection of GMOs will become more complicated in the near future, being one of the mayor challenges for the future will be to develop analytical identification methods that facilitate screening for all the promoters used worldwide.

#### **6. Acknowledgment**

MCH-S thanks the Fulbright program and the Spanish Ministry of Education for a postdoctoral fellowship (FMECD-2010).

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### **Control of** *Salmonella* **in Poultry Through Vaccination and Prophylactic Antibody Treatment**

Anthony Pavic1,2,4, Peter J. Groves2,3 and Julian M. Cox4 *1Birling Avian Laboratories; 2School of Veterinary Science, University of Sydney; 3Zootechny; 4School of Biotechnology and Biomolecular Sciences, Faculty of Science, University of New South Wales Australia* 

#### **1. Introduction**

*Salmonella* are ubiquitous, host-adapted or zoonotic human and animal pathogens and, after *Campylobacter*, the genus is the second most predominant bacterial cause of foodborne gastroenteritis Worldwide (Giannella, 1996; Smith, 2003). Numerous foods (meat, seafood, eggs, fresh horticulture, table eggs and poultry) have been associated with *Salmonella* carriage or contamination (FAO/WHO, 2002; Jay *et al.*, 2003; Foley and Lynn, 2008).

Poultry meat has been associated frequently and consistently with the transmission to humans of enteric pathogens, including *Salmonella* and *Campylobacter* (Food and Agriculture Organisation of the United Nations and the World Health Organisation [FAO/WHO], 2002; Codex Alimentarius Commission, 2004; EFSA, 2007; Lutful Kabir, 2010; Vanderplas *et al.,*  2010 and Cox and Pavic, 2010). Moreover, Callaway *et al.* (2008) stated that the "link between human salmonellosis and host animals is most clear in poultry" and that raw eggs and undercooked poultry are considered by the entire community to be hazardous. Eggs have been implicated as vehicles in numerous outbreaks of salmonellosis; in particular, eggs have been a major vehicle of transmission of strains of *Salmonella* serovar Enteritidis, though the incidence of disease associated with this particular mode of transmission has decreased dramatically (Braden, 2006).

There have been large increases, in Australia and the United States of America (6.5 times), in *per capita* consumption of poultry products since 1910, compared with modest increases (20%) in consumption of beef and pork (Buzby and Farah 2006; ABARE, 2008). Chicken meat is close to replacing beef as Australia's preferred meat, largely because of the industry's success in reducing real (net of inflation) supply costs and hence price to consumers, and through continuous product innovation. The chicken meat industry has reduced supply costs by consistently raising both on-farm and off-farm productivity over several decades through a combination of better management, genetic improvement, economies of scale and mechanisation in processing (ABARE, 2008).

With projected increases in poultry consumption and the association of *Salmonella* with poultry products, the industry needs to employ specific and economic strategies to minimise risk to public health. The production of commercial poultry uses a pyramidal multiplier structure, with breeders at the apex and broilers at the base.

The two broad approaches to *Salmonella* control are 'top down' and 'bottom up', with the latter used widely in Australia. This approach uses spin chiller chlorination within the processing plant for reduction of microbial populations. This method has limited success in reduction of *Salmonella* prevalence and, with consumer choices moving towards 'natural' (chemical free) products, has lead to experimentation with and application of 'top down' intervention strategies. A 'top down' approach, using vaccination at breeder level, can be very cost-effective and is a proven method of flock protection for both viral and bacterial pathogens such as Infectious Bursal Disease Virus, Inclusion Body Hepatitis and *Mycoplasma*  (Marangon and Busani, 2007).

The increasing costs or impracticality of improvements in biosecurity, hygiene and management, coupled with the increasing problems associated with antibiotic resistance, suggests that vaccination in poultry will become more attractive as an adjunct to existing control measures (Zhang-Barber *et al.,* 1999).

Although *Salmonella* is typically not a pathogen in the gut of the chicken, systemic infection can cause serious disease (fowl paratyphoid) in the bird (Lutful Kabir, 2010). This usually results from contamination of egg shells from infected breeder hens and spread of the organism into the respiratory tract during hatching which may result in potentially high morbidity and mortality in young chicks (Lutful Kabir, 2010).

*Salmonella* vaccines available to the poultry industry are based on inactivated or killed cells or genetically attenuated strains (Methner *et al.,* 1999; Babu *et al.,* 2003). However, with concerns in Australia over the use of genetically modified organisms in the food chain, until 2008, only inactivated vaccines were available for use (Anonymous, 1999).

Vaccination against *Salmonella* was first demonstrated to be successful in decreasing poultry mortality due to the poultry-specific *Salmonella* serovar Gallinarum (Smith, 1956). This success, and the Enteritidis disaster in table eggs during the late 1980s that occurred in Europe and the United Kingdom, lead to the development of a *Salmonella* Enteritidis vaccine, which reduced the prevalence of the serovar in poultry. This was followed by a combined (dual) vaccine containing serovars Enteritidis and Typhimurium that again aided in the reduction of carriage in flocks (Clifton-Hadley *et al.,* 2002; Woodward *et al*., 2002).

Van Den Bosch (2003) reported success with inactivated Salenvac® vaccines in the European poultry industry, which has been associated with a decline in the presence of *S*. Enteritidis and Typhimurium in England. Salenvac®T is a bivalent *S.* Typhimurium and *S.* Enteritidis bacterin-based vaccine, developed from the monovalent Salenvac® *S.* Enteritidis bacterin (Clifton-Hadley *et al.,* 2002).

Based on these studies, production of a trivalent vaccine was initiated in 2004 in association with Intervet Schering-Plough Australia Pty Limited, incorporating the three most predominant *Salmonella* serovars, Typhimurium (group B), Mbandaka (group C) and Orion (group E), from a commercial poultry meat producer, determined through in-house analysis of environmental samples from breeder houses.

Australian poultry companies need to ensure that their operations are free of the specific serogroup D serovars that are present in poultry, including the host-specific chicken pathogens, Gallinarum and Pullorum (Barrow *et al*., 1994) as well as Enteritidis (Arzey, 2005), a serovar of significant public health concern. This is achieved by ongoing monitoring of hatcheries, feed mills and poultry rearing houses, and culling all positive flocks; therefore these serovars are no longer endemic, with only sporadic cases in Australian broiler and layer flocks (Davos, 2007).

On the other hand, *Salmonella* Typhimurium, which is associated typically with at least 35% of human cases of salmonellosis in Australia, is present in 14% of broiler and 17% of layers isolates (Davos, 2007). Furthermore, other predominant *Salmonella* serovars isolated from broilers and layers include Sofia (38 % broiler, 0% layer), Infantis (10% broiler, 8.9% layer), Montevideo (8.8% broiler, 5.7% layer), Muenster (7.8% broiler, 0% layer) and Mbandaka (<1.7% broilers, 10.5% layers), (Davos, 2007).

The vaccinated or naturally challenged hen protects her progeny through maternal antibodies (IgY) transported into the egg yolk (Kowalczyk *et al.,* 1985). These maternal antibodies offer some protection to the hatchling until its own immune system is fully functioning (Kowalczyk *et al.,* 1985). The IgY suspended in egg yolk can be easily extracted via chemical methods, and purified (Ko and Ahn, 2007). Therefore *Salmonella* vaccinated hens can transfer to the yolk IgY that can be extracted, measured and used prophylactically (in feed) to protect young chicks from initial *Salmonella* colonisation (Vanderplas *et al.,* 2010; Cox and Pavic, 2010).

The objectives of this research were to develop an autogenous tri-vaccine to prevalent poultry serovars, test whether heterologus protection is possible, and if specific anti-*Salmonella* IgY, extracted from the eggs from vaccinated hens, confers prophylactic protection to day old chicks.

### **2. Material and methods**

The methods summarised in this section are divided into three sections, with general, vaccine trial and prophylactic methods individually described.

#### **2.1 General methods**

86 Biochemical Testing

With projected increases in poultry consumption and the association of *Salmonella* with poultry products, the industry needs to employ specific and economic strategies to minimise risk to public health. The production of commercial poultry uses a pyramidal multiplier

The two broad approaches to *Salmonella* control are 'top down' and 'bottom up', with the latter used widely in Australia. This approach uses spin chiller chlorination within the processing plant for reduction of microbial populations. This method has limited success in reduction of *Salmonella* prevalence and, with consumer choices moving towards 'natural' (chemical free) products, has lead to experimentation with and application of 'top down' intervention strategies. A 'top down' approach, using vaccination at breeder level, can be very cost-effective and is a proven method of flock protection for both viral and bacterial pathogens such as Infectious Bursal Disease Virus, Inclusion Body Hepatitis and *Mycoplasma* 

The increasing costs or impracticality of improvements in biosecurity, hygiene and management, coupled with the increasing problems associated with antibiotic resistance, suggests that vaccination in poultry will become more attractive as an adjunct to existing

Although *Salmonella* is typically not a pathogen in the gut of the chicken, systemic infection can cause serious disease (fowl paratyphoid) in the bird (Lutful Kabir, 2010). This usually results from contamination of egg shells from infected breeder hens and spread of the organism into the respiratory tract during hatching which may result in potentially high

*Salmonella* vaccines available to the poultry industry are based on inactivated or killed cells or genetically attenuated strains (Methner *et al.,* 1999; Babu *et al.,* 2003). However, with concerns in Australia over the use of genetically modified organisms in the food chain, until

Vaccination against *Salmonella* was first demonstrated to be successful in decreasing poultry mortality due to the poultry-specific *Salmonella* serovar Gallinarum (Smith, 1956). This success, and the Enteritidis disaster in table eggs during the late 1980s that occurred in Europe and the United Kingdom, lead to the development of a *Salmonella* Enteritidis vaccine, which reduced the prevalence of the serovar in poultry. This was followed by a combined (dual) vaccine containing serovars Enteritidis and Typhimurium that again aided in the reduction of carriage in flocks (Clifton-Hadley *et al.,* 2002; Woodward *et al*., 2002).

Van Den Bosch (2003) reported success with inactivated Salenvac® vaccines in the European poultry industry, which has been associated with a decline in the presence of *S*. Enteritidis and Typhimurium in England. Salenvac®T is a bivalent *S.* Typhimurium and *S.* Enteritidis bacterin-based vaccine, developed from the monovalent Salenvac® *S.* Enteritidis bacterin

Based on these studies, production of a trivalent vaccine was initiated in 2004 in association with Intervet Schering-Plough Australia Pty Limited, incorporating the three most predominant *Salmonella* serovars, Typhimurium (group B), Mbandaka (group C) and Orion (group E), from a commercial poultry meat producer, determined through in-house analysis

structure, with breeders at the apex and broilers at the base.

(Marangon and Busani, 2007).

(Clifton-Hadley *et al.,* 2002).

of environmental samples from breeder houses.

control measures (Zhang-Barber *et al.,* 1999).

morbidity and mortality in young chicks (Lutful Kabir, 2010).

2008, only inactivated vaccines were available for use (Anonymous, 1999).

The methods summarised in this section describe the common techniques that were used throughout the experimental work. These methods include: the *Salmonella* strains, challenge suspension and isolation testing; blood and yolk collection and their subsequent testing with the anti-Typhimurium ELISA; trial farm setup and the animal ethics requirements.

#### **2.1.1** *Salmonella* **strains**

All *Salmonella enterica* subsp. *enterica* strains used in this research (Table 1) were from Birling Avian Laboratories Reference Collection, isolated from the field (production environment), and typed by the Institute of Medical and Veterinary Science (IMVS) in accordance with the White-Kauffmann-Le Minor scheme (Grimont and Weill, 2007).


Table 1. *Salmonella* challenge strains belonging to serogroups B, C and E with their respective abbreviation used in the text.

Pure cultures on nutrient agar (NA, Oxoid Thermo Fisher, CM3) were harvested with a cotton swab (Copan, Ref 8155CIS, Italy.) and preserved (10 per serovar) in Cryovials (PRO-LAB Diagnostic, REFPL.170/M, Ontario, Canada) stored at -70ºC.

#### **2.1.2** *Salmonella* **suspension for challenge**

For each *Salmonella* strain, a bead from a Cryovial (PRO-LAB Diagnostic, REFPL.170/M, Ontario, Canada) was incubated in 100 mL of buffered peptone water (BPW, Oxoid Thermo Fisher, CM509, Hampshire, UK) to produce a seed culture. Purity of the culture was checked on NA and identity confirmed serologically using antisera (PRO-LAB Diagnostic Ontario, Canada; Refs TL6002 [O], TKL6001 [H], RL6011-04 [B], PL6013 [C] and PL6017 [E]).

After purity was confirmed and serology determined, isolated colonies were selected and suspended in BPW (4 mL) to give a 75% transmittance (1.0 McFarland) equating to 2 x 108 cfu / mL (bioMérieux, 47100-00 DR 100 Colorimeter, Marcy l'Etoile, France). The target dosage required was achieved through decimal dilution, with the target dilution used as inocula in challenge or recovery experiments, in accordance with AS/NZS5013.11.1 (2004) and confirmed by spread plate enumeration on SM®ID2 (bioMérieux, Ref 43621, Marcy l'Etoile, France).

#### **2.1.3** *Salmonella* **isolation, confirmation and serological testing**

All testing was performed at a laboratory accredited by the National Association of Testing Authorities (Australia) in accordance with ISO 6579:2002. The *Salmonella* serovars, listed in Section 2.1.1, were isolated from commercial poultry houses (from visceral emulsions, drag swabs and faeces) on the Eastern seaboard of Australia.

Samples were initially emulsified 1:10 in BPW and incubated at 37 ºC for 24 h (standard incubation temperature unless otherwise stated). Aliquots of 1000 µL and 100 µL were transferred into selective Muller Kauffman (MK, bioMérieux, Ref 42114, Marcy l'Etoile, France) and Rappaport-Vassiliadis (RV, bioMérieux, Ref 42110) (incubated at 42 °C for 24 h) broths respectively.

The following validated modifications to ISO 6579:2002 were used. The selective-differential plating agars were Hektoen and XLD (Oxoid Thermo Fisher, PP2027, Adelaide, Australia) and suspect positives (black colonies with clear edges) were confirmed on chromogenic SM®ID2 instead of the standard biochemical tests (urease, sorbitol fermentation and iron agar reactions).

Presumptive *Salmonella* were serologically confirmed with poly-O and poly-H antisera (Pro-Labs Diagnostic, Refs TL6002 and TKL6001, Ontario, Canada) after subculture onto two slopes of NA and employing the slide agglutination technique. The confirmed *Salmonella* isolates (each on a NA slope) were forwarded to the Australian *Salmonella* Reference Laboratory at the Insitute of Medical and Veterinary Science (IMVS) for complete serological and phage typing.

#### **2.1.4 Blood collection**

88 Biochemical Testing

**Serogroup Antigenic structure Serovar Abbreviation**  B 1,4,[5],12 f,g,s [1,2] [z27],[z45] Agona SA1 B 1, 4 ,[5],12 i 1,2 Typhimurium ST12 C 6,7,14, r 1, 5 [R1..], [z37], [z45], [z49] Infantis SI1 C 6,7,14 z10 e,n,z15 [z37],[z45] Mbandaka SM1 E 3, {10}, {15, 34}, y 1, 5 Orion SO1 E 3,{10}{15} k 1,5 Zanzibar SZ1

Pure cultures on nutrient agar (NA, Oxoid Thermo Fisher, CM3) were harvested with a cotton swab (Copan, Ref 8155CIS, Italy.) and preserved (10 per serovar) in Cryovials (PRO-

For each *Salmonella* strain, a bead from a Cryovial (PRO-LAB Diagnostic, REFPL.170/M, Ontario, Canada) was incubated in 100 mL of buffered peptone water (BPW, Oxoid Thermo Fisher, CM509, Hampshire, UK) to produce a seed culture. Purity of the culture was checked on NA and identity confirmed serologically using antisera (PRO-LAB Diagnostic Ontario,

After purity was confirmed and serology determined, isolated colonies were selected and suspended in BPW (4 mL) to give a 75% transmittance (1.0 McFarland) equating to 2 x 108 cfu / mL (bioMérieux, 47100-00 DR 100 Colorimeter, Marcy l'Etoile, France). The target dosage required was achieved through decimal dilution, with the target dilution used as inocula in challenge or recovery experiments, in accordance with AS/NZS5013.11.1 (2004) and confirmed by spread plate enumeration on SM®ID2 (bioMérieux, Ref 43621, Marcy

All testing was performed at a laboratory accredited by the National Association of Testing Authorities (Australia) in accordance with ISO 6579:2002. The *Salmonella* serovars, listed in Section 2.1.1, were isolated from commercial poultry houses (from visceral emulsions, drag

Samples were initially emulsified 1:10 in BPW and incubated at 37 ºC for 24 h (standard incubation temperature unless otherwise stated). Aliquots of 1000 µL and 100 µL were transferred into selective Muller Kauffman (MK, bioMérieux, Ref 42114, Marcy l'Etoile, France) and Rappaport-Vassiliadis (RV, bioMérieux, Ref 42110) (incubated at 42 °C for 24 h)

The following validated modifications to ISO 6579:2002 were used. The selective-differential plating agars were Hektoen and XLD (Oxoid Thermo Fisher, PP2027, Adelaide, Australia) and suspect positives (black colonies with clear edges) were confirmed on chromogenic SM®ID2 instead of the standard biochemical tests (urease, sorbitol fermentation and iron

Canada; Refs TL6002 [O], TKL6001 [H], RL6011-04 [B], PL6013 [C] and PL6017 [E]).

Table 1. *Salmonella* challenge strains belonging to serogroups B, C and E with their

LAB Diagnostic, REFPL.170/M, Ontario, Canada) stored at -70ºC.

**2.1.3** *Salmonella* **isolation, confirmation and serological testing** 

swabs and faeces) on the Eastern seaboard of Australia.

respective abbreviation used in the text.

**2.1.2** *Salmonella* **suspension for challenge** 

l'Etoile, France).

broths respectively.

agar reactions).

Blood was collected using a 21-gauge needle (Greiner bio-one, 450072, Germany.) into a 2 mL serum collection tube (Vacuette, 454096, Greiner bio-one, Austria) and, after clotting, transported to the laboratory in chilled containers. The serum was decanted into 1200 μL plasma tubes (Scientific Specialist, 1750-00, CA., USA) and the presence of antibodies to the vaccine was determined using a commercially available *Salmonella* Typhimurium ELISA kit (Guildhay, {trading as x-OVO since 2008, Castle Court, UK}, Flockscreen™, Cat. No V020- 43308), according to the manufacturer's instructions (2.1.7).

Initially, the serum was diluted (1:500) by adding 5 μL of serum into 2.5 mL reconstituted sample diluent (2.1.7) in plastic 5 mL tubes (Techno-Plas, 10255001, SA, Aust) and inverted a total of three times to mix.

#### **2.1.5 Egg yolk collection**

Eggs were collected after laying by mature hens and transported whole to the lab where they were broken and the yolk separated from the white using a domestic egg separator. The yolk was then poured into a 70 mL sterile jar (Techno-Plas, 10431011, SA, Aust) from which a 200 μL aliquot was removed and added to 1.8 mL of reconstituted wash buffer and mixed by repeated (5 x) aspiration. The yolk was further diluted (1:50) by pipetting 50 μL into 2.5 mL of reconstituted sample diluent buffer (2.1.7).

#### **2.1.6 Immunoglobulin Y extraction**

Only Typhimurium ELISA-positive or suspect (2.1.7) egg yolks from Section 2.1.5 were used for IgY extraction. The method selected was the water dilution method described by Staak *et al.* (2001). Initially, the weighed ELISA positive egg yolks were diluted 1:5 w/v with distilled water, mixed vigorously by vortexing (15 s) and frozen at -20 ºC for 72 h. Postfreezing the yolk suspension was thawed slowly in a refrigerator at 4 ºC.

The thawed yolk water suspension was transferred into centrifuge tubes (50 mL) (Greiner®, T2318, Sigma-Aldrich, St Louis, MO, USA) and centrifuged (Eppendorf, 5810R, Hamburg, Germany) at 2,800 x *g* (Equation 1) for 20 minutes at room temperature.

> RCF = 1.118 x 10-5*r*N2 *g* = *r*(2πN)2 / RCF RCF = Relative centrifugal force. *r* = rotation radius in centimetres. N = revolution per minute *g =* gravitational force π = *Pi*

Equation 1. Calculation of centrifugal *g* forces.

Post-centrifugation, the supernatant was decanted into a volumetric cylinder and the precipitate discarded. To each millilitre of supernatant, 0.27 g of ammonium sulphate ('salt') was added, mixed by vortexing (15 s) and incubated at room temperature for 2 h. Postincubation the 'salt'-yolk suspension was centrifuged as mentioned previously and the supernatant was discarded.

The precipitate containing IgY was resuspended, in 24 mL of ammonium sulphate (2 M), vortexed (15 s) and incubated at room temperature for 40 min. After incubation, the saltyolk suspension was re-centrifuged and the supernatant discarded. The final precipitate of crude IgY was resuspended (vortex 15 s) in 5 mL of phosphate buffered saline, transferred to a 10 mL sealable test tube (Techno-Plas, 10281003, SA, Aust.) and stored at 4 ºC.

#### **2.1.7 Typhimurium ELISA method**

The ELISA was performed in accordance with the manufacturer's instructions (Guildhay, Castle Court, UK). Briefly, kits were allowed to reach room temperature and the wash buffer (100 mL {phosphate buffer with ProClin 0.63 % v/v} to 1900 mL deionised water) and sample diluent (100 mL {phosphate buffer with protein stabiliser and ProClin 0.63% v/v} to 900 mL deionised water) prepared.

Into individually pre-coated (Typhimurium somatic liposaccaride antigen) ELISA strips containing eight wells, 50 μL was dispensed of positive controls (x2), followed by negative controls (x2) and finally the test sample(s). The ELISA plate(s) were covered with an adhesive plastic film (Sealplate, 100-seal-PLT, Excel Scientific Inc, Victorville, CA, USA), mixed by gently tapping the side and then incubated at 37 ºC for 30min.

Post-incubation, after removal of the adhesive cover, each well was washed four times (100 μL per well/per wash cycle) in a pre-programmed ELISA Plate washer (Immunowash, 1575, BioRad, CA, USA) with the reconstituted wash buffer. After washing, the plates were dried (five firm taps) by inverting them over paper towel. Once dry, 50 μL of antibody-enzyme conjugate (donkey anti-chicken IgG {Guildhay, Castle Court, UK}) was pipetted into each well. The plate was resealed, mixed and incubated as described previously.

Following incubation, the wells were washed as above, then 50 μL of ELISA substrate reagent (alkaline phenolphthalein monophosphate and enzyme co-factors in a diethanolamine buffer) were added, and the plates covered, mixed and incubated at 37 ºC for 15min. The final step was to add 50 μL of ELISA stop solution (1 M sodium hydroxide), ensuring that any bubbles formed were removed, then the plates were analysed in a blanked Microtitre Plate Reader (Vmax Kinetic microplate reader, Molecular Devices, CA, USA) at λ550 nm.

The Guildhay ELISA can detect anti-Typhimurium antibodies, within serum or egg yolk, at an initial dilution of 1:500. After the optical density (OD) all wells was measured at λ550 nm, the sample OD was compared to the mean positive controls OD (Equation 2) to produce the Sample / Positive (S/P) ratio. Using Equation 3 the S/P ratio was converted into a titre.

```
Sample Optical Density – mean Negative Optical Density 
Mean of Positive Optical Density – mean Negative Optical Density
```
Equation 2. Sample to Positive ratio calculation.

log10 titre = 1.046 x ( log10 S/P) + 3.524 Titre = antilog of log10 titre

Equation 3. *Salmonella* Typhimurium titre calculation from S/P ratio.

The Typhimurium titres were initially calculated manually, though software (Guildhay, Castle Court, UK) is available that performs all the calculations and reports the values as OD, SP or titres. According to the kit manufacturer (Guildhay) the lowest threshold for a positive was an SP ratio > 0.25 (titre > 785, OD > 0.173), negative SP < 0.15 (titre < 459, OD < 0.15) and a suspect band SP 0.15 to 0.25 (titre 460 to 784, OD 0.15 to 0.173).

#### **2.1.8 Trial farm set up**

90 Biochemical Testing

Post-centrifugation, the supernatant was decanted into a volumetric cylinder and the precipitate discarded. To each millilitre of supernatant, 0.27 g of ammonium sulphate ('salt') was added, mixed by vortexing (15 s) and incubated at room temperature for 2 h. Postincubation the 'salt'-yolk suspension was centrifuged as mentioned previously and the

The precipitate containing IgY was resuspended, in 24 mL of ammonium sulphate (2 M), vortexed (15 s) and incubated at room temperature for 40 min. After incubation, the saltyolk suspension was re-centrifuged and the supernatant discarded. The final precipitate of crude IgY was resuspended (vortex 15 s) in 5 mL of phosphate buffered saline, transferred

The ELISA was performed in accordance with the manufacturer's instructions (Guildhay, Castle Court, UK). Briefly, kits were allowed to reach room temperature and the wash buffer (100 mL {phosphate buffer with ProClin 0.63 % v/v} to 1900 mL deionised water) and sample diluent (100 mL {phosphate buffer with protein stabiliser and ProClin 0.63% v/v} to

Into individually pre-coated (Typhimurium somatic liposaccaride antigen) ELISA strips containing eight wells, 50 μL was dispensed of positive controls (x2), followed by negative controls (x2) and finally the test sample(s). The ELISA plate(s) were covered with an adhesive plastic film (Sealplate, 100-seal-PLT, Excel Scientific Inc, Victorville, CA, USA),

Post-incubation, after removal of the adhesive cover, each well was washed four times (100 μL per well/per wash cycle) in a pre-programmed ELISA Plate washer (Immunowash, 1575, BioRad, CA, USA) with the reconstituted wash buffer. After washing, the plates were dried (five firm taps) by inverting them over paper towel. Once dry, 50 μL of antibody-enzyme conjugate (donkey anti-chicken IgG {Guildhay, Castle Court, UK}) was pipetted into each

Following incubation, the wells were washed as above, then 50 μL of ELISA substrate reagent (alkaline phenolphthalein monophosphate and enzyme co-factors in a diethanolamine buffer) were added, and the plates covered, mixed and incubated at 37 ºC for 15min. The final step was to add 50 μL of ELISA stop solution (1 M sodium hydroxide), ensuring that any bubbles formed were removed, then the plates were analysed in a blanked Microtitre Plate Reader (Vmax Kinetic microplate reader, Molecular Devices, CA, USA) at

The Guildhay ELISA can detect anti-Typhimurium antibodies, within serum or egg yolk, at an initial dilution of 1:500. After the optical density (OD) all wells was measured at λ550 nm, the sample OD was compared to the mean positive controls OD (Equation 2) to produce the Sample / Positive (S/P) ratio. Using Equation 3 the S/P ratio was converted into a titre.

> Sample Optical Density – mean Negative Optical Density Mean of Positive Optical Density – mean Negative Optical Density

mixed by gently tapping the side and then incubated at 37 ºC for 30min.

well. The plate was resealed, mixed and incubated as described previously.

to a 10 mL sealable test tube (Techno-Plas, 10281003, SA, Aust.) and stored at 4 ºC.

supernatant was discarded.

**2.1.7 Typhimurium ELISA method** 

900 mL deionised water) prepared.

Equation 2. Sample to Positive ratio calculation.

λ550 nm.

The trial house used was an insulated broiler house with side curtains for ventilation control, equipped with two gas-fired space heaters for brooding, and internal fans and roof sprinklers for cooling. The house was divided into 32 individually numbered floor pens, 2.5 x 3 m, with individual bell drinkers and two tube feeders.

To minimise cross-contamination and identify the presence of non-inoculant *Salmonella* the following procedure was used in all trials. Prior to the trial all pens were disinfected using a synthetic phenol (Farm Fluid™, Antec International, Suffolk, UK), drag-swabbed and tested for the presence of *Salmonella*. Fresh litter (wood shavings) was spread evenly across each pen and drag-swabbed again. A footbath, containing a commercially available iodophorbased sanitizer (Sanichick, Ranvet, Sydney, Australia), was placed outside each of the pens. An empty pen was left between all populated pens, with controls located furthest from the entrance; all routine maintenance started from the controls and worked backwards. Vermin baits, pest strips and an Insectocuter were placed around the house.

The pens were divided into treatment sets, i.e. vaccination, (north side of house) and control, i.e. non-vaccinated (south side of house), with feed and water supplied *ad libitum*. Disposable overalls (Fabri-cell, 05250XL, Vic. Aust.), dust masks (3M, 9320, UK) and gloves (Livingstone Int, GLVLPF100LG-T, NSW, Aust.) were worn inside the house at all times and changed between treatment and control groups. Biohazard bags (Bacto, BCWB66112, Sydney, Aust.) were used to remove contaminated waste, litter and to transport culled or naturally deceased chickens. Hands were washed with an iodophor-based disinfectant, prior to leaving the house. Post-trial the house was disinfected with phenolic-based chemicals and drag-swabbed.

At the termination date, the chickens were euthanized by lethal intraperitoneal injection with 0.5 mL/kg pentobarbitone (Lethabarb®, Virbac Pty Ltd, 1PO643-1, Carros Cedex, France). The carcasses were transported in biohazard bags back to the laboratory and the caeca were removed aseptically by a veterinarian, cut into ten pieces and placed into a single sterile 250 mL sample jar (Techno-Plas, 10453003, SA, Aust.), diluted as per specific procedure with BPW, then incubated for detection as described in Section 2.1.3.

#### **2.1.9 Animal ethics**

The Birling Animal Ethics Committee (BAEC), supervised all experimental work in accordance with the Animal Research Act of NSW (1985) and Regulations (2005), following the NHMRC (National Health and Medical Research Council) guidelines (2008) and NHMRC/ARC (National Health and Medical Research Council, Australian Research Council and Universities Australia) Code of Conduct (2007). When the project was approved, it was designated with a unique BAEC number and a time period for completion.

#### **2.2 Vaccine trial**

This section describes the process from the original controlled animal pen to field trials, which occurred over a period of three years.

#### **2.2.1 Vaccine manufacture and administration**

Intervet Schering-Plough Australia was commissioned to produce an autologous trivalent inactivated vaccine using proprietary Salenvac® technology from poultry field isolates of Typhimurium, Mbandaka and Orion. Strains were grown on iron-depleted agar, improving specifically the expression of the antigenic iron regulatory proteins (IRPs), further stimulating the humoral response and increasing antibody titres (Van Den Bosch, 2003). The trivalent vaccine was produced using equal amounts of cell suspension (3x108 cfu / mL), combined with an aluminium hydroxide adjuvant, and administered to hens intramuscularly into the breast, at 12 and 17 weeks of age.

#### **2.2.2 Experimental animals**

In Experiment 1 a total of 50 vaccinated and 50 non-vaccinated, 20-week-old Cobb breeders were placed into trial pens at a minimum of 12 per group, with the remainder used as negative controls. These were obtained from a commercial broiler breeder farm where the vaccine regimes were administered under commercial conditions. All birds where individually labelled with leg or wing tags and blood and rectal swabs collected and tested five days prior to challenge.

In Experiment 2 a total of 100 non-vaccinated, 12 week old Cobb breeders were placed into the trial pens (12 per group) as mentioned previously. In Experiment 3 Cobb broiler day old chicks (*n* = 100) were sourced from a commercial hatchery from vaccinated (*n* = 50) and nonvaccinated parents (*n* = 50).

#### **2.2.3 Adult hen challenge design and trial (experiment 1 and 2)**

The number of birds required for each experiment was calculated using statistical tables as described by Martin *et al.,* (1988) to determine the lowest number of repeats required to demonstrate a difference of 20% prevalence between vaccinated (expected 30% prevalence) and non-vaccinated (expected 50% prevalence) groups, at 90% confidence.

As this study involved a new vaccine, the standard deviation of titres following vaccination was unknown. Therefore, 12 birds per group allowed estimation of the average result within a bound of 0.5 x standard deviation from a flock of >300 birds, at 90% confidence (Hancock *et al.,* 1988).

Each trial required the use of only half the trial house (2.1.8) and therefore the other half was sectioned off. The controls were placed furthermost away from the entrance to minimise any cross-contamination. The challenge groups were placed in pens that were opposite each other. Each bird was challenged with 250 µl of the 107 cfu *Salmonella* suspension via oral gavage using a 2 mL variable volume pipette (Finnpipette stepper, 4540000, Thermo Electron Corp. Waltham, MA, USA).

The initial experiment (Experiment 1) involved autologous challenge (*i.e.* with the parent vaccine strains) and the subsequent experiment (Experiment 2) involved heterologous challenge, using alternative, poultry-associated serovars from the same respective serogroups as the vaccine strains: Agona (SA1, serogroup B); Infantis (SI1, serogroup C), and; Zanzibar (SZ1, serogroup E). An additional group, challenged with Typhimurium, was used to show repeatability. Cloacal swabs were taken at days 0, 3 and 14 post-challenge. At day 21 post-challenge each bird was bled, humanely euthanized and their caeca removed for culture.

Prior to the heterologous (Experiment 2) trial, the hens were sourced as 10-week old birds, prior to vaccination (12 weeks of age), from a commercial broiler breeder farm that was *Salmonella*-free (confirmed by testing of drag swabs and faeces). These hens were individually tagged and bled, then hand-vaccinated via intra-muscular injection and bled again at 14, 16, 18 and 20 weeks of age.

#### **2.2.4 Progeny challenge trial (experiment 3)**

In this trial, 100 chicks were obtained from a commercial hatchery, from eggs produced by vaccinated (*n =* 50) and non-vaccinated birds (*n =* 50). The chicks were vaccinated according to current broiler practices (Marek's Disease, Infectious Bronchitis and Newcastle Disease). Chicks were identified as to their dams' vaccination status by toe web marking and were placed in the trial house at 10 chicks per pen. Pen dividers were used to keep birds close to feed and water, with 2 pens of chicks for each progeny group and challenge (total of 10 pens).

At arrival, blood samples were collected from 12 euthanized (cervical dislocation) chicks of vaccinated and non-vaccinated groups, for *S*. Typhimurium antibody assay (2.1.7). The paper from each box of chicks delivered to the farm was cultured for *Salmonella* as described in Section 2.1.3. The remaining chicks were challenged with *S*. Typhimurium ST12, 104 or 108 cfu per bird by oral gavage, with controls receiving the diluent buffered peptone water

On days 0, 3 and 14, cloacal swabs were collected, from a random sample of five birds per pen, for individual *Salmonella* culture, with blood samples collected via wing bleeding, on days 7, 10 and 14. On day 21 all birds were bled prior to being euthanized by lethal injection (Lethabarb) and their caeca removed for *Salmonella* testing. All birds were weighed on days 7, 14 and 21.

Any bird that appeared sick, as described in the bird health monitoring sheet, was euthanized immediately, weighed, necropsied and the caeca cultured for *Salmonella* (2.1.3).

#### **2.2.5 Serology**

92 Biochemical Testing

NHMRC/ARC (National Health and Medical Research Council, Australian Research Council and Universities Australia) Code of Conduct (2007). When the project was approved, it was designated with a unique BAEC number and a time period for completion.

This section describes the process from the original controlled animal pen to field trials,

Intervet Schering-Plough Australia was commissioned to produce an autologous trivalent inactivated vaccine using proprietary Salenvac® technology from poultry field isolates of Typhimurium, Mbandaka and Orion. Strains were grown on iron-depleted agar, improving specifically the expression of the antigenic iron regulatory proteins (IRPs), further stimulating the humoral response and increasing antibody titres (Van Den Bosch, 2003). The trivalent vaccine was produced using equal amounts of cell suspension (3x108 cfu / mL), combined with an aluminium hydroxide adjuvant, and administered to hens

In Experiment 1 a total of 50 vaccinated and 50 non-vaccinated, 20-week-old Cobb breeders were placed into trial pens at a minimum of 12 per group, with the remainder used as negative controls. These were obtained from a commercial broiler breeder farm where the vaccine regimes were administered under commercial conditions. All birds where individually labelled with leg or wing tags and blood and rectal swabs collected and tested

In Experiment 2 a total of 100 non-vaccinated, 12 week old Cobb breeders were placed into the trial pens (12 per group) as mentioned previously. In Experiment 3 Cobb broiler day old chicks (*n* = 100) were sourced from a commercial hatchery from vaccinated (*n* = 50) and non-

The number of birds required for each experiment was calculated using statistical tables as described by Martin *et al.,* (1988) to determine the lowest number of repeats required to demonstrate a difference of 20% prevalence between vaccinated (expected 30% prevalence)

As this study involved a new vaccine, the standard deviation of titres following vaccination was unknown. Therefore, 12 birds per group allowed estimation of the average result within a bound of 0.5 x standard deviation from a flock of >300 birds, at 90% confidence (Hancock

Each trial required the use of only half the trial house (2.1.8) and therefore the other half was sectioned off. The controls were placed furthermost away from the entrance to minimise any cross-contamination. The challenge groups were placed in pens that were opposite each

**2.2 Vaccine trial** 

**2.2.2 Experimental animals** 

five days prior to challenge.

vaccinated parents (*n* = 50).

*et al.,* 1988).

which occurred over a period of three years.

**2.2.1 Vaccine manufacture and administration** 

intramuscularly into the breast, at 12 and 17 weeks of age.

**2.2.3 Adult hen challenge design and trial (experiment 1 and 2)** 

and non-vaccinated (expected 50% prevalence) groups, at 90% confidence.

Blood was collected, using a 20-gauge needle, into a serum collection tube and, after clotting, transported to the laboratory in containers chilled with ice bricks. The presence of antibodies to the vaccine was determined using a commercially available *Salmonella*  Typhimurium ELISA kit, according to the manufacturer's instructions (2.1.7).

An additional commercial antigen-based ELISA (Idexx, Art.Nr. 99-44100, Liebefeld-Bern, Switzerland), for determination of titres against *Salmonella* serovars prevalent in swine (Typhimurium, Infantis and Enteritidis), was sourced and used according to manufacturer's instructions, with the following modification. All serum dilutions were 1:500, the previously mentioned x-OVO Guildhay conjugate, substrate and stop solution were used, and the ELISA plates read at 550nM.

#### **2.2.6 Longitudinal analysis**

The prevalence of serovars over time was determined by performing drag swabs on all broiler breeder flocks from two Australian poultry companies, in three states (New South Wales, Victoria and South Australia), which implemented the *Salmonella* tri-vaccine protocol described in 2.2.2. Swabs were taken at 6, 14, 18, 22, 33, 43 and 53 weeks of age. The *Salmonella* prevalence data were calculated for the years 2003 (pre vaccination), 2004 (during vaccination) and 2005 (post vaccination).

These data (2003 to 2005) were analysed initially for annual prevalence (total positives / total samples received) and the monthly prevalence was calculated by dividing the monthly positive by total tested. This same data set was used to calculate the age based prevalence and determine the serovars present.

The flocks mentioned previously were also bled (2.1.4) regularly (22, 32, 42 and 52 weeks of age) and the serum (*n* = 12) was tested for anti-Typhimurium antibodies using ELISA (2.1.7). From these data the flock immunity, the number of positive sera from the total tested (*n* = 12), and the flock mean was calculated. The flock serum data were sorted by flock age (weeks) and descriptive statistics (mean, medium, standard deviation and 95% confidence limits) were calculated.

The final analysis was to compare the effects of vaccination upon Typhimurium colonised chicks (<12 weeks of age). This analysis was performed by reviewing flock data (Typhimurium antibodies titre and corresponding drag swabs) that contain Typhimurium colonised birds, based on the whole of life cycle throughout 2004/2005. These colonised flocks were sorted upon age (weeks) with the corresponding serology and drag swab data added. A flock was deemed negative for Typhimurium if it had two consecutive negative results (i.e. 18 woa positive, 22 woa negative, 33 woa negative then the flock was deemed negative at 22 woa) and that initial age was plotted against the flock mean Typhimurium titre. These data were also used to evaluate if there was a statistical (χ2) relationship between high (>1000) and low (<1000) mean antibody titre and flock Typhimurium status at point of lay (25 woa).

#### **2.2.7 Statistical analysis**

Qualitative data were converted to numerical data, by assigning (0) for non-detection and (1) for detection, and analysed in a 2 x 2 contingency table, as described by Petrie and Watson (1999). Statistical significance (*P* ≤ 0.05) was determined using either the Fisher exact test (any cell with ≤5 observations) or the *Chi* squared test (all cells had >5 observations).

The Mantel-Haenszel stratified contingency table test was employed to compare similar treatments stratified across experiments (Thrusfield, 2005). The Student *t*-test was used to

#### **2.3 Immunoprohylaxis trial**

94 Biochemical Testing

An additional commercial antigen-based ELISA (Idexx, Art.Nr. 99-44100, Liebefeld-Bern, Switzerland), for determination of titres against *Salmonella* serovars prevalent in swine (Typhimurium, Infantis and Enteritidis), was sourced and used according to manufacturer's instructions, with the following modification. All serum dilutions were 1:500, the previously mentioned x-OVO Guildhay conjugate, substrate and stop solution were used, and the

The prevalence of serovars over time was determined by performing drag swabs on all broiler breeder flocks from two Australian poultry companies, in three states (New South Wales, Victoria and South Australia), which implemented the *Salmonella* tri-vaccine protocol described in 2.2.2. Swabs were taken at 6, 14, 18, 22, 33, 43 and 53 weeks of age. The *Salmonella* prevalence data were calculated for the years 2003 (pre vaccination), 2004 (during

These data (2003 to 2005) were analysed initially for annual prevalence (total positives / total samples received) and the monthly prevalence was calculated by dividing the monthly positive by total tested. This same data set was used to calculate the age based prevalence

The flocks mentioned previously were also bled (2.1.4) regularly (22, 32, 42 and 52 weeks of age) and the serum (*n* = 12) was tested for anti-Typhimurium antibodies using ELISA (2.1.7). From these data the flock immunity, the number of positive sera from the total tested (*n* = 12), and the flock mean was calculated. The flock serum data were sorted by flock age (weeks) and descriptive statistics (mean, medium, standard deviation and 95% confidence

The final analysis was to compare the effects of vaccination upon Typhimurium colonised chicks (<12 weeks of age). This analysis was performed by reviewing flock data (Typhimurium antibodies titre and corresponding drag swabs) that contain Typhimurium colonised birds, based on the whole of life cycle throughout 2004/2005. These colonised flocks were sorted upon age (weeks) with the corresponding serology and drag swab data added. A flock was deemed negative for Typhimurium if it had two consecutive negative results (i.e. 18 woa positive, 22 woa negative, 33 woa negative then the flock was deemed negative at 22 woa) and that initial age was plotted against the flock mean Typhimurium titre. These data were also used to evaluate if there was a statistical (χ2) relationship between high (>1000) and low (<1000) mean antibody titre and flock Typhimurium status at point of

Qualitative data were converted to numerical data, by assigning (0) for non-detection and (1) for detection, and analysed in a 2 x 2 contingency table, as described by Petrie and Watson (1999). Statistical significance (*P* ≤ 0.05) was determined using either the Fisher exact test (any cell with ≤5 observations) or the *Chi* squared test (all cells had >5

The Mantel-Haenszel stratified contingency table test was employed to compare similar treatments stratified across experiments (Thrusfield, 2005). The Student *t*-test was used to

ELISA plates read at 550nM.

**2.2.6 Longitudinal analysis** 

vaccination) and 2005 (post vaccination).

and determine the serovars present.

limits) were calculated.

lay (25 woa).

observations).

**2.2.7 Statistical analysis** 

#### **2.3.1 Dried egg yolk preparation**

Non-fertile eggs were sourced from a commercial poultry company that routinely administers the trivaccine (2.2.1) to their flocks. The eggs (*n* = 400) from three different farms were sourced from young hens with high (> 1000) anti-Typhimurium serum antibody titres. Upon arrival at the laboratory, 20 eggs were tested for the presence of yolk antibodies to *Salmonella* using the *Salmonella* Typhimurium ELISA kit (2.1.5 and 2.1.7). The remaining egg yolks were pooled into approximately 100 g lots and homogenised by vortexing for 15 seconds. These pooled eggs were then frozen and freeze dried (Avanti JE, Beckman Coulter, Bree, CA. USA).

The dried egg yolk lots were resuspended (weight/volume) in PBS buffer (2.1.5) and tested for the presence of anti-*Salmonella* Typhimurium antibodies using ELISA (2.1.7). The lots were composited into one container and homogenised by vigorous shaking and tested (*n* = 20) for anti-*Salmonella* antibody, as previously described (2.1.7). The dried egg yolk powder was then stored in an air-tight container until required.

#### **2.3.2 Crude extraction of IgY**

The freeze-thaw technique (Staak *et al.,* 2001) with ammonium salt precipitation, described fully in Section 2.1.6, was used.

#### **2.3.3 Provision in feed**

The feed was divided into three lots: the first lot incorporated 3% w/w IgY egg yolk (dT-IgY) as described by Gurtler *et al.* (2004). Dried egg yolk powder was mixed through a standard commercial broiler breeder starter ration supplied by a local mill. The second lot included dT-IgY re-composited in water (w : v) and, in the final lot, the dT-IgY was recomposited (w : v) in crude IgY extract.

The dosage of dT-IgY was calculated at 3% of total daily feed intake (1.42 g/chick) from the trial mid-point age (11 days of age) according to breeder specifications (Anonymous, 2007). All the feeds were prepared in 250 mL containers and fed to the chicks. The re-composited dT-IgY was initially smeared onto the beaks of individual chicks and the remainder spread in a straight line on chick paper, prior to supplying the standard ration.

The residual from each of the lots (in 250 mL containers) was weighed and an initial dilution (1 : 10) was made with *Salmonella* Typhimurium ELISA buffer. This suspension was diluted (1:2) in plasma tubes and Typhimurium IgY was tested using the ELISA method (2.1.7). The endpoint titre was converted to titre per gram of feed.

#### **2.3.4 Challenge strain**

A cryobead of *Salmonella* Typhimurium (ST12), isolated and prepared as in Section 2.1.2, was used as the challenge strain The target density of 104 and 105 cfu/mL was achieved by decimal dilution of the colorimetrically confirmed 2 x 108 cfu/mL initial suspension (2.1.2).

#### **2.3.5 Animal trials**

The trial farm was prepared as stated in Section 2.1.8. Groups of 20 chicks (non-*Salmonella* vaccinated flocks), individually identified by wing tags, were given one of three lots of feed formulation at one day of age and throughout the trial (15 days). On day 3 post-hatch, each chick was challenged (104 or 105 cfu/mL) by oral gavage (0.250 mL) with *Salmonella* Typhimurium (2.1.2). Faecal samples (*n =* 5) were collected from each pen on days 3 (prechallenge), 4, 5, 7 and 14 and cultured for the presence of *S.* Typhimurium (2.1.3). The birds were individually weighed at days 0, 7 and 14. At 15 days of age (doa), all birds were humanely euthanized and their caeca removed for culture and enumeration (2.1.3 and 2.3.6).

#### **2.3.6 Enumeration of** *Salmonella*

The method employed to enumerate caecal salmonellae was the miniMPN as described in Pavic *et al.,* (2010). Briefly, the removed caeca were cut into sections, to which BPW (w : v) was added (100 dilution). A millilitre of this dilution (100) was added to a 1250 μL plasma tube and subsequent decimal serial dilutions were prepared (100 μL into 900 μL) in plasma tubes.

Into appropriately labelled microtitre trays, 100 μL of each dilution was added into the corresponding well using a multi-channel pipette. This resulted in the formation of a 3-tube MPN, which was covered with a plastic film (SealPlate®, Excel Scientific, Inc, Victorville, CA, US) and incubated at 37 ºC for 24 h. Post-incubation, 100 μL from each microtitre well was added to 200 μL of modified semi-solid RV (MSRV) via a multi-channel pipette and incubated at 42 ºC 24 h.

All pale/white wells post incubation was confirmed using SM®ID2 and typical colonies were confirmed serologically with Poly O, poly H and anti-serogroup B antisera. The confirmed data set was converted to cfu/mL, using the MPN charts produced by the United States FDA (2006) from the 3 lowest positive dilutions, and calculated to MPN (cfu) per gram of caeca.

#### **3. Results**

#### **3.1 Vaccine trial**

#### **3.1.1 Autologous (experiment 1) and heterologous (experiment 2) challenge trials**

A challenge with *S.* Typhimurium, used in Experiments 1 and 2 (Table 2) to demonstrate repeatability, showed a significant difference (Mantel-Haenszel Stratified *Chi* squared *P* < 0.05) between non-vaccinated (colonisation rates of 25% and 50%) and vaccinated (colonisation rates of 0% and 9%) hens. *S.* Typhimurium was also used in both experiments to evaluate seroconversion to the Typhimurium component of the vaccine (Table 4 and 5). The vaccinated flocks exhibited significantly higher (Student *t*-test *P* < 0.05) titres, with 16% and 33% of blood samples having titres 85-6570 (>785 = kit positive threshold), while nonvaccinated hens gave titres of 27-176 (<460 = kit negative threshold).

After challenge, the rates of caecal colonisation (Table 2) in the non-vaccinated hens were 25%, 58% and 17% for serovars Typhimurium, Mbandaka and Orion respectively (Experiment 1), with an average colonisation rate of 33%. In the heterologous trial

The trial farm was prepared as stated in Section 2.1.8. Groups of 20 chicks (non-*Salmonella* vaccinated flocks), individually identified by wing tags, were given one of three lots of feed formulation at one day of age and throughout the trial (15 days). On day 3 post-hatch, each chick was challenged (104 or 105 cfu/mL) by oral gavage (0.250 mL) with *Salmonella* Typhimurium (2.1.2). Faecal samples (*n =* 5) were collected from each pen on days 3 (prechallenge), 4, 5, 7 and 14 and cultured for the presence of *S.* Typhimurium (2.1.3). The birds were individually weighed at days 0, 7 and 14. At 15 days of age (doa), all birds were humanely euthanized and their caeca removed for culture and enumeration (2.1.3 and 2.3.6).

The method employed to enumerate caecal salmonellae was the miniMPN as described in Pavic *et al.,* (2010). Briefly, the removed caeca were cut into sections, to which BPW (w : v) was added (100 dilution). A millilitre of this dilution (100) was added to a 1250 μL plasma tube and subsequent decimal serial dilutions were prepared (100 μL into 900 μL) in plasma

Into appropriately labelled microtitre trays, 100 μL of each dilution was added into the corresponding well using a multi-channel pipette. This resulted in the formation of a 3-tube MPN, which was covered with a plastic film (SealPlate®, Excel Scientific, Inc, Victorville, CA, US) and incubated at 37 ºC for 24 h. Post-incubation, 100 μL from each microtitre well was added to 200 μL of modified semi-solid RV (MSRV) via a multi-channel pipette and

All pale/white wells post incubation was confirmed using SM®ID2 and typical colonies were confirmed serologically with Poly O, poly H and anti-serogroup B antisera. The confirmed data set was converted to cfu/mL, using the MPN charts produced by the United States FDA (2006) from the 3 lowest positive dilutions, and calculated to MPN (cfu) per gram of

**3.1.1 Autologous (experiment 1) and heterologous (experiment 2) challenge trials** 

vaccinated hens gave titres of 27-176 (<460 = kit negative threshold).

A challenge with *S.* Typhimurium, used in Experiments 1 and 2 (Table 2) to demonstrate repeatability, showed a significant difference (Mantel-Haenszel Stratified *Chi* squared *P* < 0.05) between non-vaccinated (colonisation rates of 25% and 50%) and vaccinated (colonisation rates of 0% and 9%) hens. *S.* Typhimurium was also used in both experiments to evaluate seroconversion to the Typhimurium component of the vaccine (Table 4 and 5). The vaccinated flocks exhibited significantly higher (Student *t*-test *P* < 0.05) titres, with 16% and 33% of blood samples having titres 85-6570 (>785 = kit positive threshold), while non-

After challenge, the rates of caecal colonisation (Table 2) in the non-vaccinated hens were 25%, 58% and 17% for serovars Typhimurium, Mbandaka and Orion respectively (Experiment 1), with an average colonisation rate of 33%. In the heterologous trial

**2.3.5 Animal trials** 

tubes.

caeca.

**3. Results** 

**3.1 Vaccine trial** 

incubated at 42 ºC 24 h.

**2.3.6 Enumeration of** *Salmonella* 



Table 2. Mantel-Haenszel stratified contingency table analysis of *Salmonella* caecal culture results comparing non-vaccinated and Tri-valent vaccinated Cobb™ adult breeder hens challenged (107 cfu / mL) with serovar (CI = confidence Interval; M-H = Mantel-Haenszel; OR = odds ratio).

The cloacal swab results (Table 3) suggested that no hens were colonised by *Salmonella* prior to challenge (Day 0). Three days post challenge (Day 3) the prevalence in non-vaccinated hens ranged from zero to 40 % compared to zero to 10 % for the vaccinated flocks, changing to 0-83 % and 0-36 % respectively 14 days post-challenge. The cloacal swabs (Table 3) showed that the serovar Agona colonised non-vaccinated hens the best, with prevalence of 40 % (at day 3) and 83 % (at day 14), and a caecal prevalence of 92 %. In contrast, Zanzibar did not appear to colonise (day 3 and 14), based on cloacal swabs, and a caecal prevalence of only 9%.

Due to low colonisation rates (17 % and 9 %), combined with the low number of replicates (*n*  = 12 and 11), statistically valid (*P* ≤ 0.05) results could not be obtained for serogroup E (Table 2d). The null hypothesis could not be rejected. The statistically desired colonisation rate for non-vaccinated (50 %) was only achieved with serovars Mbandaka (Table 2c), Typhimurium and Agona (Table 2b) which all demonstrated a significant difference between vaccinated and non-vaccinated hens.

By using a stratified Mantal-Haenszal *Chi* squared test, comparing autologous to heterologous serovars within the same serogroup, significant differences (*P =* 0.003 and 0.01) were demonstrated for serogroups B (Table 2b) and C (Table 2c), and respective odds ratios of 6.6 and 12.5 times for colonisation among non-vaccinated compared with vaccinated groups. However, serogroup E serovars did not demonstrate a significant difference due to the low colonisation rate of the non-vaccinated hens.


The serovar Typhimurium was used to demonstrate (Table 2a) a repeatable significant difference between non-vaccinated and vaccinated hens (Mantel-Haenszal *P =* 0.017). All non-challenged controls were negative for the challenge and wild serovars of *Salmonella.* 

Prevalence without bracketed numerals were calculated from *n* = 12 as compared to *n* = (x). All groups initially contained 12 birds and bird death was due to non-trial related illness.

Table 3. *Salmonella* serovars prevalence from cloacal swabs (0, 3 and 14 days) and caeca (21 days) post challenge (107 cfu) from adult Cobb™ breeder broiler hens in Experiments 1 and 2.

#### **3.1.2 Experiment 1 autologous serology**

The blood samples taken from all hens prior to challenge demonstrated that vaccinated hens contained variable levels of antibodies to *Salmonella.* This is illustrated by the wide range of titres (1161 ± 623 at the 95 % confidence intervals from mean) from the x-OVO Guildhay *Salmonella* Typhimurium ELISA test (Table 4). All the unvaccinated flocks had titres well below the negative cutoff value. Blood samples from tri-vaccine vaccinated hens (*n =* 30) were tested, using the x-OVO Guildhay Typhimurium and Idexx Typhimurium-Infantis-Enteritidis ELISA, showing a 1 : 0.8 ratio respectively (x-OVO Guildhay Typhimurium ELISA average OD = 0.39 and Idexx Typhimurium: Infantis: Enteritidis ELISA average OD = 0.70 in positive wells), which was highly significant (Student *t*-test *P* = 0.0001). All blood sera positive for anti-Infantis antibodies contained anti-Typhimurium antibodies.


A Means without common superscripts differ significantly (Student *t*-test <0.05).

Table 4. Anti-*Salmonella* Typhimurium antibody titres means, with 95% confidence limits, for vaccinated and non-vaccinated Cobb™ adult breeder hens (Experiment 1).

The large co-efficient of variation reflects variation in seroconversion among the vaccinated birds, with 16% of those vaccinated demonstrating titres > 785 and 25% with titres > 460 (true negative). There was a statistically significant difference (Student's two-tailed *t*-test, *P*  < 0.05) in anti-*Salmonella* antibody levels between vaccinated and non-vaccinated flocks.

#### **3.1.3 Experiment 2 heterologous serology**

98 Biochemical Testing

The cloacal swab results (Table 3) suggested that no hens were colonised by *Salmonella* prior to challenge (Day 0). Three days post challenge (Day 3) the prevalence in non-vaccinated hens ranged from zero to 40 % compared to zero to 10 % for the vaccinated flocks, changing to 0-83 % and 0-36 % respectively 14 days post-challenge. The cloacal swabs (Table 3) showed that the serovar Agona colonised non-vaccinated hens the best, with prevalence of 40 % (at day 3) and 83 % (at day 14), and a caecal prevalence of 92 %. In contrast, Zanzibar did not appear to colonise (day 3 and 14), based on cloacal swabs, and a caecal prevalence of

Due to low colonisation rates (17 % and 9 %), combined with the low number of replicates (*n*  = 12 and 11), statistically valid (*P* ≤ 0.05) results could not be obtained for serogroup E (Table 2d). The null hypothesis could not be rejected. The statistically desired colonisation rate for non-vaccinated (50 %) was only achieved with serovars Mbandaka (Table 2c), Typhimurium and Agona (Table 2b) which all demonstrated a significant difference

By using a stratified Mantal-Haenszal *Chi* squared test, comparing autologous to heterologous serovars within the same serogroup, significant differences (*P =* 0.003 and 0.01) were demonstrated for serogroups B (Table 2b) and C (Table 2c), and respective odds ratios of 6.6 and 12.5 times for colonisation among non-vaccinated compared with vaccinated groups. However, serogroup E serovars did not demonstrate a significant difference due to

The serovar Typhimurium was used to demonstrate (Table 2a) a repeatable significant difference between non-vaccinated and vaccinated hens (Mantel-Haenszal *P =* 0.017). All non-challenged controls were negative for the challenge and wild serovars of *Salmonella.* 

**Exp serotype Day 0 Day 3 Day 14 Day 21 caeca** 

1 Typhimurium 0% / 0% 0% / 16% 0% / 16% 0 % / 25% 1 Mbandaka 0% / 0% 0% / 16% 0% /16 % 8 % / 58 % 1 Orion 0% / 0% 0% / 16% 0% / 16% 0% / 16% 2 Typhimurium 0% / 0% 8% / 25% 9% (11) / 25% 9% (11) / 50% 2 Agona 0% / 0% 10% /40% 36% / 83% 42% / 92% 2 Infantis 0% / 0% 0% / 20% 0% (10) / 8% 0% (10) / 17% 2 Zanzibar 0% (10) / 0%(11) 0% (10) / 0% (11) 0% (10) / 0% (11) 0% (10) / 9% (11) Prevalence without bracketed numerals were calculated from *n* = 12 as compared to *n* = (x). All groups

Table 3. *Salmonella* serovars prevalence from cloacal swabs (0, 3 and 14 days) and caeca (21 days) post challenge (107 cfu) from adult Cobb™ breeder broiler hens in Experiments 1

The blood samples taken from all hens prior to challenge demonstrated that vaccinated hens contained variable levels of antibodies to *Salmonella.* This is illustrated by the wide range of

initially contained 12 birds and bird death was due to non-trial related illness.

**3.1.2 Experiment 1 autologous serology** 

**Prevalence of vaccinated (***n =12***) / non-vaccinated (***n = 12***) hens at various days post challenge.** 

only 9%.

and 2.

between vaccinated and non-vaccinated hens.

the low colonisation rate of the non-vaccinated hens.

Half of the vaccinated group in Experiment 2 were individually tagged and bled prior to vaccination at 12 and 17 weeks of age (Table 5). Hens at 18 weeks had developed significant (Fisher exact *P =* 0.001) serum antibodies to *S*. Typhimurium compared to hens prior to vaccination (12 weeks), but not to hens in other age groups. Hens at 20 weeks of age had developed a significant difference in antibody to all age groups (*P =* 0.0005 and 0.01) with the exception of 18 week olds.


AB Percentages without common superscripts differ significantly (Fisher Exact *P* < 0.05). Mortalities of hens at 14 and 18 weeks were due to non vaccine-related causes (Femoral Head Necrosis).

Table 5. Anti-*Salmonella* Typhimurium antibody titres in vaccinated hens (*n* = 25) prior to (12 weeks), during (12 to 17 weeks) and after (18 to 20 weeks) the vaccination protocol (Experiment 2).

After the first vaccination, initial sero-conversion was observed in two (8.33 %) of the hens (*n* = 24). Unfortunately, one of those hens died at 18 weeks, whereas the other hen had titres of 812, 1222, 1200 and 1114 at weeks 14, 16, 18 and 20 respectively, and this hen was also negative for presence of *Salmonella* in its caeca post-challenge. The percentage 'true' (titre > 785) positive was 26 % or 34 % if the ELISA (2.1.7) kits 'suspect' interpretations were included. When these hens were randomised and challenged at 20 weeks of age the percentage of serum positive birds was 43 %, or 60 %, including 'suspect' reactions. The titre range in the second experiment was more precise with a mean titre of 1016 ± 546(95 % CI) and a low co-efficient of variation (26 %).

The percentage of eggs that had antibodies against *S.* Typhimurium was 16 % (titre > 785), or 48 % (*n* = 61) if 'suspects' (titre 460 to 784) were included. Titres ranged from 145 to 1890 for egg yolks from vaccinated hens compared with a range of 332 to 427 (below ELISA kit negative < 460) for non-vaccinated hens eggs. Regardless of the low number of ELISA positives (titres > 785) from eggs originating from vaccinated hens, there was still a significant difference (*P = 0.001)* between egg yolk anti-Typhimurium titres from nonvaccinated as compared to vaccinated hens.

#### **3.1.4 Maternal antibody protection (experiment 3)**

All chicks (except non-vaccinated and non challenged control and vaccinated nonchallenged control) were challenged at day 0 and appeared healthy and gained weight.

Initial serological testing from a culled group (*n* = 24) resulted in negative titres (< 460) and chick paper culture was negative for *Salmonella* for both non-vaccinated and vaccinated flocks*.* There was one chick from the vaccinated 108 and non-vaccinated 104 (Table 6) which died from conditions unrelated to the trial (Femoral Head Necrosis).


The vaccinated / non challenged (*n* = 10) and non-vaccinated / non challenged (*n* = 10) controls were all negative for *Salmonella*. Groups without common superscripts differ significantly (Fisher Exact *P* ≤ 0.05). One chick died from a non-trial related cause (Femoral Head Necrosis) in both the vaccinated 108 and non-vaccinated 104 group. There were no differences between cloacal swab results from days 3, 14 and 21.

Table 6. *Salmonella* vaccinated (*n* = 50) and non-vaccinated (n = 50) progeny protection from *S.* Typhimurium challenge at high (108 cfu) and low (104 cfu) dose.

The cloacal swab results from randomly chosen chicks (*n* = 5) were all positive 3 days post challenge for *S.* Typhimurium, during the grow-out phase (day 14 swabs) and caecal culture results (day 24) when the birds were challenged with 108 cfu *Salmonella*.

The cloacal swabs and caecal cultures revealed a significant difference (Fisher exact *P* = 0.047) between chicks challenged with 104 cfu of *S.* Typhimurium, which were progeny, of vaccinated hens (75% positive) compared to progeny from non-vaccinated (100 % positive) hens at 3, 14 and 24 days of age (Table 6). The serology results, initially and at 21 days post challenge, indicated negative *S.* Typhimurium antibody titres (< 460) for both vaccinated and non-vaccinated chicks.

#### **3.1.5 Longitudinal analysis**

100 Biochemical Testing

After the first vaccination, initial sero-conversion was observed in two (8.33 %) of the hens (*n* = 24). Unfortunately, one of those hens died at 18 weeks, whereas the other hen had titres of 812, 1222, 1200 and 1114 at weeks 14, 16, 18 and 20 respectively, and this hen was also negative for presence of *Salmonella* in its caeca post-challenge. The percentage 'true' (titre > 785) positive was 26 % or 34 % if the ELISA (2.1.7) kits 'suspect' interpretations were included. When these hens were randomised and challenged at 20 weeks of age the percentage of serum positive birds was 43 %, or 60 %, including 'suspect' reactions. The titre range in the second experiment was more precise with a mean titre of 1016 ± 546(95 % CI)

The percentage of eggs that had antibodies against *S.* Typhimurium was 16 % (titre > 785), or 48 % (*n* = 61) if 'suspects' (titre 460 to 784) were included. Titres ranged from 145 to 1890 for egg yolks from vaccinated hens compared with a range of 332 to 427 (below ELISA kit negative < 460) for non-vaccinated hens eggs. Regardless of the low number of ELISA positives (titres > 785) from eggs originating from vaccinated hens, there was still a significant difference (*P = 0.001)* between egg yolk anti-Typhimurium titres from non-

All chicks (except non-vaccinated and non challenged control and vaccinated nonchallenged control) were challenged at day 0 and appeared healthy and gained weight.

Initial serological testing from a culled group (*n* = 24) resulted in negative titres (< 460) and chick paper culture was negative for *Salmonella* for both non-vaccinated and vaccinated flocks*.* There was one chick from the vaccinated 108 and non-vaccinated 104 (Table 6) which

Vaccine status / challenge level Detected Not detected AVaccinated / 108 cfu 19 0 ANon-vaccinated / 108 cfu 20 0 BVaccinated / 104 cfu 15 5 ANon-vaccinated / 104 cfu 19 0 The vaccinated / non challenged (*n* = 10) and non-vaccinated / non challenged (*n* = 10) controls were all negative for *Salmonella*. Groups without common superscripts differ significantly (Fisher Exact *P* ≤ 0.05). One chick died from a non-trial related cause (Femoral Head Necrosis) in both the vaccinated 108 and non-vaccinated 104 group. There were no differences between cloacal swab results from days 3, 14

Table 6. *Salmonella* vaccinated (*n* = 50) and non-vaccinated (n = 50) progeny protection from

The cloacal swab results from randomly chosen chicks (*n* = 5) were all positive 3 days post challenge for *S.* Typhimurium, during the grow-out phase (day 14 swabs) and caecal culture

The cloacal swabs and caecal cultures revealed a significant difference (Fisher exact *P* = 0.047) between chicks challenged with 104 cfu of *S.* Typhimurium, which were progeny, of

**Groups 21 day old caecal** *Salmonella*

and a low co-efficient of variation (26 %).

vaccinated as compared to vaccinated hens.

and 21.

**3.1.4 Maternal antibody protection (experiment 3)** 

died from conditions unrelated to the trial (Femoral Head Necrosis).

*S.* Typhimurium challenge at high (108 cfu) and low (104 cfu) dose.

results (day 24) when the birds were challenged with 108 cfu *Salmonella*.

The comparison of the prevalence of *Salmonella*, at genus level and within flocks*,* was performed by plotting the monthly drag swab relative prevalence (positive flocks / total flocks tested in each month) one year prior to vaccination (2003), during vaccination (2004) and subsequent vaccination (2005).

The prevalence of *Salmonella* in 2003 was 52 %, decreasing to 41 % in 2004 and 40 % in 2005 (Figures 1 and 2). The first flocks vaccinated were 12 weeks of age as of the 1/1/2004, designated with an arrow (Figure 1). For each month post vaccination the 'forecast' relative prevalence (Figure 1) was calculated (Excel, forecast function using the previous 12 months prevalence data to calculate the predicted value).

The year prior to vaccination (Figure 1) showed peaks and troughs in flock prevalence, which followed seasons (lowest prevalence in winter and higher prevalence in summer). As the flocks were vaccinated, the monthly prevalence levels decreased and the monthly variation flattened; a moderate linear association (R2 = 0.56) was found, compared to 'forecast' values (R2 = 0.16) (Figure 1).

Fig. 1. *Salmonella* flock monthly prevalence (%) in Breeders during the years 2003 (*n* = 794) to 2004 (*n* = 1024) with the arrow indicating when vaccination started and forecast data (Red line) used to show the predicted prevalence if vaccination was not introduced.

Fig. 2. *Salmonella* flock monthly prevalence (%) in Breeders during the vaccinations years 2004 (*n* = 1024) to 2005 (*n* = 968). The two arrows indicate the start of vaccination and the month when all flocks were vaccinated.

Fig. 3. The prevalence rate in tri-valent vaccine-relevant serogroups in breeders, based upon flock age for the pre-vaccinated year 2003 (*n* = 794) and the vaccinated years 2004 (*n* = 1024) and 2005 (*n* = 968) with vaccination occurring at 12 and 18 weeks of age.

JAN feb mar apr may jun july aug sep oct nov dec JAN feb mar apr may jun july aug sep oct nov dec month

Fig. 2. *Salmonella* flock monthly prevalence (%) in Breeders during the vaccinations years 2004 (*n* = 1024) to 2005 (*n* = 968). The two arrows indicate the start of vaccination and the

Fig. 3. The prevalence rate in tri-valent vaccine-relevant serogroups in breeders, based upon flock age for the pre-vaccinated year 2003 (*n* = 794) and the vaccinated years 2004 (*n* = 1024)

and 2005 (*n* = 968) with vaccination occurring at 12 and 18 weeks of age.

percentage positive from annual total Linear (percentage positive from annual total)

0% 1% 2% 3% 4% 5% 6% 7%

month when all flocks were vaccinated.

monthly component of annual flock

*Salmonella* prevalence

y = -0.0005x + 0.0398 R2

= 0.0593

The variation in sample size, 794 (2003), 1024 (2004) and 968 (2005), was due to more intensive testing in the first year of vaccination. The flock prevalence rate between 2004 (40 %) and 2005 (41 %) remained static, which was corroborated by a R2 (linear) of almost zero (Figure 2). The spikes observed in the latter half of 2005 (Figure 2) were due to the unplanned introduction of non-vaccinated *Salmonella*-positive males, at 20 weeks of age, to other breeder farms.

The results in Figure 3 demonstrate an interesting trend in the prevalence for the years in which the vaccine e was administered (2004 and 2005, blue and green bars) compared to the year prior (2003, red bar). The prevalence of *Salmonella* from serogroups B, C and E decreased post-vaccination while increases among non-vaccine serogroups were observed, with serovar subsp 1 rough:r:1,5 predominating (Table 7).


Table 7. Flock *Salmonella* serovar trend for the year prior, 2003 (*n* = 794), and the years postvaccination, 2004 (*n* = 1024) and 2005 (*n =* 968).

During and subsequent to vaccination there was a change in the profile of serovars isolated from breeder flocks (Table 7). The vaccine serogroups all decreased with the exception of Mbandaka (serogroup C). This increase could be explained by early colonisation prior to vaccination (Figure 4). Table 7 shows serovar succession, which may have occurred post vaccination.

Prior to vaccination (2003) there were 13 serovars that accounted for 97 % of all positives (Table 7). This figure decreased to 88.9 % (2004) and 71.9 % (2005) when all flocks were vaccinated. There was also a major change in the predominant serovar with rough: r: 1:5 increasing from 0.6 % (2003) to 23 % (2004) and 14.6 % (2005), both significantly different (χ<sup>2</sup> *P* = 0.0001). Beside those in Table 7, the serovars isolated were: in 2003 Lille (1.2 %), Ohio (0.6 %), subsp. 1 ser 16:I;v;- (0.6 %) and Tennessee (0.6 %); 2004 and 2005 Anatum (0.5 % {2004}, 1 % {2005}), Chester (0.6 %, 1.2 %), Give (1.6 %, 2.0 %), Havana (0.6 %, 5 %), Javiana (0.6 %, 1.5 %), Kiambu (1.5 %, 3 %), Lillee (1.5 %, 1 %), Ohio (1.2 %, 1.9 %), Subsps 1 ser 16:I;v;- (1.5 %, 3.5 %), Subsps 1 ser 6,7:r:- (0.6 %, 0 %), Worthington (1.6 %, 0 %), Bredeney (0 %, 2 %), Cubana (0 %, 3.5 %) and Tennessee (0 %, 2 %).

There was no overall change in prevalence of serogroup C1 (Table 7); while the C1 positive flocks were all positive prior to vaccination at a steady 5 % prevalence rate, there was a shift in serovar prevalence, with a decrease in Infantis, the vaccine-homologous serovar.

Results suggest marginal heterologous serovar protection as shown by the steady decrease in Agona (serogroup B) post-vaccination. This decrease was similar to that for Typhimurium, observed over the same time period, and may explain the reduction in total prevalence observed during the field trial (Figure 1).

The seroconversion of birds to the vaccine Typhimurium component was routinely measured (ELISA) at the ages mentioned in Figure 4, and the mean (*n =* 12), with 95 % confidence limits, was recorded. The green (line) and the red (line) indicated the Typhimurium ELISA positive and negative titres levels respectively (Figure 4).

The ELISA results (Figure 4) showed a high degree of variability in seroconversion, indicated by the very large error bars (95 % confidence). The level of flock immunity (number of sera tested that had Typhimurium ELISA titres > 785) for each age group (Figure 4) during 2004 (2005) was 23 % (27 %); 15 % (17 %); 11 % (11 %) and 3 % (6.4 %). The average flock immunity for 2004 and 2005 was (titre > 785 positive level) 28 % and 27 % respectively or 40 % and 37 % if the titre was calculated at > 460 (negative level).

The 2005 trend line had a dip at 23 to 32 weeks of age; only 21 flocks (272 sera) were tested in 2004 compared to 135 flocks (1345 serums) tested in 2005, which affected the average. This large discrepancy in flocks tested in this age group (2004 and 2005) was not observed in any other age group during the same period (2004 to 2005).

The titre profile shown in Figure 4 for all flocks was also apparent when the titres of the individual flocks were examined over the same time period (categorized by weeks of age{woa}). They all showed a similar age titre profile (Figure 4; year 2004) with a shoulder period (ages 13 to 22 and 23 to 32) and then a decrease with age.

An investigation into the efficacy of the tri-vaccine on juvenile (< 6 woa) breeder flocks (grown day old to death, rearing and production), already colonized by serovar Typhimurium (*n* = 32), was undertaken during 2004 to 2005. All these Typhimurium positive flocks (determined using drag swabs) were routinely sampled, as mentioned previously, and the drag swabs were compared to the anti-Typhimurium titre for the same age groups. A flock was deemed negative if two consecutive drag swabs did not contain the Typhimurium serovar, and the initial age recorded (i.e. 6 woa = Typhimurium positive; 12 woa = positive; 18 woa = positive; 22 woa = positive; 33 woa = negative; 43 woa = negative and 53 woa = negative, the age negative is 33 woa).

The corresponding Typhimurium titre was also measured and the flock average over the sampling period was recorded. The data summarized in Figure 5 show a random and variable Typhimurium clearance rate in flocks with low (< 1000) anti-Typhimurium antibody means, with only two flocks clearing the serovar prior to production age (> 25

(0.6 %), subsp. 1 ser 16:I;v;- (0.6 %) and Tennessee (0.6 %); 2004 and 2005 Anatum (0.5 % {2004}, 1 % {2005}), Chester (0.6 %, 1.2 %), Give (1.6 %, 2.0 %), Havana (0.6 %, 5 %), Javiana (0.6 %, 1.5 %), Kiambu (1.5 %, 3 %), Lillee (1.5 %, 1 %), Ohio (1.2 %, 1.9 %), Subsps 1 ser 16:I;v;- (1.5 %, 3.5 %), Subsps 1 ser 6,7:r:- (0.6 %, 0 %), Worthington (1.6 %, 0 %), Bredeney (0

There was no overall change in prevalence of serogroup C1 (Table 7); while the C1 positive flocks were all positive prior to vaccination at a steady 5 % prevalence rate, there was a shift

Results suggest marginal heterologous serovar protection as shown by the steady decrease in Agona (serogroup B) post-vaccination. This decrease was similar to that for Typhimurium, observed over the same time period, and may explain the reduction in total

The seroconversion of birds to the vaccine Typhimurium component was routinely measured (ELISA) at the ages mentioned in Figure 4, and the mean (*n =* 12), with 95 % confidence limits, was recorded. The green (line) and the red (line) indicated the

The ELISA results (Figure 4) showed a high degree of variability in seroconversion, indicated by the very large error bars (95 % confidence). The level of flock immunity (number of sera tested that had Typhimurium ELISA titres > 785) for each age group (Figure 4) during 2004 (2005) was 23 % (27 %); 15 % (17 %); 11 % (11 %) and 3 % (6.4 %). The average flock immunity for 2004 and 2005 was (titre > 785 positive level) 28 % and 27 % respectively

The 2005 trend line had a dip at 23 to 32 weeks of age; only 21 flocks (272 sera) were tested in 2004 compared to 135 flocks (1345 serums) tested in 2005, which affected the average. This large discrepancy in flocks tested in this age group (2004 and 2005) was not observed in any

The titre profile shown in Figure 4 for all flocks was also apparent when the titres of the individual flocks were examined over the same time period (categorized by weeks of age{woa}). They all showed a similar age titre profile (Figure 4; year 2004) with a shoulder

An investigation into the efficacy of the tri-vaccine on juvenile (< 6 woa) breeder flocks (grown day old to death, rearing and production), already colonized by serovar Typhimurium (*n* = 32), was undertaken during 2004 to 2005. All these Typhimurium positive flocks (determined using drag swabs) were routinely sampled, as mentioned previously, and the drag swabs were compared to the anti-Typhimurium titre for the same age groups. A flock was deemed negative if two consecutive drag swabs did not contain the Typhimurium serovar, and the initial age recorded (i.e. 6 woa = Typhimurium positive; 12 woa = positive; 18 woa = positive; 22 woa = positive; 33 woa = negative; 43 woa = negative

The corresponding Typhimurium titre was also measured and the flock average over the sampling period was recorded. The data summarized in Figure 5 show a random and variable Typhimurium clearance rate in flocks with low (< 1000) anti-Typhimurium antibody means, with only two flocks clearing the serovar prior to production age (> 25

in serovar prevalence, with a decrease in Infantis, the vaccine-homologous serovar.

Typhimurium ELISA positive and negative titres levels respectively (Figure 4).

or 40 % and 37 % if the titre was calculated at > 460 (negative level).

period (ages 13 to 22 and 23 to 32) and then a decrease with age.

other age group during the same period (2004 to 2005).

and 53 woa = negative, the age negative is 33 woa).

%, 2 %), Cubana (0 %, 3.5 %) and Tennessee (0 %, 2 %).

prevalence observed during the field trial (Figure 1).

#### 2004 2005

Fig. 4. Flock seroconversion of the Typhimurium component (positive {green line} and negative {red line} ELISA thresholds) of the Tri-vaccine as measure by anti-Typhimurium ELISA (95 % confidence limits) from flock (*n* = 12) bloods taken at various ages between 2004 and 2005.


Fig. 5. Naturally Typhimurium colonised chicks (2004 to 2005), grown in day old to death houses, clearance age (weeks) in relation to the flock mean (*n* = 12) anti-Typhimurium titre trend (exponential R2), measured post vaccination (12 and 18 weeks), with critical limits set at hen age of 25 weeks (green line) and mean titre > 1000 (blue line).

woa). Six of seven flocks with high titre (> 1000) were clear of Typhimurium at production age (< 25 woa). These flocks with high titre means (> 1000) also had high (75 %, *n* =12) flock immunity (anti-Typhimurium titre > 850).

The data in Figure 5 were analyzed using an exponential trend line, which showed a moderate association (R2 = 0.45) that, when converted to a 2 x 2 contingency table (Table 8), was highly significant (χ<sup>2</sup> *P* = 0.0002) between titre and Typhimurium clearance. Additionally, Typhimurium colonized flocks were 17.5 times more likely to remain colonized after 25 woa, if their titres were below 1000.


Fisher exact *P* = 0.0002 with an odds ratio of 140 and a relative risk of 17.5.

Table 8. The *Chi* squared analysis comparing high (> 1000) and low (< 1000) mean (*n* = 12) anti-Typhimurium titres in pre-vaccinated Typhimurium positive flocks that had consecutive negative drag swabs at 25 weeks of age.

#### **3.2 Immunoprophylaxis trial**

#### **3.2.1 IgY analysis**

ELISA analysis of the breeder egg yolks from three different farms showed that all the flocks had high flock immunity (number of samples titre > 785 in serum sample divided by total tested) and a high mean flock titre (Table 9). The crude IgY yield (Figure 6) ELISA results (Figure 7) showed a high variance in Typhimurium antibodies (15 % to 80 % > 785 and means of 343 to 2518) deposited in egg yolks tested (*n* = 20) which was further investigated using Farm 1 yolks (Figure 8).


Table 9. Summary of *Salmonella* Typhimurium vaccinated donor flocks for mean (95% CI) antibody titres, from serum (*n =* 12), egg yolk (*n =* 20) and crude extract (*n =* 20), flock immunity (percentage of sera with titre > 785), yolk positive rate (No. of yolks > 785 titre) and crude IgY yield (mg/g).

woa). Six of seven flocks with high titre (> 1000) were clear of Typhimurium at production age (< 25 woa). These flocks with high titre means (> 1000) also had high (75 %, *n* =12) flock

The data in Figure 5 were analyzed using an exponential trend line, which showed a moderate association (R2 = 0.45) that, when converted to a 2 x 2 contingency table (Table 8), was highly significant (χ<sup>2</sup> *P* = 0.0002) between titre and Typhimurium clearance. Additionally, Typhimurium colonized flocks were 17.5 times more likely to remain

**Typhimurium titre Negative at < 25 Positive at >26 Total** High >1000 6 1 **7** Low <1000 2 23 **25 total 8 24 32**

Table 8. The *Chi* squared analysis comparing high (> 1000) and low (< 1000) mean (*n* = 12)

ELISA analysis of the breeder egg yolks from three different farms showed that all the flocks had high flock immunity (number of samples titre > 785 in serum sample divided by total tested) and a high mean flock titre (Table 9). The crude IgY yield (Figure 6) ELISA results (Figure 7) showed a high variance in Typhimurium antibodies (15 % to 80 % > 785 and means of 343 to 2518) deposited in egg yolks tested (*n* = 20) which was further investigated

mean (95% CI) 1118 (+502) 1844 (+528) 2104 (+564) Flock Immunity rate 70 % 85 % 100 % Egg Typhimurium IgY positive rate 15 % 26 % 80 % Egg Typhimurium IgY titre mean (95% CI) 343 (+143) 761 (+276) 2518 (+1361) Crude IgY yield mg/g yolk (95% CI) 28 (+8) 32 (+12) 26 (+4) Crude Typhimurium IgY mean (95% CI) titre 153 (+118) 386 (+286) 690 (+390)

Table 9. Summary of *Salmonella* Typhimurium vaccinated donor flocks for mean (95% CI) antibody titres, from serum (*n =* 12), egg yolk (*n =* 20) and crude extract (*n =* 20), flock immunity (percentage of sera with titre > 785), yolk positive rate (No. of yolks > 785 titre)

**Farm 1 Farm 2 Farm 3** 

anti-Typhimurium titres in pre-vaccinated Typhimurium positive flocks that had

immunity (anti-Typhimurium titre > 850).

colonized after 25 woa, if their titres were below 1000.

consecutive negative drag swabs at 25 weeks of age.

**3.2 Immunoprophylaxis trial** 

using Farm 1 yolks (Figure 8).

Flock Typhimurium serum (IgY)

and crude IgY yield (mg/g).

**3.2.1 IgY analysis** 

Fisher exact *P* = 0.0002 with an odds ratio of 140 and a relative risk of 17.5.

Fig. 6. Salt precipitated crude IgY containing Typhimurium antibodies, which were extracted from vaccinated breeder hen's eggs.

Fig. 7. *Salmonella* Typhimurium ELISA plate, using doubling dilution, to determine the crude IgY titre from egg yolk extract.

Fig. 8. Variability in anti-Typhimurium ELISA (positive > 785, green line, and negative < 459, red line) titre means (*n* = 8, 95 % CI) in eggs (*n* = 20) from Farm 1. The data portrayed in Figure 8 show a very large variance in the Typhimurium antibody levels present in Farm 1 egg yolks. Only egg yolks 4, 8 and 19 had Typhimurium titres levels close to the ELISA positive range of > 785. The majority of yolks, with the exception of yolks 1, 5 and 15, were below the negative level of < 459.

Of the yolks from the three farms, composited post-freeze drying, anti-Typhimurium antibodies (titre > 785) were present in 90 % of the samples tested. The titres in the composites ranged from 16 to 1024 with a mean (+ 95 % CI) of 386 (+ 203). The egg yolk composited crude extract (Figure 7) resuspended in PBS (5 mL per yolk), had an IgY titre of 256.

#### **3.2.2 IgY feed trial**

The results of the net body weight gain for these four feeding regimes are summarised in Figure 9 which showed that there was no significant difference (Student *t*-test > 0.05) for all the feeds regardless of Typhimurium challenge dose and at various ages (0, 7, and 14 doa).

Typhimurium Challenge 10^4 cfu/ml Typhimurium challenge 10^5 cfu/ml No Challenge

Fig. 9. The net body weight gain (after 15 days) of challenged (104 or 105 cfu/mL of *S. typhimurium*) chicks fed with four rations.

0 2 4 6 8 10 12 14 16 18 20 Farm 1 egg yolk sample mean (*n* = 8 repeats)

Fig. 8. Variability in anti-Typhimurium ELISA (positive > 785, green line, and negative < 459, red line) titre means (*n* = 8, 95 % CI) in eggs (*n* = 20) from Farm 1. The data portrayed in Figure 8 show a very large variance in the Typhimurium antibody levels present in Farm 1 egg yolks. Only egg yolks 4, 8 and 19 had Typhimurium titres levels close to the ELISA positive range of > 785. The majority of yolks, with the exception of yolks 1, 5 and 15, were

Of the yolks from the three farms, composited post-freeze drying, anti-Typhimurium antibodies (titre > 785) were present in 90 % of the samples tested. The titres in the composites ranged from 16 to 1024 with a mean (+ 95 % CI) of 386 (+ 203). The egg yolk composited crude extract (Figure 7) resuspended in PBS (5 mL per yolk), had an IgY titre of

The results of the net body weight gain for these four feeding regimes are summarised in Figure 9 which showed that there was no significant difference (Student *t*-test > 0.05) for all the feeds regardless of Typhimurium challenge dose and at various ages (0, 7, and 14 doa).

chick feed (CF) CF plus 3 % dried

anti-Typhimurium IgY (dT-IgY) egg yolk

Fig. 9. The net body weight gain (after 15 days) of challenged (104 or 105 cfu/mL of *S.* 

treatment

Typhimurium Challenge 10^4 cfu/ml Typhimurium challenge 10^5 cfu/ml No Challenge

CF plus dT-IgY resuspended in water

CF plus dT-IgY resuspended in crude Typhimurium IgY extract

below the negative level of < 459.

Typhimurium titre

256.

**3.2.2 IgY feed trial** 

bird weight (g)

*typhimurium*) chicks fed with four rations.

The rehydrated dT-IgY was fed to the chicks initially by hand (smearing of dT-IgY paste on the beak) with the remainder of the feed spread out in a line on chick paper. Only when all this feed was consumed was the standard ration introduced. This ensured that all the rehydrated dT-IgY was consumed.

The residual treated feed was tested for the presence of Typhimurium antibody. The results, summarised in Figure 10, show that the chicks in the treatment groups received anti-Typhimurium IgY, in various degrees, throughout the trial.

Fig. 10. Typhimurium antibody titre (95% CI) of three treated feed rations: (CF) plus 3% (w:w) freeze dried Typhimurium IgY (dt-IgY) yolk (green line); CF plus dT-IgY resuspended in water (w:v) (red line) and dT-IgY resuspended in crude Typhimurium IgY extract (w:v) (blue line) feed prior (3 days) and post (4 days of age+) Typhimurium (104 or 105 cfu/mL) challenge.

The data summarised in Figure 10 showed that the dT-IgY resuspended in crude IgY extract (blue line) had the highest and most consistent titres, with a range 80 to 160 and a mean of 110, throughout the trial. The dT-IgY resuspended in water (red line) had the highest titre range of 50 to 130 and a mean of 60. Finally, the dT-IgY egg yolk powder (green line) treatment was the most inconsistent with a titre range of 0 to 150 and a low mean of 61.

The final analysis was to measure the effect of these feed treatments on *S.* Typhimurium levels in the caeca. The data summarised in Figure 11 show that, regardless of treatment, there was no significant (Student *t* test > 0.05) reduction in caecal Typhimurium populations as compared to the control. This was also observed in faecal (days 4, 5, 7 and 14) and caecal samples that were 100 % positive when tested qualitatively for *Salmonella* presence.

Fig. 11. *S*. Typhimurium caecal counts (log10 cfu/g, 95 % CI) from challenged (104 or 105 cfu/mL) chicks on three feed treatments.

#### **4. Discussion**

#### **4.1 Vaccine trial**

One of the objectives behind developing and using a multivalent vaccine was to reduce carriage and shedding of otherwise prevalent salmonellae, and thus to protect the progeny from egg contamination and from colonisation by low level (< 100 cfu) environmental and / or feed exposure (Anonymous, 2004; Franco, 2005; Volkova *et al*., 2009). The protection of poultry against *Salmonella* infection is largely empirical, based upon experimental trial work and field studies (Anonymous, 2004). Knowledge relating to the course of the innate and adaptive response to colonisation with various *Salmonella* types is beginning to increase (Anonymous 2004; Beal and Smith 2007). To be suitable for use in industry, a vaccine must be safe for poultry and other species, and not interfere with detection of *Salmonella*, as well as effective in eliminating shedding and enhancing clearance (Anonymous 2004).

With the above definition in mind the decision to use the predominant serovar from each of serogroups B, C and E was based upon success of this approach with serovars Typhimurium and Enteritidis in a European study (Van Den Bosch 2003), and serogroups C and E were added primarily to offer protection against the prominent Australian poultry serogroups (Davos, 2005) and, secondarily, to expand the knowledge base (Anonymous, 2004,). There were no serovars from serogroup D included in this vaccine as Australian commercial flocks are free of this serogroup (due to zero tolerance), including serovars Enteritidis, Pullorum and Gallinarum (Arzey, 2005).

0

**4. Discussion 4.1 Vaccine trial** 

cfu/mL) chicks on three feed treatments.

Enteritidis, Pullorum and Gallinarum (Arzey, 2005).

chick feed (CF) CF plus 3% dried

anti-Typhimurium IgY (dT-IgY egg yolk)

Fig. 11. *S*. Typhimurium caecal counts (log10 cfu/g, 95 % CI) from challenged (104 or 105

One of the objectives behind developing and using a multivalent vaccine was to reduce carriage and shedding of otherwise prevalent salmonellae, and thus to protect the progeny from egg contamination and from colonisation by low level (< 100 cfu) environmental and / or feed exposure (Anonymous, 2004; Franco, 2005; Volkova *et al*., 2009). The protection of poultry against *Salmonella* infection is largely empirical, based upon experimental trial work and field studies (Anonymous, 2004). Knowledge relating to the course of the innate and adaptive response to colonisation with various *Salmonella* types is beginning to increase (Anonymous 2004; Beal and Smith 2007). To be suitable for use in industry, a vaccine must be safe for poultry and other species, and not interfere with detection of *Salmonella*, as well

With the above definition in mind the decision to use the predominant serovar from each of serogroups B, C and E was based upon success of this approach with serovars Typhimurium and Enteritidis in a European study (Van Den Bosch 2003), and serogroups C and E were added primarily to offer protection against the prominent Australian poultry serogroups (Davos, 2005) and, secondarily, to expand the knowledge base (Anonymous, 2004,). There were no serovars from serogroup D included in this vaccine as Australian commercial flocks are free of this serogroup (due to zero tolerance), including serovars

as effective in eliminating shedding and enhancing clearance (Anonymous 2004).

Treatment

10^4 Challenge 10^5 Challenge

CF plus dT-IgY resuspended in water

CF plus dT-IgY resuspended in crude IgY extract

1

2

3

Caecal Typhimurim log10 cfu/g

4

5

6

According to Grimont and Weill (2007) only 30 serovars account for 90 % of *Salmonella* isolates in any given country. In the Australian broiler industry, typically ten different serovars account for 84 % of all isolates (Davos, 2007). To suit inevitable changes in serovar prevalence, killed vaccines such as the trivaccine in the present study are most suitable for industrial use as they can be more easily prepared and therefore more readily adapted than attenuated strain vaccines.

Prior to 2008, due to consumer fears about genetically modified organisms (GMOs), in the food supply, only killed *Salmonella* vaccines were available for commercial use in Australia (Anonymous, 1999). The next stage of development would include vaccination with killed and attenuated *Salmonella*e to target both cell mediated and humoral immunity.

Bailey *et al.* (2007a) showed increased IgG and IgA production in serum, the gut and crop of adult broiler breeders after administered of a live followed by a killed vaccine, when compared to a killed or attenuated vaccine alone. Attenuated vaccines can be given to dayold chicks, providing competitive exclusion and stimulating polymorphonucleated cell migration to the intestinal cell walls, thus preventing initial colonisation by wild strains (Van Immerseel *et al.,* 2005).

In the present study, a killed multi-serotype vaccine was injected into hens after 12 weeks of age to provide humoral protection. Bailey *et al.* (2007a) administered an *aroA* attenuated Enteritidis vaccine at 1 and 21 days followed by an autogenous killed trivalent vaccine to 11 and 17 week old broiler breeder parents. Following post-vaccination autologous challenge, the reduction in caecal counts and increase in antibody titre were similar in the study of Bailey *et al.* (2007ab) and the present study, suggesting the killed vaccine alone is effective.

However, Deguchi *et al.* (2009) administered an autologous trivalent (Enteritidis, Typhimurium and Infantis) killed vaccine at only 6 weeks of age and was able to show an increased antibody titre and reduced caecal carriage, for challenge (109 cfu) with all autologous serovars. The variable results across these studies suggest that the effectiveness of vaccination may vary with the nature and timing of administration of the vaccine. The efficacy of a killed vaccine can be enhanced by using improved adjuvants that may target different parts of the immune system (Barrow 2007).

The modest total colonisation rates in non-vaccinated hens of 33 % and 42 % (Experiment 1 and 2) illustrated that the challenge dose (107 cfu) may have been too low or the strains were attenuated. Other studies (Byrd *et al.*, 2003; Babu *et al.,* 2004) showed high rates of colonisation (> 80%) with high challenge doses of Typhimurium (108 to 109 cfu and 1010 cfu respectively). However, the 107 cfu challenge dose was chosen as this is a more realistic, field-appropriate, natural challenge, than 1010 cfu.

Numerous studies indicate avian susceptibility to *Salmonella* is not uniform and many influencing factors exist. The literature suggests that a stable anaerobic microflora, low house density, bacterial interference, breed resistance, antagonism, colonisation resistance, barrier effects, challenge strain virulence, competitive exclusion and passive immunity could all contribute to low challenge rates (Lloyd *et al.*, 1977; Fuller, 1989; Mead and Barrow, 1990; Kottom *et al.,* 1995*;* Duchet-Suchaux *et al.*, 1997; Nisbet *et al.*, 2000; Quinn *et al.,* 2000; Chambers and Lu, 2002; Kinde *et al.*, 2005).

The hens used in this study were derived from a field trial situation, stocked at low densities and were healthy, thus decreasing their susceptibility to colonisation to levels less than expected than under the stressful conditions of industry practices. When this vaccine was used under field conditions, over a four year period, the incidence of Typhimurium was reduced in vaccinated flocks with an anti-Typhimurium ELISA titre of > 1000 (Figure 5).

The results summarised in Tables 2.1b and 2.1c highlight autologous and heterologous serotype protection conferred by the multivalent vaccine and these trial results were similar to those of Clifton-Hadley *et al.* (2002) during trial of the first Salenvac® bi-vaccine. The repeatability with *S.* Typhimurium in both trials was expected and heterologous protection was demonstrated with the serogroup B serotype Agona. Protection against heterologous serotypes from serogroup B was also demonstrated by Beal *et al.* (2006).

The serovars Orion and Zanzibar (serogroup E) were both chosen due to their field prevalence at the time in chicken litter and meat (Davos, 2005). However, these serovars both failed to colonise caeca 21 days after challenge. While detection in flocks may suggest colonisation, such serovars may derive from the environment or feed and be transitory or only capable of minimal colonisation in the birds; still detectable by drag swabs of flocks.

The x-OVO Guildhay *Salmonella* Typhimurium lipopolysaccharide ELISA was used in this study to measure the immune response to the vaccine, as it was used during development of the first Salenvac killed vaccine (McMullin *et al.,* 1997). The trivaccine contains 1x108 cfu of each of the three serovars added in a ratio of 1:1:1.

The assumption is that the hen develops antibodies to each serovar at titres similar to those determined for *S.* Typhimurium. Okamura *et al.* (2007) demonstrated similar *Salmonella* lipopolysaccharide ELISA antibody titres in hens, vaccinated with a killed bivalent vaccine containing serovars Enteritidis and Typhimurium.

A comparison using the x-OVO Guildhay Typhimurium kit and a commercial swine *Salmonella* (Typhimurium, Infantis and Enteritidis) kit, at a 1:500 dilution with anti-chicken conjugate and substrate added to both kits and read at 550nM showed a 1:0.8 ratio, which was highly significant (Student *t*-test *P* < 0.0001). While the ratio was not the theorised 1:1 ratio, there was a consistently higher OD observed in the Idexx trivalent kit, which may be attributed to the presence of serogroup C1 antibodies.

The *S.* Typhimurium ELISA results in experiment 1 and 2 were significantly different (Student *t*-test *P* = 0.001) between vaccinated and non-vaccinated hens (titre < 460) (Table 4) and results from this trial are very similar to the cited trials and therefore protective levels of antibodies have to be present in vaccinated hens caeca (Clifton-Hadley *et al.,* 2002; Beal *et al.*, 2006; Okamura *et al.* 2007; Deguchi *et al.* 2009).

As this was a new vaccine an extremely high co-efficient of variance (127 %) was observed in, the commercial field, vaccinated flocks used in Experiment 1. This may have been due to phase separation of the adjuvant and the killed cells gravity settled to the bottom of the vial. The repeat Experiment (2) the vaccine suspension was vigorously shaken prior to hand vaccination under trial conditions. This resulted in a decreased co-efficient of variation in vaccinated hens to 26 % and a higher percentage positive (36 % >785 titre).

Prior to Experiment 2 (heterologous challenge), all hens were sourced unvaccinated, then manually vaccinated at 12 and 17 weeks to measure the seroconversion rate. The data

The hens used in this study were derived from a field trial situation, stocked at low densities and were healthy, thus decreasing their susceptibility to colonisation to levels less than expected than under the stressful conditions of industry practices. When this vaccine was used under field conditions, over a four year period, the incidence of Typhimurium was reduced in vaccinated flocks with an anti-Typhimurium ELISA titre of > 1000 (Figure 5).

The results summarised in Tables 2.1b and 2.1c highlight autologous and heterologous serotype protection conferred by the multivalent vaccine and these trial results were similar to those of Clifton-Hadley *et al.* (2002) during trial of the first Salenvac® bi-vaccine. The repeatability with *S.* Typhimurium in both trials was expected and heterologous protection was demonstrated with the serogroup B serotype Agona. Protection against heterologous

The serovars Orion and Zanzibar (serogroup E) were both chosen due to their field prevalence at the time in chicken litter and meat (Davos, 2005). However, these serovars both failed to colonise caeca 21 days after challenge. While detection in flocks may suggest colonisation, such serovars may derive from the environment or feed and be transitory or only capable of minimal colonisation in the birds; still detectable by drag swabs of flocks. The x-OVO Guildhay *Salmonella* Typhimurium lipopolysaccharide ELISA was used in this study to measure the immune response to the vaccine, as it was used during development of the first Salenvac killed vaccine (McMullin *et al.,* 1997). The trivaccine contains 1x108 cfu of

The assumption is that the hen develops antibodies to each serovar at titres similar to those determined for *S.* Typhimurium. Okamura *et al.* (2007) demonstrated similar *Salmonella* lipopolysaccharide ELISA antibody titres in hens, vaccinated with a killed bivalent vaccine

A comparison using the x-OVO Guildhay Typhimurium kit and a commercial swine *Salmonella* (Typhimurium, Infantis and Enteritidis) kit, at a 1:500 dilution with anti-chicken conjugate and substrate added to both kits and read at 550nM showed a 1:0.8 ratio, which was highly significant (Student *t*-test *P* < 0.0001). While the ratio was not the theorised 1:1 ratio, there was a consistently higher OD observed in the Idexx trivalent kit, which may be

The *S.* Typhimurium ELISA results in experiment 1 and 2 were significantly different (Student *t*-test *P* = 0.001) between vaccinated and non-vaccinated hens (titre < 460) (Table 4) and results from this trial are very similar to the cited trials and therefore protective levels of antibodies have to be present in vaccinated hens caeca (Clifton-Hadley *et al.,* 2002; Beal *et al.*,

As this was a new vaccine an extremely high co-efficient of variance (127 %) was observed in, the commercial field, vaccinated flocks used in Experiment 1. This may have been due to phase separation of the adjuvant and the killed cells gravity settled to the bottom of the vial. The repeat Experiment (2) the vaccine suspension was vigorously shaken prior to hand vaccination under trial conditions. This resulted in a decreased co-efficient of variation in

Prior to Experiment 2 (heterologous challenge), all hens were sourced unvaccinated, then manually vaccinated at 12 and 17 weeks to measure the seroconversion rate. The data

vaccinated hens to 26 % and a higher percentage positive (36 % >785 titre).

serotypes from serogroup B was also demonstrated by Beal *et al.* (2006).

each of the three serovars added in a ratio of 1:1:1.

containing serovars Enteritidis and Typhimurium.

attributed to the presence of serogroup C1 antibodies.

2006; Okamura *et al.* 2007; Deguchi *et al.* 2009).

summarised in Table 5 indicated seroconversion (> 785 titre) which, while slow between the priming dose at 12 weeks of age (8%) and the booster at 17 weeks (18 %), peaked at 43 % by 20 weeks of age. Deguchi *et al.* (2009) also observed significantly high titre values four weeks post vaccination and McMullin *et al.* (1997) indicated that a minimum of 30 % of hens positive serologically by 22 weeks of age was required to confer flock immunity (to colonisation by *S.* Enteritidis using Salenvac vaccine and the Guildhay ELISA for *S.* Enteritidis). In the present study, 43 % of hens were seropositive (titre > 785) after 20 weeks, indicating flock immunity.

The Guildhay method requires an initial serum dilution of 1:500 whereas similar work with killed Typhimurium used initial serum dilution of 1:250, 1:400 and 1:400 respectively (Beal *et al.,* 2005; Withanage *et al.,* 2005; Bailey *et al.,* 2007a). The higher initial dilution rate may have greatly reduced the number of ELISA positives in this trial.

In the vaccinated flock challenged with *S.* Typhimurium (Table 2a) the strain was recovered from the caeca of only one hen (*n* = 11) compared to six hens in the non-vaccinated group (*n* = 12). The vaccination of hens against *Salmonella* may aid in the decrease of *Salmonella* numbers house from the caeca, and thus decrease shell contamination, which may, in turn, prevent chick colonisation during hatching.

The amount of IgY transferred to the egg yolk is proportional to maternal serum IgG concentrations (Loeken and Roth, 1983; Al-Natour *et al.*, 2004; Hamal *et al.,* 2006). The transfer of circulating anti-*Salmonella*e antibody (IgG) into egg yolk (IgY), was demonstrated by Chalghoumi *et al.* (2008) and Bailey *et al.* (2007a), after vaccination with serovars Enteritidis and Typhimurium.

Therefore, it is expected that antibodies against the three vaccine strains used in this study would have been transferred to the yolk. In the present study, the detection of serovar Typhimurium antibodies (titre > 785) were measured in 16 % of egg yolks, and 52 % contained no Typhimurium antibody (titre < 460). In addition, 32 % of yolks gave titres that were in the 'suspect' range for the ELISA (460 to 784).

The data suggest maternal transfer of IgY of Typhimurium antibodies from vaccinated hens. The level of detection in this study was 16 % (> 760) or 48 % (> 460) and was similar to the 30 % achieved in a previous study (Hamal *et al.,* 2006).

The variable level of titre measured in egg yolk may be an artefact from initial dilution (1:500), as discussed previously, especially when compared to the study of Hassan and Curtiss (1996) egg yolk dilution of 1:100. The titres of anti-*Salmonella* antibody in this study ranged from 538-1784 and 145-1890 in serum and yolk respectively (at the 95th percentile), indicating that sero-conversion was not uniform among hens.

Therefore, low level of circulating antibody would likely result in transfer of a low level of antibody into the egg yolk. Also, as the eggs were sampled from pens and not individual hens, only broad comparisons could be made. The presence of anti-Typhimurium IgY in egg yolk from vaccinated hens was not observed as increased anti-Typhimurium IgG in their serum of their progeny.

The final experiment (Experiment 3) measured the effects of maternal anti-*Salmonella* antibodies in day-old chicks, challenged with different doses of *S.* Typhimurium. Reduction in colonisation by *S.* Typhimurium is problematic, even in vaccinated chicks. While good reduction has been observed when chicks are challenged with 104 cfu, reduction is poor at higher challenge doses (106 and 108 cfu; Nisbet *et al.,* 2000; Clifton-Hadley *et al.,* 2002).

Methner *et al.* (1999) showed a 0.5-1.5 log10 cfu reduction in carriage in vaccinated chicks challenged with variable populations of *S.* Typhimurium. Bailey, *et al.* (2007b) showed a 0.5 log10 reduction in day old progeny from vaccinated hens, challenged with 107 cfu, which may be due to maternal IgY.

In the present experiment, in vaccinated chicks, a 25 % clearance from caeca was observed in chicks challenged with 104 cfu, while no reduction was evident using a 108 cfu challenge (Table 6), indicating a limit to protection by maternal antibodies.

The literature showed conflicting results with respect to maternal antibody protection against *Salmonella* colonisation; Hassan and Curtiss (1996) showed protection whereas Bailey *et al.* (2007b) showed no protection. There is a need for more experimental work in this area using standardised ELISA, challenge and measurement techniques.

Regardless of challenge dose, Nisbet *et al.* (2000) showed a 2 log10 reduction in *Salmonella* establishing in the caeca whereas Clifton-Hadley *et al.* (2002) suggested that high challenge doses of highly invasive and virulent *S.* Typhimurium may overcome those components of the immune system associated with the systemic phase of infection. Therefore, the 104 cfu challenge is the minimum dosage that will not overwhelm the immune system and still results in colonisation of non-vaccinated controls.

Based on the results of this study, vaccination of all breeder flocks by several Australian commercial broiler growers was introduced as an adjunct to other *Salmonella* control measures. The effectiveness of this vaccine in the field was tested using routine monitoring data, for blood and drag swabs for weeks 6, 14, 18, 22, 33, 43, 53 for the year prior (2003) to vaccination, during vaccination (2004) and post vaccination (2005). The results showed an overall decrease in flock prevalence from 51% (2003) to 40 % and 41 % (2004 and 2005 respectively) which, while not statistically significant (χ<sup>2</sup> *P* = >0.05), was an important reduction. The comprehensive risk assessment produced by the FAO/WHO (2002) stated that "there was a one to one relationship (assuming that everything else remains constant) between percentage change in prevalence to expected risk of illness". The example cited by the FAO/WHO (2002) stated that if prevalence reduced from 20 % to 10 %, this would result in a similar 50 % reduction of risk in illness per serving.

The reduction in flock prevalence was noticed across all flock ages (Figure 3) with a reduction in chick (<12 woa) prevalence, which may be attributed to maternal tri-vaccine antibody (Hassan and Curtiss, 1996). The increase in *Salmonella* prevalence during weeks 13 to 22 may be due to the hen sexual maturity, which may be associated with a situation akin to the peri-parturient relaxation of resistance (Kelly, 1973) seen in mammals.

This condition is contributory to susceptibility of sheep to parasite infection after birth and has been attributed to the release of prolactin (Kelly, 1973). Hens also produce prolactin, initiating broodiness, which may limit early egg production and may increase their susceptibility to *Salmonella* colonisation (Leboucher *et al.,* 1990; Talbot *et al.,* 1991; March, *et al.,* 1994; Berry, 2003).

in colonisation by *S.* Typhimurium is problematic, even in vaccinated chicks. While good reduction has been observed when chicks are challenged with 104 cfu, reduction is poor at

Methner *et al.* (1999) showed a 0.5-1.5 log10 cfu reduction in carriage in vaccinated chicks challenged with variable populations of *S.* Typhimurium. Bailey, *et al.* (2007b) showed a 0.5 log10 reduction in day old progeny from vaccinated hens, challenged with 107 cfu, which

In the present experiment, in vaccinated chicks, a 25 % clearance from caeca was observed in chicks challenged with 104 cfu, while no reduction was evident using a 108 cfu challenge

The literature showed conflicting results with respect to maternal antibody protection against *Salmonella* colonisation; Hassan and Curtiss (1996) showed protection whereas Bailey *et al.* (2007b) showed no protection. There is a need for more experimental work in

Regardless of challenge dose, Nisbet *et al.* (2000) showed a 2 log10 reduction in *Salmonella* establishing in the caeca whereas Clifton-Hadley *et al.* (2002) suggested that high challenge doses of highly invasive and virulent *S.* Typhimurium may overcome those components of the immune system associated with the systemic phase of infection. Therefore, the 104 cfu challenge is the minimum dosage that will not overwhelm the immune system and still

Based on the results of this study, vaccination of all breeder flocks by several Australian commercial broiler growers was introduced as an adjunct to other *Salmonella* control measures. The effectiveness of this vaccine in the field was tested using routine monitoring data, for blood and drag swabs for weeks 6, 14, 18, 22, 33, 43, 53 for the year prior (2003) to vaccination, during vaccination (2004) and post vaccination (2005). The results showed an overall decrease in flock prevalence from 51% (2003) to 40 % and 41 % (2004 and 2005 respectively) which, while not statistically significant (χ<sup>2</sup> *P* = >0.05), was an important reduction. The comprehensive risk assessment produced by the FAO/WHO (2002) stated that "there was a one to one relationship (assuming that everything else remains constant) between percentage change in prevalence to expected risk of illness". The example cited by the FAO/WHO (2002) stated that if prevalence reduced from 20 % to 10 %, this would result

The reduction in flock prevalence was noticed across all flock ages (Figure 3) with a reduction in chick (<12 woa) prevalence, which may be attributed to maternal tri-vaccine antibody (Hassan and Curtiss, 1996). The increase in *Salmonella* prevalence during weeks 13 to 22 may be due to the hen sexual maturity, which may be associated with a situation akin

This condition is contributory to susceptibility of sheep to parasite infection after birth and has been attributed to the release of prolactin (Kelly, 1973). Hens also produce prolactin, initiating broodiness, which may limit early egg production and may increase their susceptibility to *Salmonella* colonisation (Leboucher *et al.,* 1990; Talbot *et al.,* 1991; March, *et* 

to the peri-parturient relaxation of resistance (Kelly, 1973) seen in mammals.

higher challenge doses (106 and 108 cfu; Nisbet *et al.,* 2000; Clifton-Hadley *et al.,* 2002).

(Table 6), indicating a limit to protection by maternal antibodies.

results in colonisation of non-vaccinated controls.

in a similar 50 % reduction of risk in illness per serving.

*al.,* 1994; Berry, 2003).

this area using standardised ELISA, challenge and measurement techniques.

may be due to maternal IgY.

The increase in flock prevalence of *Salmonella*e in that age bracket (13 to 22 weeks) may also be due to the introduction of males from other rearing houses. These males were not vaccinated and may have been a source of *Salmonella*.

There was a decrease in *Salmonella* prevalence post-vaccination, (2004-2005) across all age groups, for serogroups B and C but not for serogorup E (Figure 3 and Table 7). The flock horizontal data are very similar to the observations noticed in the trial (Table 2) and support the hypothesis that serogroup E is transitory and does not persistently colonise the avian intestinal tract.

There was also a change in *Salmonella* serovar profile (Table 7), with 13 serovars in 2003 accounting for 97 % of all flock isolates. When these same serovars were tracked through the vaccination years (2004 to 2005) they accounted for only 88.6 % and 71.9 % respectively. The biggest change in prevalence was observed in the non-vaccine serovars Senftenberg (serogroup E4) from 0.6 % in 2003 to 4.7 % in 2005 and subsp. 1 ser rough:r:1,5 from 0.6 % to 14.6 % for the same time period (Figure 3 and Table 7).

The serovar changes observed may have been introduction through feed, litter, vermin, and equipment (FAO/WHO, 2002; Arsenault *et al.,* 2007). The presence of this rough:r:1, 5 serovar has identical flagellar antigens to Infantis, suggesting these isolates are variants of that serovar. The term "rough" means that this *Salmonella* is devoid of somatic antigens (lipopolysaccharide). However, its flagellar antigenic structure is very similar to that of serovar Infantis (Davos, personal communication).

According to the complete Le Minor-White-Kauffman scheme there are 11 other serovars that have the same flagellar structure: Bradford (serogroup [B]); Czernyring [054]; Abertbanju [V]; Lubumbashi [S]; Hindmarsh [C2-C3]; Linde [P]; Jamaica [D1]; Ughelli [E1]; Senegal [F]; Tennenlohe [K] and Gege [N] (Grimont and Weill, 2007). However, none of these serovars have been isolated in Australia (Davos, 2008) thus supporting the initial hypothesis that this rough strain is a variant of Infantis.

The flock seroconversion of the Typhimurium component was measured and reported in Figure 4. The annual (2004 and 2005) flock positive (> 785) titre rates (flock immunity) were 28 % and 27 % respectively, with peak rates recorded between 13 and 22 woa. These figures were less than the trial figure of 43 % at 20 woa and slightly lower than the 30 % seroconversion described by McMullin *et al.* (1997). This may be due to difficultly in hand vaccinating thousands of hens per day (shown by the wide 95 % confidence range) and the stress placed upon the hen which may act as an immunosuppressant and compromise seroconversion (Shini *et al.*, 2010).

The data summarised in Figure 5 suggest that a high average titre (> 1000 mean) may aid in reduced shedding of serovar Typhimurium within the flock. This may be due to the increased production of IgY and IgA within the gut of the hen (Bailey *et al.,* 2007a). The mean titre of 1000 was chosen as its S/P ratio was 0.35 which was 0.1 larger than the cut-off positive S/P ratio of 0.25. The cut off age of 25 weeks was chosen as that is approximately the age when the hens are in full fertile egg production.

An interesting observation was seen on a day old to death (Aug to Jun) farm with six houses (flocks) on which all were colonised with Typhimurium prior to vaccination. The flock in the fourth house had a high mean titre (2566 and 75 % flock immunity) and was clear of Typhimurium colonisation at 23, 33 and 43 woa (no 53 woa swabs taken) whereas the adjoining flocks in houses three and five, with low titres (266 and 786 respectively), were still positive for Typhimurium after 33 and 43 weeks.

In another State, a four-house day old to death (Oct to Sep) complex had the first house with a high mean titre (1246) and was negative for Typhimurium after 25 weeks whereas the adjoining three houses with low titres (426, 828 and 533) were positive after 25 weeks. More controlled research is required to understand the factors contributing to clearance.

As these flocks were on the same farm and the adjoining houses were positive, then it may be safe to assume that the feed and water were *Salmonella* free and biosecurity prevented cross-contamination. However, both of these farms had relatively young flocks over the Australian summer (Nov to Feb). These poultry farms have curtained sides, and the presence of flies is very high. Flies are known carriers of *Salmonella* (Wales *et al*., 2010) and could continuously reinfect the flocks, therefore limiting the effectiveness of biosercurity in preventing *Salmonella* colonisation and spread.

Another possible explanation is that the isolation of *Salmonella* using a drag swab may favour the dominant serovar within the flock and the sub-dominant serovars may be masked or have a lower probability of detection. However, this error should be consistent throughout the farm and a reduced number of Typhimurium would also mean a reduced risk as mentioned previously.

The two most consistent observations are that both these flocks, in different locations and States, had high (> 1000) Typhimurium titres; as Occam's (or Ockham's) Razor states, "with all things being equal the simplest solution is often the correct one" (Anonymous, 2010). Therefore high titre and flock immunity is very important in clearance of Typhimurium.

One of the problems associated with horizontal field trials is the absence of contemporary controls. Therefore, assumptions are made that all growing conditions, feed, water supply and management are the same (constant variable), with vaccination (controlled variable) and *Salmonella* exposure (uncontrolled variable) the measured variables. Unfortunately, when using commercial broiler flocks, in real world situations, as the time frame increases so too does the possibility that flock management practices may change due to flock illness or economic necessities thus altering crucial parameters. Therefore, any conclusions made from historical data need to be guarded and very general.

In conclusion, the vaccination of Cobb™ breeders with an autologous vaccine demonstrated statistically significant reduction in serogroup B and C colonisation following challenge at 107 cfu per bird. Seroconversion of the vaccinated hens, as well as maternal transfer of antibodies to the egg yolk, was shown. Challenge (104 cfu per bird) trials with day-old chicks demonstrated a significant difference in colonisation of progeny from vaccinated versus non-vaccinated parents. The horizontal analysis over a three year period (2003 to 2005) showed that the number of colonised flocks decreased by 10 % following vaccination and, if the vaccine titre mean was greater than 1000 the likelihood of persistent colonisation was reduced by 17 times.

#### **4.2 Prophylaxis trial**

It is well documented that young (< 3 woa) chicks lack a mature gut flora and immune system and therefore are more susceptible to *Salmonella* colonisation (Nurmi and Ratala

Typhimurium colonisation at 23, 33 and 43 woa (no 53 woa swabs taken) whereas the adjoining flocks in houses three and five, with low titres (266 and 786 respectively), were

In another State, a four-house day old to death (Oct to Sep) complex had the first house with a high mean titre (1246) and was negative for Typhimurium after 25 weeks whereas the adjoining three houses with low titres (426, 828 and 533) were positive after 25 weeks. More

As these flocks were on the same farm and the adjoining houses were positive, then it may be safe to assume that the feed and water were *Salmonella* free and biosecurity prevented cross-contamination. However, both of these farms had relatively young flocks over the Australian summer (Nov to Feb). These poultry farms have curtained sides, and the presence of flies is very high. Flies are known carriers of *Salmonella* (Wales *et al*., 2010) and could continuously reinfect the flocks, therefore limiting the effectiveness of biosercurity in

Another possible explanation is that the isolation of *Salmonella* using a drag swab may favour the dominant serovar within the flock and the sub-dominant serovars may be masked or have a lower probability of detection. However, this error should be consistent throughout the farm and a reduced number of Typhimurium would also mean a reduced

The two most consistent observations are that both these flocks, in different locations and States, had high (> 1000) Typhimurium titres; as Occam's (or Ockham's) Razor states, "with all things being equal the simplest solution is often the correct one" (Anonymous, 2010). Therefore high titre and flock immunity is very important in clearance of Typhimurium.

One of the problems associated with horizontal field trials is the absence of contemporary controls. Therefore, assumptions are made that all growing conditions, feed, water supply and management are the same (constant variable), with vaccination (controlled variable) and *Salmonella* exposure (uncontrolled variable) the measured variables. Unfortunately, when using commercial broiler flocks, in real world situations, as the time frame increases so too does the possibility that flock management practices may change due to flock illness or economic necessities thus altering crucial parameters. Therefore, any conclusions made

In conclusion, the vaccination of Cobb™ breeders with an autologous vaccine demonstrated statistically significant reduction in serogroup B and C colonisation following challenge at 107 cfu per bird. Seroconversion of the vaccinated hens, as well as maternal transfer of antibodies to the egg yolk, was shown. Challenge (104 cfu per bird) trials with day-old chicks demonstrated a significant difference in colonisation of progeny from vaccinated versus non-vaccinated parents. The horizontal analysis over a three year period (2003 to 2005) showed that the number of colonised flocks decreased by 10 % following vaccination and, if the vaccine titre mean was greater than 1000 the likelihood of persistent colonisation

It is well documented that young (< 3 woa) chicks lack a mature gut flora and immune system and therefore are more susceptible to *Salmonella* colonisation (Nurmi and Ratala

controlled research is required to understand the factors contributing to clearance.

still positive for Typhimurium after 33 and 43 weeks.

preventing *Salmonella* colonisation and spread.

from historical data need to be guarded and very general.

risk as mentioned previously.

was reduced by 17 times.

**4.2 Prophylaxis trial** 

1973; Martin *et al.,* 2000;). The transfer of maternal antibodies from the yolk to the chick may prevent colonisation of chicks by *Salmonella* (Hassan *et al.,* 1996).

The data summarised in Table 9 shows that maternal antibodies for *Salmonella*  Typhimurium are transferred from hen serum to the egg yolk and these antibodies can be chemically extracted (Staak *et al.,* 2001). However, there is a large variation in these data (15 % to 80 %) in terms of the number of eggs that were positive for *S.* Typhimurium antibodies. This may be due to the level of seroconversion and the subsequent production of circulating antibody within the donor hen. Hamal *et al.* (2006) showed a relationship between plasma antibody levels and transfer into the yolk. These authors also noticed a farm difference in the serum antibody levels to the same vaccine, though, the serum to egg transfer rate was similar in both farms.

The data in Table 9 shows that hens from farm 3, with a high flock mean Typhimurium serum (ELISA titre of 2104), also had 80 % of their yolks positive. These yolks, when chemically extracted, had a high crude extract mean anti-Typhimurium antibody titre (ELISA titre of 6900). The extract titres were higher than the initial serum level and similar observations have been made in numerous studies (Rose *et al*., 1974; Kariyawasam *et al.,*  2004; Malik *et al.,* 2006; Yegani and Korver, 2010).

The farm (1) that had the lowest levels of Typhimurium IgY was further investigated by performing repeats (*n* = 8) of each individual yolk tested (Figure 8). These data (Figure 8) showed that there was a lot of variation within the egg yolk as signified by the large 95% confidence limits error bars. This may be due to either mechanical error, such as difficultly in accurately pipetting 100 µl of highly viscous egg yolk, or that the amount of *S.*  Typhimurium IgY present was so low that it reduced the probability of detection. An alternative method would be to weigh approximately 100 µg of egg yolk instead of pipetting.

The yields of crude IgY which was chemically extracted (Staak *et al.,* 2001) from egg yolks was higher (26 to 32 mg/g) than expected (20 to 25 mg/g) from another study employing the same method (Rose and Orlans, 1981). This higher yield may be due to the extraction of some low molecular weight proteins. This observation could be further investigated through electrophoretic analysis of the extracts to determine the size of the impurities.

In the previous Section (4.1) it was shown that maternal antibody to *S.* Typhimurium can confer some protection to chicks challenged with low levels (104) of *S.* Typhimurium. This led to the hypothesis that feeding day old chicks rations supplemented with anti-Typhimurium IgY may provide passive immunity. This hypothesis was based upon the review by Schade *et al.* (2005), which described many examples of transfer of antibody against specific pathogens from hyperimmunized hens into the egg. In recent years, there have been many trials that tested the efficacy of such antibodies against *Salmonella in vitro* (Lee *et al.,* 2002; Chalghoumi *et al.,* 2009a) and *in vivo* (Gurtler *et al.,* 2004; Rahimi *et al.,* 2007; Chalghoumi *et al.,* 2009b).

In the present trial the effects of IgY supplemented feeds on the growth rate, as well as protection from colonisation upon challenge with 104 and 105 cfu of *S.* Typhimurium, were evaluated; the challenge dose was based upon populations likely to be encountered naturally. The initial part of the trial was to observe and measure whether the chicks would consume the treated feeds and the effects of these on weight gain.

One of the concerns was the salt content in the crude yolk extract may make the feed unpalatable or that other antibodies in the crude yolk extract may interact adversely within the gastrointestinal tract thus affecting feed conversion. The results for this aspect of the trial are summarised in Figure 9 and show that there was no significant (Student *t-*test *P* = 0.56) difference in weight gain between the treated and control chicks, as shown by Kassaify and Mine (2004) in field trials.

Chicks consumed a standard dose of 1.42 g antibody extract/chick/day. This dosage was calculated from Anonymous (2007) and was based upon 3 % (Gurtler *et al.*, 2004) of daily intake at 11 days (trial midpoint) of age.

The titre of the Typhimurium IgY in the treated feeds was measured daily (Figure 10) and showed that there was a high degree of variation in dosage. The feed treated with dried anti-Typhimurium egg yolk (dT-IgY), resuspended in crude *S.* Typhimurium containing IgY extract, and had the most consistent titre. The dried feed supplemented with 3 % dried egg yolk was the most variable. This may be due to the difficulty of evenly distributing the dried egg yolk throughout the ration; the titres in reconstituted (wet) feeds were much more consistent.

Finally, after 15 days of age, the chicks were euthanized and their caeca removed for enumeration of *S.* Typhimurium (Figure 11). The results showed, regardless of the treatment and challenge level, that there was no significant difference (Student *t*-test *P* = 0.65) between treatment and control groups.

The work performed by Kassaify and Mine (2004) measured the effects of egg yolk powder at 2.5 %, 5 %, 7.5 % and 10 % w : w and showed that *Salmonella* Typhimurium colonisation in poultry was eliminated at only the 10 % w : w concentration. The authors suggested that egg yolk containing anti-infection and adhesive factors, which may agglutinate the pathogen, stimulates the immune system or factors compete for adhesion sites (Kassaify and Mine, 2004).

Based on this result the concentration of egg yolk in the current trial, at 3 %, may have been too low and an insufficient amount of egg yolk containing anti-Typhimurium IgY reached the caeca. The former is supported by the work of Chalghoumi *et al.* (2009b) who also showed no significant caecal reduction in four-day-old chicks challenged with 106 cfu Typhimurium or Enteritidis fed 1 to 5 % (w:w) freeze dried egg yolk containing anti-*Salmonella* IgY.

The 3 % dose rate was based on the work of Gurtler *et al.* (2004), which demonstrated a significant reduction in *S.* Enteritidis contamination of eggs from hens that were challenged with high levels of Enteritidis (108 and 109). This challenge rate was not used in the present study as it was not considered to represent a natural infective dose.

An alternate delivery method, IgY administered for 28 days in drinking water, led to a reduction in caecal colonisation of Enteritidis, administered at 106 cfu, to 0.27 log10 compared to controls, at 3.98 log10/g (Rahimi *et al.,* (2007). This method may be preferable to adding IgY directly into feed, as the water lines can be set to dose at a specific concentration. In standard Australian veterinary practice antimicrobials are added into water to treat disease and in feed for prophylactic treatment.

The initial concentration of egg yolk containing IgY may be critical as these orallyadministered antibodies, like any other protein molecule, are susceptible to denaturation by the acidic pH of the proventriculus and gizzard and degradation by proteases (Yegani and Korver, 2010). However, there are reports in the literature that suggest that the IgY Fab fragments maintain their ability to bind antigen, even after exposure to pepsin and trypsin and at pH 4.0 (Shimizu *et al.,* 1988; Carlander *et al*., 2000; Gurtler *et al.,* 2004). Therefore, there may be a percentage of the yolk containing IgY digested with only a limited portion still being active post-digestion.

These antibodies, post-digestion, would decline in immunological activity and concentration as they progressed from the proximal to distal regions of the intestine, due to viscosity and passage rate, but remain detectable in the caecum and therefore reducing the probability of antibody encountering and binding the antigen (Reilly *et al.,* 1997; Wilkie *et al*., 2006; Yegani and Korver, 2010). These factors may influence the ability of IgY to prevent colonisation by specific pathogens in the lower parts of the intestinal tract. It may be possible to develop a protease-resistant oral dosage form of IgY in order to increase the fraction of immunoreactive antibody delivered locally in the gastrointestinal tract (Reilly *et al*., 1997).

The easiest method to ensure that effective concentration of IgY reaches the caeca is to ensure that the initial concentration of dried egg yolk is high enough to deliver a dosage of antibody inhibitory toward *Salmonella* into the caeca. The literature reviewed currently suggested that 10 % (w : w) would be the optimum dosage.

In conclusion, this study demonstrated the successful chemical extraction of Typhimurium IgY transferred from serum to egg yolk for three different flocks. However, when yolk containing Typhimurium IgY was added to dry chick feed at 3 % (w:w) and fed prophylactically to dayold chicks there was no protection observed when these chicks were challenged with *S.*  Typhimurium (105 cfu/mL). This was possibly due to the feed dosage being too low or an inability of the orally administered antibodies to reach the site of activity required.

### **5. Conclusion**

118 Biochemical Testing

One of the concerns was the salt content in the crude yolk extract may make the feed unpalatable or that other antibodies in the crude yolk extract may interact adversely within the gastrointestinal tract thus affecting feed conversion. The results for this aspect of the trial are summarised in Figure 9 and show that there was no significant (Student *t-*test *P* = 0.56) difference in weight gain between the treated and control chicks, as shown by Kassaify and

Chicks consumed a standard dose of 1.42 g antibody extract/chick/day. This dosage was calculated from Anonymous (2007) and was based upon 3 % (Gurtler *et al.*, 2004) of daily

The titre of the Typhimurium IgY in the treated feeds was measured daily (Figure 10) and showed that there was a high degree of variation in dosage. The feed treated with dried anti-Typhimurium egg yolk (dT-IgY), resuspended in crude *S.* Typhimurium containing IgY extract, and had the most consistent titre. The dried feed supplemented with 3 % dried egg yolk was the most variable. This may be due to the difficulty of evenly distributing the dried egg yolk throughout the ration; the titres in reconstituted (wet) feeds were much more

Finally, after 15 days of age, the chicks were euthanized and their caeca removed for enumeration of *S.* Typhimurium (Figure 11). The results showed, regardless of the treatment and challenge level, that there was no significant difference (Student *t*-test *P* = 0.65) between

The work performed by Kassaify and Mine (2004) measured the effects of egg yolk powder at 2.5 %, 5 %, 7.5 % and 10 % w : w and showed that *Salmonella* Typhimurium colonisation in poultry was eliminated at only the 10 % w : w concentration. The authors suggested that egg yolk containing anti-infection and adhesive factors, which may agglutinate the pathogen, stimulates the immune system or factors compete for adhesion sites (Kassaify and Mine,

Based on this result the concentration of egg yolk in the current trial, at 3 %, may have been too low and an insufficient amount of egg yolk containing anti-Typhimurium IgY reached the caeca. The former is supported by the work of Chalghoumi *et al.* (2009b) who also showed no significant caecal reduction in four-day-old chicks challenged with 106 cfu Typhimurium or Enteritidis fed 1 to 5 % (w:w) freeze dried egg yolk containing anti-

The 3 % dose rate was based on the work of Gurtler *et al.* (2004), which demonstrated a significant reduction in *S.* Enteritidis contamination of eggs from hens that were challenged with high levels of Enteritidis (108 and 109). This challenge rate was not used in the present

An alternate delivery method, IgY administered for 28 days in drinking water, led to a reduction in caecal colonisation of Enteritidis, administered at 106 cfu, to 0.27 log10 compared to controls, at 3.98 log10/g (Rahimi *et al.,* (2007). This method may be preferable to adding IgY directly into feed, as the water lines can be set to dose at a specific concentration. In standard Australian veterinary practice antimicrobials are added into water to treat

study as it was not considered to represent a natural infective dose.

disease and in feed for prophylactic treatment.

Mine (2004) in field trials.

treatment and control groups.

consistent.

2004).

*Salmonella* IgY.

intake at 11 days (trial midpoint) of age.

The autologus *Salmonella* tri-vaccine was shown to convey protection from colonisation of autologous and heterologous *Salmonella* challenge in the hen and to convey maternal antibodies to progeny. These maternal antibodies helped protect day-old chicks from colonisation after low-level challenge with *Salmonella* Typhimurium (at an age of 14 days). However, when these maternal antibodies were extracted from vaccinated hens' eggs and fed to non-vaccinated day old chicks for prophylaxis there was no difference in caecal colonisation rates between treated and untreated groups.

Reduction of the prevalence of *Salmonella* in flocks will benefit society by a potential reduction in human foodborne illness, as highlighted by a FAO/WHO (2002) risk assessment, which showed that for every 50 % reduction in prevalence there is also a 50 % reduction in risk.

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### **Biochemical and Histopathological Toxicity by Multiple Drug Administration**

Zeeshan Feroz1 and Rafeeq Alam Khan2\*

*1Ziauddin College of Pharmacy, Ziauddin University, Karachi, 2Department of Basic Medical Sciences, King Saud Bin Abdul Aziz University of Health Sciences, Jeddah, 1Pakistan 2Kingdom of Saudi Arabia* 

#### **1. Introduction**

126 Biochemical Testing

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With the increase in ways and means to improve health care there has been an increase in miseries of humanity a patient is often presented with several pathological situations that greatly necessitate the need for multiple drug administration and this in turn increases the chances of drug toxicity. Hence there is an immense need to explore such drug combinations that could be given safely to patients with multiple disorders.

Multiple drug administration increases chances of drug interaction, altering the responses of drugs either increased or decreased pharmacological effects, or a new pharmacological response. Generally drug interactions should be avoided, due to the possibility of poor or unexpected outcomes and can be prevented with access to current, comprehensive and reliable information which may improve the safe and cost-effective patient care. Most countries face an augmented load of cardiovascular diseases (CVD) and epilepsy along with chronic non-communicable disorders such as diabetes mellitus. Hence it is essential to recognize the probable toxicities that might occur due to multiple drug administration. Occasionally these toxicities are predictable on the basis of known pharmacology of the drugs used, thus combinations require separate investigations with animal toxicity studies.

#### **1.1 Epilepsy**

Epilepsy is the leading neurological disorder in the world categorized by abnormal hyper excitability of the neurons causing seizures with or without loss of consciousness. These seizures are of short-term and an indication of unusual, extreme or synchronous neuronal activity in the brain (Fisher et al., 2005).

Epilepsy represents the 3rd most common neurologic disorder in developed countries after stroke and dementias, encountered in elderly (Lim, 2004). The prevalence of epilepsy is around 0.4 to 0.8 % (Brodie and Dichter, 1996) and its overall occurrence is around 50-70 cases per 100, 000 in developed countries and 100 per 100, 000 in developing countries (Lim, 2004). Epilepsy

<sup>\*</sup> Corresponding Author

is a significant, but often underappreciated, health problem in Asia (Mac et al., 2007). It is estimated that approximately 50 million people worldwide have epilepsy (Kwan and Brodie, 2000). This figure had recently reduced and it has been estimated that approximately 45 million of population globally have epilepsy (French and Pedley, 2008). In Pakistan its predominance is approximately 1 % (Aziz et al., 1994 and 1997; Khatri et al., 2003). The utmost occurrence rate of 1.25% was found at the age group 20-29 years. The incidence rate gradually dropped, reaching the lowest of 0.49% in the age group of 50-59. Conversely the prevalence rate augmented again reaching to 1.1% at age > 60 years (Aziz et al., 1994).

Etiology of epilepsy is age related, in children, approximately 20% are remote symptomatic, 50% are cryptogenic while 30% are idiopathic. On the other hand, in elderly, approximately 55% are remote symptomatic whereas 45% are idiopathic/ cryptogenic. In elderly causes and risk factors for seizures are significantly variable (LIM, 2004). The cumulative lifetime risks for epilepsy and unprovoked seizure in industrialized countries are 3.1% and 4.1%, respectively (McHugh and Norman, 2008). In most of the cases (62%) the reason is unidentified, stroke (9.0%), head trauma (9.0%), alcohol (6.0%), neurodegenerative disease (4.0%), static encephalopathy (3.5%), brain tumors (3.0%), and infection (2.0%) account for remaining cases. Although cerebrovascular reasons are more widespread in the old age, the reason is yet to be explored in 25% to 40% of patients who are 65 years of age or older (French and Pedley, 2008). Majority of the patients are well controlled on a single antiepileptic drug (Nadkarni et al., 2005). Since the early 1990s, a number of latest antiepileptic drugs have arrived in the market that proposed considerable benefits in terms of their favorable pharmacokinetics, enhanced tolerability and decrease probability for drug-drug interactions (Bialer and White, 2010). Every new drug offers a special profile of pharmacokinetics, undesirable effects, and mechanisms of action, making best utilization of these agents even more difficult (LaRoche and Helmers, 2004). Seizures can be managed in patients with epilepsy by means of conventional antiepileptic drugs, but regardless of optimal therapy 25% to 30% of patients continue to have seizures and others have undesirable side effects. Hence there is a need for additional drugs as well as new approaches for preventing epilepsy (Dichter and Brodie, 1996; Bialer and White, 2010).

#### **1.2 Hypertension**

Hypertension is a widespread human disease that badly affects approximately 1 billion people globally (Varon and Marik, 2003; Dickson and Sigmond, 2006; Chobanian, 2008). Unfortunately, regardless of current progresses in understanding and treating hypertension, its occurrence keeps on increasing. Worldwide 26% of the adult population suffers from hypertension (Dickson and Sigmond, 2006) and its occurrence is projected to boost up to 60% by 2025, when a total of 1.56 billion people may be exaggerated. Pakistan ranks at number sixth in terms of its population (165million in 2007) which is constantly increasing at a rate about 1.83% per year, national heath surveys reveals that 33% of Pakistan's population beyond the age of 45 has hypertension (Wasay and Jabbar, 2009). Hypertension is the foremost threat for cardiovascular disease (Lardinois, 1995; Peralta et al., 2007; Chobanian, 2008) responsible for one half of the coronary heart disease and about two third of the cerebrovascular disease load (Cutler et al., 2008) and is accountable for the most of deaths globally (Adrogue and Madias, 2007), even moderate increase in arterial blood pressure results in reduced life span.

#### **1.3 Diabetes**

128 Biochemical Testing

is a significant, but often underappreciated, health problem in Asia (Mac et al., 2007). It is estimated that approximately 50 million people worldwide have epilepsy (Kwan and Brodie, 2000). This figure had recently reduced and it has been estimated that approximately 45 million of population globally have epilepsy (French and Pedley, 2008). In Pakistan its predominance is approximately 1 % (Aziz et al., 1994 and 1997; Khatri et al., 2003). The utmost occurrence rate of 1.25% was found at the age group 20-29 years. The incidence rate gradually dropped, reaching the lowest of 0.49% in the age group of 50-59. Conversely the prevalence

Etiology of epilepsy is age related, in children, approximately 20% are remote symptomatic, 50% are cryptogenic while 30% are idiopathic. On the other hand, in elderly, approximately 55% are remote symptomatic whereas 45% are idiopathic/ cryptogenic. In elderly causes and risk factors for seizures are significantly variable (LIM, 2004). The cumulative lifetime risks for epilepsy and unprovoked seizure in industrialized countries are 3.1% and 4.1%, respectively (McHugh and Norman, 2008). In most of the cases (62%) the reason is unidentified, stroke (9.0%), head trauma (9.0%), alcohol (6.0%), neurodegenerative disease (4.0%), static encephalopathy (3.5%), brain tumors (3.0%), and infection (2.0%) account for remaining cases. Although cerebrovascular reasons are more widespread in the old age, the reason is yet to be explored in 25% to 40% of patients who are 65 years of age or older (French and Pedley, 2008). Majority of the patients are well controlled on a single antiepileptic drug (Nadkarni et al., 2005). Since the early 1990s, a number of latest antiepileptic drugs have arrived in the market that proposed considerable benefits in terms of their favorable pharmacokinetics, enhanced tolerability and decrease probability for drug-drug interactions (Bialer and White, 2010). Every new drug offers a special profile of pharmacokinetics, undesirable effects, and mechanisms of action, making best utilization of these agents even more difficult (LaRoche and Helmers, 2004). Seizures can be managed in patients with epilepsy by means of conventional antiepileptic drugs, but regardless of optimal therapy 25% to 30% of patients continue to have seizures and others have undesirable side effects. Hence there is a need for additional drugs as well as new approaches for preventing epilepsy (Dichter and Brodie, 1996; Bialer and White, 2010).

Hypertension is a widespread human disease that badly affects approximately 1 billion people globally (Varon and Marik, 2003; Dickson and Sigmond, 2006; Chobanian, 2008). Unfortunately, regardless of current progresses in understanding and treating hypertension, its occurrence keeps on increasing. Worldwide 26% of the adult population suffers from hypertension (Dickson and Sigmond, 2006) and its occurrence is projected to boost up to 60% by 2025, when a total of 1.56 billion people may be exaggerated. Pakistan ranks at number sixth in terms of its population (165million in 2007) which is constantly increasing at a rate about 1.83% per year, national heath surveys reveals that 33% of Pakistan's population beyond the age of 45 has hypertension (Wasay and Jabbar, 2009). Hypertension is the foremost threat for cardiovascular disease (Lardinois, 1995; Peralta et al., 2007; Chobanian, 2008) responsible for one half of the coronary heart disease and about two third of the cerebrovascular disease load (Cutler et al., 2008) and is accountable for the most of deaths globally (Adrogue and Madias, 2007), even moderate increase in arterial blood

rate augmented again reaching to 1.1% at age > 60 years (Aziz et al., 1994).

**1.2 Hypertension** 

pressure results in reduced life span.

Diabetes mellitus and hypertension are widespread that exist together at a larger rate than the individual one (Sowers and Zemel, 1990; Epstein and Sowers, 1992; Tenenbaum et al., 1999; Zanella et al., 2001). The occurrence of hypertension in the diabetic individual noticeably enhances the threat and hastens the course of cardiac disease, peripheral vascular disease, stroke, retinopathy, and nephropathy (Epstein and Sowers, 1992; Zanella et al., 2001). The occurrence of simultaneous hypertension and diabetes appears to be growing in developed nations because populations are aging and both hypertension and non-insulin dependent diabetes mellitus occurrence increases as the age progresses (Sowers and Epstein, 1995). People with diabetes faces two to four times augmented risk of CVD in contrast to the general population, simultaneous hypertension triples the already high risk of coronary artery disease, doubles total mortality and stroke risk, and may be accountable for up to 75% of all CVD in people with diabetes (Stults and Jones, 2006).

Rates of diabetes are increasing around the world (Kassab et al., 2001) which now becomes one of the major public health challenges for the 21st century. The increase occurrence in diabetes is because of aging population, obesity and stressing life style. Poverty has been under recognized as a contributor to prevalence of diabetes but it is strongly associated with the unhealthy alimentary habits (Krier et al., 1999; Riste et al., 2001). The incidence of diabetes get higher in the last decade because of factors that are strongly related to the life style as is inactivity and population aging (Muchmore et al., 1994; Keen, 1998). Studies show that type 2 diabetes affects 3% to 5% of the population in some countries and type 1 moves towards the younger ages (Dixon, 2002; Petkova et al., 2006).

The increase rate of diabetes will be noticeably higher in developing countries, between 1995 and 2025, the number of persons with diabetes is predictable to enhance by 170% in the developing world, in contrast with 42% in developed nations. Hence, by the year 2025 above 75% of the people with diabetes will exist in developing countries (Nicolucci et al., 2006). A national health survey of Pakistan reveals that 25% of patients above 45 years have diabetes mellitus and Pakistan ranks number six globally in terms of prevalence of diabetes. It was projected that in 2000 there were 5.2 million diabetic patients and this will increase to 13.9 million by 2020, leading Pakistan to 4th most populous country for diabetes mellitus (Wasay and Jabbar, 2009). However a survey conducted in 2010 by Hayat and Shaikh reveals that Pakistan ranks number seven on diabetes prevalence and figures show that about 6.9 million people have diabetes. The International Diabetes Federation predicts that this number will rise to 11.5 million by 2025 if effective procedures are not taken to control the disease (Jawad, 2003).

The occurrence of diabetes is continually growing and rising at a distressing rate, and it is projected that, unless successful prevention and control measures are put into practice, this disease will soon involve 300 million persons worldwide (Sowers, 2004). Globally more than 170 million people have diabetes, and this figure is expected to be more than double by the year 2030, if existing trends continue (Boden and Taggart, 2009, Hoque et al., 2009).

#### **1.4 Arrhythmia**

Hypertension is usually linked with arrhythmias in patients with and without simultaneous CVD. There are studies which show the possible links between hypertension and atrial and ventricular arrhythmias, though the principal pathophysiological mechanism remains unclear (Yiu and Tse, 2008). The prevalence and risk factors for arrhythmias vary among men and women (Wolbrette et al., 2002). The most prevalent arrhythmia seen in clinical practice is atrial fibrillation which currently influences more than 2 million Americans, with an expected rise to 10 million by the year 2050 (Zimetbaum, 2007).

A patient is often presented with several other pathological states along with epilepsy; such as hypertension, arrhythmias, and diabetes, therefore it is essential to discover the drugdrug interaction upon simultaneous use of anti-epileptic with antihypertensive, antiarrhythmic and antidiabetic. A well reported example is the increase in serum phenytoin levels when used concomitantly with amiodarone and therefore resulting in phenytoin toxicity (Lesko, 1989; Nolan et al., 1990) thus there is an massive need to assess the toxicities of multiple drug administration and to explore relatively safe combination for individuals with multiple disorders, not to predict but rather to warn the users and prescribers, of the possible dangers, to discourage the use of combination which have high cumulative toxicities in animals and to suggest more useful combination in countries where drug regulatory control is very poor.

#### **2. Biochemical testing and histopathological examination of liver toxicities**

Serum biochemical parameters can provide important and useful information in assessing not only the extent and severity of liver damage, but also the type of liver damage (Ramaiah, 2007). Histopathological assessments also take part in the diagnosis of liver disease; moreover evaluation of morphological changes may provide additional information that may be useful for clinical management for example, grading of inflammatory activity and staging of fibrosis in chronic viral hepatitis, and the distinction between simple steatosis and steatohepatitis in alcoholic and non-alcoholic fatty liver disease (Hubscher, 2006).

Liver function tests (LFT) are helpful screening tools to detect hepatic dysfunction (Kim, 2008; Thapa and Walia, 2007; Astegiano et al., 2004). Since liver performs a variety of functions, no single test is sufficient enough to provide complete estimate of liver functions (Kim, 2008; Astegiano et al., 2004).

Table 1A and 1B reveals the comparison of γ–glutamyl transferase (γ-GT), alkaline phosphatase (ALP), alanine transaminases (ALT), total bilirubin (TBR) and direct bilirubin (DBR) levels between control animals and animals kept on individual drugs and their combinations for a period of 60 days and then after drug free interval of 15 days in normal therapeutic doses. The administration of amiodarone (4.285 mg/kg) in rabbits shows highly significant elevation in the levels of serum γ–GT, ALP, ALT and DBR (Feroz et al., 2011a). There are studies in which long-term administration of amiodarone was associated with fatal hepatotoxicity (Richer and Robert, 1995; Usdin et al., 1996; Mendez et al., 1999) although most hepatic adverse effects were transient and reversible; however deaths have also been reported from amiodarone-induced hepatotoxicity (Richer and Robert, 1995). Microscopic examination of the hepatic tissue has shown mild diffuse cellular swelling in hepatocytes (Fig 1B). Moreover the administration of losartan potassium (0.892 mg/kg) and verapamil (1.714 mg/kg) revealed highly significant elevation in serum ALP, elevations in serum ALP initiate predominantly from liver and bone (Renner and Dallenbach, 1992). There was also a significant elevation in TBR in animals kept on verapamil alone (Feroz et al., 2011a). However no significant changes have been found in animals kept on glibenclamide (0.125 mg/kg), oxcarbazepine (18.5 mg/kg) and captopril (0.512 mg/Kg) alone.

Data from animal's studies also shows that the administration of amiodarone-glibenclamidelosartanpotassium- oxcarbazepine (AGLO) combination causes highly significant elevation in serum ALP. The transaminases, ALP and γ -GT are most widely used as indicators of hepatobiliary disease (Renner and Dallenbach, 1992). Increase in ALP might be due to cholestasis (Giannini et al., 2005) or it may also suggest a biliary tract disorder (Herlong, 1994). This explains that rise in ALP might be due to chloestatic diseases or partial obstruction of bile ducts or primary biliary cirrhosis (Pratt and Kaplan, 2000). Microscopic examination of hepatic tissue has shown moderate portal inflammation (Fig 1C). There was also a significant decreased in TBR in animals kept on AGLO combination (Feroz et al., 2011a).

The administration of amiodarone-glibenclamide-verapamil-oxcarbazepine (AGVO) combination in animals shows highly significant elevation in γ -GT. Its level is elevated in a number of pathological conditions such as pancreatic disease, myocardial infarction, renal failure, chronic obstructive pulmonary disease, diabetes, and alcoholism. Measurement of serum γ -GT offers the presence or absence of hepatobiliary disease. Microscopic examination of hepatic tissue has shown congestion and mild mononuclear inflammatory infiltrate (Fig 1D). There was also a significant decreased in TBR in animals kept on AGVO combination (Feroz et al., 2011a). There has been a highly significant and significant decreased in ALT and TBR in animals kept on amiodarone-glibenclamide-captopriloxcarbazepine (AGCO) combination (Feroz et al., 2011a). Aminotransferase levels are sensitive indicators of liver-cell injury and are useful in identifying the hepatocellular disease (Pratt and Kaplan, 2000). Abnormal AST and ALT point to a hepatocyte disorder (Herlong, 1994). However microscopic examination shows congestion only (Fig 1E) illustrating no remarkable changes in the hepatic tissue of these animals (Feroz et al., 2011a).


n=9

130 Biochemical Testing

ventricular arrhythmias, though the principal pathophysiological mechanism remains unclear (Yiu and Tse, 2008). The prevalence and risk factors for arrhythmias vary among men and women (Wolbrette et al., 2002). The most prevalent arrhythmia seen in clinical practice is atrial fibrillation which currently influences more than 2 million Americans, with

A patient is often presented with several other pathological states along with epilepsy; such as hypertension, arrhythmias, and diabetes, therefore it is essential to discover the drugdrug interaction upon simultaneous use of anti-epileptic with antihypertensive, antiarrhythmic and antidiabetic. A well reported example is the increase in serum phenytoin levels when used concomitantly with amiodarone and therefore resulting in phenytoin toxicity (Lesko, 1989; Nolan et al., 1990) thus there is an massive need to assess the toxicities of multiple drug administration and to explore relatively safe combination for individuals with multiple disorders, not to predict but rather to warn the users and prescribers, of the possible dangers, to discourage the use of combination which have high cumulative toxicities in animals and to suggest more useful combination in countries where drug

**2. Biochemical testing and histopathological examination of liver toxicities**  Serum biochemical parameters can provide important and useful information in assessing not only the extent and severity of liver damage, but also the type of liver damage (Ramaiah, 2007). Histopathological assessments also take part in the diagnosis of liver disease; moreover evaluation of morphological changes may provide additional information that may be useful for clinical management for example, grading of inflammatory activity and staging of fibrosis in chronic viral hepatitis, and the distinction between simple steatosis and

steatohepatitis in alcoholic and non-alcoholic fatty liver disease (Hubscher, 2006).

Liver function tests (LFT) are helpful screening tools to detect hepatic dysfunction (Kim, 2008; Thapa and Walia, 2007; Astegiano et al., 2004). Since liver performs a variety of functions, no single test is sufficient enough to provide complete estimate of liver functions

Table 1A and 1B reveals the comparison of γ–glutamyl transferase (γ-GT), alkaline phosphatase (ALP), alanine transaminases (ALT), total bilirubin (TBR) and direct bilirubin (DBR) levels between control animals and animals kept on individual drugs and their combinations for a period of 60 days and then after drug free interval of 15 days in normal therapeutic doses. The administration of amiodarone (4.285 mg/kg) in rabbits shows highly significant elevation in the levels of serum γ–GT, ALP, ALT and DBR (Feroz et al., 2011a). There are studies in which long-term administration of amiodarone was associated with fatal hepatotoxicity (Richer and Robert, 1995; Usdin et al., 1996; Mendez et al., 1999) although most hepatic adverse effects were transient and reversible; however deaths have also been reported from amiodarone-induced hepatotoxicity (Richer and Robert, 1995). Microscopic examination of the hepatic tissue has shown mild diffuse cellular swelling in hepatocytes (Fig 1B). Moreover the administration of losartan potassium (0.892 mg/kg) and verapamil (1.714 mg/kg) revealed highly significant elevation in serum ALP, elevations in serum ALP initiate predominantly from liver and bone (Renner and Dallenbach, 1992). There was also a significant elevation in TBR in animals kept on verapamil alone (Feroz et al., 2011a). However

an expected rise to 10 million by the year 2050 (Zimetbaum, 2007).

regulatory control is very poor.

(Kim, 2008; Astegiano et al., 2004).

Mean + S.E.M

\*p < 0.05 significant with respect to control

\*\*p <0.005 highly significant with respect to control

Table 1A.Comparison of hepatic parameters following 60 days administration of individual drugs and their combinations {Adopted from Feroz et al., 2011(a)}.


n=9

Mean + S.E.M

\*p < 0.05 significant with respect to control

\*\*p <0.005 highly significant with respect to control

Table 1B. Comparison of hepatic parameters following drug-free interval of 15 days of individual drugs and their combinations {Adopted from Feroz et al., 2011(a)}.

Fig. 1A. Hepatic tissue showing no microscopic change {Adopted from Feroz et al., 2011(a)}.

**Control** 11.30+1.21 55.30+5.90 82.70+5.20 0.31+0.03 0.15+0.01 **Amiodarone** 19.16+1.42\*\* 88.0+5.70\*\* 96.56+1.50\* 0.26+0.02 0.30+0.04\*\* **Glibenclamide** 10.58+0.82 62.42+2.10 67.80+4.80 0.22+0.03 0.16+0.01 **Los. Pot** 8.51+ 0.78 79.63+3.10\*\* 80.80+8.0 0.17+0.05 0.13+0.02 **Oxcarbazepine** 14.17+1.40 53.96+1.30 78.72+2.11 0.23+0.01 0.16+0.01 **Verapamil** 10.39+1.30 81.0+6.80\*\* 79.4+3.60 0.50+0.12\* 0.12+0.01 **Captopril** 13.17+0.51 56.93+2.20 73.39+3.30 0.30+0.06 0.14+0.01 **AGLO** 10.79+1.10 79.0+5.10\*\* 76.10+4.20 0.12+0.01\* 0.17+0.03 **AGVO** 16.42+0.92\*\* 66.0+7.70 70.51+4.22 0.11+0.02\* 0.18+0.03 **AGCO** 11.43+1.50 46.90+4.0 54.0+5.10\*\* 0.13+0.04\* 0.18+0.03

Table 1B. Comparison of hepatic parameters following drug-free interval of 15 days of

Fig. 1A. Hepatic tissue showing no microscopic change {Adopted from Feroz et al., 2011(a)}.

individual drugs and their combinations {Adopted from Feroz et al., 2011(a)}.

**ALT (µ/l)** 

**TBR**  (mg/dl) **DBR**  (mg/dl)

**ALP (µ/l)** 

Parameters/ Groups

n=9

Mean + S.E.M

**γ-GT (µ/l)** 

\*p < 0.05 significant with respect to control

\*\*p <0.005 highly significant with respect to control

Fig. 1B. Hepatic tissue showing cellular swelling {Adopted from Feroz et al., 2011(a)}.

Fig. 1C. Hepatic tissue showing moderate portal inflammation {Adopted from Feroz et al., 2011(a)}.

Fig. 1D. Hepatic tissue showing congestion and mild mononuclear inflammatory infiltrate {Adopted from Feroz et al., 2011(a)}.

Fig. 1E. Hepatic tissue showing congestion {Adopted from Feroz et al., 2011(a)}.

Fig. 1D. Hepatic tissue showing congestion and mild mononuclear inflammatory infiltrate

Fig. 1E. Hepatic tissue showing congestion {Adopted from Feroz et al., 2011(a)}.

{Adopted from Feroz et al., 2011(a)}.

### **3. Biochemical testing and histopathological examination of renal toxicities**

Drugs are a frequent cause of acute kidney injury particularly in patients older than 60 years and have a higher occurrence of diabetes and CVD (Naughton, 2008). Renal function test are important in assessing the amount and severity of renal damage.

Table 2A and 2B reveals the comparison of urea and creatinine levels between control animals and animals kept on individual drugs and their combinations for a period of 60 days and then after drug free interval of 15 days in normal therapeutic doses. The administration of amiodarone in animals causes increase in the levels of urea and creatinine, whereas animals received verapamil showed significant increase in the level of urea only. However biochemical changes do not correlate to histopathological changes in renal tissue (Fig 2A) hence it is not an indication of renal damage (Feroz et al., 2010). Animals received glibenclamide showed no significant changes at biochemical level; however microscopic examination of renal tissue reveals mild tubulointerestial nephritis (Fig 2B), which together with insignificant rise in serum urea might be an indicative of developing renal damage. Significant elevation in serum urea level in animals received amiodarone and verapamil alone after drug free interval might be due to slow excretion rate of amiodarone and verapamil from the body.

Animals received AGVO combination showed highly significant rise in urea level though it was reversed after the drug-free interval, while animals kept on AGCO combination showed highly significant elevation of serum urea and creatinine. Increased creatinine level suggests decreased creatinine clearance which is a reliable indicator of decreased glomerular filtration rate due to renal damage. However after dug-free interval urea level remained highly significant while creatinine level was changed from highly significant to significant (Feroz et al., 2010).


n=9 Mean + S.E.M \*p < 0.05 significant with respect to control

\*\*p <0.005 highly significant with respect to control

Table 2A. Comparison of renal parameters following 60 days administration of individual drugs and their combinations (Adopted from Feroz et al., 2010).


n=9

Mean + S.E.M

\*p < 0.05 significant with respect to control

\*\*p <0.005 highly significant with respect to control

Table 2B. Comparison of renal parameters following drug-free interval of 15 days of individual drugs and their combinations (Adopted from Feroz et al., 2010).

Fig. 2A. Renal tissue showing no microscopic change (Adopted from Feroz et al., 2010).

**Urea (mg/dl)** 

**Control** 51.97+4.37 0.63+0.02 **Amiodarone** 68.15+1.30\* 0.70+0.03 **Glibenclamide** 52.40+2.59 0.66+0.03 **Los. Pot** 54.61+1.41 0.73+0.05 **Oxcarbazepine** 49.0+1.41 0.54+0.03 **Verapamil** 61.88+1.46\* 0.64+0.03 **Captopril** 60.77+1.57 0.67+0.02 **AGLO** 54.66+2.45 0.68+0.07 **AGVO** 59.20+4.48 0.63+0.05 **AGCO** 120.65+6.42\*\* 0.75+0.03\*

Table 2B. Comparison of renal parameters following drug-free interval of 15 days of

Fig. 2A. Renal tissue showing no microscopic change (Adopted from Feroz et al., 2010).

individual drugs and their combinations (Adopted from Feroz et al., 2010).

**Creatinine (mg/dl)** 

**Parameters/ Groups**

\*p < 0.05 significant with respect to control \*\*p <0.005 highly significant with respect to control

n=9

Mean + S.E.M

Fig. 2B. Renal tissue showing mild tubulointerestial nephritis (Adopted from Feroz et al., 2010).

#### **4. Biochemical testing and histopathological examination of cardiac toxicities**

Cardiac enzymes are proteins that escape out of injured myocardial cells resulting in elevated levels in blood. Table 3A and 3B reveals the comparison of creatinine kinase (CK) and aspartate transaminases (AST) levels between control animals and animals kept on individual drugs and their combinations for a period of 60 days and then after drug free interval of 15 days in normal therapeutic doses. Animals received amiodarone, losartan potassium, oxcarbazepine and captopril alone revealed highly significant elevation in the level of CK but these changes do not correlate with histological changes (Fig 3A) (Feroz et al., 2010). However animals kept on oxcarbazepine alone revealed significant elevation in CK level even after drug free interval which might be an indication of developing neuroleptic malignant syndrome (Pelonero et al., 1998).

The administration of AGLO combination in animals causes significant elevation in CK and AST levels, moreover inflammatory changes in the cardiac tissues (Fig 3B) suggest possible cardiac injury (Feroz et al., 2010). There are studies which suggest that raise in CK level increases the risk of myocardial infarction (Smith et al., 1976; Kumar et al., 2003; Watanabe et al., 2009). Thus simultaneous elevation of AST along with CK and histological changes might be indicative of severe myocardial cellular damage (Kratz et al., 2002). There was significant decrease in CK level in animal received AGCO combination at the end of dosing and after drug-free interval which may be due to reduced muscle mass, wasting or cachexia (Feroz et al., 2010).


n=9

Mean + S.E.M

\*p < 0.05 significant with respect to control

\*\*p <0.005 highly significant with respect to control

Table 3A. Comparison of cardiac parameters following 60 days administration of individual drugs and their combinations (Adopted from Feroz et al., 2010).


n=9

Mean + S.E.M

\*p < 0.05 significant with respect to control

\*\*p <0.005 highly significant with respect to control

Table 3B. Comparison of cardiac parameters following drug-free interval of 15 days of individual drugs and their combinations (Adopted from Feroz et al., 2010).

**CK (µ/l)** 

Table 3A. Comparison of cardiac parameters following 60 days administration of individual

**CK (µ/l)** 

**Control** 311.01+12.59 53.78+1.02 **Amiodarone** 304.16+9.45 54.63+2.32 **Glibenclamide** 319.67+12.81 53.24+5.45 **Los. Pot** 310.81+3.57 51.33+0.94 **Oxcarbazepine** 354.12+15.38\* 56.03+1.93 **Verapamil** 305.35+6.63 49.28+2.96 **Captopril** 346.63+17.37 56.42+0.73 **AGLO** 270.56+13.33\* 56.57+0.88 **AGVO** 307.34+23.83 53.36+1.65 **AGCO** 271.31+6.70\* 50.75+1.29

Table 3B. Comparison of cardiac parameters following drug-free interval of 15 days of

individual drugs and their combinations (Adopted from Feroz et al., 2010).

drugs and their combinations (Adopted from Feroz et al., 2010).

**Control** 311.35+12.57 54.07+1.03 **Amiodarone** 567.42+29.61\*\* 54.81+2.37 **Glibenclamide** 321.26+13.48 48.63+4.69 **Los. Pot** 400.84+4.24\*\* 51.36+0.99 **Oxcarbazepine** 392.61+14.86\*\* 56.05+1.99 **Verapamil** 307.28+6.49 49.42+3.01 **Captopril** 590.12+12.59\*\* 57.13+0.80 **AGLO** 393.91+21.54\*\* 60.66+1.93\* **AGVO** 330.11+38.41 53.05+1.48 **AGCO** 275.40+7.30\* 50.87+1.29

**AST (µ/l)** 

**AST (µ/l)** 

**Parameters/ Groups**

\*p < 0.05 significant with respect to control \*\*p <0.005 highly significant with respect to control

> **Parameters/ Groups**

\*p < 0.05 significant with respect to control \*\*p <0.005 highly significant with respect to control

n=9

n=9

Mean + S.E.M

Mean + S.E.M

Fig. 3A. Cardiac tissue showing no microscopic change (Adopted from Feroz et al., 2010).

Fig. 3B. Cardiac tissue showing focal pericardial inflammation (Adopted from Feroz et al., 2010).

#### **5. Biochemical testing of lipid profile**

Cholesterol and triglycerides are the most important plasma lipids, crucial for formation of cell membrane, synthesis of hormones and offer a source of free fatty acids (Dietschy, 1998). Table 4A and 4B reveals the comparison of cholesterol, triglyceride, HDL-C (high density lipoprotein cholesterol) and LDL-C (low density lipoprotein cholesterol) levels between control animals and animals kept on individual drugs and their combinations for a period of 60 days and then after drug free interval of 15 days in normal therapeutic doses. The administration of losartan potassium and captopril in animals showed highly significant decrease in the level of triglyceride, whereas animals kept on AGLO and AGCO combinations showed highly significant increase in cholesterol at the end of dosing which remained significant even after drug-free interval. There was also highly significant increase in LDL-C level in animals kept on AGLO and AGCO combinations which remained significant even after drug-free interval (Feroz et al., 2011b). Elevated levels of cholesterol and LDL-C are undoubtedly associated with enhanced threat of coronary heart disease (Brown, 1984) and cerebrovascular morbidity and mortality. There has been a correlation among increased LDL-C and atherosclerosis. Since LDL-C gets deposited in the walls of the blood vessel forming atherosclerotic plaque. There are studies which recommend that pathological process could be inverted by dropping the serum LDL-C level (Ross, 1993). There was also significant increase in HDL-C in animals kept on oxcarbazepine alone and AGCO in combination at the end of dosing and following drug-free interval; however reason of elevated HDL-C is yet to be explored (Feroz et al., 2011b).


n=9

Mean + S.E.M

\*p < 0.05 significant with respect to control

\*\*p <0.005 highly significant with respect to control

Table 4A. Comparison of lipid profile following 60 days administration of individual drugs and their combinations {Adopted from Feroz et al., 2011(b)}.


n=9

140 Biochemical Testing

Cholesterol and triglycerides are the most important plasma lipids, crucial for formation of cell membrane, synthesis of hormones and offer a source of free fatty acids (Dietschy, 1998). Table 4A and 4B reveals the comparison of cholesterol, triglyceride, HDL-C (high density lipoprotein cholesterol) and LDL-C (low density lipoprotein cholesterol) levels between control animals and animals kept on individual drugs and their combinations for a period of 60 days and then after drug free interval of 15 days in normal therapeutic doses. The administration of losartan potassium and captopril in animals showed highly significant decrease in the level of triglyceride, whereas animals kept on AGLO and AGCO combinations showed highly significant increase in cholesterol at the end of dosing which remained significant even after drug-free interval. There was also highly significant increase in LDL-C level in animals kept on AGLO and AGCO combinations which remained significant even after drug-free interval (Feroz et al., 2011b). Elevated levels of cholesterol and LDL-C are undoubtedly associated with enhanced threat of coronary heart disease (Brown, 1984) and cerebrovascular morbidity and mortality. There has been a correlation among increased LDL-C and atherosclerosis. Since LDL-C gets deposited in the walls of the blood vessel forming atherosclerotic plaque. There are studies which recommend that pathological process could be inverted by dropping the serum LDL-C level (Ross, 1993). There was also significant increase in HDL-C in animals kept on oxcarbazepine alone and AGCO in combination at the end of dosing and following drug-free interval; however

> **Triglyceride (mg/dl)**

**Control** 91.84+2.65 102.65+2.45 3.10+0.13 28.15+1.90 **Amiodarone** 88.80+0.62 100.51+2.97 3.07+0.04 27.04+1.13 **Glibenclamide** 93.37+3.07 106.77+4.71 3.28+0.06 23.25+2.08 **Los. Pot** 92.77+0.57 91.94+2.86\* 3.15+0.07 34.87+1.70 **Oxcarbazepine** 95.08+1.26 97.08+1.45 3.37+0.05\* 29.38+2.03 **Verapamil** 96.90+1.13 108.53+3.32 3.31+0.08 25.24+1.95 **Captopril** 96.06+2.89 93.40+4.47\* 3.32+0.07 34.18+2.26 **AGLO** 105.15+3.94\*\* 100.20+2.90 2.93+0.05 46.35+3.50\*\* **AGVO** 95.93+1.21 104.46+1.31 3.27+0.04 27.41+1.80 **AGCO** 173.53+4.22\*\* 95.35+3.17 3.40+0.08\*\* 108.83+6.13\*\*

Table 4A. Comparison of lipid profile following 60 days administration of individual drugs

**HDL-C (mg/dl)** 

**LDL-C (mg/dl)** 

**5. Biochemical testing of lipid profile** 

reason of elevated HDL-C is yet to be explored (Feroz et al., 2011b).

**Cholesterol (mg/dl)** 

and their combinations {Adopted from Feroz et al., 2011(b)}.

Parameters/ Groups

n=9

Mean + S.E.M

\*p < 0.05 significant with respect to control \*\*p <0.005 highly significant with respect to control Mean + S.E.M

\*p < 0.05 significant with respect to control

\*\*p <0.005 highly significant with respect to control

Table 4B. Comparison of lipid profile following drug-free interval of 15 days of individual drugs and their combinations {Adopted from Feroz et al., 2011(b)}.

#### **6. Biochemical testing of glucose**

Table 5A and 5B reveals the comparison of glucose level between control animals and animals kept on individual drugs and their combinations for a period of 60 days and then after drug free interval of 15 days in normal therapeutic doses. AGCO combination showed significant increase in glucose level in rabbits at the completion of dosing period of 60 days and following drug-free interval (Feroz et al., 2011b). Elevated blood glucose level may be due to elevation in the level of cholesterol and LDL-C, because diabetes mellitus is a group of heterogenous, autoimmune, hormonal and metabolic disorders, frequently occurs along with hypertension, hyperlipidemia and obesity (Mahomed and Ojewole, 2003), which also augmented the possibility of coronary heart disease (Howard et al., 2000), however threat of CVD fatality in diabetic persons may be as high as that in non-diabetic persons with prior myocardial infarction (Haffner et al., 1998). There was also a significant elevation in glucose level in animals kept on captopril and oxcarbazepine alone, however it has to be elucidated. Conversely animal kept on glibenclamide alone revealed highly significant decrease in glucose level because the major mechanism of action of glibenclamide is the stimulation of insulin release and the inhibition of glucagon secretion; conversely it was inverted following drug-free interval (Feroz et al., 2011b).


n=9

Mean + S.E.M

\*p < 0.05 significant with respect to control

\*\*p <0.005 highly significant with respect to control

Table 5A. Comparison of glucose following 60 days administration of individual drugs and their combinations {Adopted from Feroz et al., 2011(b)}


n=9

Mean + S.E.M

\*p < 0.05 significant with respect to control

\*\*p <0.005 highly significant with respect to control

Table 5B. Comparison of glucose following drug-free interval of 15 days of individual drugs and their combinations {Adopted from Feroz et al., 2011(b)}.

#### **7. Biochemical testing of electrolytes**

Table 6A and 6B reveals the comparison of sodium, potassium and calcium concentrations between control animals and animals kept on individual drugs and their combinations for a period of 60 days and then after drug free interval of 15 days in normal therapeutic doses. The balance of electrolytes in our bodies is essential for normal cellular function, since it promotes fluid balance, maintain blood volume, facilitate fluid absorption and generate impulses. Significant alterations may occur in electrolytes following multiple drug administration. There has been significant decrease in concentration of sodium in animals received amiodarone (Feroz et al., 2009), which has potential for significant morbidity and mortality (Goh, 2004). However this decrease became insignificant following drug-free interval. Similarly there was highly significant decrease in serum calcium in animal received glibenclamide, losartan potassium, verapamil, oxcarbazepine, captopril and combination of these drugs (Feroz et al., 2009). Calcium is essentially required for development and maintenance of bones, not only regulate nerve function, but also contributes to the contraction of the muscles and heart. There are studies which suggest that amiodarone induces vitamin D deficiency in individuals not exposed to sunlight (Campbell and Allain, 2006). Vitamin D is essentially required for absorption of calcium; hence in the study by Feroz et al 2009 hypocalcaemia in animals on amiodarone alone or in combination might be due to the deficiency of vitamin D (Cooper and Gittoes, 2008). However reason for hypocalcaemia in other animal groups is yet to be explored. There was highly significant increase in calcium after drug free interval in animals received AGVO combination, this increase in calcium might be due to increase bone resorption, or gastrointestinal absorption or decreased elimination by the kidneys (Strewler, 2000). Hypercalcemia is always a concern, because elevated concentrations can result in renal failure, mineralization of the other soft tissues, cardiac arrhythmia and dysfunction (Sakals et al., 2006). Animals received AGCO combination also showed decrease in potassium level at the end of dosing as well as following drug-free interval. A decreased serum potassium concentration points to disturbance in normal homeostasis which might be an indication of muscle necrosis. However potassium level in animals received AGVO combination was significantly increase after drug-free interval. Hyperkalemia because of drugs most frequently occurs from impaired renal potassium excretion. On the other hand, disturbed cellular uptake of a potassium load as well as unnecessary intake or infusion of potassium-containing substances may also induce hyperkalemia. Therefore prescribing physicians must be conscious about medications that can precipitate hyperkalemia (Perazella, 2000).


n=5

142 Biochemical Testing

**(mg/dl)** 

**(mg/dl)** 

**Parameter/ Groups Glucose** 

**Control** 122.20+7.60 **Amiodarone** 111.84+3.30 **Glibenclamide** 75.20+5.79\*\* **Los. Pot** 119.67+4.0 **Oxcarbazepine** 142.17+4.54\*\* **Verapamil** 123.33+2.31 **Captopril** 146.06+4.72\*\* **AGLO** 102.50+6.12 **AGVO** 111.88+3.11 **AGCO** 145.44+2.93\*\*

Table 5A. Comparison of glucose following 60 days administration of individual drugs and

**Parameter/ Groups Glucose** 

**Control** 123.40+7.40 **Amiodarone** 113.76+3.10 **Glibenclamide** 104.50+5.40 **Los. Pot** 122.30+4.30 **Oxcarbazepine** 141.50+4.70 **Verapamil** 124.39+2.30 **Captopril** 144.30+4.30\* **AGLO** 102.60+6.10 **AGVO** 112.11+3.10 **AGCO** 137.90+3.60\*

Table 5B. Comparison of glucose following drug-free interval of 15 days of individual drugs

Table 6A and 6B reveals the comparison of sodium, potassium and calcium concentrations between control animals and animals kept on individual drugs and their combinations for a period of 60 days and then after drug free interval of 15 days in normal therapeutic doses. The balance of electrolytes in our bodies is essential for normal cellular function, since it promotes fluid balance, maintain blood volume, facilitate fluid absorption and generate

n=9

n=9

Mean + S.E.M

Mean + S.E.M

\*p < 0.05 significant with respect to control \*\*p <0.005 highly significant with respect to control

\*p < 0.05 significant with respect to control \*\*p <0.005 highly significant with respect to control

**7. Biochemical testing of electrolytes** 

and their combinations {Adopted from Feroz et al., 2011(b)}.

their combinations {Adopted from Feroz et al., 2011(b)}

Mean + S.E.M

\*p < 0.05 significant with respect to control

\*\*p <0.005 highly significant with respect to control

Table 6A. Comparison of sodium, potassium and calcium following 60 days administration of individual drugs and their combinations (Adopted from Feroz et al., 2009).


n=5

Mean + S.E.M

\*p < 0.05 significant with respect to control

\*\*p <0.005 highly significant with respect to control

Table 6B. Comparison of sodium, potassium and calcium following drug-free interval of 15 days of individual drugs and their combinations (Adopted from Feroz et al., 2009).

#### **8. Hematological testing**

Table 7A and 7B reveals the comparison of hemoglobin concentration, platelet, leucocytes and erythrocytes count between control animals and animals kept on individual drugs and their combinations for a period of 60 days and then after drug free interval of 15 days in normal therapeutic doses. Changes in hematological parameters such as erythrocytes, leucocytes and platelet count and hemoglobin had always a serious concern following administration of drugs individually as well as in combination. There has been significant increase in platelet count in animal group received captopril and oxcarbazepine alone (Feroz et al., 2011a). Increased in platelet might be due to inflammatory disorder or iron deficiency anemia (Schafer, 2004), however there was also a significant increase in leucocytes count in animal group kept on oxcarbazepine alone, on the other hand animal group received amiodarone alone showed significant decrease in leucocytes count which might be due to disturbance in immune system, where as platelet count was not changed significantly, though amiodarone is known to produce thrombocytopenia (Weinberger et al., 1987).

Study conducted by Feroz et al 2011a revealed more severe hematological changes in animals received drugs in combination throughout the experimental period in comparison to animals received the drugs individually. Concurrent administration of AGLO combination showed a significant increased in leucocytes count which might be an indicator of an infection, inflammation, or allergy. On the other hand concurrent administration of AGVO combination showed highly significant increase in erythrocytes count while the other hematological parameters were not altered significantly.

There was significant increase and decrease in leucocytes and platelet count respectively in animals kept on AGCO combination. Decrease in platelet count may be due to insufficient production of platelet in bone marrow, a variety of reasons such as leukemia, lymphomas and several bone marrow disorders may have this effect on platelet count (McMillan, 2007).


Spleen enlargement may also decrease platelet count, or it may probably due to folic acid deficiency (Mant et al., 1979).

n=9

144 Biochemical Testing

**Control** 178.70+5.80 5.90+0.37 16.80+1.25 **Amiodarone** 162.72+2.30 5.40+0.65 15.70+1.20 **Glibenclamide** 181.46+2.90 6.38+0.59 17.04+0.91 **Los. Pot** 172.50+2.80 5.28+0.31 17.70+0.33 **Oxcarbazepine** 182.90+6.0 5.26+0.24 16.58+0.33 **Verapamil** 181.40+9.40 6.0+0.44 17.72+0.52 **Captopril** 180.60+10.0 5.48+0.33 16.72+0.49 **AGLO** 175.70+7.40 5.22+0.10 16.30+0.49 **AGVO** 175.56+3.10 7.0+0.07\* 20.38+0.28\*\* **AGCO** 170.40+5.90 3.94+0.15\*\* 13.16+0.87\*\*

Table 6B. Comparison of sodium, potassium and calcium following drug-free interval of 15

Table 7A and 7B reveals the comparison of hemoglobin concentration, platelet, leucocytes and erythrocytes count between control animals and animals kept on individual drugs and their combinations for a period of 60 days and then after drug free interval of 15 days in normal therapeutic doses. Changes in hematological parameters such as erythrocytes, leucocytes and platelet count and hemoglobin had always a serious concern following administration of drugs individually as well as in combination. There has been significant increase in platelet count in animal group received captopril and oxcarbazepine alone (Feroz et al., 2011a). Increased in platelet might be due to inflammatory disorder or iron deficiency anemia (Schafer, 2004), however there was also a significant increase in leucocytes count in animal group kept on oxcarbazepine alone, on the other hand animal group received amiodarone alone showed significant decrease in leucocytes count which might be due to disturbance in immune system, where as platelet count was not changed significantly, though amiodarone is known to produce thrombocytopenia (Weinberger et al., 1987).

Study conducted by Feroz et al 2011a revealed more severe hematological changes in animals received drugs in combination throughout the experimental period in comparison to animals received the drugs individually. Concurrent administration of AGLO combination showed a significant increased in leucocytes count which might be an indicator of an infection, inflammation, or allergy. On the other hand concurrent administration of AGVO combination showed highly significant increase in erythrocytes count while the

There was significant increase and decrease in leucocytes and platelet count respectively in animals kept on AGCO combination. Decrease in platelet count may be due to insufficient production of platelet in bone marrow, a variety of reasons such as leukemia, lymphomas and several bone marrow disorders may have this effect on platelet count (McMillan, 2007).

other hematological parameters were not altered significantly.

days of individual drugs and their combinations (Adopted from Feroz et al., 2009).

**Potassium (µg/ml)** 

**Calcium (µg/ml)** 

**Sodium (µg/ml)** 

**Parameters/ Groups**

n=5

Mean + S.E.M

\*p < 0.05 significant with respect to control \*\*p <0.005 highly significant with respect to control

**8. Hematological testing** 

Mean + S.E.M

\*p < 0.05 significant with respect to control

\*\*p <0.005 highly significant with respect to control

Table 7A. Comparison of hematological parameters following 60 days administration of individual drugs and their combinations {Adopted from Feroz et al., 2011(a)}.


n=9

Mean + S.E.M

\*p < 0.05 significant with respect to control

\*\*p <0.005 highly significant with respect to control

Table 7B. Comparison of hematological parameters following drug-free interval of 15 days of individual drugs and their combinations {Adopted from Feroz et al., 2011(a)}.

#### **9. Conclusion**

The problems associated with drug therapy are a significant challenge to health care providers, especially in developing countries where health care system is poor. Minimizing the risk for drug interactions is the desirable aim in drug therapy, since interactions can leads to significant morbidity, mortality and patient quality of life. Individuals taking multiple medications are at increased threat of adverse drug reactions; hence when ever multiple drugs are to be administered in case of multiple disorders such as epilepsy, hypertension, diabetes mellitus and arrhythmias drug treatment should be monitored to avoid adverse effects of the drugs. Studies conducted by Feroz et al not only provides valuable information pertaining to gross toxicities, microscopic changes and toxic effects on hepatic, renal, cardiac, lipid profile, glucose, electrolytes and hematological parameters but also give clues about the drug combination having higher incidence of cumulative toxicities.

These studies in general has revealed that animals received AGCO combination comparatively showed higher toxicities with marked decrease in ALT, TBR, CK, potassium, calcium and platelet count and increase in urea, creatinine, cholesterol, LDL-C, glucose and leucocytes count. However further studies on more animals and human beings are necessary to defend the utilization of multiple drugs.

These studies provides detailed evaluation of dug interaction and adverse effect of cumulative drug therapy; such observations are of undisputed importance but it should not be disregarded that pathway of drug metabolism in man may be quite dissimilar from that which has been determined in many species of laboratory animal, hence trial in man is the only valid way of establish drug interactions, before reaching to any final conclusion. However the risk of adverse drug reactions and drug interactions can be reduced by forming drug information centers, continuous medical education and incorporation of adverse drug reaction reporting into the clinical activities of the physicians (Oshikoya and Awobusuyi, 2009).

#### **10. References**


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These studies provides detailed evaluation of dug interaction and adverse effect of cumulative drug therapy; such observations are of undisputed importance but it should not be disregarded that pathway of drug metabolism in man may be quite dissimilar from that which has been determined in many species of laboratory animal, hence trial in man is the only valid way of establish drug interactions, before reaching to any final conclusion. However the risk of adverse drug reactions and drug interactions can be reduced by forming drug information centers, continuous medical education and incorporation of adverse drug reaction reporting into the clinical activities of the physicians (Oshikoya and

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### **Lipoxygenase-Quercetin Interaction: A Kinetic Study Through Biochemical and Spectroscopy Approaches**

Veronica Sanda Chedea1,2, Simona Ioana Vicaş2, Carmen Socaciu3, Tsutomu Nagaya4, Henry Joseph Oduor Ogola4,5, Kazushige Yokota4, Kohji Nishimura4 and Mitsuo Jisaka4\*

#### **1. Introduction**

150 Biochemical Testing

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736

#### **1.1 Lipoxygenase – definition, structure, reaction mechanism and metabolic functions**

Lipoxygenases (EC 1.13.11.12, linoleate:oxygen, oxidoreductases, LOXs) which are widely found in plants, fungi, and animals, are a large monomeric protein family with non-heme, non-sulphur, iron cofactor containing dioxygenases that catalyze the oxidation of polyunsaturated fatty acids (PUFA) as substrate with at least one 1*Z*, 4*Z*-pentadiene moiety such as linoleic, linolenic and arachidonic acid to yield hydroperoxides (Gardner, 1991).

Fig. 1. Lipoxygenase substrates, linoleic, -linolenic and arachidonic acid.

<sup>\*</sup> *1 Laboratory of Animal Biology, National Research Development Institute* 

*for Animal Biology and Nutrition Baloteşti (IBNA), Romania* 

*<sup>2</sup> Faculty of Environmental Protection, University of Oradea, Romania* 

*<sup>3</sup> Department of Chemistry and Biochemistry, University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, Romania*

*<sup>4</sup> Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University, Japan* 

*<sup>5</sup> School of Agriculture, Food Security and Biodiversity, Bondo University College, Kenya* 

The crystal structures of soybean LOX-1 (Minor et al., 1996), LOX-3 (Skrzypczak-Jankun et al., 1997), rabbit 15S-LOX-1 (Gillmor et al., 1997), and coral 8R-LOX (Oldham et al., 2005) have been elucidated thus far and has helped in understanding the properties of LOXs at the molecular level. Several structures of soybean LOX in complex with its product or inhibitor are also known (Skrzypczak-Jankun et al., 2001; Skrzypczak-Jankuna et al., 2003; Skrzypczak-Jankunb et al., 2003; Borbulevych et al., 2004; Skrzypczak-Jankun et al., 2004). There are significant differences in size, sequence, and substrate preference between the plant and animal LOXs, but the overall folding and geometry of the nonheme iron-binding site are conserved (Kühn et al., 2005; Skrzypczak-Jankun et al., 2006). All LOXs are folded in a twodomain structure that is composed of a smaller -barrel domain (N-terminal domain) and a larger -helical catalytic domain (C-terminal domain) (Choi et al., 2008). The nonheme iron essential for activity is positioned deep in a large cavity that accommodates the substrate (Choi et al., 2008). The regio- and stereospecificities of the various LOX isozymes are believed to be determined by the shape and depth of the cavity as well as the binding orientation of the substrate in the cavity (Borngräber et al., 1999; Kühn, 2000; Coffa et al., 2005).

The initial step of LOX reaction is removal of a hydrogen atom from a methylene unit between double bonds in substrate fatty acids (Fig. 2A). The resulting carbon radical is stabilized by electron delocalization through the double bonds. Then, a molecular oxygen is added to the carbon atom at +2 or –2 position from the original radical carbon, forming a peroxy radical as well as a conjugated *trans*, *cis*-diene chromophore. The peroxy radical is then hydrogenated to form a hydroperoxide. The initial hydrogen removal and the following oxygen addition occur in opposite (or antarafacial) sides related to the plane formed by the 1*Z*, 4*Z*-pentadiene unit. In most LOX reactions, particularly those in plants, the resulting hydroperoxy groups are in *S*-configuration, while one mammalian LOX and some marine invertebrate LOXs produce *R*-hydroperoxides. Even in the reactions of such "*R*-LOXs", the antarafacial rule of hydrogen removal and oxygen addition is conserved (Chedea & Jisaka, 2011).

In cases of plant LOXs, including soybean LOXs, the usual substrates are C18 polyunsaturated fatty acids (linoleic and -linolenic acids), and the products are their 9*S*- or 13*S*-hydroperoxides (Fig. 2B). Most plant LOXs react with either one of the regio-specificity, while some with both. Therefore, based on the regio-specificty, plant LOXs are classified into 9-LOXs, 13-LOXs, or 9/13-LOXs (Chedea & Jisaka, 2011).

Fig. 2. LOX reaction showing the principal steps of LOX reaction (Panel A), and the actual reactions of plant LOXs and -linolenic acid (Panel B). HPOTE: hydroperoxyoctadecatrienoic acid (Chedea & Jisaka, 2011).

The crystal structures of soybean LOX-1 (Minor et al., 1996), LOX-3 (Skrzypczak-Jankun et al., 1997), rabbit 15S-LOX-1 (Gillmor et al., 1997), and coral 8R-LOX (Oldham et al., 2005) have been elucidated thus far and has helped in understanding the properties of LOXs at the molecular level. Several structures of soybean LOX in complex with its product or inhibitor are also known (Skrzypczak-Jankun et al., 2001; Skrzypczak-Jankuna et al., 2003; Skrzypczak-Jankunb et al., 2003; Borbulevych et al., 2004; Skrzypczak-Jankun et al., 2004). There are significant differences in size, sequence, and substrate preference between the plant and animal LOXs, but the overall folding and geometry of the nonheme iron-binding site are conserved (Kühn et al., 2005; Skrzypczak-Jankun et al., 2006). All LOXs are folded in a twodomain structure that is composed of a smaller -barrel domain (N-terminal domain) and a larger -helical catalytic domain (C-terminal domain) (Choi et al., 2008). The nonheme iron essential for activity is positioned deep in a large cavity that accommodates the substrate (Choi et al., 2008). The regio- and stereospecificities of the various LOX isozymes are believed to be determined by the shape and depth of the cavity as well as the binding orientation of the

The initial step of LOX reaction is removal of a hydrogen atom from a methylene unit between double bonds in substrate fatty acids (Fig. 2A). The resulting carbon radical is stabilized by electron delocalization through the double bonds. Then, a molecular oxygen is added to the carbon atom at +2 or –2 position from the original radical carbon, forming a peroxy radical as well as a conjugated *trans*, *cis*-diene chromophore. The peroxy radical is then hydrogenated to form a hydroperoxide. The initial hydrogen removal and the following oxygen addition occur in opposite (or antarafacial) sides related to the plane formed by the 1*Z*, 4*Z*-pentadiene unit. In most LOX reactions, particularly those in plants, the resulting hydroperoxy groups are in *S*-configuration, while one mammalian LOX and some marine invertebrate LOXs produce *R*-hydroperoxides. Even in the reactions of such "*R*-LOXs", the antarafacial rule of hydrogen removal and oxygen addition is conserved

In cases of plant LOXs, including soybean LOXs, the usual substrates are C18 polyunsaturated fatty acids (linoleic and -linolenic acids), and the products are their 9*S*- or 13*S*-hydroperoxides (Fig. 2B). Most plant LOXs react with either one of the regio-specificity, while some with both. Therefore, based on the regio-specificty, plant LOXs are classified

Fig. 2. LOX reaction showing the principal steps of LOX reaction (Panel A), and the actual

substrate in the cavity (Borngräber et al., 1999; Kühn, 2000; Coffa et al., 2005).

into 9-LOXs, 13-LOXs, or 9/13-LOXs (Chedea & Jisaka, 2011).

reactions of plant LOXs and -linolenic acid (Panel B). HPOTE: hydroperoxyoctadecatrienoic acid (Chedea & Jisaka, 2011).

(Chedea & Jisaka, 2011).

Theorell et al. (1947) succeeded in crystallizing and characterizing lipoxygenase (LOX) from soybeans and since then among plant LOXs, soybean LOX-1 can be regarded as the mechanistic paradigm for these nonheme iron dioxygenases (Coffa et al., 2005; Minor et al., 1996; Fiorucci et al., 2008).

LOX isoenzymes of soybean seed are 94–97 kDa monomeric proteins with distinct isoelectric points ranging from about 5.7 to 6.4, and can be distinguished by optimum pH, substrate specificity, product formation and stability (Siedow, 1991). LOX-1 is the smallest in size (838 amino acids; 94 kDa), exhibits maximal activity at pH 9.0, and converts linoleic acid preferentially into the 13-hydroperoxide derivative. LOX-2 is characterized by a larger size (865 amino acids; 97 kDa), by a peak of activity at pH 6.8, and forms equal amounts of the 13- and 9-hydroperoxide compounds (Loiseau et al., 2001). LOX-2 oxygenates the esterified unsaturated fatty acid moieties in membranes in contrast to LOX-1 which only uses free fatty acids as substrates (Maccarrone et al., 1994). LOX-3 (857 amino acids; 96.5 kDa) exhibits its maximal activity over a broad pH range centred around pH 7.0 and displays a moderate preference for producing a 9-hydroperoxide product. It is the most active isoenzyme with respect to both carotenoid cooxidation and production of oxodienoic acids (Ramadoss, 1978).

Arachidonic acid metabolism by LOX in platelets was demonstrated in 1974 (Hamberg & Samuelsson, 1974). Rabbit reticulocyte LOX was described for the first time in 1975 (Schewe et al., 1975). There are 6 functional LOXs in humans: 5-LOX; 12/15-LOX (15-LOX-1); platelet-type 12-LOX; 12R-LOX; epidermis-type 15-LOX (15-LOX-2); and epidermis-Alox3. Each of these genes expresses a catalytically active enzyme except epidermis-Alox3, which encodes an enzyme that has hydroperoxidase activity. Thus, the main LOX enzymes with fatty acid oxygenase activity found in humans are 5-LOX, 15-LOX-1, 15-LOX-2, platelet-type 12-LOX and 12R-LOX (Bhattacharya et al., 2009).

In their review concerning the metabolic functions of LOX, Ivanov et al. (2010) present two aspects of LOX biology: (i) 30 years ago, leukotrienes generated by 5-LOX were identified as potent pro-inflammatory mediators (Samuelsson et al., 1980; Jakschik & Lee, 1980). Since then, additional pro-inflammatory products have been discovered (Feltenmark et al., 2008). On the other hand, anti-inflammatory and/or pro-resolving lipids generated by mammalian isoforms have also been identified, including lipoxins (Serhan et al., 1984; Maderna & Gordon, 2009), resolvins (Serhan et al., 2004), protectins (Ariel et al., 2005; Schwab et al., 2007) and maresins (Serhan et al., 2009). Therefore, LOX products play important roles in the development of acute inflammation but they have also been implicated in inflammatory resolution. (ii) Mice deficient in 12R-LOX develop normally during pregnancy but die immediately after birth due to excessive dehydration (Furstenberger et al., 2007; Epp et al., 2007). Although the molecular mechanisms of postpartum mortality are unknown, the enzyme was implicated in the formation of the epidermal water barrier. Genetic polymorphisms of the corresponding human gene have been related to ichthyosis (Eckl et al., 2009), a disease characterized by dry, thickened, scaly or flaky skin (Ivanov et al., 2010).

Available data also suggest that lipoxygenases contribute to *in vivo* metabolism of endobiotics and xenobiotics in mammals (Kulkarni, 2001). Recent reviews describe the role of lipoxygenase in cancer (Bhattacharya et al., 2009; Pidgeon et al., 2007; Moreno, 2009), inflammation (Duroudier et al., 2009; Hersberger, 2010) and vascular biology (Chawengsub et al., 2009; Mochizuki & Kwon, 2008) and for an extensive presentation of the role of eicosanoids in prevention and management of diseases the reader is referred to the review of Szefel et al. (2011).

#### **1.2 Quercetin**

Quercetin (3,3',4',5,7-pentahydroxyflavone) (Fig. 3) is an important dietary flavonoid, present in different vegetables, fruits, seeds, nuts, tea and red wine (Beecher et al., 1999; Formica, 1995; Hollman & Katan, 1999).

Fig. 3. Chemical structure of quercetin.

Quercetin has been discussed for several decades as a multipotent bioflavonoid with great potential for the prevention and treatment of disease (Bischoff, 2008). Its documented impact on human health includes cardiovascular protection, anticancer, antiviral, antiinflammatory activities, antiulcer effects and cataract prevention. The study of quercetin as potential chemopreventer is assuming increasing importance considering its involvement in the suppression of many tumor-related processes including oxidative stress, apoptosis, proliferation and metastasis. Quercetin has also received greater attention as pro-apoptotic flavonoid with a specific and almost exclusive activity on tumor cell lines rather than normal, non-transformed cells (Lugli et al., 2009).

Among the biological effects of particular relevance, the antihypertensive effects of quercetin in humans and the improvement of endothelial function should also be emphasized (Bischoff, 2008). Together with its antithrombotic and anti-inflammatory effects, the latter mainly mediated through the inhibition of cytokines and nitric oxide, quercetin is a candidate for preventing obesity-related diseases (Bischoff, 2008). Most exciting findings are that quercetin enhances physical power by yet unclear mechanisms. The anti-infectious and immunomodulatory activities of quercetin might be related to this effect (Bischoff, 2008).

Like other flavonoids, quercetin appears to combine both lipoxygenase-inhibitory activities and free radical-scavenging properties in one agent and thus belongs to a family of very effective natural antioxidants (Sadik et al., 2003). Quercetin is a flavonol that can be easily oxidized in an aqueous environment, and in the presence of iron and hydroxyl free radicals (Borbulevych et al., 2004). More specifically, in an aqueous solution, quercetin is known to be oxidized to the relatively stable, neutral protocatechuate intermediate 2-(3,4 dihydroxybenzoyl)-2,4,6-trihydroxybenzofuran-3(2H)-one (Fig. 5). This protocatechuate derivative is a common oxidized intermediate mediated by various oxidases, such as lipoxygenase, tyrosinase (Kubo et al., 2004), and peroxidase (Awad et al., 2000), as well as by diphenylpicrylhydrazyl (DPPH) and azobisisobutyronitrile (AIBN) (Krishnamachari et al., 2002) and seems to be a key intermediate to understand quercetin's diverse biological activities (Ha et al., 2010).

#### **1.3 LOX inhibition by quercetin**

154 Biochemical Testing

et al., 2009; Mochizuki & Kwon, 2008) and for an extensive presentation of the role of eicosanoids in prevention and management of diseases the reader is referred to the review

Quercetin (3,3',4',5,7-pentahydroxyflavone) (Fig. 3) is an important dietary flavonoid, present in different vegetables, fruits, seeds, nuts, tea and red wine (Beecher et al., 1999;

Quercetin has been discussed for several decades as a multipotent bioflavonoid with great potential for the prevention and treatment of disease (Bischoff, 2008). Its documented impact on human health includes cardiovascular protection, anticancer, antiviral, antiinflammatory activities, antiulcer effects and cataract prevention. The study of quercetin as potential chemopreventer is assuming increasing importance considering its involvement in the suppression of many tumor-related processes including oxidative stress, apoptosis, proliferation and metastasis. Quercetin has also received greater attention as pro-apoptotic flavonoid with a specific and almost exclusive activity on tumor cell lines rather than

Among the biological effects of particular relevance, the antihypertensive effects of quercetin in humans and the improvement of endothelial function should also be emphasized (Bischoff, 2008). Together with its antithrombotic and anti-inflammatory effects, the latter mainly mediated through the inhibition of cytokines and nitric oxide, quercetin is a candidate for preventing obesity-related diseases (Bischoff, 2008). Most exciting findings are that quercetin enhances physical power by yet unclear mechanisms. The anti-infectious and immunomodulatory activities of quercetin might be related to this effect (Bischoff,

Like other flavonoids, quercetin appears to combine both lipoxygenase-inhibitory activities and free radical-scavenging properties in one agent and thus belongs to a family of very effective natural antioxidants (Sadik et al., 2003). Quercetin is a flavonol that can be easily oxidized in an aqueous environment, and in the presence of iron and hydroxyl free radicals (Borbulevych et al., 2004). More specifically, in an aqueous solution, quercetin is known to be oxidized to the relatively stable, neutral protocatechuate intermediate 2-(3,4 dihydroxybenzoyl)-2,4,6-trihydroxybenzofuran-3(2H)-one (Fig. 5). This protocatechuate

of Szefel et al. (2011).

Formica, 1995; Hollman & Katan, 1999).

Fig. 3. Chemical structure of quercetin.

normal, non-transformed cells (Lugli et al., 2009).

**1.2 Quercetin** 

2008).

Because of its unique capability of the direct catalysis the enzymatic lipid peroxidation, 15- LOX-1 belongs to the endogenous prooxidants the action of which may be favored under conditions of oxidative stress (Schewe, 2002). Consequently, the inhibition of 15-LOX-1 may contribute to the universal antioxidant activities of dietary flavonoids (Sadik et al., 2003). Flavonoids appear to combine both lipoxygenase-inhibitory activities and free radicalscavenging properties in one agent and thus constitute a family of very effective natural antioxidants (Sadik et al., 2003). The literature data indicate that quercetin represents one of the most potent inhibitors of different LOXs (Schneider & Bucara, 2005; Schneider & Bucarb, 2005).

The inhibition of rabbit 15-LOX-1 and of soybean LOX-1 by quercetin was studied in detail (Sadik et al., 2003). Quercetin modulates the time course of the lipoxygenase reaction in a complex manner by exerting three distinct effects: (i) prolongation of the kinetic lag period, (ii) instant decrease in the initial rate after the lag phase being overcome, (iii) timedependent inactivation of the enzyme during reaction, but not in the absence of substrate (Schewe & Sies, 2003). Competitive reversible and irreversible inhibition schemes, as well as inhibition via reduction of the enzyme-bound radical intermediate have been considered to explain the activity of polyphenolic compounds (Fiorucci et al., 2008). Moreover, heterogeneity in the interpretation of the experimental results of the inhibition processes, for example concerning kinetic data, prevents converging toward a general way of inhibition (Fiorucci et al., 2008). It may be supposed that the inactivation is due to combined action of quercetin and intermediates of the catalytic cycle on the active site of the enzyme (Sadik et al., 2003). In previous work, Redrejo-Rodriguez et al. (2004) reported by semiempirical studies that the interaction of quercetin with lipoxygenase is related to the spatial adaptation of the flavonoid to the hydrophobic cavity that constitutes the channel of access of substrate to the catalytic site.

Structural analysis reveals that quercetin entrapped within LOX undergoes degradation and the resulting compound has been identified by X-ray analysis as protocatechuic acid (3,4 dihydroxybenzoic acid) positioned near the iron site (Borbulevych et al., 2004).

Fig. 4. Chemical structure of protocatechuic acid, the product of quercetin degradation by soybean LOX-3 as identified by Borbulevych et al. (2004).

The finding that LOX can turn different compounds, like quercetin and epigallocatechin gallate, into simple catechol derivatives (with one aromatic ring only) might be of importance as an additional small piece of a "jigsaw puzzle" in the much bigger picture of drug metabolism. Their interactions with LOX can be more complicated than simply blocking the access to the enzyme's active site (Borbulevych et al., 2004). Ha et al. (2010) have studied the inhibitory activity of protocatechuic acid and of dodecyl protocatechuate on soybean LOX-1 oxidizing linoleic acid. Their results show that the protocatechuate derivative, dodecyl protocatechuate, inhibited the enzymatic peroxidation of linoleic acid as a competitive inhibitor, but its parent compound, protocatechuic acid did not show any activity up to 200 M. In this way it was shown that the catechol moiety alone is not sufficient to elicit the inhibitory activity and that the hydrophobic dodecyl group is associated with inhibitory activity as it was also reported for dodecyl gallate (Ha & Kubo, 2007; Ha et al., 2010).

Fiorucci et al. (2008) report theoretical investigations concerning three binding modes between quercetin and LOX-3 enzyme. Thus O3, O7, and O4' oxygen atoms have been considered to bind the iron center (Fiorucci et al., 2008). These specific interactions lead then, through electron transfer from the substrate to the cation, to semiquinone forms, and related tautomeric structures that are compatible with an addition of the triplet spin state dioxygen to the flavonol squeleton (Fiorucci et al., 2008). Among the three considered modes of binding, it appears that quercetin should be linked to the metal center via its 3-OH functional group. The most favorable term to the binding free energy is due to electrostatic interactions (Fiorucci et al., 2008).

In their proposed mechanism of oxidative degradation of quercetin by soybean LOX-1, Ha et al. (2010) indicate that quercetin might first be oxidized to the corresponding o-quinone after the abstraction of 2 e- and 2 H+ from the OH groups at C3' and C4' (Jungbluth et al., 2000). The enzymatically oxidized o-quinone might be subsequently isomerized to a pquinone methide type intermediate, followed by the addition of H2O at C2, yielding the relatively stable intermediate 2-(3,4-dihydroxybenzoyl)-2,4,6-trihydroxybenzofuran-3(2H) one (Ha et al., 2010). This enzymatically-generated intermediate appears to be relatively stable, and hence, prolongs the inhibitory activity (Ha et al., 2010). The results indicate that the soybean LOX-1 generated intermediate of quercetin is the same as the DPPH (2,2 diphenyl--picrylhydrazyl) generated intermediate, 2-(3,4-dihydroxybenzoyl)-2,4,6 trihydroxybenzofuran-3(2H)-one (Krishnamachari et al., 2002). In addition, the same oxidized intermediate was also characterized as the relatively stable intermediate generated by mushroom tyrosinase (Ha et al., 2010).

The finding that LOX can turn different compounds, like quercetin and epigallocatechin gallate, into simple catechol derivatives (with one aromatic ring only) might be of importance as an additional small piece of a "jigsaw puzzle" in the much bigger picture of drug metabolism. Their interactions with LOX can be more complicated than simply blocking the access to the enzyme's active site (Borbulevych et al., 2004). Ha et al. (2010) have studied the inhibitory activity of protocatechuic acid and of dodecyl protocatechuate on soybean LOX-1 oxidizing linoleic acid. Their results show that the protocatechuate derivative, dodecyl protocatechuate, inhibited the enzymatic peroxidation of linoleic acid as a competitive inhibitor, but its parent compound, protocatechuic acid did not show any activity up to 200 M. In this way it was shown that the catechol moiety alone is not sufficient to elicit the inhibitory activity and that the hydrophobic dodecyl group is associated with inhibitory activity as it was also reported for dodecyl gallate (Ha & Kubo,

Fiorucci et al. (2008) report theoretical investigations concerning three binding modes between quercetin and LOX-3 enzyme. Thus O3, O7, and O4' oxygen atoms have been considered to bind the iron center (Fiorucci et al., 2008). These specific interactions lead then, through electron transfer from the substrate to the cation, to semiquinone forms, and related tautomeric structures that are compatible with an addition of the triplet spin state dioxygen to the flavonol squeleton (Fiorucci et al., 2008). Among the three considered modes of binding, it appears that quercetin should be linked to the metal center via its 3-OH functional group. The most favorable term to the binding free energy is due to electrostatic

In their proposed mechanism of oxidative degradation of quercetin by soybean LOX-1, Ha et al. (2010) indicate that quercetin might first be oxidized to the corresponding o-quinone after the abstraction of 2 e- and 2 H+ from the OH groups at C3' and C4' (Jungbluth et al., 2000). The enzymatically oxidized o-quinone might be subsequently isomerized to a pquinone methide type intermediate, followed by the addition of H2O at C2, yielding the relatively stable intermediate 2-(3,4-dihydroxybenzoyl)-2,4,6-trihydroxybenzofuran-3(2H) one (Ha et al., 2010). This enzymatically-generated intermediate appears to be relatively stable, and hence, prolongs the inhibitory activity (Ha et al., 2010). The results indicate that the soybean LOX-1 generated intermediate of quercetin is the same as the DPPH (2,2 diphenyl--picrylhydrazyl) generated intermediate, 2-(3,4-dihydroxybenzoyl)-2,4,6 trihydroxybenzofuran-3(2H)-one (Krishnamachari et al., 2002). In addition, the same oxidized intermediate was also characterized as the relatively stable intermediate generated

Fig. 5. Product of oxidative degradation of quercetin by soybean LOX-1 as proposed by Ha

2007; Ha et al., 2010).

interactions (Fiorucci et al., 2008).

by mushroom tyrosinase (Ha et al., 2010).

et al. (2010).

The studies on LOX and quercetin contribute to the understanding of biocatalytic properties of this enzyme and its role in the metabolism of this popular (as a medicinal remedy) flavonol and possibly other, similar compounds (Borbulevych et al., 2004).

#### **1.4 O-quinone formation during quercetin oxidation by LOX**

Because flavonoids, such as quercetin, and oxidases are present simultaneously in fruits and vegetables, the generation of quinoid derivatives in biological systems is plausible (Pinto & Macias, 2005). This process is of great relevance from a biological point of view, because the conversion of supposed beneficial antioxidants such as flavonoids to electrophilic prooxidants may constitute a possible toxicological risk (Boersma et al., 2000).

On the other hand, it is to be noted that, in addition to dioxygenase activity, lipoxygenase, possesses a peroxidase activity toward a wide range of compounds (Gardner, 1991). Dioxygenase activity produces the insertion of oxygen into a polyunsaturated fatty acid containing a 1,4-*cis*,*cis*-pentadiene moiety, producing the corresponding lipid hydroperoxide. In the process, an intermediate peroxyl radical is generated. This compound, or the peroxide, supports the cooxidase activity of lipoxygenase toward a suitable electron donor, which is transformed into a radical (Pinto & Macias, 2005). The hydroperoxidase activity of LOX also can be observed in the presence of hydrogen peroxide instead of lipid hydroperoxide, being related to xenobiotic oxidation processes (Hover & Kulkarni, 2000; Santanoa et al., 2002; Santanob et al., 2002). In addition, it is known that a variety of phenolic compounds and flavonoids with antioxidant properties are inhibitors of lipoxygenase (Prasad et al., 2004).

On the basis of the results obtained, Pinto & Macias (2005) concluded that in the presence of hydrogen peroxide or hydroperoxylinoleic acid, lipoxygenase produces a quinoid product as a result of the enzymatic oxidation (in the presence of hydrogen peroxide) or cooxidation (in the presence of linoleic acid) of quercetin (Pinto & Macias, 2005).

During lipoxygenase catalysis enzyme-bound prooxidant intermediates such as fatty acid peroxyl radical (ROO•) are formed (Schewe, 2002). It is tempting to speculate, therefore, that the flavonoids are co-oxidized in this system to a semi-quinone or quinone (with flavonoids containing a catechol B ring) or a phenoxy radical (with noncatechol flavonoids) which in turn may covalently bind to sulfhydryl or amino groups of the lipoxygenase, thus rendering its inhibition irreversible (Sadik et al., 2003). In the case of quercetin and other flavonols, the intermediate formation of corresponding quinone methides (Awad et al., 2001) may be involved (Sadik et al., 2003).

Quercetin is considered an excellent free radical scavenging antioxidant owing to the high number of hydroxyl groups and conjugated orbitals by which quercetin can donate electrons or hydrogen, and scavenge H2O2 and superoxide anion (*•*O2*-*) (Heijnen et al., 2001). The reaction of quercetin with *•*O2*-* leads to the generation of the semiquinone radical and H2O2 (Metodiewa et al., 1999). Quercetin also reacts with H2O2 in the presence of peroxidases, and thus it decreases H2O2 levels and protects cells against H2O2 damage; nevertheless, during the same process potentially harmful reactive oxidation products are also formed. The first oxidation product of quercetin is a semiquinone radical (Metodiewa et al., 1999). This radical is unstable and rapidly undergoes a second oxidation reaction that produces another quinone (quercetin-quinone, QQ) (Metodiewa et al., 1999). Since QQ can react with proteins, lipids and DNA, it is responsible for protein and DNA damage as well as lipid peroxidation.

The oxidative decomposition of quercetin by hydroperoxidase activity of lipoxygenase has been reported, suggesting that in the presence of the lipoxygenase/H2O2 system quercetin is oxidized to a quinoid product. It is remarkable that this behavior is not shown by naringenin or resveratrol, other bioactive antioxidant phenolics, probably due to the different redox potentials of these compounds (Pinto & Macias, 2005).

UV-Vis spectroscopy is a widespread and commonly used technique that has been successfully used for the determination of catalytic mechanism, including enzymesubstrate/inhibitor interaction profiles. In this report, we used UV-Vis spectroscopy to help elucidate possible interacting/inhibitory effect of quercetin with soybean LOX-1, oxidizing or not the linoleic acid. The UV-Vis spectral analysis of the mixture quercetin and LOX-1 and the kinetic parameters (Km and Vmax) of LOX-1-catalyzed oxidation of linoleic acid in the absence and presence of different quercetin concentrations revealed that:


#### **2. Biochemical analysis of LOX interaction with quercetin**

#### **2.1 UV-Vis spectroscopy**

When a sample of an unknown compound is exposed to light, certain functional groups within the molecule absorb light of different wavelengths. In UV/Visible Spectroscopy, the term chromophore is used to indicate a functional group that absorbs electromagnetic radiation, usually in the UV or visible region. The type of functional groups that absorb ultraviolet light can be conjugated species, such as alkenes, aromatics, etc., making UV/Visible spectroscopy useful for distinguishing conjugated dienes from conjugated trienes, and so forth (Perkampus, 1992).

The reference beam in the spectrometer travels from the light source to the detector without interacting with the sample. The sample beam interacts with the sample exposing it to ultraviolet light of continuously changing wavelength. When the emitted wavelength corresponds to the energy level which promotes an electron to a higher molecular orbital, energy is absorbed. The detector records the ratio between reference and sample beam intensities (I0/I). At the wavelength where the sample absorbs a large amount of light, the detector receives a very weak sample beam. Once intensity data has been collected by the spectrometer, it is sent to the computer as a ratio of reference beam and sample beam

react with proteins, lipids and DNA, it is responsible for protein and DNA damage as well

The oxidative decomposition of quercetin by hydroperoxidase activity of lipoxygenase has been reported, suggesting that in the presence of the lipoxygenase/H2O2 system quercetin is oxidized to a quinoid product. It is remarkable that this behavior is not shown by naringenin or resveratrol, other bioactive antioxidant phenolics, probably due to the

UV-Vis spectroscopy is a widespread and commonly used technique that has been successfully used for the determination of catalytic mechanism, including enzymesubstrate/inhibitor interaction profiles. In this report, we used UV-Vis spectroscopy to help elucidate possible interacting/inhibitory effect of quercetin with soybean LOX-1, oxidizing or not the linoleic acid. The UV-Vis spectral analysis of the mixture quercetin and LOX-1 and the kinetic parameters (Km and Vmax) of LOX-1-catalyzed oxidation of linoleic acid in the

i. quercetin (max= 370 nm) was oxidized to a new compound having max= 321 nm by

iv. quercetin concentration significantly affected its partitioning level as a substrate or an

v. the ratio substrate:inhibitor might be a factor determining the type of inhibition

When a sample of an unknown compound is exposed to light, certain functional groups within the molecule absorb light of different wavelengths. In UV/Visible Spectroscopy, the term chromophore is used to indicate a functional group that absorbs electromagnetic radiation, usually in the UV or visible region. The type of functional groups that absorb ultraviolet light can be conjugated species, such as alkenes, aromatics, etc., making UV/Visible spectroscopy useful for distinguishing conjugated dienes from conjugated

The reference beam in the spectrometer travels from the light source to the detector without interacting with the sample. The sample beam interacts with the sample exposing it to ultraviolet light of continuously changing wavelength. When the emitted wavelength corresponds to the energy level which promotes an electron to a higher molecular orbital, energy is absorbed. The detector records the ratio between reference and sample beam intensities (I0/I). At the wavelength where the sample absorbs a large amount of light, the detector receives a very weak sample beam. Once intensity data has been collected by the spectrometer, it is sent to the computer as a ratio of reference beam and sample beam

ii. a mixed inhibition occurred for quercetin concentrations in the range of 10~50 M. iii. at 100 M, the highest quercetin concentration tested, the Kmapp decreased by half while reaction rate increased indicative of a cooxidation of quercetin in addition to the LOX classical reaction illustrating the switch in quercetin's role from inhibitor towards

different redox potentials of these compounds (Pinto & Macias, 2005).

absence and presence of different quercetin concentrations revealed that:

observed in the case of lipoxygenase and quercetin interaction.

**2. Biochemical analysis of LOX interaction with quercetin** 

as lipid peroxidation.

LOX-1 in absence of substrate.

substrate.

inhibitor of LOX.

**2.1 UV-Vis spectroscopy** 

trienes, and so forth (Perkampus, 1992).

intensities. The computer determines at what wavelength the sample absorbed a large amount of ultraviolet light by scanning for the largest gap between the two beams (Perkampus, 1992).

When a large gap between intensities is found, where the sample beam intensity is significantly weaker than the reference beam, the computer plots this wavelength as having the highest ultraviolet light absorbance when it prepares the ultraviolet absorbance spectrum. Once the spectrometer has collected data from sample exposure to the UV beam, the data is transmitted to an attached computer which processes the intensity/wavelength data to produce an absorbance spectrum (Perkampus, 1992).

Various techniques have been devised for the determination of lipoxidase activity, including colorimetric, polarographic and spectrophotometric methods (Holman, 1955). Firstly, the spectrophotometric method was developed after Holman and Burr (1945) and Bergström (1946). They had independently observed an increase in ultraviolet light absorption, at 234 nm, when lipoxidase acted upon essential fatty acids. The increase in UV-peak absorption was then related to the amount of peroxide formation which, was found to be proportional to time and to enzyme concentration (Tappel, 1962; Theorell et al., 1944). The polyunsaturated fatty acid is solubilized by the addition of a detergent, and with this soluble substrate the activities of purified and crude lipoxidase are demonstrated over a wide range of pH (Tappel, 1962).

In the '50s the research done for the elucidation of lipoxygenase activity was strongly linked to the UV-Vis spectrometry and until today the UV-Vis spectrometry is an essential tool in probing the lipoxygenase activity. Thus in 1952 in an article published in the Journal of Biological Chemistry, Tappel et al. (1952), using a Beckman DU quartz spectrophotometer that was equipped with a temperature-controlled cell compartment, showed that with sodium linoleate as a substrate, under suitable conditions the reaction velocity was linear with respect to the enzyme concentration and the reaction did not show an induction period. With methyl linoleate the reaction velocity was not directly proportional to enzyme concentration unless a Tween preparation was added.

Antioxidants inhibited linoleate oxidation as a result of a direct effect on lipoxidase and of a preferential oxidation of the antioxidant. Rapid oxidation of nordihydroguaiaretic acid under suitable conditions was obtained in the absence of net linoleate oxidation, the linoleate having a function analogous to that of a coenzyme (Tappel et al., 1952).

The inactivation of soybean lipoxygenase during oxygenation of fatty acid substrates was first described by Theorell et al. (1944). It was shown that velocity of the lipoxygenasecatalyzed reaction decreases as a linear function of substrate utilization with all substrates tested.

#### **2.2 UV-Vis spectra of LOX reaction with quercetin**

We demonstrated by UV-Vis spectroscopy that pH values may influence the molecular interactions between soybean LOX-1 and quercetin, and especially the alkaline pH favors the ionic display of quercetin in order to interact with LOX better (Chedea et al., 2006). After 60 min of incubation of soybean LOX and quercetin (50 M) the UV-Vis spectra showed the formation of a new product (max = 321 nm) indicating the formation of an intermolecular complex between LOX and quercetin (Fig. 6) (Chedea et al., 2006).

Fig. 6. The absorption spectra of the mixture soybean LOX-1 and quercetin (50 M) at the initial moment (min 0), blue line, and after 60 min of incubation, green line. For measurements 160 l standard LOX-1 (2300 enzymatic units/ml) and quercetin to the final concentration of 50 M were added to 790 l 0.2 M phosphate buffer pH 9. The spectra were registered on a Jasco-V 500 spectrophotometer and the blank contained 950 l buffer and 50 l ethanol (Chedea et al., 2006).

Fig. 6 shows the spectra of the mixture of LOX and quercetin. Three peaks (1, 2, 3) registered at t=0 and t=60 min of incubation were identified. Peak 1 corresponds to Band II, peak 3 to Band I, both bands characteristic for flavonoids, while peak 2 indicated the formation of a new compound as the result of reaction between lipoxygenase and quercetin.

In the spectral range of 240-450 nm, flavone and their hydroxy substituted derivatives show two main absorption bands commonly referred to as Band I (300-400 nm) and Band II (240- 280 nm) (Mabry et al., 1970). Band I is supposed to be associated with the light absorption of the cinnamoyl system (B+C ring), and Band II with the absorption of the benzoyl moiety formed by the A+C ring (Fig. 7) (Zsila et al., 2003).

Fig. 7. Chemical structure of quercetin. Frame highlights the cinnamoyl part of the molecule (Zsila et al., 2003).

The results of Chedea et al. (2006) are confirmed by the study of Ha et al. (2010). They observed the decrease in the absorbance of the band centered at 272 and 386 nm with the concomitant increase in the absorbance at 330 nm, and the presence of two isosbestic points at 286 and 357 nm, respectively, which is also in agreement with the report of Takahama (Takahama, 1985), and the hypsochromic shift likely indicates that quercetin was oxidized (Ha et al., 2010). The absorbance at 385 nm mainly results from the *n*\* transition, and hence, the shift may be caused by structural changes including the ketone moiety, besides the change of the catechol on the ring B to the corresponding -quinone (Ha et al., 2010). In the study of Pinto and Macias, when quercetin is incubated in the presence of lipoxygenase and linoleic acid, a decrease of the band centered at 375 nm is produced, together with a slight increase of absorbance in the region of 330-340 nm (Pinto & Macias, 2005).

Lipoxygenase inhibition, as a result of intermolecular interaction between the enzyme and quercetin, is dependent on the quercetin's concentration. As the concentration of inhibitor increases the formation of a new compound as a result of this reaction increases as well, showing that a greater concentration of quercetin determines a more intense interaction between lipoxygenase and inhibitor (Vicaş et al., 2006). Quercetin exhibited a dosedependent inhibitory effect, and the lipoxygenase-catalyzed oxidation of linoleic acid to 13- HPOD was inhibited with an IC50 value of 4.8 ± 4 M (Ha et al., 2010).

Pinto at al. (2011) have shown also the existence of the interaction between lipoxygenase and quercetin. They also investigated the formation of an intermolecular complex between quercetin and lipoxygenase (Pinto et al., 2011). The acting forces between lipoxygenase and quercetin include mainly hydrogen bond and van der Waals, electrostatic and hydrophobic forces (Pinto et al., 2011).

#### **2.3 Enzyme kinetics**

160 Biochemical Testing

Fig. 6. The absorption spectra of the mixture soybean LOX-1 and quercetin (50 M) at the

measurements 160 l standard LOX-1 (2300 enzymatic units/ml) and quercetin to the final concentration of 50 M were added to 790 l 0.2 M phosphate buffer pH 9. The spectra were registered on a Jasco-V 500 spectrophotometer and the blank contained 950 l buffer and 50

Fig. 6 shows the spectra of the mixture of LOX and quercetin. Three peaks (1, 2, 3) registered at t=0 and t=60 min of incubation were identified. Peak 1 corresponds to Band II, peak 3 to Band I, both bands characteristic for flavonoids, while peak 2 indicated the formation of a

In the spectral range of 240-450 nm, flavone and their hydroxy substituted derivatives show two main absorption bands commonly referred to as Band I (300-400 nm) and Band II (240- 280 nm) (Mabry et al., 1970). Band I is supposed to be associated with the light absorption of the cinnamoyl system (B+C ring), and Band II with the absorption of the benzoyl moiety

Fig. 7. Chemical structure of quercetin. Frame highlights the cinnamoyl part of the molecule

initial moment (min 0), blue line, and after 60 min of incubation, green line. For

new compound as the result of reaction between lipoxygenase and quercetin.

l ethanol (Chedea et al., 2006).

(Zsila et al., 2003).

formed by the A+C ring (Fig. 7) (Zsila et al., 2003).

Enzyme kinetics is the investigation of how enzymes bind substrates and turn them into products. The rate data used in kinetic analyses are obtained from enzyme assays. In 1913 Leonor Michaelis and Maud Menten proposed a quantitative theory of enzyme kinetics, which is referred to as Michaelis-Menten kinetics. Their work was further developed by G. E. Briggs and J. B. S. Haldane, who derived kinetic equations that are still widely used today.

The major contribution of Michaelis and Menten was to think of enzyme reactions in two stages. In the first, the substrate binds reversibly to the enzyme, forming the enzymesubstrate complex. This is sometimes called the Michaelis-Menten complex in their honor. The enzyme then catalyzes the chemical step in the reaction and releases the product (Rogers & Gibon, 2009).

To find the maximum speed of an enzymatic reaction, the substrate concentration is increased until a constant rate of product formation is seen. Saturation happens because, as substrate concentration increases, more and more of the free enzyme is converted into the substrate-bound ES form. At the maximum velocity (Vmax) of the enzyme, all enzyme active sites are saturated with substrate, and the amount of ES complex is the same as the total amount of enzyme.

However, Vmax is only one kinetic constant of enzymes. The amount of substrate needed to achieve a given rate of reaction is also important. This is given by the Michaelis-Menten constant (Km), which is the substrate concentration required for an enzyme to reach one-half its maximum velocity. For finding the substrate concentration required for an enzyme to reach one-half its maximum velocity, the reaction speed is measured at different substrate concentrations. Each enzyme has a characteristic Km for a given substrate, and this can show how tight the binding of the substrate is to the enzyme (Rogers & Gibon, 2009).

The effect of substrate concentration ([S]) on activity is usually expressed using a Michaelis-Menten plot, such as the one shown below, and enzymes which generate such a plot are said to obey Michaelis-Menten kinetics. Michaelis-Menten plots show three distinct regions which correspond to reaction order. At low [S], the reaction accelerates as more substrate is added, reflecting first-order kinetics. At high [S], the concentration of enzyme becomes limiting, and additional substrate cannot accelerate the reaction. This situation is known as zero-order kinetics. Finally, there is a transition period between first order and zero order where kinetics are mixed (http://wiz2.pharm.wayne.edu/biochem/enz.html).

Fig. 8. A Michaelis-Menten plot.

If one draws a line across from the level (zero order) region of the plot to the Y-axis, this data point is Vmax, the maximum rate of reaction for a given concentration of enzyme. The second kinetic constant is also derived by drawing a line from the Y-axis at 1/2 Vmax to the curve, and then down to the X-axis. Each substrate will generate a unique Km and Vmax for a given enzymatic process.

A standard equation used to express the kinetic constants under the Michaelis-Menten hypothesis is aptly called the Michaelis-Menten equation, and is shown below. Later, two other investigators rearranged this equation to generate a second useful equation, the Lineweaver-Burk equation, also shown below.

Two things should be noticed about the Lineweaver-Burk equation: first, it is in the form y = mx + b, and as such, a plot of this equation will generate a straight line for enzymes obeying simple Michaelis Menten kinetics. In addition, the x and y values for the plot are both inverted, and as such, the plot is often referred to as the double reciprocal plot. The Lineweaver-Burk plot has two advantages over the Michaelis-Menten plot, in that it gives a more accurate estimate of Vmax, and it gives more accurate information about inhibition as

constant (Km), which is the substrate concentration required for an enzyme to reach one-half its maximum velocity. For finding the substrate concentration required for an enzyme to reach one-half its maximum velocity, the reaction speed is measured at different substrate concentrations. Each enzyme has a characteristic Km for a given substrate, and this can show

The effect of substrate concentration ([S]) on activity is usually expressed using a Michaelis-Menten plot, such as the one shown below, and enzymes which generate such a plot are said to obey Michaelis-Menten kinetics. Michaelis-Menten plots show three distinct regions which correspond to reaction order. At low [S], the reaction accelerates as more substrate is added, reflecting first-order kinetics. At high [S], the concentration of enzyme becomes limiting, and additional substrate cannot accelerate the reaction. This situation is known as zero-order kinetics. Finally, there is a transition period between first order and zero order

If one draws a line across from the level (zero order) region of the plot to the Y-axis, this data point is Vmax, the maximum rate of reaction for a given concentration of enzyme. The second kinetic constant is also derived by drawing a line from the Y-axis at 1/2 Vmax to the curve, and then down to the X-axis. Each substrate will generate a unique Km and Vmax for a

A standard equation used to express the kinetic constants under the Michaelis-Menten hypothesis is aptly called the Michaelis-Menten equation, and is shown below. Later, two other investigators rearranged this equation to generate a second useful equation, the

Two things should be noticed about the Lineweaver-Burk equation: first, it is in the form y = mx + b, and as such, a plot of this equation will generate a straight line for enzymes obeying simple Michaelis Menten kinetics. In addition, the x and y values for the plot are both inverted, and as such, the plot is often referred to as the double reciprocal plot. The Lineweaver-Burk plot has two advantages over the Michaelis-Menten plot, in that it gives a more accurate estimate of Vmax, and it gives more accurate information about inhibition as

how tight the binding of the substrate is to the enzyme (Rogers & Gibon, 2009).

where kinetics are mixed (http://wiz2.pharm.wayne.edu/biochem/enz.html).

Fig. 8. A Michaelis-Menten plot.

Lineweaver-Burk equation, also shown below.

given enzymatic process.

$$\mathbf{v} = \frac{\mathbf{v} \mathbf{max} \text{ [\ $]}}{\mathbf{x} \mathbf{m} + \text{[\$ ]}}$$


well. A typical Lineweaver-Burk plot appears below. Note that Vmax is derived from the yintercept, and Km can be derived either from the slope, or from extrapolating the line to the negative X-axis (http://wiz2.pharm.wayne.edu/biochem/enz.html).

Fig. 9. A typical Lineweaver-Burk plot.

#### **2.3.1 Enzyme inhibition**

There are two main classes of enzyme inhibitors, reversible and irreversible, that are differentiated by the magnitude of their affinity for enzyme. Reversible enzyme inhibitors bind and dissociate with their enzyme in an equilibrium process. Irreversible inhibitors bind tightly to an enzyme to form an essentially permanent complex. Reversible inhibitors can be classified as competitive, mixed, or noncompetitive inhibitors. If the detailed mechanism of inhibition is known, then the classification can be made by identifying where on the enzyme the inhibitor binds, or the order with which it binds, relative to substrate. Alternatively, a determination of simple kinetic parameters can generally be used to classify the inhibitor (http://www.wiley.com/college/pratt/0471393878/student/animations/enzyme\_inhibitio n/index.html)

#### **2.3.1.1 Competitive inhibitors**

Competitive inhibitors compete with substrate for an enzyme's active site, lowering the enzyme's likelihood of binding substrate and slowing the observed reaction velocity. Kinetic studies can be used to determine the type and potency of inhibition for an unknown inhibitor. Typical steady-state kinetic experiments can be performed where reaction velocity is measured in the presence of varying concentrations of substrate. If inhibitor is then added, and the data shows an increase in Km, yet the Vmax is unaffected, this is the signature of a competitive inhibitor (http://www.wiley.com/college/pratt/0471393878/student/ animations/enzyme\_inhibition/index.html)

#### **2.3.1.2 Mixed inhibition**

A mixed inhibitor binds to a site on the enzyme and interferes with both apparent substrate affinity and catalytic turnover, thus affecting the observed Km for the enzyme-catalyzed reaction. Mixed inhibitors do not bind directly in the active site, and therefore do not block substrate binding, but instead bind at sites that can be proximal or distal from the active site. Mixed inhibitors can therefore bind to free enzyme prior to substrate, distorting the active site to a nonoptimal conformation for catalysis. The inhibitor-distorted active site has trouble converting the substrate to product before it dissociates, resulting in a lowered apparent substrate binding affinity. Steady-state experiments performed in the presence of a mixed inhibitor demonstrate an increase or decrease in KM, and a decrease in Vmax (http://www.wiley.com/college/pratt/0471393878/student/animations/enzyme\_inhibitio n/index.html)

#### **2.3.1.3 Noncompetitive inhibition**

Noncompetitive inhibition is a special case of mixed inhibition where the affinity of inhibitor for E and ES is the same. Steady-state experiments performed in the presence of a noncompetitive inhibitor demonstrate a decrease in Vmax, yet Km is unaffected (http://www.wiley.com/college/pratt/0471393878/student/animations/enzyme\_inhibitio n/index.html)

#### **2.4 The influence of pure quercetin on sodium linoleate oxidation by pure soybean LOX-1**

#### **2.4.1 The influence of quercetin on the LOX-1 oxidation of sodium linoleate for different experimental protocols**

It is of great interest to check if the oxidative decomposition of quercetin by lipoxygenase is produced in the presence of linoleic acid. It is known that the hydroperoxidase activity of lipoxygenase produces the cooxidation of suitable electron donors in the presence of the hydroperoxides of linoleic or arachidonic acid, the natural substrates for this enzyme (Pinto & Macias, 2005). When linoleic acid is used as the substrate the primary LOX dioxygenation product obtained is (9Z,11Z,13S)-hydroperoxyoctadeca-9,11-dienoic acid (13-HPOD) (Grechkin, 1998).

Dioxygenase activity of lipoxygenase was measured by recording the increase in absorbance at 234 nm (formation of 13-HPOD), the incubation mixture containing 50 l quercetin in ethanol to a final concentration of 100 M, 8.4 l sodium linoleate at different concentrations (2.2 mM, 4.39 mM, 6.6 mM and 20 mM) and 160 l lipoxygenase (2300 units/ml) in 782 l 0.2 M borate buffer pH 9. The final substrate concentrations were 18.5 M, 36.9 M, 55.4 M respectively 168 M. The reaction components were mixed following three protocols as Fig. 10 indicates. The differences between these protocols are given by the order in which the enzyme, the substrate and the inhibitor were mixed and the incubation time.

As already presented in section 2.2, lipoxygenase in the absence of linoleic acid interacts with quercetin, thus Protocol 1 was designed to establish how the inhibition of substrate oxidation occurs when LOX-1 is initially incubated with the inhibitor. On the hand, Protocol 2 follows the classical way of LOX assay where the enzyme reacts with the substrate in the presence of quercetin. In the typical LOX reaction, the oxidation of the iron atom occurs with consumption of one molecule of fatty acid hydroperoxide (Zheng & Brash, 2010), therefore, the objective of the protocol was to explore the possibility of quercetin reacting with the LOX reaction product, the 13-hydroperoxide. Finally, the objective of Protocol 3 was to check for the existence of competition between the substrate and inhibitor in a reaction environment where both linoleic acid and quercetin-also oxidized by lipoxygenase-exist.

164 Biochemical Testing

inhibitor. Typical steady-state kinetic experiments can be performed where reaction velocity is measured in the presence of varying concentrations of substrate. If inhibitor is then added, and the data shows an increase in Km, yet the Vmax is unaffected, this is the signature of a competitive inhibitor (http://www.wiley.com/college/pratt/0471393878/student/

A mixed inhibitor binds to a site on the enzyme and interferes with both apparent substrate affinity and catalytic turnover, thus affecting the observed Km for the enzyme-catalyzed reaction. Mixed inhibitors do not bind directly in the active site, and therefore do not block substrate binding, but instead bind at sites that can be proximal or distal from the active site. Mixed inhibitors can therefore bind to free enzyme prior to substrate, distorting the active site to a nonoptimal conformation for catalysis. The inhibitor-distorted active site has trouble converting the substrate to product before it dissociates, resulting in a lowered apparent substrate binding affinity. Steady-state experiments performed in the presence of a mixed inhibitor demonstrate an increase or decrease in KM, and a decrease in Vmax (http://www.wiley.com/college/pratt/0471393878/student/animations/enzyme\_inhibitio

Noncompetitive inhibition is a special case of mixed inhibition where the affinity of inhibitor for E and ES is the same. Steady-state experiments performed in the presence of a noncompetitive inhibitor demonstrate a decrease in Vmax, yet Km is unaffected (http://www.wiley.com/college/pratt/0471393878/student/animations/enzyme\_inhibitio

**2.4 The influence of pure quercetin on sodium linoleate oxidation by pure soybean** 

It is of great interest to check if the oxidative decomposition of quercetin by lipoxygenase is produced in the presence of linoleic acid. It is known that the hydroperoxidase activity of lipoxygenase produces the cooxidation of suitable electron donors in the presence of the hydroperoxides of linoleic or arachidonic acid, the natural substrates for this enzyme (Pinto & Macias, 2005). When linoleic acid is used as the substrate the primary LOX dioxygenation product obtained is (9Z,11Z,13S)-hydroperoxyoctadeca-9,11-dienoic acid (13-HPOD)

Dioxygenase activity of lipoxygenase was measured by recording the increase in absorbance at 234 nm (formation of 13-HPOD), the incubation mixture containing 50 l quercetin in ethanol to a final concentration of 100 M, 8.4 l sodium linoleate at different concentrations (2.2 mM, 4.39 mM, 6.6 mM and 20 mM) and 160 l lipoxygenase (2300 units/ml) in 782 l 0.2 M borate buffer pH 9. The final substrate concentrations were 18.5 M, 36.9 M, 55.4 M respectively 168 M. The reaction components were mixed following three protocols as Fig. 10 indicates. The differences between these protocols are given by the order in which the

enzyme, the substrate and the inhibitor were mixed and the incubation time.

**2.4.1 The influence of quercetin on the LOX-1 oxidation of sodium linoleate for** 

animations/enzyme\_inhibition/index.html)

**2.3.1.2 Mixed inhibition** 

n/index.html)

n/index.html)

(Grechkin, 1998).

**LOX-1** 

**2.3.1.3 Noncompetitive inhibition** 

**different experimental protocols** 

Fig. 10. Schematic representation of experimental protocols (1,2,3).

Fig. 11. presents comparatively the inhibitory effect of quercetin 100 M on the oxidation of sodium linoleate by LOX-1 using all 3 experimental protocols. The kinetic curves of the reaction in the presence of quercetin are compared with the one of lipoxygenase without inhibitor (red curve).

It can be seen from Fig. 11 that the strongest inhibition is reached in the case of protocol 2 (pink line) when the inhibitor was added after the incubation for 15 seconds of sodium linoleate 55.4 M with the enzyme. For protocol 1 the typical Michaelis-Menten kinetic plot shape was maintained like for the LOX reaction with substrate without inhibitor (Fig. 11 blue curve vs. red plot). A different shape has the plot in the case of protocols 2 (pink line) and 3 (green line). At low substrate concentrations for both protocols, 2 and 3 (Fig. 11 curves green and pink), the kinetic plots start with a burst phase-a very fast increase of the reaction velocity. It follows a decrease which is fast in the case of protocol 3 (green curve) and slower in the case of protocol 2 (pink curve). For protocol 2 the kinetic curve continues with a slight increase (starting with 55.4 M concentration of substrate), which in our situation didn't reach the plateau phase. In the case of protocol 3 a new increase of the reaction rate -not so high compared with the first burst- follows at substrate concentration between 36.9 M and 55.4 M, continuing with a slight decrease at concentrations of substrate higher than 55.4 M.

Fig. 11. The inhibitory effect of quercetin on the lipoxygenase reaction according to protocol 3- dark green line; to protocol 2- pink line; to protocol 1- blue line. The red curve represents the kinetic without quercetin. The reaction components, 782 l 0.2 M borate buffer, (pH = 9.0), 160 l of LOX solution (2300 U enzyme/ml) 8.4 l of sodium linoleate, of different concentrations (2.2 mM, 4.39 mM, 6.6 mM and 20 mM) having the final substrate concentration in the reaction mixture of 18.5 M, 36.9 M, 55.4 M and 168 M) and 50 l quercetin in ethanol to the final concentration of 100 M were added and mixed as Fig. 10. presents. The absorption at 234 nm (A234) against the blank was recorded.

When both the substrate and the inhibitor were mixed and left for few seconds and then LOX was added the kinetic curve (Fig. 11 protocol 3), the first phase of fast increase shows that the hydroperoxides are formed as oxidation product of the linoleic acid even though the quercetin is present in the reaction mixture for very low substrate concentrations. LOX is partially inhibited by the quercetin at 36.9 M substrate concentration but then, at higher concentrations of substrate the enzyme becomes active again, but not as much as in the case of very low substrate concentration. Rapid inhibition followed by time dependent inactivation of soybean LOX-1 was also observed by Sadik et al. when quercetin was added to the reaction set-up after the substrate (Sadik et al., 2003).

When LOX was incubated with the substrate for 15 seconds and then the quercetin was added, the kinetic plot shows that LOX oxidizes the substrate having the highest product yield for very low substrate concentrations, that quercetin partially inhibits the reaction at linoleate concentrations between 30 M and 55 M.

Careful examination of the three protocols leads to speculation the quercetin-based inhibition of the soybean LOX-1 oxidation of linoleic acid does not follow the typical competitive inhibition model under the experimental conditions used (comparatively, protocol 3 gave the lowest inhibition than the other protocols tested). In Protocol I, it was observed that the new compound, a LOX-1-catalyzed by-product of quercetin, effectively inhibited the linoleic acid's oxidation. In the case where quercetin is added the last, the LOX reaction inhibition pattern suggested that at certain substrate concentrations (between 45 M and 75 M) quercetin would react with the hydroperoxide responsible for the initiation

Fig. 11. The inhibitory effect of quercetin on the lipoxygenase reaction according to protocol 3- dark green line; to protocol 2- pink line; to protocol 1- blue line. The red curve represents the kinetic without quercetin. The reaction components, 782 l 0.2 M borate buffer, (pH = 9.0), 160 l of LOX solution (2300 U enzyme/ml) 8.4 l of sodium linoleate, of different concentrations (2.2 mM, 4.39 mM, 6.6 mM and 20 mM) having the final substrate

concentration in the reaction mixture of 18.5 M, 36.9 M, 55.4 M and 168 M) and 50 l quercetin in ethanol to the final concentration of 100 M were added and mixed as Fig. 10.

When both the substrate and the inhibitor were mixed and left for few seconds and then LOX was added the kinetic curve (Fig. 11 protocol 3), the first phase of fast increase shows that the hydroperoxides are formed as oxidation product of the linoleic acid even though the quercetin is present in the reaction mixture for very low substrate concentrations. LOX is partially inhibited by the quercetin at 36.9 M substrate concentration but then, at higher concentrations of substrate the enzyme becomes active again, but not as much as in the case of very low substrate concentration. Rapid inhibition followed by time dependent inactivation of soybean LOX-1 was also observed by Sadik et al. when quercetin was added

When LOX was incubated with the substrate for 15 seconds and then the quercetin was added, the kinetic plot shows that LOX oxidizes the substrate having the highest product yield for very low substrate concentrations, that quercetin partially inhibits the reaction at

Careful examination of the three protocols leads to speculation the quercetin-based inhibition of the soybean LOX-1 oxidation of linoleic acid does not follow the typical competitive inhibition model under the experimental conditions used (comparatively, protocol 3 gave the lowest inhibition than the other protocols tested). In Protocol I, it was observed that the new compound, a LOX-1-catalyzed by-product of quercetin, effectively inhibited the linoleic acid's oxidation. In the case where quercetin is added the last, the LOX reaction inhibition pattern suggested that at certain substrate concentrations (between 45 M and 75 M) quercetin would react with the hydroperoxide responsible for the initiation

presents. The absorption at 234 nm (A234) against the blank was recorded.

to the reaction set-up after the substrate (Sadik et al., 2003).

linoleate concentrations between 30 M and 55 M.

of the LOX cycle, and thus implying the possibility of the quercetin undergoing nonenzymatic oxidation leading to the reaction inhibition.

#### **2.4.2 Determination of kinetic parameters Km and Vmax of soybean LOX-1 standard towards linoleic acid as substrate**

The kinetic parameters for LOX-1 oxidation of sodium linoleate were calculated as control values and compared with LOX-1 oxidation of sodium linoleate in the presence of different concentrations of quercetin. For the measurements of pure LOX activity, to 832 l 0.2 M borate buffer, (pH = 9.0), 160 l of LOX solution (2300 U enzyme/ml) and 8.4 l of sodium linoleate of different concentrations (2.2 mM, 4.39 mM, 6.6 mM and 20 mM), having the final substrate concentration in the reaction mixture of 18.5 M, 36.9 M, 55.4 M and 168 M, were added. The absorption at 234 nm (A234) against the blank was recorded. The blank contained a mixture of 840 l 0.2 M borate buffer, (pH = 9.0) and 160 l of LOX solution (2300 U enzyme/ml). In order to obtain the Lineweaver-Burk plot, the v, 1/[S] and 1/v were calculated for different substrate concentrations (from 18.5- 168 M).

Fig. 12. Michaelis-Menten plot (A) and Lineweaver-Burk plot (B) for different sodium linoleate concentrations oxidized by standard LOX-1. For the measurements of pure LOX activity, to 832 l 0.2 M borate buffer, (pH = 9.0), 160 l of LOX solution (2300 U enzyme/ml) and 8.4 l of sodium linoleate of different concentrations (2.2 mM, 4.39 mM, 6.6 mM and 20 mM), having the final substrate concentration in the reaction mixture of 18.5 M, 36.9 M, 55.4 M and 168 M, were added. The absorption at 234 nm (A234) against the blank was recorded. The blank contained a mixture of 840 l 0.2 M borate buffer, (pH = 9.0) and 160 l of LOX solution (2300 U enzyme/ml) (Chedea et al., 2008). Excel program was used to draw the graphs.

The kinetic parameters calculated for LOX-1 oxidizing sodium linoleate show that the enzyme has a great affinity towards substrate (Km = 1.1 M) and that its oxidation is fast as well (Vmax= 2.7 Ms-1).

#### **2.4.3 Kinetic parameters for LOX-1 oxidizing sodium linoleate in presence of different quercetin concentrations**

The reaction velocities in the case of this first protocol for the three quercetin concentrations were calculated as the ratio of absorption and correspondent time registered at 234 nm. For the absorption measurements of pure LOX activity to 782 l 0.2 M borate buffer, (pH = 9.0), 160 l of LOX solution (2300 U enzyme/ml) 50 l quercetin (10 M, 50 M and 100 M final concentrations) and 8.4 l of sodium linoleate of different concentrations (2.2 mM, 4.39 mM, 6.6 mM and 20 mM), having the final substrate concentration in the reaction mixture of 18.5 M, 36.9 M, 55.4 M and 168 M, were added. The absorption at 234 nm (A234) against the blank was recorded. The blank contained a mixture of 840 l 0.2 M borate buffer, (pH = 9.0) and 160 l of LOX solution (2300 U enzyme/ml).

The inhibitory effect of quercetin at different concentrations (100, 50 and 10 M) is presented in Fig. 13.

Fig. 13. Lipoxygenase inhibition by quercetin at different concentrations (10 M, 50 M and 100 M). Protocol 1 (Fig. 10) was followed for measurements as indicated in the legend of Fig. 11.

Plotting the reaction velocity in function of substrate concentration it can be seen that the classical Michaelis-Menten shape of the curve is not registered in the case of LOX reaction inhibition by quercetin at 50 M. For this reason, it was taken into calculation a larger range of substrate and quercetin concentration and for those the reaction velocity was calculated.

The kinetic curves representing the reaction velocity of the sodium linoleate oxidation by LOX-1, in presence of different concentrations of quercetin (Fig. 13) show that quercetin has an inhibitory effect on lipoxygenase for all the concentrations tested. To obtain these results, the reaction components were mixed as protocol 1 indicates, the enzyme being incubated with quercetin for 5 minutes and then the substrate is added. From Fig. 14 it can be seen that none of the kinetic plots follows the classical Michaelis-Menten shape. To get more information about the quercetin inhibitory action on soybean LOX-1, the kinetic parameters were calculated.

the absorption measurements of pure LOX activity to 782 l 0.2 M borate buffer, (pH = 9.0), 160 l of LOX solution (2300 U enzyme/ml) 50 l quercetin (10 M, 50 M and 100 M final concentrations) and 8.4 l of sodium linoleate of different concentrations (2.2 mM, 4.39 mM, 6.6 mM and 20 mM), having the final substrate concentration in the reaction mixture of 18.5 M, 36.9 M, 55.4 M and 168 M, were added. The absorption at 234 nm (A234) against the blank was recorded. The blank contained a mixture of 840 l 0.2 M borate buffer, (pH = 9.0)

The inhibitory effect of quercetin at different concentrations (100, 50 and 10 M) is presented

Fig. 13. Lipoxygenase inhibition by quercetin at different concentrations (10 M, 50 M and 100 M). Protocol 1 (Fig. 10) was followed for measurements as indicated in the legend of

Plotting the reaction velocity in function of substrate concentration it can be seen that the classical Michaelis-Menten shape of the curve is not registered in the case of LOX reaction inhibition by quercetin at 50 M. For this reason, it was taken into calculation a larger range of substrate and quercetin concentration and for those the reaction velocity was calculated. The kinetic curves representing the reaction velocity of the sodium linoleate oxidation by LOX-1, in presence of different concentrations of quercetin (Fig. 13) show that quercetin has an inhibitory effect on lipoxygenase for all the concentrations tested. To obtain these results, the reaction components were mixed as protocol 1 indicates, the enzyme being incubated with quercetin for 5 minutes and then the substrate is added. From Fig. 14 it can be seen that none of the kinetic plots follows the classical Michaelis-Menten shape. To get more information about the quercetin inhibitory action on soybean LOX-1, the kinetic parameters

and 160 l of LOX solution (2300 U enzyme/ml).

in Fig. 13.

Fig. 11.

were calculated.

Fig. 14. Kinetic plots of the sodium linoleate (336 M, 252 M, 168 M, 126 M, 84 M, 63 M, 42 M, 21 M, 10.5 M and 5.25 M), oxidation by LOX-1, in presence of different concentrations of quercetin 10 M (A), 25 M (B), 50 M (C) and 100 M (D).

Fig. 14 indicates different behaviours of LOX-1 quercetin inhibition determined by the substrate's and inhibitor's concentrations. For quercetin at 100 M (Fig. 14D), 50 M (Fig. 14C), at low concentrations of substrate (up to 21 M a fast increase of the reaction velocity is recorded, followed by a slight decrease and increase again (concentration of substrate between 21 M and 42 M). This "oscillatory" behaviour continues at higher substrate concentrations in the case of quercetin at 100 M and 50 M with a tendency of increasing the reaction's velocity at concentrations of substrate higher than 336 M for 100 M quercetin and of reaching the plateau phase for 50 M quercetin.

A fast decrease followed by an increase until the maximum velocity, characterises the kinetic plot of LOX-1 oxidizing the sodium linoleate at low substrate concentrations up to 84 M (Fig. 14B) in the presence of quercetin 25 M. At this concentration of substrate (84 M) the reaction velocity reaches its maximum. It follows a significant decrease of the reaction velocity at substrate concentrations between 84 M and 134.4 M, the reaction rate increasing again and having the tendency to follow the classical Michael-Menten kinetic shape till at 252 M, substrate's concentration. At this point a slight decrease of the reaction rate is registered, showing that for quercetin 25 M the inhibition is given by the high substrate concentration as well, when the sodium linoleate has a concentration higher than 252 M (Fig. 14B). The same behaviour is registered in the case of quercetin 10 M at linoleate concentrations higher than 252 M (Fig. 14A). For quercetin 10 M the kinetic plot follows the tendency of the classical Michaelis-Menten shape, with a slight decrease in reaction velocity at concentrations of substrate between 63 M and 126 M.

The observation that diminution of the degree of inhibition occurred by the decreasing of substrate concentration may imply that the substrate oxidation also plays a role in the process of LOX-1 inhibition by quercetin. This involvement may be one of the initial steps in the LOX inhibition, where quercetin is non-enzymatically oxidized by the reaction's product, the hydroperoxide, resulting in the formation of the quercetin o-quinone. Once the "activation" of quercetin through its oxidation is triggered, it can be assumed that the enzymatic inhibition would proceed via a quercetin oxidation product.

Km, Kmapp and Vmax calculated from the Lineweaver-Burk plots, for LOX-1 oxidizing sodium linoleate in the absence and presence of quercetin at 10 M, 25 M, 50 M and 100 M are presented in Table 1.


Table 1. Km, Kmapp and Vmax calculated from the Lineweaver-Burk plots, for LOX-1 oxidizing sodium linoleate in the absence and presence of quercetin at 10 M, 25 M, 50 M and 100 M.

The determined Kmapp and Vmax show a mixed inhibition for quercetin concentrations in the range of 10-50 M. For 100 M the Kmapp is half decreased and the reaction rate increases, reaching its highest value.

In a previous study LOX activity was measured spectrophotometrically at 234 nm using 15 mU of enzyme in the presence of 100 M linoleic acid in 50 mM potassium phosphate buffer, pH 7.5. For each scan 2 l of quercetin solution was added to 3 mL lipoxygenase solution to give a final concentration in the range 0.8 to 4.0 M and an inhibitory effect concentration dependent was detected (Pinto et al., 2011). This effect shows the characteristics of a competitive mechanism as it was deduced from Lineweaver-Burk plot (Pinto et al., 2011). On the basis of the competitive inhibition detected, the interaction should be located near or at the catalytic site (Pinto et al., 2011). The results obtained from the evaluation of three dimensional florescence spectra suggest a conformational modification of the protein in the region of the coupling with quercetin (Pinto et al., 2011).

The degree of inhibition was paradoxically diminished with decreasing substrate concentration which reveals an unusual mode of the inhibitory effect (Sadik et al., 2003). In this case, a competitive type of inhibition appears to be excluded (Sadik et al., 2003). The rabbit reticulocyte 15-LOX-1 samples were pre-incubated for 2 min at 20° with 10 M quercetin and the reactions were started by addition of 0.265 mM potassium linoleate and the formation of conjugated dienoic fatty acids was recorded spectrophotometrically at 234 nm (Sadik et al., 2003). Ha et al. report that quercetin inhibited soybean LOX-1 in a nonclassical manner. The progress curves of O2 consumption showed that quercetin inhibited soybean LOX-1 by a slow-binding inhibition mechanism (Ha et al., 2010).

### **3. Conclusions**

170 Biochemical Testing

follows the tendency of the classical Michaelis-Menten shape, with a slight decrease in

The observation that diminution of the degree of inhibition occurred by the decreasing of substrate concentration may imply that the substrate oxidation also plays a role in the process of LOX-1 inhibition by quercetin. This involvement may be one of the initial steps in the LOX inhibition, where quercetin is non-enzymatically oxidized by the reaction's product, the hydroperoxide, resulting in the formation of the quercetin o-quinone. Once the "activation" of quercetin through its oxidation is triggered, it can be assumed that the

Km, Kmapp and Vmax calculated from the Lineweaver-Burk plots, for LOX-1 oxidizing sodium linoleate in the absence and presence of quercetin at 10 M, 25 M, 50 M and 100 M are

> 100 0.55 3.8 - 50 0.83 2.4 mixed 25 0.78 1.8 mixed 10 261.78 0.5 mixed

Table 1. Km, Kmapp and Vmax calculated from the Lineweaver-Burk plots, for LOX-1 oxidizing sodium linoleate in the absence and presence of quercetin at 10 M, 25 M, 50 M and 100

The determined Kmapp and Vmax show a mixed inhibition for quercetin concentrations in the range of 10-50 M. For 100 M the Kmapp is half decreased and the reaction rate increases,

In a previous study LOX activity was measured spectrophotometrically at 234 nm using 15 mU of enzyme in the presence of 100 M linoleic acid in 50 mM potassium phosphate buffer, pH 7.5. For each scan 2 l of quercetin solution was added to 3 mL lipoxygenase solution to give a final concentration in the range 0.8 to 4.0 M and an inhibitory effect concentration dependent was detected (Pinto et al., 2011). This effect shows the characteristics of a competitive mechanism as it was deduced from Lineweaver-Burk plot (Pinto et al., 2011). On the basis of the competitive inhibition detected, the interaction should be located near or at the catalytic site (Pinto et al., 2011). The results obtained from the evaluation of three dimensional florescence spectra suggest a conformational modification

The degree of inhibition was paradoxically diminished with decreasing substrate concentration which reveals an unusual mode of the inhibitory effect (Sadik et al., 2003). In this case, a competitive type of inhibition appears to be excluded (Sadik et al., 2003). The rabbit reticulocyte 15-LOX-1 samples were pre-incubated for 2 min at 20° with 10 M quercetin and the reactions were started by addition of 0.265 mM potassium linoleate and the formation of conjugated dienoic fatty acids was recorded spectrophotometrically at 234 nm (Sadik et al., 2003). Ha et al. report that quercetin inhibited soybean LOX-1 in a non-

of the protein in the region of the coupling with quercetin (Pinto et al., 2011).

Vmax

(Ms-1/s) Type of inhibition

reaction velocity at concentrations of substrate between 63 M and 126 M.

enzymatic inhibition would proceed via a quercetin oxidation product.

Kmapp (M)

0 1.103 2.7

presented in Table 1.

M.

Quercetin conc. M

reaching its highest value.

The aim of this study was to show how a widespread analysis tool like UV-Vis spectroscopy shapes the enzyme inhibition research having as an example the lipoxygenase interaction with quercetin. Almost all therapeutic drugs are enzyme inhibitors, from old medicine box standards such as aspirin and penicillin to the newest compounds used to treat HIV infection. Understandably, enzyme kinetics plays an outstanding role in this effort to produce effective therapeutics, for kinetic studies can quantify the degree that inhibitors inactivate or slow down the targeted enzyme's catalytic rate and describe its potential efficacy as a drug.

Since its characterisation in 1947 lipoxygenase is strongly related to the UV-Vis spectrometry as a valuable tool for its activity assay. Due to its implications in food chemistry and medicine LOX inhibition attracted up to date, the interest of researchers in the field. A highly functionalized flavonoid, quercetin proves to be a potent inhibitor of lipoxygenase acting both as a substrate and a source of inhibition, quercetin seems to play an antinomic role (Fiorucci et al., 2008). The partitioning level between quercetin as a substrate or as an inhibitor is dependent on its concentration. Different reversible inhibition schemes (competitive, noncompetitive, or mixed) as well as inhibition via reduction of the enzymebound radical intermediate have been considered to explain the activity of quercetin as LOX inhibitor. Moreover, heterogeneity in the interpretation of the experimental results of the inhibition processes, for example concerning kinetic data, prevents converging toward a general way of inhibition (Fiorucci et al., 2008). The ratio substrate:inhibitor might be a factor determining the type of inhibition observed in the case of lipoxygenase and quercetin interaction.

In our present study the UV-Vis spectra show the oxidation of quercetin and the formation of a new compound. The absorption maximum of this new formed molecule is centered around max= 321 nm, different from quercetin (max= 370 nm), suggesting loss of -electron delocalisation, i.e. interruption of the quercetin B and C ring -bond extended conjugated system (Bors et al., 1990; Abou Samra et al., 2011). The determined KMapp and vmax show a mixed inhibition for quercetin concentrations in the range of 10-50 M. For the highest quercetin concentration tested, 100 M, the Kmapp decreased by half but the reaction rate increases which might indicate a cooxidation of quercetin besides the LOX classical reaction, proving the switch in quercetin role from inhibitor towards substrate. Recent literature data show that quercetin itself inhibits the LOX reaction and also its oxidation products are inhibitors of LOX oxidation. For instance, quercetin may first act as a lipoxygenase inhibitor by reducing the ferric form of the enzyme to an inactive ferrous form, and then, the oxidized metabolite becomes a more potent inhibitor (Ha et al., 2010).

In our study, the Michaelis-Menten (M-M), Lineweaver-Burk kinetics and UV spectral analysis have detected both the mixed inhibition and cooxidation of quercetin during soybean LOX-1-mediated metabolism of polyunsaturated fatty acid, linoleic acid. Though not yet convincingly proven, the mechanisms underlying the distinct kinetic behaviors are, at least empirically, believed to be due to the existence of divergent interactions between the quercetin molecules and/or its intermediates and the active site of the enzymes. Furthermore, the atypical kinetics might be interpreted using the model of two or possibly more binding regions within the enzyme active site(s) for the quercetin and intermediates. The finding that LOX can turn different compounds, like quercetin and epigallocatechin gallate, into simple catechol derivatives (with one aromatic ring only) might be of importance as an additional small piece of a "jigsaw puzzle" in the much bigger picture of drug metabolism (Borbulevych et al., 2004). However, the interactions of these flavanols with LOX can be more complicated than simply blocking the access to the enzyme's active site as observed in this study. Therefore, it warrants future endeavours to thoroughly understand thus reliably predict interaction mechanism between the LOX proteins and therapeutic agents at the molecular level. This will be important in fully understanding the exact role of lipoxygenase inhibition by quercetin in therapy targeting and possibly identifying new bioactive molecules which would be used as drugs.

#### **4. Acknowledgment**

V.S. Chedea is a Japan Society for the Promotion of Science (JSPS) postdoctoral fellow.

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### **Extending the Shelf Life of Fresh Marula (***Sclerocarya birrea***) Juice by Altering Its Physico-Chemical Parameters**

S. Dube1, N.R. Dlamini2,I. Shereni1 and T. Sibanda1 *1Department of Applied Biology and Biochemistry, National University of Science and Technology, Ascot, Bulawayo, 2CSIR Biosciences, Pretoria 1Zimbabwe 2South Africa* 

#### **1. Introduction**

#### **1.1 Taxonomy and distribution**

*Sclerocarya birrea* (A. Rich.) Hochst. subsp. *caffra* (Sond.) (marula) is a very common and widespread throughout much of sub-Saharan Africa and is a member of the Anacardiaceae family, along with 650 species and 70 genera of mainly tropical or subtropical evergreen or deciduous trees, shrubs and woody vines along side with the mango (*Mangifera indica* L.). *S. birrea* has three recognised subspecies; fruit-bearing species of which *Sclerocarya birrea* subsp. *caffra* is the most ubiquitous and occurs in east tropical Africa (Kenya, Tanzania), south tropical Africa (Angola, Malawi, Mozambique, Zambia and Zimbabwe) and southern Africa (Botswana, Namibia, South Africa and Swaziland) as well as Madagascar. *Sclerocarya birrea*  subsp. *multifoliolata* (Engl.) Kokwaro subspecies occurs in mixed deciduous woodland and wooded grassland in Tanzania. The tree grows in a wide variety of soils but prefers welldrained soil. It exists at altitudes varying from sea level to 1800 m and an annual rainfall range of 200 – 1500 mm. The key factor limiting its distribution is its sensitivity to frost (Von, 1983; Palgrave, 1990; Mizrahi and Nerd, 1996; Aganga and Mosase, 2001; Emanuel, *et al*., 2005).

#### **1.2 Flowering and fruiting**

*S. birrea* is a dioecious species. Male and female flowers occur separately, but not always, on separate trees. The flowers are small, with red sepals and yellow petals, and are borne in 50 – 80 mm-long sprays of small oblong clusters (Venter and Venter, 1996; van Wyk and van Wyk, 1997; van Wyk *et al*., 2002).

#### **1.3 Traditional uses of marula fruit**

It has many uses, including the fruits that are eaten fresh or after fermentation to make a beer, the kernels are eaten or the oil extracted, the leaves are browsed by animals and have medicinal properties, as does the bark. The wood is carved into traditional utensils such as spoons and plates as well as decorative curios. Because of the widespread occurrence and use of *S. birrea* it has frequently been identified as a key species in the development of rural businesses using the fruit, beer, nuts or oil and therefore a good candidate for domestication (Fox and Norwood, 1982; Nerd and Mizrahi, 1993; Mizrahi and Nerd, 1996; Shackleton *et al*., 2002). The fruit and nuts have extensive commercial value. The fruit is described as having an exotic flavor and high nutritive value (for example, vitamin C content is up to 3 times higher in 'marula' fruit than equivalent weight of oranges. The fruit is often fermented to give an alcoholic beverage. Some tribes, such as the Pedi, prepare a relish from the leaves of *S. birrea*. The Zulu people of South Africa regard *S. birrea* fruit as a potent insecticide (van Wyk, *et al*., 2002; Mdluli, 2005). The whole fruit is used in many parts of southern Africa for brewing beer and distilling spirits. In Mozambique, South Africa and Zambia, the fruit of *S. birrea* is used to flavor liqueur. The gum obtained from the tree is rich in tannins, and hence, it is employed in the production of an ink substitute. Zimbabwean and South African villagers benefit from the customary usage of *S. birrea* wood in the manufacture of dishes, maize stamping mortars, drums, toys, curios, divining bowls and carvings. The peel from *S. birrea* fruit is of paramount importance in the production of oil for cosmetic purposes (Ojewole, *et al*., 2010).

#### **1.4 The marula fruit**

Female trees bear pale-yellow, round fruit with thick leather-like rind, and fibrous, juicy, mucilaginous flesh. The fruit, which is about the size of a golf ball, is pale-yellow when ripe, with a diameter of 30 – 40 mm (Nerd and Mizrahi, 1993; Ojewole *et al*., 2010). The fruit has sour taste, and is much sought-after by birds, game mammals and humans, because of its delicious pulp, and edible, tasty nuts (van Wyk, *et al*., 2002). The fruit pulp contains citric acid, vitamin C and sugar, while the nut is rich in non-drying oil and protein (van Wyk, *et al*., 2002; Hillman, *et al*., 2008; Ojewole, *et al*., 2010). The only way to make this fruit juice available to children before fermentation and is by finding ways to extend its shelf life. The average *Sclerocarya birrea* (marula) tree under the climatic conditions of Matebeleland South produces 20000±5000 fruits per season. The fruits ripen between February and April. The time from ripening onset to rotting of the fruit was 16±4 days. Skin colour starts to change on day 3 after abscission, and a completely yellow colour is obtained by day 12.

#### **1.5 Fruit ripening**

Marula fruits abscise before ripening; at this stage the skin color is green and the fruit is firm. The time for fruit abscission varies among trees. Of the studied trees, fruits abscised mainly in March and in late April. This can be attributed to genetic variation, which can be exploited for expanding the harvest period by planting clones that ripen at different times. (Palgrave, 1990; Mdluli, 2005; Dlamini and Dube, 2008).

#### **2. Marula pulp characteristics**

Of the wide range of nutrients in the Marula pulp the Vitamin C content has attracted the most attention. Indeed the vitamin C content is important to local communities who know well that it prevents scurvy. The fruit has small amounts of other vitamins such as thiamine, riboflavin and nicotinic acid. It is 85% moisture and 14% carbohydrate, mostly sucrose.

medicinal properties, as does the bark. The wood is carved into traditional utensils such as spoons and plates as well as decorative curios. Because of the widespread occurrence and use of *S. birrea* it has frequently been identified as a key species in the development of rural businesses using the fruit, beer, nuts or oil and therefore a good candidate for domestication (Fox and Norwood, 1982; Nerd and Mizrahi, 1993; Mizrahi and Nerd, 1996; Shackleton *et al*., 2002). The fruit and nuts have extensive commercial value. The fruit is described as having an exotic flavor and high nutritive value (for example, vitamin C content is up to 3 times higher in 'marula' fruit than equivalent weight of oranges. The fruit is often fermented to give an alcoholic beverage. Some tribes, such as the Pedi, prepare a relish from the leaves of *S. birrea*. The Zulu people of South Africa regard *S. birrea* fruit as a potent insecticide (van Wyk, *et al*., 2002; Mdluli, 2005). The whole fruit is used in many parts of southern Africa for brewing beer and distilling spirits. In Mozambique, South Africa and Zambia, the fruit of *S. birrea* is used to flavor liqueur. The gum obtained from the tree is rich in tannins, and hence, it is employed in the production of an ink substitute. Zimbabwean and South African villagers benefit from the customary usage of *S. birrea* wood in the manufacture of dishes, maize stamping mortars, drums, toys, curios, divining bowls and carvings. The peel from *S. birrea* fruit is of paramount importance in the production of oil for cosmetic purposes

Female trees bear pale-yellow, round fruit with thick leather-like rind, and fibrous, juicy, mucilaginous flesh. The fruit, which is about the size of a golf ball, is pale-yellow when ripe, with a diameter of 30 – 40 mm (Nerd and Mizrahi, 1993; Ojewole *et al*., 2010). The fruit has sour taste, and is much sought-after by birds, game mammals and humans, because of its delicious pulp, and edible, tasty nuts (van Wyk, *et al*., 2002). The fruit pulp contains citric acid, vitamin C and sugar, while the nut is rich in non-drying oil and protein (van Wyk, *et al*., 2002; Hillman, *et al*., 2008; Ojewole, *et al*., 2010). The only way to make this fruit juice available to children before fermentation and is by finding ways to extend its shelf life. The average *Sclerocarya birrea* (marula) tree under the climatic conditions of Matebeleland South produces 20000±5000 fruits per season. The fruits ripen between February and April. The time from ripening onset to rotting of the fruit was 16±4 days. Skin colour starts to change

Marula fruits abscise before ripening; at this stage the skin color is green and the fruit is firm. The time for fruit abscission varies among trees. Of the studied trees, fruits abscised mainly in March and in late April. This can be attributed to genetic variation, which can be exploited for expanding the harvest period by planting clones that ripen at different times.

Of the wide range of nutrients in the Marula pulp the Vitamin C content has attracted the most attention. Indeed the vitamin C content is important to local communities who know well that it prevents scurvy. The fruit has small amounts of other vitamins such as thiamine, riboflavin and nicotinic acid. It is 85% moisture and 14% carbohydrate, mostly sucrose.

on day 3 after abscission, and a completely yellow colour is obtained by day 12.

(Palgrave, 1990; Mdluli, 2005; Dlamini and Dube, 2008).

**2. Marula pulp characteristics** 

(Ojewole, *et al*., 2010).

**1.4 The marula fruit** 

**1.5 Fruit ripening** 

Citric acid is the most abundant acid excluding ascorbic acid, but malic and tartaric acid have also been noted. The mineral composition of the fruit shows high concentrations of Potassium, Calcium and Magnesium (Shackleton, *et al*., 2010). The aroma of Marula is perceived by other people to be like that of the grapefruit and there are in fact compounds that the two fruits have in common. However, the similarity in taste between grapefruit and marula is probably because of a dominant bitter taste caused by non volatiles. Others have likened the smell of marula juice to pineapples but this is probably also only in part due to complimentary volatile components such as ethyl acetate, benzaldehyde, linalool. With a unique and pleasurable taste, an attractive colour and odour and health properties such as high vitamin C and potassium, this fruit has all the physical requirements of the growing it for industrial purposes. Marula, *Scelerocarya birrea*, subspecies *caffera*, is one of Africa's botanical treasures. The flesh of the fruit clings onto to its brown stone. The flesh is very fibrous and juicy. Inside the woody stone are two to three seeds, which are very rich in oil and protein. (Shackleton, *et al*., 2010).

Generally edible fruits constitute a commercially important and nutritionally indispensable food commodity. Being a part of a balanced diet, fruits play a vital role in human nutrition by supplying the necessary growth regulating factors essential for maintaining normal health. Fruits are widely distributed in nature. One of the limiting factors that influence their economic value is the relatively short ripening period and reduced post-harvest life. Fruit ripening is a highly coordinated, genetically programmed, and an irreversible phenomenon involving a series of physiological, biochemical, and organoleptic changes, that finally leads to the development of a soft edible ripe fruit with desirable quality attributes. Excessive textural softening during ripening leads to adverse effects/spoilage upon storage. Carbohydrates play a major role in the ripening process, by way of depolymerization leading to decreased molecular size with concomitant increase in the levels of ripening inducing specific enzymes, whose target differ from fruit to fruit. (Hillman, *et al* 2008; Brumell and Harpster, 2001; Prasanna, *et al*., 2007; Prescott, 2011) The major classes of cell wall polysaccharides that undergo modifications during ripening are starch, pectins, cellulose, and hemicelluloses. Pectins are the common and major components of primary cell wall and middle lamella, contributing to the texture and quality of fruits. Their degradation during ripening seems to be responsible for tissue softening of a number of fruits. Structurally pectins are a diverse group of heteropolysaccharides containing partially methylated D-galacturonic acid residues with side chain appendages of several neutral polysaccharides. The degree of polymerization/esterification and the proportion of neutral sugar residues/side chains are the principal factors contributing to their (micro-) heterogeneity. Pectin degrading enzymes such as polygalacturonase, pectin methyl esterase, lyase, and rhamnogalacturonase are the most implicated in fruit-tissue softening. Recent advances in molecular biology have provided a better understanding of the biochemistry of fruit ripening as well as providing a hand for genetic manipulation of the entire ripening process. It is desirable that significant breakthroughs in such related areas will come forth in the near future, leading to considerable societal benefits(Brumell and Harpster, 2001; Prasanna, *et al*., 2007; Prescott, 2011).

Excessive softening is the main factor limiting fruit shelf life and storage. Transgenic plants modified in the expression of cell wall modifying proteins have been used to investigate the role of particular activities in fruit softening during ripening, and in the manufacture of processed fruit products. Transgenic experiments show that polygalacturonase (PG) activity is largely responsible for pectin depolymerization and solubilization, but that PG-mediated pectin depolymerization requires pectin to be de-methyl-esterified by pectin methylesterase (PME), and that the PG beta-subunit protein plays a role in limiting pectin solubilization. Suppression of PG activity only slightly reduces fruit softening (but extends fruit shelf life), suppression of PME activity does not affect firmness during normal ripening, and suppression of beta-subunit protein accumulation increases softening. All these pectinmodifying proteins affect the integrity of the middle lamella, which controls cell-to-cell adhesion and thus influences fruit texture (Arthachinta, 2000; Brumell and Harpster, 2001; Prasanna, *et al*., 2007; Prescott, 2011). Diminished accumulation of either PG or PME activity considerably increases the viscosity of tomato juice or paste, which is correlated with reduced polyuronide depolymerization during processing. In contrast, suppression of betagalactosidase activity early in ripening significantly reduces fruit softening, suggesting that the removal of pectic galactan side-chains is an important factor in the cell wall changes leading to ripening-related firmness loss. (Brumell and Harpster, 2001; Prasanna, *et al*., 2007; Prescott, 2011). Suppression or over expression of endo-(1--4) beta-D-glucanase activity has no detectable effect on fruit softening or the depolymerization of matrix glycans, and neither the substrate nor the function for this enzyme has been determined. The role of xyloglucan endotransglycosylase activity in softening is also obscure, and the activity responsible for xyloglucan depolymerization during ripening, a major contributor to softening, has not yet been identified. However, ripening-related expansion protein abundance is directly correlated with fruit softening and has additional indirect effects on pectin depolymerization, showing that this protein is intimately involved in the softening process. (Brumell and Harpster, 2001; Prasanna, *et al*., 2007; Prescott, 2011). Transgenic work has shown that the cell wall changes leading to fruit softening and textural changes are complex, and involve the coordinated and interdependent activities of a range of cell wall-modifying proteins. It is suggested that the cell wall changes caused early in ripening by the activities of some enzymes, notably beta-galactosidase and ripening-related expansin, may restrict or control the activities of other ripening-related enzymes necessary for the fruit softening process (Brumell and Harpster, 2001; Prasanna *et al*., 2007; Prescott, 2011). Whether the sugar of fruits is formed within them, or introduced through the stem, and, if formed in the fruits, from what substance formed, are questions which have been investigated, but not wholly settled. It has been pretty generally held that starch in the unripe fruits is converted into sugar in the ripe fruits; the fruit acids inducing the change, as we know they have power to do. But starch is not found in the unripe stage of all fruits, and, in the cases where found, its quantity is sometimes too small to serve as the source of all the sugar of the ripened fruit The maturity of fruit is the period of its maximum quantity of sugar. Sooner or later, the quantity of sugar begins to diminish, and then the fruit is overripe. It is safe to say that the sugar often begins to decompose during the life of the fruit; that is to say, fruit becomes overripe during its life. It would be difficult, however, to fix on the termination of the life of fruit. We certainly cannot say that life ceases when the circulation with the plant is cut off; and we cannot say that life continues in the sarcocarp until it is wholly disintegrated. (Brumell and Harpster, 2001; Prasanna, *et al*., 2007; Prescott, 2011). Now it is within the limits of our subject to inquire by what changes the sugar begins to disappear*.* In general terms, sugar suffers oxidation in ripe fruits, small portions being oxidized away even during the production of larger portions, and before perfect maturity. We do not know what fruit constituents, if any, result in this oxidation. The final products of oxidation, carbonic acid and water, are exhaled during ripening, and with greater rapidity after maturity has been

is largely responsible for pectin depolymerization and solubilization, but that PG-mediated pectin depolymerization requires pectin to be de-methyl-esterified by pectin methylesterase (PME), and that the PG beta-subunit protein plays a role in limiting pectin solubilization. Suppression of PG activity only slightly reduces fruit softening (but extends fruit shelf life), suppression of PME activity does not affect firmness during normal ripening, and suppression of beta-subunit protein accumulation increases softening. All these pectinmodifying proteins affect the integrity of the middle lamella, which controls cell-to-cell adhesion and thus influences fruit texture (Arthachinta, 2000; Brumell and Harpster, 2001; Prasanna, *et al*., 2007; Prescott, 2011). Diminished accumulation of either PG or PME activity considerably increases the viscosity of tomato juice or paste, which is correlated with reduced polyuronide depolymerization during processing. In contrast, suppression of betagalactosidase activity early in ripening significantly reduces fruit softening, suggesting that the removal of pectic galactan side-chains is an important factor in the cell wall changes leading to ripening-related firmness loss. (Brumell and Harpster, 2001; Prasanna, *et al*., 2007; Prescott, 2011). Suppression or over expression of endo-(1--4) beta-D-glucanase activity has no detectable effect on fruit softening or the depolymerization of matrix glycans, and neither the substrate nor the function for this enzyme has been determined. The role of xyloglucan endotransglycosylase activity in softening is also obscure, and the activity responsible for xyloglucan depolymerization during ripening, a major contributor to softening, has not yet been identified. However, ripening-related expansion protein abundance is directly correlated with fruit softening and has additional indirect effects on pectin depolymerization, showing that this protein is intimately involved in the softening process. (Brumell and Harpster, 2001; Prasanna, *et al*., 2007; Prescott, 2011). Transgenic work has shown that the cell wall changes leading to fruit softening and textural changes are complex, and involve the coordinated and interdependent activities of a range of cell wall-modifying proteins. It is suggested that the cell wall changes caused early in ripening by the activities of some enzymes, notably beta-galactosidase and ripening-related expansin, may restrict or control the activities of other ripening-related enzymes necessary for the fruit softening process (Brumell and Harpster, 2001; Prasanna *et al*., 2007; Prescott, 2011). Whether the sugar of fruits is formed within them, or introduced through the stem, and, if formed in the fruits, from what substance formed, are questions which have been investigated, but not wholly settled. It has been pretty generally held that starch in the unripe fruits is converted into sugar in the ripe fruits; the fruit acids inducing the change, as we know they have power to do. But starch is not found in the unripe stage of all fruits, and, in the cases where found, its quantity is sometimes too small to serve as the source of all the sugar of the ripened fruit The maturity of fruit is the period of its maximum quantity of sugar. Sooner or later, the quantity of sugar begins to diminish, and then the fruit is overripe. It is safe to say that the sugar often begins to decompose during the life of the fruit; that is to say, fruit becomes overripe during its life. It would be difficult, however, to fix on the termination of the life of fruit. We certainly cannot say that life ceases when the circulation with the plant is cut off; and we cannot say that life continues in the sarcocarp until it is wholly disintegrated. (Brumell and Harpster, 2001; Prasanna, *et al*., 2007; Prescott, 2011). Now it is within the limits of our subject to inquire by what changes the sugar begins to disappear*.* In general terms, sugar suffers oxidation in ripe fruits, small portions being oxidized away even during the production of larger portions, and before perfect maturity. We do not know what fruit constituents, if any, result in this oxidation. The final products of oxidation, carbonic acid and water, are exhaled during ripening, and with greater rapidity after maturity has been passed. The quantity of acids in fruits usually diminishes during ripening*.* The diminution is not, however, nearly so great as it appears to the taste, because the acid of ripe fruits is masked to the taste by the larger proportions of sugar and the pectous substances then present. The removal of acids is chiefly due to oxidation. It is not found that acids are neutralized, to any considerable extent, during ripening, by alkalies conveyed through the stem (Brumell and Harpster, 2001; Prasanna *et al*., 2007; Prescott, 2011). It is stated that the acids continue to oxidize away, after the sugar has reached its maximum and before it begins to diminish. Hence, perfect ripeness in fruit has been defined as that period during the maximum quantity of sugar when the quantity of acid is least. This will be, of course, just before the sugar begins to diminish. It has been stated that both citric and malic acids are often found in unripe grapes, and are substituted by tartaric acid during the ripening. Oxalic acid is more often found in unripe than in ripe fruits. It is to be desired that closer determinations should be made as to the presence and proportion of oxalic acid in tomatoes and some other fruits (Brumell and Harpster, 2001; Prasanna *et al*., 2007; Prescott, 2011).

#### **3. Fresh marula fruit juice shelf life extension by high-pressure processing – pascalization and aseptic methods**

The application of high hydrostatic pressure in processing of food is of great interest because of its ability to inactivate food related microorganisms and enzymes, at low temperature, without the need for chemical preservatives. High hydrostatic pressures, around 650 MPa reduces the microbial load in foods such as fruits. Pressure-treated foods have organoleptic properties similar to fresh products, which is a major advantage in juice processing as it matches consumer demand for healthy, nutritious and ''natural'' products. However, an important issue arises when we consider the acceptance of such products by the consumer (Jay, 1986; Adams and Moss, 1995; Deliza *et al*., 2004). It is also noted that the microbicidal activity of high pressure is enhanced by low pH or temperatures above and below ambient. High hydrostatic pressure acts primarily on non-covalent linkages, such as ionic bonds, hydrogen bonds and hydrophobic interactions, and it promotes reactions in which there is an overall decrease in volume (Adams and Moss, 1995; Indrawati *et al*., 2008; Sampedro *et al.,* 2008; Chao, *et al* 2011). It can have profound effects on proteins, where such interactions are critical to structure and function, although the effect is variable and depends on individual protein structure. Other proteins are relatively unaffected and this can cause problems when they have enzymic activity which limits product shelf-life. Pectin esterase in orange juice, for instance, must be inactivated to stabilize the desired product cloudiness, but is very stable to pressures up to 1000 MPa. Non-protein macromolecules can also be affected by high pressures so that pascalized starch products often taste sweeter due to conformational changes in the starch which allow salivary amylase greater access. Adverse effects on protein structure and activity obviously contribute to the antimicrobial effect of high pressures, although the cell membrane also appears to be an important target. Membrane lipid bilayers have been shown to compress under pressure and this alters their permeability. As a general rule vegetative bacteria and fungi can be reduced by at least one log cycle by 400 MPa applied for 5 min. (Indrawati *et al*., 2008; Sampedro *et al*., 2008; Chao *et al*., 2011)

Bacterial endospores are more resistant to hydrostatic pressure, tolerating pressures as high as 1200 MPa. Their susceptibility can be increased considerably by modest increases in temperature, when quite low pressures (100 MPa) can produce spore germination, a process in which the spores lose their resistance to heat and to elevated pressure. Hydrostatic processing has a number of appealing features for the food preparations such as Marula fruit juice. (Indrawati, *et al*., 2008; Sampedro, *et al*., 2008; Chao, *et al*., 2011)

It acts instantly and uniformly throughout a substance so that the processing time is not related to container size and there is none of the penetration problems associated with heat processing. Nutritional quality, flavour, appearance and texture resemble the fresh material very closely. To the consumer where it has been used it is regarded as a 'natural' process with none of the negative associations of processes such as irradiation or chemical preservatives. ((Indrawati, *et al*., 2008; Sampedro, *et al*., 2008; Chao, *et al*., 2011)

Appertized foods include those which are hermetically sealed into containers, usually cans, and then subjected to heat process in-pack. While this has been hugely successful as a long term method of food preservation, it does require extended heating periods in which a food's functional and chemical properties can be adversely affected. (Adams and Moss, 1995)

#### **3.1 Fresh marula fruit juice shelf life extension by temperature control**

Low-temperature storage - chilling and freezing the rates of most chemical reactions are temperature dependent; as the temperature is lowered so the rate decreases. Since food spoilage is usually a result of chemical reactions mediated by microbial and endogenous enzymes, the useful life of many foods can be increased by storage at low temperatures. Chilled foods are those foods stored at temperatures near, but above their freezing point, typically 0-5°C. Chill storage can change both the nature of spoilage and the rate at which it occurs. There may be qualitative changes in spoilage characteristics as low temperatures exert a selective effect preventing the growth of mesophiles and leading to a microflora dominated by psychrotrophs (Paine and Paine, 1992; Blakestone, 1999; Cheikh, *et al*., 2009; Philip, 2010).

**Blanching** is achieved either by brief immersion of foods into hot water or the use of steam. Its primary functions are as follows: inactivation of enzymes that might cause undesirable changes during freezing storage, enhancement or fixing of the green color of certain vegetables, reduction in the numbers of microorganisms on the foods, facilitating the packing of leafy vegetables by inducing wilting, displacement of entrapped air in the plant tissues. When water is used, it is important that bacterial spores not be allowed to build up sufficiently to contaminate the juice. Reductions of initial microbial loads as high as 99% have been claimed upon blanching (Blakestone, 1999; Cheikh, *et al*., 2009; Philip 2010).

According to Adams and Moss,1995 the term Pasteurization is given to heat processes typically in the range 60-80 °C and applied for up to a few minutes, is used for two purposes. First is the elimination of a specific pathogen or pathogens associated with a product. This type of pasteurization is often a legal requirement introduced as a public health measure when a product has been frequently implicated as a vehicle of illness. The second reason for pasteurizing a product is to eliminate a large proportion of potential spoilage organisms, thus extending its shelf-life. This is normally the objective when acidic products such as beers, fruit juices, pickles, and sauces are pasteurized. Where pasteurization is introduced to improve safety, its effect can be doubly beneficial (Adams and Moss, 1995; Blakestone, 1999; Cheikh, *et al*., 2009; Philip, 2010). The process cannot

in which the spores lose their resistance to heat and to elevated pressure. Hydrostatic processing has a number of appealing features for the food preparations such as Marula

It acts instantly and uniformly throughout a substance so that the processing time is not related to container size and there is none of the penetration problems associated with heat processing. Nutritional quality, flavour, appearance and texture resemble the fresh material very closely. To the consumer where it has been used it is regarded as a 'natural' process with none of the negative associations of processes such as irradiation or chemical

Appertized foods include those which are hermetically sealed into containers, usually cans, and then subjected to heat process in-pack. While this has been hugely successful as a long term method of food preservation, it does require extended heating periods in which a food's functional and chemical properties can be adversely affected. (Adams and Moss,

Low-temperature storage - chilling and freezing the rates of most chemical reactions are temperature dependent; as the temperature is lowered so the rate decreases. Since food spoilage is usually a result of chemical reactions mediated by microbial and endogenous enzymes, the useful life of many foods can be increased by storage at low temperatures. Chilled foods are those foods stored at temperatures near, but above their freezing point, typically 0-5°C. Chill storage can change both the nature of spoilage and the rate at which it occurs. There may be qualitative changes in spoilage characteristics as low temperatures exert a selective effect preventing the growth of mesophiles and leading to a microflora dominated by psychrotrophs (Paine and Paine, 1992; Blakestone, 1999; Cheikh, *et al*., 2009;

**Blanching** is achieved either by brief immersion of foods into hot water or the use of steam. Its primary functions are as follows: inactivation of enzymes that might cause undesirable changes during freezing storage, enhancement or fixing of the green color of certain vegetables, reduction in the numbers of microorganisms on the foods, facilitating the packing of leafy vegetables by inducing wilting, displacement of entrapped air in the plant tissues. When water is used, it is important that bacterial spores not be allowed to build up sufficiently to contaminate the juice. Reductions of initial microbial loads as high as 99% have been claimed upon blanching (Blakestone, 1999; Cheikh, *et al*., 2009; Philip 2010).

According to Adams and Moss,1995 the term Pasteurization is given to heat processes typically in the range 60-80 °C and applied for up to a few minutes, is used for two purposes. First is the elimination of a specific pathogen or pathogens associated with a product. This type of pasteurization is often a legal requirement introduced as a public health measure when a product has been frequently implicated as a vehicle of illness. The second reason for pasteurizing a product is to eliminate a large proportion of potential spoilage organisms, thus extending its shelf-life. This is normally the objective when acidic products such as beers, fruit juices, pickles, and sauces are pasteurized. Where pasteurization is introduced to improve safety, its effect can be doubly beneficial (Adams and Moss, 1995; Blakestone, 1999; Cheikh, *et al*., 2009; Philip, 2010). The process cannot

fruit juice. (Indrawati, *et al*., 2008; Sampedro, *et al*., 2008; Chao, *et al*., 2011)

preservatives. ((Indrawati, *et al*., 2008; Sampedro, *et al*., 2008; Chao, *et al*., 2011)

**3.1 Fresh marula fruit juice shelf life extension by temperature control** 

1995)

Philip, 2010).

discriminate between the target pathogen(s) and other organisms with similar heat sensitivity so a pasteurization which destroys say *Salmonella* will also improve shelf-life. The converse does not normally apply since products pasteurized to improve keeping quality are often intrinsically safe due to other factors such as low pH. On its own, the contribution of pasteurization to extension of shelf-life can be quite small; particularly if the pasteurized food lacks other contributing preservative factors such as low pH, where as appertization refers to processes where the only organisms that survive processing are non-pathogenic and incapable of developing within the product under normal conditions of storage. As a result, appertized products have a long shelf-life even when stored at ambient temperatures. An appertized or commercially sterile food is not necessarily sterile - completely free from viable organisms (Adams and Moss, 1995; Blakestone, 1999; Cheikh, *et al*., 2009; Philip, 2010).

**Sterilization:** This is the process of destroying all forms of microbial life. A sterile object is free from living organism. killing bacteria is the irreversible loss of the bacteria's ability to reproduce. The cells are killed over a period of time at a constant exponential rate that is the inverse of exponential growth rate. Some portion of population dies during any given time. The graph of logarithm of number of survivor's v/s time in hours shows that the death rate is constant. Slope of this curve is a measure of death-rate (Jay, 1986; Adams and Moss, 1995). The probability of killing the organisms is also proportional to concentration of chemical agent or intensity of physical agent. It takes time to kill the population and if we have many cells, we must treat them for a longer time. Although imperfect, cooking and canning are the most common applications of heat sterilization. Boiling water kills the vegetative stage of all common microbes. Cooking food does not sterilize food but simply reduces the number of disease-causing micro-organisms to a level that is not dangerous for people with normal digestive and immune systems. Pressure cooking is analogous to autoclaving and when performed correctly renders food sterile. (Jay, 1986; Adams and Moss, 1995).

In UHT processing the food is heat processed before it is packed and then sealed into sterilized containers in a sterile environment. This approach allows more rapid heating of the product, the use of higher temperatures than those employed in canning, typically 130- 140°C, and processing times of seconds rather than minutes (Jay, 1986; Adams and Moss, 1995; Michael and Hepell 2000).The advantage of using higher temperatures is that the *z*  value for chemical reactions such as vitamin loss, browning reactions and enzyme inactivation is typically 25-40°C compared with 10°C for spore inactivation. This means that they are less temperature sensitive so that higher temperatures will increase the microbial death rate more than they increase the loss of food quality associated with thermal reactions. A common packing system used in conjunction with UHT processing is a form/ fill/seal operation in which the container is formed in the packaging machine from a reel of plastic or laminate material, although some systems use preformed containers. In order to obtain commercial sterility it is given a bactericidal treatment, usually with hydrogen peroxide, sometimes coupled with UV irradiation (Jay, 1986; Adams and Moss, 1995).

#### **3.2 Fresh marula fruit juice shelf life extension by chemical methods**

Chemicals that can possibly be used for sterilization marula juice include the gases ethylene oxide, Ozone, hydrogen peroxide which are examples of chemical sterilization techniques based on oxidative capabilities.

Ethylene oxide (ETO) is the most commonly used form of chemical sterilization. Due to its low boiling point of 10.4ºC at atmospheric pressure, Ethylene oxide behaves as a gas at room temperature. Ethylene oxide chemically reacts with amino acids, proteins, and DNA to prevent microbial reproduction. The sterilization process is carried out in a specialized gas chamber. After sterilization, products are transferred to an aeration cell, where they remain until the gas disperses and the product is safe to handle. Ethylene oxide is used for cellulose and plastics irradiation, usually in hermetically sealed packages. Ethylene oxide can be used with a wide range of plastics and other materials without affecting their integrity. Ethylene oxide vapours are inflammable so a mixture of ethylene oxide 10 to 20 % with 80 to 90 % CO2, or Feron is used, CO2 or Feron serve as inert diluent which prevent inflammability. It is a unique and powerful sterilizing agent. Bacterial spores show little resistance to destruction by this agent. (Morga, *et al.,* 1979; Adams and Moss, 1995) It has got very good penetrating power. It passes through the sterilizes large packets of materials and even certain plastics. It should be used with caution. The concentration of ethylene oxide and temperature and humidity are critical factors which determine the time required for sterilization. The apparatus used for its application is an autoclave modified. It is effective at low temperature and does not damage the material exposed to it but it is slow in action. The mode of action is believed to be alkylation reactions with organic compounds such enzymes and other proteins. (Jay, 1986; Adams and Moss, 1995). Ozone sterilization has been recently approved for use in the U.S. It uses oxygen that is subjected to an intense electrical field that separates oxygen molecules into atomic oxygen, which then combines with other oxygen molecules to form ozone. Ozone is used as a disinfectant for water and food. It is used in both gas and liquid forms as an antimicrobial agent in the treatment, storage and processing of foods (Adams and Moss, 1995; Cullen *et al*., 2010; Philip, 2010).

Low Temperature Gas Plasma (LTGP) is used as an alternative to ethylene oxide. It uses a small amount of liquid hydrogen peroxide (H2O2), which is energized with radio frequency waves into gas plasma. This leads to the generation of free radicals and other chemical species, which destroy organisms (Jay, 1986; Adams and Moss, 1995).

Hypochlorites: Calcium hypochlorite Ca(OCl)2 and sodium hypochlorite NaOCl are widely used. They are in powder or liquid forms and in various concentrations from 5-70%. CaCl2 is used to sanitize equipment and 1% NaOCl for personal hygiene and household disinfection. (Adams and Moss, 1995; Cullen *et al.,* 2010; Philip, 2010). This could find application in the extension of marula juice shelf life extension.

#### **3.3 Preservatives**

Although some would regard all chemical additions to food as synonymous with adulteration, many are recognized as useful and are allowed. Additives may be used to aid processing, to modify a food's texture, flavour, nutritional quality or colour but, here, we are concerned with those which primarily effect keeping quality: preservatives(Jay, 1986). Preservatives are defined as 'substances capable of inhibiting, retarding or arresting the growth of micro-organisms or of any deterioration resulting from their presence or of masking the evidence of any such deterioration'. They do not therefore include substances which act by inhibiting a chemical reaction which can limit shelf-life, such as the control of rancidity or oxidative discoloration by antioxidants. Neither does it include a number of food additives which are used primarily for other purposes. Preservatives may be

Ethylene oxide (ETO) is the most commonly used form of chemical sterilization. Due to its low boiling point of 10.4ºC at atmospheric pressure, Ethylene oxide behaves as a gas at room temperature. Ethylene oxide chemically reacts with amino acids, proteins, and DNA to prevent microbial reproduction. The sterilization process is carried out in a specialized gas chamber. After sterilization, products are transferred to an aeration cell, where they remain until the gas disperses and the product is safe to handle. Ethylene oxide is used for cellulose and plastics irradiation, usually in hermetically sealed packages. Ethylene oxide can be used with a wide range of plastics and other materials without affecting their integrity. Ethylene oxide vapours are inflammable so a mixture of ethylene oxide 10 to 20 % with 80 to 90 % CO2, or Feron is used, CO2 or Feron serve as inert diluent which prevent inflammability. It is a unique and powerful sterilizing agent. Bacterial spores show little resistance to destruction by this agent. (Morga, *et al.,* 1979; Adams and Moss, 1995) It has got very good penetrating power. It passes through the sterilizes large packets of materials and even certain plastics. It should be used with caution. The concentration of ethylene oxide and temperature and humidity are critical factors which determine the time required for sterilization. The apparatus used for its application is an autoclave modified. It is effective at low temperature and does not damage the material exposed to it but it is slow in action. The mode of action is believed to be alkylation reactions with organic compounds such enzymes and other proteins. (Jay, 1986; Adams and Moss, 1995). Ozone sterilization has been recently approved for use in the U.S. It uses oxygen that is subjected to an intense electrical field that separates oxygen molecules into atomic oxygen, which then combines with other oxygen molecules to form ozone. Ozone is used as a disinfectant for water and food. It is used in both gas and liquid forms as an antimicrobial agent in the treatment, storage and processing of foods

Low Temperature Gas Plasma (LTGP) is used as an alternative to ethylene oxide. It uses a small amount of liquid hydrogen peroxide (H2O2), which is energized with radio frequency waves into gas plasma. This leads to the generation of free radicals and other chemical

Hypochlorites: Calcium hypochlorite Ca(OCl)2 and sodium hypochlorite NaOCl are widely used. They are in powder or liquid forms and in various concentrations from 5-70%. CaCl2 is used to sanitize equipment and 1% NaOCl for personal hygiene and household disinfection. (Adams and Moss, 1995; Cullen *et al.,* 2010; Philip, 2010). This could find application in the

Although some would regard all chemical additions to food as synonymous with adulteration, many are recognized as useful and are allowed. Additives may be used to aid processing, to modify a food's texture, flavour, nutritional quality or colour but, here, we are concerned with those which primarily effect keeping quality: preservatives(Jay, 1986). Preservatives are defined as 'substances capable of inhibiting, retarding or arresting the growth of micro-organisms or of any deterioration resulting from their presence or of masking the evidence of any such deterioration'. They do not therefore include substances which act by inhibiting a chemical reaction which can limit shelf-life, such as the control of rancidity or oxidative discoloration by antioxidants. Neither does it include a number of food additives which are used primarily for other purposes. Preservatives may be

(Adams and Moss, 1995; Cullen *et al*., 2010; Philip, 2010).

extension of marula juice shelf life extension.

**3.3 Preservatives** 

species, which destroy organisms (Jay, 1986; Adams and Moss, 1995).

microbicidal and kill the target organisms or they may be microbistatic in which case they simply prevent them growing. This is very often a dose-dependent feature; higher levels of antimicrobial proving lethal while the lower concentrations that are generally permitted in foods tend to be microbistatic (Jay, 1986). For this reason chemical preservatives are useful only in controlling low levels of contamination and are not a substitute for good hygiene practices, effect on flavour and on product pH, thus potentiating their own action by increasing the proportion of undissociated acid present.

*Benzoic acid* occurs naturally in cherry bark, cranberries, greengage plums, tea and anise but is prepared synthetically for food use. Its antimicrobial activity is principally in the undissociated form and since it is a relatively strong acid (pH4.19) it is effective only in acid foods. As a consequence, its practical use is to inhibit the growth of spoilage yeasts and moulds (Jay, 1986).

#### **3.4 Fresh marula fruit juice shelf life extension by radiation methods**

Gamma rays and X-rays which have ionizing energy enough to pull electrons away from molecules and ionize them. When such radiation passes through cells it creates free hydrogen and hydroxyl radical and some peroxides which in turn can cause different kinds of intracellular damage. U.V. light does not ionize; it is absorbed quite specifically by different chemical species that can engage in a variety of chemical reactions not possible for unexcited molecules. Organisms may be subjected to acoustic radiation (sound waves). Ionizing radiation is also used to sterilize biological materials. This method is called Cold Sterilization because ionizing radiations produced relatively little heat in the material being irradiated. Thus it is possible to sterilize heat-sensitive substances by radiations and such techniques are being developed in the food and pharmaceutical industries (Jay, 1986; Adams and Moss, 1995; Chao *et al*., 2011).

Ultraviolet light has the bactericidal activity. Although the radiant energy of sunlight, is partly composed of UV light, most of the shorter wavelength of this are filtered by the earth's atmosphere is restricted. Many lamps are available which emit a high concentration of UV light in the most effective region. UV light has very little ability to penetrate matter. UV light is absorbed by many cellular materials but most significantly by the nucleic acids where it does the greatest damage. The absorption and subsequent reactions are predominantly in the pyrimidines of the nucleotide bases which result in killing of cells. Death of a population of UV-irradiated cells demonstrates log-linear kinetics (Jay, 1986; Adams and Moss, 1995; Chao *et al.,* 2011).

Similar to thermal death and, in an analogous way, D values can be determined. These give the dose required to produce a tenfold reduction in surviving numbers where the dose, expressed in ergs, is the product of the intensity of the radiation and the time for which it is applied.

Determination of UV D values is not usually a straightforward affair since the incident radiation can be absorbed by other medium components and has very low penetration. Passage through 5 cm of clear water will reduce the intensity of UV radiation by two-thirds. This effect increases with the concentration of solutes and suspended material so that in milk 90% of the incident energy will be absorbed by a layer only 0.1 mm thick. This low penetrability limits application of UV radiation in the food industry to disinfection of air and surfaces (Jay, 1986; Adams and Moss, 1995; Chao *et al*., 2011).

Low-pressure mercury vapor discharge lamps are used: 80% of their UV emission is at a wavelength of 254 ohm which has 85% of the biological activity of 260 om. Wavelengths below 200nm are screened out by surrounding the lamp with an absorbent glass since these wavelengths are absorbed by oxygen in the air producing ozone which is harmful. The output of these lamps falls off over time and they need to be monitored regularly. Process water can be disinfected by UV; this avoids the risk of tainting sometimes associated with chlorination, although the treated water will not have the residual antimicrobial properties of chlorinated water. Process workers must also be protected from UV since the wavelengths used can cause burning of the skin and eye disorders. (Jay, 1986; Adams and Moss, 1995; Chao *et al*., 2011).

#### **3.5 Gamma-rays**

These are high energy radiations emitted from radioactive isotopes such as 60 C. they are similar to X-rays but of shorter wavelength. They have great penetration power and are lethal to all life including microbes. They are used for sterilization of materials of considerable thickness or volume. (Jay, 1986; Adams and Moss, 1995).

**Microwave Radiation** The microwave region of the e.m. spectrum occupies frequencies between 109 Hz up to 1012 Hz and so has relatively low quantum energy. Unlike the other forms of radiation, microwaves act indirectly on micro-organisms through the generation of heat. When a food containing water is placed in a microwave field, the dipolar water molecules align themselves with the field. As the field reverses its polarity 2 or 5 x 1 09 times each second, depending on the frequency used, the water molecules are continually oscillating. This kinetic energy is transmitted to neighbouring molecules leading to a rapid rise in temperature throughout the product. Microwaves are generated using a magnetron (Jay, 1986; Adams and Moss, 1995). The principal problem associated with the domestic use of microwaves is non-uniform heating of foods, due to the presence of cold spots in the oven, and the non-uniform dielectric properties of the food. These can lead to cold spots in some microwaved foods and concern over the risks associated with consumption of inadequately heated meals has led to more explicit instructions on microwaveable foods. These often specify a tempering period after heating to allow the temperature to equilibrate. (Jay, 1986; Adams and Moss, 1995; Prescott, 2011).

#### **3.6 Fresh marula fruit juice shelf life extension by hormonal application**

Most fruit first become ripened because of the release of a hormone within the fruit and the plant called ethylene (H2C=CH2). Ethylene causes the breakdown which in itself causes a production of enzymes that break down the structures of the fruit (e.g. amylase, pictenase).The ethylene gas also destroys the green pigment of the fruit chlorophyll. But for any fruit to become ripened there needs to be a high enough concentration of ethylene around the fruit to even begin to ripen. The process of the fruit spoiling is just the decomposition of the fruit itself. That is mostly caused by the two enzymes previously mentioned -amylase and pectinase. The role that these enzymes play are like biological scissors. The structure of plants are made out of carbohydrates - mostly starches (Jay, 1986;

penetrability limits application of UV radiation in the food industry to disinfection of air

Low-pressure mercury vapor discharge lamps are used: 80% of their UV emission is at a wavelength of 254 ohm which has 85% of the biological activity of 260 om. Wavelengths below 200nm are screened out by surrounding the lamp with an absorbent glass since these wavelengths are absorbed by oxygen in the air producing ozone which is harmful. The output of these lamps falls off over time and they need to be monitored regularly. Process water can be disinfected by UV; this avoids the risk of tainting sometimes associated with chlorination, although the treated water will not have the residual antimicrobial properties of chlorinated water. Process workers must also be protected from UV since the wavelengths used can cause burning of the skin and eye disorders. (Jay, 1986; Adams and

These are high energy radiations emitted from radioactive isotopes such as 60 C. they are similar to X-rays but of shorter wavelength. They have great penetration power and are lethal to all life including microbes. They are used for sterilization of materials of

**Microwave Radiation** The microwave region of the e.m. spectrum occupies frequencies between 109 Hz up to 1012 Hz and so has relatively low quantum energy. Unlike the other forms of radiation, microwaves act indirectly on micro-organisms through the generation of heat. When a food containing water is placed in a microwave field, the dipolar water molecules align themselves with the field. As the field reverses its polarity 2 or 5 x 1 09 times each second, depending on the frequency used, the water molecules are continually oscillating. This kinetic energy is transmitted to neighbouring molecules leading to a rapid rise in temperature throughout the product. Microwaves are generated using a magnetron (Jay, 1986; Adams and Moss, 1995). The principal problem associated with the domestic use of microwaves is non-uniform heating of foods, due to the presence of cold spots in the oven, and the non-uniform dielectric properties of the food. These can lead to cold spots in some microwaved foods and concern over the risks associated with consumption of inadequately heated meals has led to more explicit instructions on microwaveable foods. These often specify a tempering period after heating to allow the temperature to equilibrate.

and surfaces (Jay, 1986; Adams and Moss, 1995; Chao *et al*., 2011).

considerable thickness or volume. (Jay, 1986; Adams and Moss, 1995).

**3.6 Fresh marula fruit juice shelf life extension by hormonal application** 

Most fruit first become ripened because of the release of a hormone within the fruit and the plant called ethylene (H2C=CH2). Ethylene causes the breakdown which in itself causes a production of enzymes that break down the structures of the fruit (e.g. amylase, pictenase).The ethylene gas also destroys the green pigment of the fruit chlorophyll. But for any fruit to become ripened there needs to be a high enough concentration of ethylene around the fruit to even begin to ripen. The process of the fruit spoiling is just the decomposition of the fruit itself. That is mostly caused by the two enzymes previously mentioned -amylase and pectinase. The role that these enzymes play are like biological scissors. The structure of plants are made out of carbohydrates - mostly starches (Jay, 1986;

(Jay, 1986; Adams and Moss, 1995; Prescott, 2011).

Moss, 1995; Chao *et al*., 2011).

**3.5 Gamma-rays** 

Adams and Moss, 1995; Allong *et al.,* 2001; Prescott, 2011). The amylase acts on the starches, which are made of amylose, which is just a long chain of glucose molecule. And what the amylase does is it cuts the bonds of the starch chain, so then the glucose molecules are free; and now the fruit tastes sweet. The other enzyme pectinase does almost the exact same thing as amylase, it cuts away the bonds of the pectin molecule so now we have a bunch of pectinic acid, and now the fruit is soft enough to eat. Pectin is extremely important for fruits/plants because it hold the cells together. Fruits become discolored due to chemical reactions as well. Like previously stated before, ethylene destroys chlorophyll. When most fruits are produced, they all initially contain chlorophyll. Fruits then become ripened due to other chemicals within the fruit and as well as chemicals outside of the fruit (Jay, 1986; Adams and Moss, 1995; Prescott, 2011).

#### **3.7 Fresh marula fruit juice shelf life extension by appropriate hybrids or genetic manipulation**

Through selection hybrid trees that are short and easy to hand or machine pick can be produced. This would minimise contamination from the soil and improve the shelf life of the juice. This has been achieved in other plants where trees were bred to achieve heights where hand or machine picking was practiced with ease (Bergh and Whitsell, 1962).

#### **4. Laboratory work on some aspects of marula juice shelf life extension**

The aim of this study was to determine some aspects of physico-chemical conditions that could contribute to the shelf life extension of fresh marula juice.

Fruit juice is defined as the fermentable but unfermented juice pressed or squeezed from the fruit excluding the peel. Fresh juice has a very short life after extraction from the whole fruit, due to enzyme or microbial actions unless it is rapidly processed and/or preserved. Steps to achieve aseptic processing involve minimizing the presence of microorganism in the environment and on the product without compromising the organoleptic quality of the product. Since all fruit juices provide an excellent medium for microbiological activity it becomes imperative that high standards of hygiene are observed from the on set. To extract the juice the fruits were washed with chlorinated water and rolled on a clean hard surface and then pieced with a toothpick and the juice then squeezed out into clean containers. The single strength juice from different fruits was, pooled, filtered and subjected to the following treatments in 100ml containers: Blanching; Pasteurization; Sterilization; and Blanching /Pasteurization and then preserved with 0.1% Sodium Benzoate, one portion stored at 4°C and the other portion at 22°C for 8 weeks as a way of extending the shelf life. The following parameters were assessed, sugar content of the berry, acidity, and the pH at the green stage of the fruit to fully ripe yellow fruits to map out strategies for shelf life extension. There amount of Vitamin C (Ascorbic acid) was assessed for the following treatments Blanching; Pasteurization; Sterilization; and Blanching /Pasteurization as an indicator of stability. The °Brix value and Browning index were determined. The sensory scores were determined to ensure that the treatments done do not result in an unpalatable product. Measurements of headspace were determined to evaluate its effect on the shelf life. Extending the life of fresh marula juice by altering its physico chemical properties were carried out in the laboratory as follows.


Other Quality parameters were also assessed


anchored with lowest value. The panelists were instructed to make a horizontal mark through the scale and write the corresponding sample number adjacent to the mark. The results were recorded on a scale of one to five. The sum of scores of each sample was divided by the number of panelist and the resulting average expressed as sensory

For microbial load the sample was diluted up to 10-4 in saline water and then plated on nutrient agar and colony forming units counted.

### **5. Results**

score.

192 Biochemical Testing

1. Blanching in which the fruit juice was subjected to a temperature of 100°C for 3

2. Pasteurization in which the fruit juice was subjected to a temperature of 82°C for 15

3. Sterilization in which the fruit juice was subjected to a temperature of 100°C for 5

4. Blanching and Pasteurization in which the fruit juice was subjected to both treatments as stated above. Each experiment had two replications to determine statistically

5. The juice from each treatment was preserved with 0.1% Sodium Benzoate, one portion

6. Brix value; The oBrix was determined on a Riechart Abbe Refractometer. Distilled water was placed on the platform and the readings adjusted to zero then a drop of sample was placed on the platform and the total soluble solids (mostly sugars) value or °Brix

7. Browning index; The browning index was determined. The brown pigments were extracted by diluting the juice 1:1 with 95% ethanol, filtering and measuring the

8. Total titratable acidity (TTA %) For total titratable acidity 0.1NaOH was titrated against 1ml sample single strength juice containing a drop of 1% phenolphthalein indicator in

9. Vitamin C content in mg/100g and vitamin C determination was done by taking 1ml of sample and titrating it with redox dye 2, 6-dichlorophenolindophenol (DCPIP) to a permanent pink colour and then concentration calculations were done. The juice of marula fruit is known to be rich in vitamin C (Hillman *et al*., 2008; Dlamini and Dube, 2008). Hillman *et al*., (2008), reported vitamin C contents of marula clones ranging from 7 to 21 mg/g dry weight, amounts that were approximately 10 times higher than orange and pomegranate fruit juice. The high ascorbic acid content of marula makes it a cheap and accessible source of nutrients and antioxidants. The protection against disease provided by fruits and vegetables has been attributed to the various antioxidants contained in these foods. Because of its strong reducing properties, ascorbic acid acts a singlet oxygen quencher, thus reducing the damaging effect of free radicals which are implicated in the etiology of a number of diseases, including cancer and heart, vascular,

10. Sensory score were monitored weekly for eight weeks Sensory score was done after previously documented methods. A trained 10-member panel holds the sample in the mouth, smell it and visualise it. The samples were coded with three-digit random numbers, placed in random order and served at 16°C in wine glasses. Unsalted crackers and water rinsing were utilized between each sample. Each panelist was asked to rate the samples for clarity, degree of browning, color, taste and flavor. A score sheet with vertical line scales for each rating was provided. Each scale was 10-cm long with anchor terms labeling each end. The top of the clarity scale was anchored with clear, and the bottom was anchored with extremely cloudy. The top of the browning scale was labeled with no browning, and the bottom was labeled with excessive browning. The tops of the color and flavor scales were anchored with highest value, and the bottoms were

minutes;

seconds;

minutes;

significant differences between samples.

Other Quality parameters were also assessed

value were read and recorded.

stored at 4°C and the other portion at 22°C for 8 weeks.

absorbance at 420nm on a Milton Roy spectrophotometer.

95% ethanol to a permanent end point pink colour at pH 8.1.

and neurodegenerative diseases (Hillman *et al*., 2008).

The fruits ripen between February and April. The time from ripening onset to rotting of the fruit was 16±4 days. Skin colour started to change on day 3 after abscission, and a completely yellow colour was obtained by day 12. The sugar content of the berry increases rapidly, acidity (TTA%) decreases, and the pH increases from 2.5 at the green stage of the fruit to 3.32 in fully ripe fruits. Berry skins lose chlorophyll and begin to develop characteristic yellow-gold colour. There was a significant decrease of Vitamin C (Ascorbic acid) due to processing in the order: Sterilize>Blanch/pasteurise>Pasteurise>Blanch. At 22°C the first week shows the greatest decline in vitamin C, which is followed by a period of improved stability. There is a decline in the °Brix value with time in Blanched juice stored at 22oC. At 4°C all treatments show minimal degradation in the °Brix values and Browning index. The sensory score increases as the fruit ripens reaching its peak at °Brix value of 13.5, which corresponds to yellow colour stage of ripening. In response to various storage containers Browning index and vitamin C content decreased in the order of liner>plastic>clear glass>brown glass with time in juice stored at 22°C. There was no significant change in sensory score of Sterilized and Blanched/Pasteurised juice stored at 4°. But at 22°C the decline in sensory score was rapid for all treatments. Measurements of headspace did not affect vitamin C content and browning index when the juice was stored at 4°C but at 22°C they did. Higher processing temperatures reduce microbial flora. Some fruits retained 80% of their vitamin C content after storage at -18°C for two years.

#### **6. Discussion**

The pattern of fruit abscission differed among trees, but in most examined individual trees, 80% of the fruits abscised within two weeks which is in agreement with previous authors (Arthachinta, 2000). Ripening of abscised fruits is affected by storage temperatures. After 14 days of storage, fruits kept at 4°C remained green and firm, while those kept at 22°C developed a yellow color and could be squeezed for juice. As ripening progressed they developed a golden yellow color, higher juice content, and lower acidity as also noted for other fruits studied. The fruits began to develop brown spots on the skin one week after the on set of the deeper yellow colour. Similar observations have been made for other fruits studied (Huang and Liu, 2002; Brumell and Harpster, 2001; Mahayothee *et al*., 2002; Prasanna *et al*., 2007; Prescott, 2011). These results indicate that climacteric processes in marula start after abscission, which is in agreement with studies on other related fruits (Brumell and Harpster, 2001; Prasanna, *et al*., 2007; Prescott, 2011). Blanching as treatment of prolonging the shelf life has been used in the canning industries. The effect of this treatment was comparable with previous results from various fruits particularly reduction in vitamin C content (Brumell and Harpster, 2001; Prasanna, *et al*., 2007; Rico *et al.,* 2007; Prescott, 2011). Sterilized marula juice remained in a stable state for extended periods but had the greatest loss of vitamin C and sensory score recorded initially which is in agreement with previous studies on these attributes(Brumell and Harpster, 2001; Prasanna *et al*., 2007; Nassu *et al.,* 2001; Prescott, 2011). °Brix and TTA were not affected by sterilization. Pasteurization least affected the sensory score, the °Brix, and Vitamin C. But the Browning index progressed rapidly under this treatment as previously recorded for other fruits(Brumell and Harpster, 2001; Prasanna *et al.,* 2007; Prescott, 2011). Storage at 4°C showed the highest stability of the juice for all treatments however at 22°C the was a rapid decline of vitamin C and rapid increase in the browning index in the first two weeks which was followed by a more stable quality maintenance for six weeks of the study period. Similar observations have been noted in some fruits previously studied (Brumell and Harpster, 2001; Prasanna *et al*., 2007; Prescott, 2011). The drop in pH as fruits ripen has been similarly observed for other fruits (Brumell and Harpster, 2001; Prasanna *et al.,* 2007; Prescott, 2011). In order to improve the shelf life of the non-alcoholic marula fruit juice, the appropriate headspace and the most suitable packaging materials should be established and then production of this juice can be embarked-on on a commercial scale. This will make the juice available to children. Thus a wider portion of the population will benefit from this nutritious resource. Efforts to improve on the shelf life of indigenous fruits have been successfully made in Malaysia and other developing countries (Brumell and Harpster, 2001; Prasanna *et al*., 2007; Prescott, 2011).

#### **7. Concluding remarks**

By modifying the physico-chemical conditions marula juice can have its shelf lie extended without compromising its organoleptic properties. The amounts of vitamin C are in agreement with previous studies and this vitamin has been used as measure of stability of fresh fruit juices a factor confirmed by this study. Sugar levels as measured by °Brix and TTA and enzymatic browning are a function ripening and storage conditions and physicochemical conditions. Temperature in particular is critical for shelf life extension of fresh marula juice.

#### **8. Acknowledgements**

This work was funded by the NUST Research Board.

#### **9. References**

Adams, M.R. & Moss, M.O. (1995). Food Microbiology Amazon UK 3rd edition.


C content (Brumell and Harpster, 2001; Prasanna, *et al*., 2007; Rico *et al.,* 2007; Prescott, 2011). Sterilized marula juice remained in a stable state for extended periods but had the greatest loss of vitamin C and sensory score recorded initially which is in agreement with previous studies on these attributes(Brumell and Harpster, 2001; Prasanna *et al*., 2007; Nassu *et al.,* 2001; Prescott, 2011). °Brix and TTA were not affected by sterilization. Pasteurization least affected the sensory score, the °Brix, and Vitamin C. But the Browning index progressed rapidly under this treatment as previously recorded for other fruits(Brumell and Harpster, 2001; Prasanna *et al.,* 2007; Prescott, 2011). Storage at 4°C showed the highest stability of the juice for all treatments however at 22°C the was a rapid decline of vitamin C and rapid increase in the browning index in the first two weeks which was followed by a more stable quality maintenance for six weeks of the study period. Similar observations have been noted in some fruits previously studied (Brumell and Harpster, 2001; Prasanna *et al*., 2007; Prescott, 2011). The drop in pH as fruits ripen has been similarly observed for other fruits (Brumell and Harpster, 2001; Prasanna *et al.,* 2007; Prescott, 2011). In order to improve the shelf life of the non-alcoholic marula fruit juice, the appropriate headspace and the most suitable packaging materials should be established and then production of this juice can be embarked-on on a commercial scale. This will make the juice available to children. Thus a wider portion of the population will benefit from this nutritious resource. Efforts to improve on the shelf life of indigenous fruits have been successfully made in Malaysia and other developing countries (Brumell and Harpster, 2001; Prasanna *et al*., 2007; Prescott, 2011).

By modifying the physico-chemical conditions marula juice can have its shelf lie extended without compromising its organoleptic properties. The amounts of vitamin C are in agreement with previous studies and this vitamin has been used as measure of stability of fresh fruit juices a factor confirmed by this study. Sugar levels as measured by °Brix and TTA and enzymatic browning are a function ripening and storage conditions and physicochemical conditions. Temperature in particular is critical for shelf life extension of fresh

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**7. Concluding remarks** 

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**9. References** 

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### **Molecular Characterization and Serotyping of**  *Listeria monocytogenes* **with a Focus on Food Safety and Disease Prevention**

I.C. Morobe1,2,4, C.L. Obi1,3, M.A. Nyila2, M.I. Matsheka4 and B.A. Gashe4 *1Walter Sisulu University, Nelson Mandela Drive, Mthatha, Eastern Cape, 2School of Agriculture and Life Sciences, Department of Life and Consumer Sciences, University of South Africa, Pretoria, 3Division of Academic Affairs and Research, Walter Sisulu University, Eastern Cape, 4Department of Biological Sciences, University of Botswana, Gaborone, 1,2,3South Africa 4Botswana* 

#### **1. Introduction**

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*Listeria monocytogenes* is a gram-positive, non-spore forming, facultative bacterium that is now an established food-borne pathogen known for causing the disease listeriosis in humans. Apart from displaying typical symptoms associated with gastrointestinal infections, listeriosis is also characterized by flu-like symptoms. The incidence of listeriosis is low in the general population, but what is significant is the very high fatality rate which can range from 20 to 30% in immune-compromised people (Mead *et al.,* 2006). In a report by Mead *et al.,* (2006) approximately 2500 cases of Listeriosis are recorded annually in the United States, resulting in 500 deaths. The primary mode of infection is through the ingestion of contaminated food. Therefore, food serves as an important medium in the transmission of infection and to date still plays a critical role in the propagation and perpetuation of cases of listeriosis the world over.

The ubiquitous nature of *L. monocytogenes* in the environment poses a challenge in reducing cases of listeriosis. The food industry is incapacitated in producing food free of this pathogen. Its wide distribution increases the chances of cross–contamination between appliances or several products during processing. *Listeria* also has the ability to colonize surfaces, forming biofilms that remain attached to equipment used in food production (Wong *et al.,* 1998). The organism ability to grow at low water activity, low pH (Buchanan *et al.,* 2000) as well as refrigerated vacuum packed food products (Duffy *et al.,* 1994) makes it difficult to eliminate from retail food products. *L. monocytogenes* is also problematic due to its resistance to multiple antibiotics, which makes it difficult to treat (Charpentier *et al.,* 1995).

*Listeria Monocytogenes* is divided into at least 12 serotypes (1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4a, 4b, 4c, 4d, 4e, and 7). The virulence of *Listeria monocytogenes* seems to be serotype dependent with serotypes 1/2a, 1/2c, 1/2b and 4b being involved in 98% of documented human listeriosis cases. The 4a and 4c serotypes of lineage are rarely associated with outbreaks of disease despite frequent isolation from a variety of food and environmental samples (Wiedmann *et al*., 1997). Therefore, serotyping can provide useful information on the potential risk posed by *Listeria monocytogenes* isolates from various sources.

The conventional serotyping slide agglutination technique has been uses with great success in diagnostic and epidemiological investigations but is not routinely used because of the cost factor associated with the requirement of purchasing the whole spectrum of type specific antisera. Lack of standardization of reagents is known to hinder reproducibility of the technique. An enzyme–linked immunosorbent assay (ELISA) with the prospect of rapid serotyping of *L. monocytogenes* has been developed, but lack of concordance between the ELISA and agglutination results have been reported (Borucki *et al*., 2003).This limitation has spurred a quest for alternative based typing techniques with universal application. Consequently *L. monocytogenes* PCR based serotyping methods have been described (Borucki *et al*., 2003; Doumith *et al*., 2004; Chen and Knabel, 2007) and chart a way into the establishment of a user friendly DNA based serotyping system. *L. monocytogenes* can now be further classified into three evolutionary lineages. Lineage I encompasses serotypes 1/2b, 3b, 4b, 4d and 4, lineage II includes serotypes 1/2a, 1/2c, 3a, and 3c; and lineage III comprises serotypes 4a, 4c as well as 4b.

While the PCR based serotyping methods play an important role in screening of subgroups of epidemiological importance, they need to be used in conjunction with sub-typing methods with higher discriminatory power in order to effectively track outbreak strains. Automated ribotyping of *Listeria monocytogenes* is an alternative DNA based typing technique of favor that has been used with great success in several investigations. However, this method has a prohibitive cost element, in that specialized equipment has to be purchased to analyze the results. PCR based typing methods remain an attractive cost effective option in sub-typing of bacterial isolates and have an added advantage of being rapid. REP-typing is a highly discriminatory typing technique has been used with success in epidemiologically surveillance of pathogenic bacteria of importance, including *Listeria*  (Jersek *et al*., 1998; Wojciech *et al.*, 2004).

Even though a past study by Manani *et al.,* (2006) reported the occurrence of *Listeria monocytogenes* in frozen vegetables in this country, there is little data on the occurrence of this pathogen in foods available to consumers in Botswana. The REP-typing method described by Jersek *et al*, (1998) together with the modified PCR serotyping method initially described by Borucki *et al.*, (2003), were used to determine the of presence of Listeria serotypes of clinical significance in various retail food products in Botswana. These methods have predominantly been used in isolates from Europe and North America (Chen and Knabel, 2007) and this study provided the opportunity to assess the robustness and appropriateness of the genetic markers underpinning these DNA based typing techniques in characterizing isolates from a geographically distinct region.

#### **2. Literature review**

#### **2.1 Listeria disease**

*Listeria* includes 6 different species, *L. monocytogenes, L. ivanovii* sub-species, *ivanovii, L. innocua, L. welshimeri, L. seegligeri* and *L. grayi.* Both *L. monocytogenes L. innocua* and *ivanovii* 

listeriosis cases. The 4a and 4c serotypes of lineage are rarely associated with outbreaks of disease despite frequent isolation from a variety of food and environmental samples (Wiedmann *et al*., 1997). Therefore, serotyping can provide useful information on the

The conventional serotyping slide agglutination technique has been uses with great success in diagnostic and epidemiological investigations but is not routinely used because of the cost factor associated with the requirement of purchasing the whole spectrum of type specific antisera. Lack of standardization of reagents is known to hinder reproducibility of the technique. An enzyme–linked immunosorbent assay (ELISA) with the prospect of rapid serotyping of *L. monocytogenes* has been developed, but lack of concordance between the ELISA and agglutination results have been reported (Borucki *et al*., 2003).This limitation has spurred a quest for alternative based typing techniques with universal application. Consequently *L. monocytogenes* PCR based serotyping methods have been described (Borucki *et al*., 2003; Doumith *et al*., 2004; Chen and Knabel, 2007) and chart a way into the establishment of a user friendly DNA based serotyping system. *L. monocytogenes* can now be further classified into three evolutionary lineages. Lineage I encompasses serotypes 1/2b, 3b, 4b, 4d and 4, lineage II includes serotypes 1/2a, 1/2c, 3a, and 3c; and lineage III

While the PCR based serotyping methods play an important role in screening of subgroups of epidemiological importance, they need to be used in conjunction with sub-typing methods with higher discriminatory power in order to effectively track outbreak strains. Automated ribotyping of *Listeria monocytogenes* is an alternative DNA based typing technique of favor that has been used with great success in several investigations. However, this method has a prohibitive cost element, in that specialized equipment has to be purchased to analyze the results. PCR based typing methods remain an attractive cost effective option in sub-typing of bacterial isolates and have an added advantage of being rapid. REP-typing is a highly discriminatory typing technique has been used with success in epidemiologically surveillance of pathogenic bacteria of importance, including *Listeria* 

Even though a past study by Manani *et al.,* (2006) reported the occurrence of *Listeria monocytogenes* in frozen vegetables in this country, there is little data on the occurrence of this pathogen in foods available to consumers in Botswana. The REP-typing method described by Jersek *et al*, (1998) together with the modified PCR serotyping method initially described by Borucki *et al.*, (2003), were used to determine the of presence of Listeria serotypes of clinical significance in various retail food products in Botswana. These methods have predominantly been used in isolates from Europe and North America (Chen and Knabel, 2007) and this study provided the opportunity to assess the robustness and appropriateness of the genetic markers underpinning these DNA based typing techniques in

*Listeria* includes 6 different species, *L. monocytogenes, L. ivanovii* sub-species, *ivanovii, L. innocua, L. welshimeri, L. seegligeri* and *L. grayi.* Both *L. monocytogenes L. innocua* and *ivanovii* 

potential risk posed by *Listeria monocytogenes* isolates from various sources.

comprises serotypes 4a, 4c as well as 4b.

(Jersek *et al*., 1998; Wojciech *et al.*, 2004).

**2. Literature review 2.1 Listeria disease** 

characterizing isolates from a geographically distinct region.

*L.* are pathogenic, but only *L. monocytogenes* is associated with humans and animal illness (Rodriguez-Lazaro *et al.,* 2004). All these species are psychotrophic and widely spread in the environment. *L. innocua* is a major contaminant of vegetable surfaces and equipment or machinery (Aguado *et al.,* 2004).

*Listeria monocytogenes* appears to be a normal resident of the intestinal tract in humans. Studies carried out by examination of faecal samples, have found out that approximately 5 to 10 % of the general population are carriers of *L. monocytogenes.* This observation is thought to partially explain why antibodies to Listeria species are common in healthy individuals. Thus, because of the high rate of clinically healthy carriers, the presence of *L. monocytogenes* in faeces is not necessarily an indication of an infection (Farber *et al.,* 1991). Listeriosis is clinically defined when the organism is isolated from blood, cerebrospinal fluid and even placenta and foetus in cases of abortion. In a study of the duration of faecal excretion, shedding patterns were found to be erratic among different individuals with some carriers found to shed the organism for long periods. Among animals the carrier rate is generally considered to be 1 to 5 %, although recent studies involving newer methods for isolating *Listeria* species have indicated much higher carriage rates (Farber *et al.,* 1991).

Ingestion of food contaminated with *L. monocytogenes* can result in symptoms characteristic of listeriosis. Studies have shown that the number of *Listeria monocytogenes* cells can rise following refrigeration from fewer than 100 cells per gram, and this is the dose that is generally accepted for healthy people (Huss *et al.,* 2000; Buchanan *et al.,* 2000). Individuals who are particularly susceptible to this condition are immune-compromised individual (as in HIV/AIDS infection), pregnant women, persons with low stomach acidity, newborn babies, cancer patients, alcoholics, drug abusers, patients with corticosteroid therapy and the elderly (McLauchlin *et al.,* 2004; Rodriguez – Lazaro et *al.,* 2004). Most healthy individuals experience flu-like symptoms. The manifestation of listeriosis includes septicemia, meningitis (meningoencephalitis), encephalitis and intrauterine or cervical infections in pregnant women. According to Mead and his colleagues (1999), Infection acquired in early pregnancy may lead to abortion, still birth or premature delivery. When listeriosis is acquired late in pregnancy it can be transmitted transplacentally and lead to neonatal listeriosis. The onset of the aforementioned disorders is usually preceded by influenza-like symptoms including persistent fever followed by nausea, vomiting and diarrhea, particularly in patients who use antacid or cimetidine. The onset time to serious forms of listeriosis ranges from a few days to 3 weeks, while the onset time to gastrointestinal symptoms is greater than 12 hours (›12hours).The severe form of the disease has a high fatality rate of 30 %.

#### **2.2 Determinants of** *Listeria* **virulence**

*Listeria monocytogenes* produces an exotoxin listeriolysin (LLO) which is major virulence factor and its secretion is essential for promoting the intracellular growth and T- cell recognition of the organism (Farber *et al.,* 1991). The hemolysin of *L. monocytogenes* is recognized as a key agent in human neutrophil activation. The stimulation of these phagocytes, however, requires additional listerial virulence factors of which PIcA may play a prominent role (Karunasagar *et al.,* 1993). The presence of the listeriolysin gene is restricted to the species *L. monocytogenes*. Beside the characterized Listeriolysin encoded by the hly gene, *L. monocytogenes* also produces two other hemolysins; phosphatidylinositol-specific phospholipase C (Pl-PLC) and phosphatidylcholine-specific phospholipase C (PC-PLC). Unlike the LLO which lyses host cells by pore formation, these virulence factors act by disrupting the membrane lipids. The bacterium also produces zinc (2+) dependent protease, which acts like an exotoxin.

There are six *Listeria monocytogenes* virulent genes, namely; *prfA*, *pclA*, *hlyA*, *mpl*, *actA*, and *plcB* located together in one virulence gene cluster between the house keeping gene *idh* and *prs*. The *actA* gene product is a surface protein required for intercellular movement and cell to cell spread through bacterially induced actin polymerization. The virulence of *L. monocytogenes* is multi-factorial. Other factors affecting the pathogenicity of *L. monocytogenes* are, iron compounds, catalase and superoxide dismutase, and surface components. The virulence of the organism may be affected by its growth temperature. Growth of *L .monocytogenes* at a reduced temperature (4C) increases its virulence intravenously. This phenomenon may affect the virulence of the organism in refrigerated foods.

#### **2.3 Treatment**

When infection occurs during pregnancy, antibiotics given promptly to the pregnant women can often prevent infection of the fetus or new born. In general, isolates of *L*. *monocytogenes*, as well as strains of other Listeria species, are susceptible to a wide range of antibiotics except tetracycline, erythromycin, streptomycin, cephalosporins, and fosfomycin (Charpentier *et al.,* 1999). The treatment of choice for listeriosis remains the administration of ampicillin, penicillin G combined with an aminoglycoside and gentamycin. The association of trimethoprim with sulphonamide, such as sulfamethaxazole in co–trimoxazole, is a second choice therapy (Charpentier *et al.,* 1999). The most active agent in the combination is trimethoprim, which is synergized by sulfamethaxazole. Most isolates from clinical as well foodborne and environmental sources are susceptible to the antibiotics active against gram positive bacteria (Yucel *et al.,* 2005).

#### **2.4 Epidemiology and occurrence of** *Listeria*

The incidence of listeriosis appears to be on the increase worldwide with a significant number of cases, especially in Europe. The annual endemic disease rate varies from 2 to 15 cases per million populations, with published rates varying from 1.6 to a high rate of 14.7 in France for 1986. *Listeria* has been isolated sporadically from wide variety of sources and listeriosis outbreaks that have occurred in the past have highlighted contaminated food as the main source of transmission. A wide range of foods such as salads, seafood's, meat, and dairy have been implicated in listeriosis (Huss *et al.,* 2000). Usually the presence of *Listeria* species in food is thought to be an indicator of poor hygiene (Manani *et al.,* 2006). A variety of ready- to–eat food products, such as frozen or raw vegetables, milk and milk products, meat and meat products and seafood support the growth of *Listeria monocytogenes*. These foods are considered of high risk due to the ability of listeria to grow and survive in them (Kunene *et al.,* 1999). However, there are other products, traditionally considered of low risk, which have recently been linked to listeriosis transmission, such as the large listeriosis outbreak reported in Italy due to the consumption of corn. Though no fatalities occurred, more than 1500 people were affected (Aguado *et al.,* 2004). There is no doubt that the susceptible population is increasing, as there is a steady increase in numbers and types of foods in which *L. monocytogenes* is isolated.

In Africa the incidence of listeriosis have also been reported in countries like Zambia where 85 cases of meningitis due to *Listeria* were reported (Chintu *et al.,* 1975). In Togo, 8 out of 342 healthy slaughter animals were positive for *L. monocytogenes* (serovars 1/2a and 4b) isolated from the intestinal lymph nodes (Hohne *et al.,* 1975). In Northern Nigeria 27% mortality due to *L. monocytogenes* (serovar; 4) was reported (Onyemelukwe *et al.,* 1983). Listeria organisms are documented to be zoonotic with one of the sources of infection being the domestic fowl. *L. monocytogenes* can be found on poultry carcasses and in poultry processing plants. The prevalence of pathogens in chickens in many countries is well documented but their presence in South African poultry products has not been extensively reported on. Two studies investigating contamination of food available from street vendors in Johannesburg have been documented (Mosupye and Von Holly 1999; 2000). The carriage of *Listeria monocytogenes* and other *Listeria* in indigenous birds has not been documented in Kenya (Njagi *et al.,* 2004).

#### **2.5 Antimicrobial resistance in** *L. monocytogenes*

200 Biochemical Testing

phospholipase C (Pl-PLC) and phosphatidylcholine-specific phospholipase C (PC-PLC). Unlike the LLO which lyses host cells by pore formation, these virulence factors act by disrupting the membrane lipids. The bacterium also produces zinc (2+) dependent protease,

There are six *Listeria monocytogenes* virulent genes, namely; *prfA*, *pclA*, *hlyA*, *mpl*, *actA*, and *plcB* located together in one virulence gene cluster between the house keeping gene *idh* and *prs*. The *actA* gene product is a surface protein required for intercellular movement and cell to cell spread through bacterially induced actin polymerization. The virulence of *L. monocytogenes* is multi-factorial. Other factors affecting the pathogenicity of *L. monocytogenes* are, iron compounds, catalase and superoxide dismutase, and surface components. The virulence of the organism may be affected by its growth temperature. Growth of *L .monocytogenes* at a reduced temperature (4C) increases its virulence intravenously. This

When infection occurs during pregnancy, antibiotics given promptly to the pregnant women can often prevent infection of the fetus or new born. In general, isolates of *L*. *monocytogenes*, as well as strains of other Listeria species, are susceptible to a wide range of antibiotics except tetracycline, erythromycin, streptomycin, cephalosporins, and fosfomycin (Charpentier *et al.,* 1999). The treatment of choice for listeriosis remains the administration of ampicillin, penicillin G combined with an aminoglycoside and gentamycin. The association of trimethoprim with sulphonamide, such as sulfamethaxazole in co–trimoxazole, is a second choice therapy (Charpentier *et al.,* 1999). The most active agent in the combination is trimethoprim, which is synergized by sulfamethaxazole. Most isolates from clinical as well foodborne and environmental sources are susceptible to the antibiotics active against gram

The incidence of listeriosis appears to be on the increase worldwide with a significant number of cases, especially in Europe. The annual endemic disease rate varies from 2 to 15 cases per million populations, with published rates varying from 1.6 to a high rate of 14.7 in France for 1986. *Listeria* has been isolated sporadically from wide variety of sources and listeriosis outbreaks that have occurred in the past have highlighted contaminated food as the main source of transmission. A wide range of foods such as salads, seafood's, meat, and dairy have been implicated in listeriosis (Huss *et al.,* 2000). Usually the presence of *Listeria* species in food is thought to be an indicator of poor hygiene (Manani *et al.,* 2006). A variety of ready- to–eat food products, such as frozen or raw vegetables, milk and milk products, meat and meat products and seafood support the growth of *Listeria monocytogenes*. These foods are considered of high risk due to the ability of listeria to grow and survive in them (Kunene *et al.,* 1999). However, there are other products, traditionally considered of low risk, which have recently been linked to listeriosis transmission, such as the large listeriosis outbreak reported in Italy due to the consumption of corn. Though no fatalities occurred, more than 1500 people were affected (Aguado *et al.,* 2004). There is no doubt that the susceptible population is increasing, as there is a steady increase in numbers and types of

phenomenon may affect the virulence of the organism in refrigerated foods.

which acts like an exotoxin.

**2.3 Treatment** 

positive bacteria (Yucel *et al.,* 2005).

**2.4 Epidemiology and occurrence of** *Listeria*

foods in which *L. monocytogenes* is isolated.

*Listeria monocytogenes*, as well as other *Listeria* spp., are usually susceptible to a wide range of antibiotics (Charpentier *et al.,* 1995). However, evolution of bacterial resistance towards antibiotics has been accelerated considerably by the selective pressure exerted by overprescription of drugs in clinical settings and their heavy use as promoters in animals husbandry (Charpentier *et al.,* 1995).Therefore, it was not unexpected when the first multiresistant strain of *L. monocytogenes* was isolated in France in 1988 (Poyart-Salmeron *et al*., 1990) and since then multi-resistant *L monocytogenes* strains have been recovered from food, the environment and sporadic cases of human listeriosis (Charpentier *et al.,* 1995). Antibiotics to which some *L. monocytogenes* strains are resistant to include tetracycline, gentamicin, penicillin, ampicillin, streptomycin, erythromycin, kanamycin, sulfonamide, trimethoprim, and rifampicin (Charpentier and Courvalin, 1999).

Tetracycline resistance has been the most frequently observed resistance phenotype among *L. monocytogenes* isolates (Charpentier *et al.,* 1995; Charpentier and Courvalin, 1999). Six classes of tetracycline-resistance genes; *tet*(K), *tet*(L), *tet*(M), *tet*(O), *tet*(P), and *tet*(S) have been described in Gram positive bacteria (Charpentier et al., 1995). However, only *tet*(L) and *tet*(S) have been identified in *L. monocytogenes* (Poyart-Salmeron *et al.,* 1992; Charpentier and Courvalin, 1999). *Tet*(M) and *tet*(S) confer resistance by ribosomal protection, whereas the *tet*(L) gene codes for a protein which promotes active efflux of tetracycline from the bacteria. Transfer of resistance between *L. monocytogenes* can occur in the gastrointestinal tract of domestic animals where both species live and where sub-inhibitory levels of tetracycline may be expected. In fact, tetracyclines are second most commonly used antibiotics worldwide. They are used extensively in animal foodstuffs, especially for poultry, and it is noteworthy that tetracycline resistance was the single most common resistance marker in food-borne *L. monocytogenes* isolated from chicken and turkey (Chopra *et al.,* 2001). Antibiotic resistance in *L. monocytogenes* is reaching an era where virtually all antibiotics will be rendered ineffective because of various mechanisms employed by *L monocytogenes* to counteract the therapeutic agents.

#### **2.6 Isolation and detection of** *L. monocytogenes*

The Genus *Listeria* includes 6 different species but only *L. monocytogenes* and *L. ivanovii* are known to be pathogenic. However, only *L .monocytogenes* is associated with humans and animal illness (Rodriguez-Lazaro *et al*., 2004).In the past strains were classified to species level using morphological characteristics and biochemical tests (suspect colonies, motility, catalase, hemolysins, CAMP and API *Listeria* identification system). Significant efforts have been dedicated to the development of enrichment media and protocols for *L. monocytogenes* isolation. Ideal enrichment media would facilitate recovery of injured *Listeria* cells and enrichment of *Listeria* species (*L*. *monocytogenes*) over competing microflora. In traditional culture–based assays, it becomes very difficult to detect *L. monocytogenes* at any level when it is greatly outnumbered by other *Listeria* species, such as *L. innocua*, which is in most cases present together with *L. monocytogenes* (Bille *et al.,* 1992). Specific–specific identification with biochemical standard methods, which include sugar fermentation or the CAMP test, is laborious and time consuming and can require 1 to 2 weeks for species identification. Moreover differentiation between species and strains is not always reached (Aguado *et al.,* 2003). A diagnostic scheme for the same day identification of food borne cells of *L. monocytogenes* has been proposed. Large representative colonies that emerge in 40 hours at 30°C are used as heavy inoculum on agar plates for the rapid determination of hemolytic activity and acidification of rhamnose and xylose. Additional tests consisting of cell phasecontrast microscopy, motility testing, the catalase production test and the KOH viscosity test in place of Gram staining have been employed in the rapid identification of *L. monocytogenes* (Borucki *et al.,* 2003).

The rapid identification of *L. monocytogenes* is important so that the appropriate antibiotic therapy can be initiated. Currently molecular methods that enable the identification of *Listeria* to the species level include; Random Amplified Polymorphic DNA Polymorphism, to discriminate *Listeria monocytogenes* from *Listeria innocua* (in the 16S rRNA genes), Polymerase Chain Reaction (PCR), real time PCR, Ligase Chain Reaction (LCR) for the detection of *Listeria monocytogenes* and Pulse-field fingerprinting of *listeria*, for detection of genomic divisions for *L. monocytogenes* and their correlation with serovars, and restriction endonuclease analysis (REA), have been employed to directly characterize the microorganism without the need for isolation (Wouters *et al*., 1999). These new molecular methods may also improve the ability to diagnose pregnancy –associated disease and permit the rapid detection and control of *L. monocytogenes* in the food supply (Wiedmann *et al.,* 1993).

The Listeriolysin genes have also been used identification. DNA hybridization studies have shown that listeriolysin genes are found in *Listeria* species, such as *L*. *monocytogenes, L. ivanovii*, *L. seeligeri*. In the analysis of genomic DNA of Listeria by southern hybridization with *hlyA* probes all strains were isolated and digested with the restriction endonuclease Hind*III*. The 0.8-kb Bam*HI* probe that was made up entirely of sequences upstream of the listeriolysin gene was found to hybridize to *L. monocytogenes* strains irrespective of serotype, as well as to the *L. seeligeri* and *L. ivanovii* strains (Borucki *et al.*, 2003). Other methods that can be employed to detect listerolysin are; hemolysin assays and polyacrylamide gel electrophoresis, imuno-magnetic beads for listeria and *Listeria* exotoxin detection kits (Borucki *et al.,* 2003). Immunoblotting performed with affinity–purified antibody to literiolysin allowed the detection of this protein in supernatants of all three species. In this immunological assay two recombinants, (pLM47 and pLM48) were found to produce a polypeptide of 60KDa which cross-reacted with the antisera to produce a hemolytic phenotype on blood agar plates (Leimeister- Wachter *et al.,* 1992).

#### **2.7 Typing of L. monocytogenes.**

202 Biochemical Testing

animal illness (Rodriguez-Lazaro *et al*., 2004).In the past strains were classified to species level using morphological characteristics and biochemical tests (suspect colonies, motility, catalase, hemolysins, CAMP and API *Listeria* identification system). Significant efforts have been dedicated to the development of enrichment media and protocols for *L. monocytogenes* isolation. Ideal enrichment media would facilitate recovery of injured *Listeria* cells and enrichment of *Listeria* species (*L*. *monocytogenes*) over competing microflora. In traditional culture–based assays, it becomes very difficult to detect *L. monocytogenes* at any level when it is greatly outnumbered by other *Listeria* species, such as *L. innocua*, which is in most cases present together with *L. monocytogenes* (Bille *et al.,* 1992). Specific–specific identification with biochemical standard methods, which include sugar fermentation or the CAMP test, is laborious and time consuming and can require 1 to 2 weeks for species identification. Moreover differentiation between species and strains is not always reached (Aguado *et al.,* 2003). A diagnostic scheme for the same day identification of food borne cells of *L. monocytogenes* has been proposed. Large representative colonies that emerge in 40 hours at 30°C are used as heavy inoculum on agar plates for the rapid determination of hemolytic activity and acidification of rhamnose and xylose. Additional tests consisting of cell phasecontrast microscopy, motility testing, the catalase production test and the KOH viscosity test in place of Gram staining have been employed in the rapid identification of *L. monocytogenes*

The rapid identification of *L. monocytogenes* is important so that the appropriate antibiotic therapy can be initiated. Currently molecular methods that enable the identification of *Listeria* to the species level include; Random Amplified Polymorphic DNA Polymorphism, to discriminate *Listeria monocytogenes* from *Listeria innocua* (in the 16S rRNA genes), Polymerase Chain Reaction (PCR), real time PCR, Ligase Chain Reaction (LCR) for the detection of *Listeria monocytogenes* and Pulse-field fingerprinting of *listeria*, for detection of genomic divisions for *L. monocytogenes* and their correlation with serovars, and restriction endonuclease analysis (REA), have been employed to directly characterize the microorganism without the need for isolation (Wouters *et al*., 1999). These new molecular methods may also improve the ability to diagnose pregnancy –associated disease and permit the rapid detection and control of *L. monocytogenes* in the food supply (Wiedmann *et* 

The Listeriolysin genes have also been used identification. DNA hybridization studies have shown that listeriolysin genes are found in *Listeria* species, such as *L*. *monocytogenes, L. ivanovii*, *L. seeligeri*. In the analysis of genomic DNA of Listeria by southern hybridization with *hlyA* probes all strains were isolated and digested with the restriction endonuclease Hind*III*. The 0.8-kb Bam*HI* probe that was made up entirely of sequences upstream of the listeriolysin gene was found to hybridize to *L. monocytogenes* strains irrespective of serotype, as well as to the *L. seeligeri* and *L. ivanovii* strains (Borucki *et al.*, 2003). Other methods that can be employed to detect listerolysin are; hemolysin assays and polyacrylamide gel electrophoresis, imuno-magnetic beads for listeria and *Listeria* exotoxin detection kits (Borucki *et al.,* 2003). Immunoblotting performed with affinity–purified antibody to literiolysin allowed the detection of this protein in supernatants of all three species. In this immunological assay two recombinants, (pLM47 and pLM48) were found to produce a polypeptide of 60KDa which cross-reacted with the antisera to produce a hemolytic

phenotype on blood agar plates (Leimeister- Wachter *et al.,* 1992).

(Borucki *et al.,* 2003).

*al.,* 1993).

*L. monocytogenes* strains are serotyped according to variation in the somatic (O) and flagellar (H) antigens (Seeliger and Hohne, 1979). Thirteen *L. monocytogenes* serotypes (serovars) have been characterized in this species by using specific and standardized sera (Seeliger & Langer, 1979). Serovar identification by serological tests has remained popular. However, numerous molecular biology methods such as multiplex PCR (Borucki *et al*., 2003; Doumith *et al*., 2004; Chen and Knabel) have come to the fore in the characterization of *L. monocytogenes* serotypes. Three genetic lineages (I, II, III) for *L. monocytogenes* have been identified using these various molecular subtyping techniques. Epidemic clone I contains serotypes 1/2b, 3b, 3c, and 4b, lineage II contained serotypes 1/2a, 1/2c, and 3a and lineage III contained serotypes 4a and 4c (Chen and Knabel, 2007). Although most clinical isolates belong to serovars 1/2a, 1/2b, and 4b, the majority of strains which have caused large outbreaks are serovars 1/2a and 1/2b (Kathariou, 2000); Jacquet *et al.,* 2002; Zhang and Knabel, 2005). Interestingly, although serotype 1/2a is the most frequently isolated from food, serotype 4b causes the majority of human epidemics (Zhang and Nabel., 2005). This suggests that serovar 4b may pose unique virulence properties. However, geographical differences in the global distribution of serotypes apparently exist.

Phage typing has proven to be a valuable epidemiological tool in investigations of many infectious diseases. Since the initial discovery phages specific for listeria species in 1945, several groups have assessed the usefulness of phage typing L*. monocytogenes*. Phages derived from both environment sources and lysogenic strains have been found. In isozyme typing, bacteria are differentiated by the variation in the electrophoretic mobility of any of a large number of metabolic enzymes. This technique is useful in either confirming or eliminating a common source as the cause of an outbreak of food-borne listeriosis (Farber *et al.,* 1991).

DNA fingerprinting using restriction enzyme analysis (REA) has recently been used to characterize strains of *L. monocytogenes* causing outbreaks of listeriosis associated with Mexican–style soft cheese in Los Angeles, as well as the Nova Scotia and Switzerland outbreaks (Aguado *et al.,* 2004). Plasmid typing has also been used in conjunction with DNA fingerprint to confirm a case of cross–infection with *L. monocytogenes*. However, this technique is of less importance since *L .monocytogenes* does not appear to carry plasmid. On the other hand *L. innocua* carry plasmids ranging in size from 3 to 55 MDa (Aguado *et al.,* 2004). Monocine typing has recently been evaluated as a typing tool for *L. monocytogenes*. Although this technique is potentially promising as an epidemiological tool, only 59 and 56 % of serovars 1/2a and 4b were found to be producers of monocines. In one instance a pair of *L. monocytogenes* strains isolated from a mother and a newborn, which could not be phage typed, but proved to be identical by monocine typing (Baloga *et al.,* 2006).

#### **3. Materials and methods**

#### **3.1 Sampling**

Samples were obtained randomly from selected supermarkets and street vendors in 5 Geographical areas of Gaborone Samples collected were; raw vegetables (cabbage) and salads, raw milk, cheese and meat (biltong). In this study 250 -300 samples per product were obtained. Samples were put in separate properly labeled sterile specimen bags and put into a cooler box containing ice packs. Gloves were worn to avoid cross- contamination between samples from different supermarkets and street vendors. Samples were transferred into properly labeled stomacher bags and then homogenized with the Stomacher (Seward 400, Tekmar, and Cincinnati Ohio, USA) set at medium speed. *Listeria monocytogenes* positive control (ATCC 19115) was purchased from South African Bureau of Standards (SABS).

#### **3.2 Enrichment, culturing, morphological observation and biochemical identification**

The homogenized samples were enriched by putting 25g of the sample into 225ml enrichment broth (Mast Diagnostics DM257) and incubated at 30ºC for 48 hours on Innova 4000 Newbrunswick Scientific incubator shaker. A loop full of culture was subcultured after 48 hours onto Listeria Selective Agar (Oxoid M009, Basingstoke, UK) plates and then incubated at 37ºC for 24 hours. Modified Listeria Selective Enrichment Supplement (SR206E, Oxoid, Basingstoke, and Hampshire, England) was added to Listeria Agar Base and dark brown colonies with black zones were subcultured on nutrient agar (Oxoid CM001) plates. A Gram stain was done on suspected colonies from a culture medium (Nutrient Agar). Gram positive short rods colonies picked were subcultured onto Tryptic Soya Agar (Merck, Darmstadt, Germany) slants. After 24 hour incubation, the slants were kept at 4C. Other broth cultures were stored in 80% Tryptic Soya Broth and 20% Glycerol ( i.e., 750µl Tryptose broth plus 250µl of 20% Glycerol) were put into a 2ml vial and kept at -82C for subsequent steps.

#### **3.2.1 Biochemical testing**

A catalase test was performed to separate the listeria (catalase positive) from listeria (catalase negative). Picking a colony with sterile loop on a slide containing 3% hydrogen peroxide does this. The evolution of gas bubbles (oxygen) indicates a positive test. Seroagglutination was carried out by a slide agglutination technique using commercially prepared *Listeria* spp. antisera (Oxoid Listeria test kit DR1126A, Basingstoke, New Hampshire, England). Agglutination patterns were linked to *Listeria* spp*.* following manufacturer's instructions. Isolates that were positive in the serology test were subjected to the API Listeria test (BioMerieux, Paris, France). This is the confirmatory test for the organism and it differentiates *L. monocytogenes* from other *Listeria* species.

#### **3.3 Serotype identification by PCR**

#### **3.3.1 DNA extraction**

*L. monocytogenes* strains were stored long term in tryptic soy broth (Merck, Darmstadt, Germany) with 20% glycerol at -82ºC. Strains were then recovered by inoculating into tryptose soy broth and were grown overnight at 37 °C. Cells were harvested on a bench top centrifuge and genomic DNA extracted using Guanidium Thiocyanate chromosomal technique (Pitcher et al., 1989). 500μl of guanidium thiocyanate solution (60g Guanidium thiocyanate, 20ml 0.5M EDTA pH8,20ml deionized water and 5ml of 10% (w/v) N-Lauryl-Sarcosine Sodium salt, made up to 100ml with deionized water) was added and briefly mixed to lyse the cells. 250μl ice-cold 7.5M ammonium acetate was added, mixed and left on ice for 10 minutes. 500μl Chloroform/ Isopropanol (24:1) was added, mixed and span at 1200g for 10 minutes. 600μl supernatant was transferred to clean Eppendorf tubes and 400μl ice –cold isopropanol was added, mixed by gentle inversion of the Eppendorf tube and span at 6500g for 20 seconds to collect DNA. The pellet was washed three times with 200μl of icecold 70% alcohol, span at 6500g for 2 minutes to remove excess Guanidium thiocyanate. The pellet was then air dried at 4 ºC for 7 minutes and resuspened in 30μl of TE buffer and kept at 4ºC for subsequent steps.

#### **3.3.2 PCR amplification**

204 Biochemical Testing

a cooler box containing ice packs. Gloves were worn to avoid cross- contamination between samples from different supermarkets and street vendors. Samples were transferred into properly labeled stomacher bags and then homogenized with the Stomacher (Seward 400, Tekmar, and Cincinnati Ohio, USA) set at medium speed. *Listeria monocytogenes* positive control (ATCC 19115) was purchased from South African Bureau of Standards (SABS).

**3.2 Enrichment, culturing, morphological observation and biochemical identification**  The homogenized samples were enriched by putting 25g of the sample into 225ml enrichment broth (Mast Diagnostics DM257) and incubated at 30ºC for 48 hours on Innova 4000 Newbrunswick Scientific incubator shaker. A loop full of culture was subcultured after 48 hours onto Listeria Selective Agar (Oxoid M009, Basingstoke, UK) plates and then incubated at 37ºC for 24 hours. Modified Listeria Selective Enrichment Supplement (SR206E, Oxoid, Basingstoke, and Hampshire, England) was added to Listeria Agar Base and dark brown colonies with black zones were subcultured on nutrient agar (Oxoid CM001) plates. A Gram stain was done on suspected colonies from a culture medium (Nutrient Agar). Gram positive short rods colonies picked were subcultured onto Tryptic Soya Agar (Merck, Darmstadt, Germany) slants. After 24 hour incubation, the slants were kept at 4C. Other broth cultures were stored in 80% Tryptic Soya Broth and 20% Glycerol ( i.e., 750µl Tryptose broth plus 250µl of 20% Glycerol) were put into a 2ml vial and kept at -82C for subsequent

A catalase test was performed to separate the listeria (catalase positive) from listeria (catalase negative). Picking a colony with sterile loop on a slide containing 3% hydrogen peroxide does this. The evolution of gas bubbles (oxygen) indicates a positive test. Seroagglutination was carried out by a slide agglutination technique using commercially prepared *Listeria* spp. antisera (Oxoid Listeria test kit DR1126A, Basingstoke, New Hampshire, England). Agglutination patterns were linked to *Listeria* spp*.* following manufacturer's instructions. Isolates that were positive in the serology test were subjected to the API Listeria test (BioMerieux, Paris, France). This is the confirmatory test for the

*L. monocytogenes* strains were stored long term in tryptic soy broth (Merck, Darmstadt, Germany) with 20% glycerol at -82ºC. Strains were then recovered by inoculating into tryptose soy broth and were grown overnight at 37 °C. Cells were harvested on a bench top centrifuge and genomic DNA extracted using Guanidium Thiocyanate chromosomal technique (Pitcher et al., 1989). 500μl of guanidium thiocyanate solution (60g Guanidium thiocyanate, 20ml 0.5M EDTA pH8,20ml deionized water and 5ml of 10% (w/v) N-Lauryl-Sarcosine Sodium salt, made up to 100ml with deionized water) was added and briefly mixed to lyse the cells. 250μl ice-cold 7.5M ammonium acetate was added, mixed and left on ice for 10 minutes. 500μl Chloroform/ Isopropanol (24:1) was added, mixed and span at 1200g for 10 minutes. 600μl supernatant was transferred to clean Eppendorf tubes and 400μl

organism and it differentiates *L. monocytogenes* from other *Listeria* species.

steps.

**3.2.1 Biochemical testing** 

**3.3 Serotype identification by PCR** 

**3.3.1 DNA extraction** 

Amplification of serotype specific *hly* gene product of 214 bp and the Serogroup identification by multiplex PCR using primer pairs D1 and D2. PCR using reverse and forward primers (D1-Forwad; 5' CGATATTTTATCTACTTTGTC 3'; D1-Reverse; 5' TTGCTCCAAAGCAGGGCAT 3' and D2-Forward; 5' GCGGAGAAAGCTATCGCA 3'; D2- Reverse; 5' TTGTTCAAACATAGGGCTA 3') as described by Borucki and Call (2003). Reaction mixtures was made up to 50μl using the high pure PCR template kit (Fermentas) and Roche PCR core kit reagent according to manufacturer's instructions. Each reaction consisted of 50pmol of each primer and 50ng of DNA template with 2.5 units of Tag polymerase.

Amplification was carried out using Applied Biosystems GeneAmp 2400 thermocycler. PCR cycling conditions were as follows; 95ºC for 3 minutes followed by 25 cycles (with D1 and D2 primers) 72ºC for 1 minute followed by a final step of 72ºC for 10 minutes after cycling was completed. The product size was resolved using electrophoresis through 1.8% agarose gels containing ethidium bromide and visualized on a UV transilluminator. During this experiment laboratory control strain of *L. monocytogenes* were used as a positive control, included in each group of samples undergoing analysis.

The strains that tested positive with D1 primers were further subjected to PCR using GLT primers ( GLT-Forward 5'- AAA GTG AGT TCT TAC GAG ATT T-3' and GLT-Reverse 5'- AAT TAG GAA ATC GAC CTT CT-3'). The PCR reaction conditions were as mentioned above but with a different PCR cycling protocol. Initial denaturation was carried out at 95ºC for 5 minutes followed by 25 cycles of 45ºC for 30 seconds and 72ºC for 1 minute, followed by a final step of 72ºC for 10 minutes after cycling was completed. PCR products were determined using electrophoresis through 1.8% agarose gel containing ethedium bromide and visualized on a UV transilluminator.

#### **3.3.3 Lineage group classification by MAMA – PCR**

MAMA primers were used to test strains that tested negative with GLT primers. The high pure PCR template kit (Fermentas) was used according to the manufacturer's instructions. Reaction mixtures contained primers (LM4-Forward (5'- CAG TTG CAA GCG CTTGGAGT-3' ) and LMB-Reverse (5'- GTA AGT CTC CGA GGT TGC AA-3') at a concentration of 50pmoles. MAMA-PCR amplification conditions were as follows; 10 minutes initial denaturation step, followed by 40 cycles of 0.5 minutes at 95ºC, 1 minute at 55ºC and 1 minute at 72ºC, with a final extension step for 10 minutes at 72ºC. Amplification product was electrophoresed on a 1.5% agarose gel containing 0.4μg/ml ethidium bromide at 60 volts for 90 minutes and visualized on a UV transilluminator.

The strains that tested positive with MAMA primers were subjected to PCR using ORF2110 primers (Forward: 5' - AGTGGACAATTGATTGGTGAA-3' and Reverse: 5'- CATCCATCCCTTACTTTGGAC-3') as described by Doumith *et al*. (2004) at a concentration of 50pmoles.ORF2110 - PCR amplification conditions were as follows; initial denaturation step at 94ºC for 3 minutes followed by 35 cycles of 94ºC for 0.40 minutes, 53ºC for 1 minute and 72ºC for 1 minute and one final cycle of 72ºC for 7 minutes. Amplification product was electrophoresed on a 1.5% agarose gel containing 0.4μg/ml ethidium bromide at 60 volts for 90 minutes and visualized on a UV transilluminator.

#### **3.4 Typing by repetitive element sequence – based PCR**

Amplification of REP-PCR products was done using REP IR – I 5'– IIIICGICGICATCIGGC-3' and REP 2-1 5'-ICGICTTATCIGGCCTAC-3' primer pairs as described by Jersek et al., (1999). Reaction mixtures made up of 50μl using the high pure PCR template kit (Fermentas) and Roche PCR core kit reagents were used according to the manufacturer's instructions. Each reaction consisted of 50pmol of each primer and 50ng of DNA template with 2.5 units of Tag polymerase. REP – PCR cycling conditions were as follows; An initial denaturation at 95°C for 3 minutes followed by 30 cycles of 90°C for 30 seconds at 40°C for 1 minute , at 72°C for 1 minute and final cycle at 72°C for 8 minutes. The REP-PCR gene products were resolved into finger printing patterns on a 1.5% agarose gel (Roche) at 60Volts for 1 hour. Gel images captured on the Syngene Gene Genius BioImaging System (Cambridge, UK). Fingerprint patterns were considered different if there was a presence or absence of a band at a particular molecular weight. Variations in the brightness of the band, was not considered to constitute a difference. The gel images were subjected to cluster analysis using GelCompar software (Applied Maths, Kortrijk, Belgium). The similarity was performed using the Dice coefficient. A band matching tolerance of 1.0% was chosen.

#### **4. Results**

#### **4.1 Isolation and identification of** *L. monocytogenes*

From the five various food products (Cheese, raw milk, Biltong, frozen cabbage and Coleslaw salad), 57 isolates of *L. monocytogenes* were recovered from all food types except biltong. These isolates were identified as *Listeria* by using the seroagglutination test and positively identifies as *L. monocytogenes* using phenotypic and biochemical testing.

#### **4.2 Serogroup identification by Polymerase Chain Reaction (PCR)**

Serogroup identification by PCR was performed on all the 57 confirmed *L. monocytogenes* isolates. Using primer pairs D1 a PCR product of 214bp was for the entire strains analyzed. The PCR product of this size suggests serotypes belonging to phylogenetic lineage of division I and III, which comprise serogroups 4a, 1/2b, 3b, 4b, 4c, 4d and 4e. No PCR product was obtained for all 57 isolates using The D2 Lineage II specific primers.

To differentiate 1/2b and 3b serotypes from the rest of the members in division I, the strains that tested positive with D1 primers were subjected to PCR using GLT primers. Only 6 (10.52%) strains gave the expected PCR product size of 483b. In one specific case the primers gave an amplicon bigger than the expected PCR product (Fig 2 lane 4).

Strains that did not give any product (amplicon) or the expected PCR product size with GLT primers were assumed to belong to either division I or division III. To differentiate the

CATCCATCCCTTACTTTGGAC-3') as described by Doumith *et al*. (2004) at a concentration of 50pmoles.ORF2110 - PCR amplification conditions were as follows; initial denaturation step at 94ºC for 3 minutes followed by 35 cycles of 94ºC for 0.40 minutes, 53ºC for 1 minute and 72ºC for 1 minute and one final cycle of 72ºC for 7 minutes. Amplification product was electrophoresed on a 1.5% agarose gel containing 0.4μg/ml ethidium bromide at 60 volts for

Amplification of REP-PCR products was done using REP IR – I 5'– IIIICGICGICATCIGGC-3' and REP 2-1 5'-ICGICTTATCIGGCCTAC-3' primer pairs as described by Jersek et al., (1999). Reaction mixtures made up of 50μl using the high pure PCR template kit (Fermentas) and Roche PCR core kit reagents were used according to the manufacturer's instructions. Each reaction consisted of 50pmol of each primer and 50ng of DNA template with 2.5 units of Tag polymerase. REP – PCR cycling conditions were as follows; An initial denaturation at 95°C for 3 minutes followed by 30 cycles of 90°C for 30 seconds at 40°C for 1 minute , at 72°C for 1 minute and final cycle at 72°C for 8 minutes. The REP-PCR gene products were resolved into finger printing patterns on a 1.5% agarose gel (Roche) at 60Volts for 1 hour. Gel images captured on the Syngene Gene Genius BioImaging System (Cambridge, UK). Fingerprint patterns were considered different if there was a presence or absence of a band at a particular molecular weight. Variations in the brightness of the band, was not considered to constitute a difference. The gel images were subjected to cluster analysis using GelCompar software (Applied Maths, Kortrijk, Belgium). The similarity was performed

From the five various food products (Cheese, raw milk, Biltong, frozen cabbage and Coleslaw salad), 57 isolates of *L. monocytogenes* were recovered from all food types except biltong. These isolates were identified as *Listeria* by using the seroagglutination test and

Serogroup identification by PCR was performed on all the 57 confirmed *L. monocytogenes* isolates. Using primer pairs D1 a PCR product of 214bp was for the entire strains analyzed. The PCR product of this size suggests serotypes belonging to phylogenetic lineage of division I and III, which comprise serogroups 4a, 1/2b, 3b, 4b, 4c, 4d and 4e. No PCR

To differentiate 1/2b and 3b serotypes from the rest of the members in division I, the strains that tested positive with D1 primers were subjected to PCR using GLT primers. Only 6 (10.52%) strains gave the expected PCR product size of 483b. In one specific case the primers

Strains that did not give any product (amplicon) or the expected PCR product size with GLT primers were assumed to belong to either division I or division III. To differentiate the

positively identifies as *L. monocytogenes* using phenotypic and biochemical testing.

product was obtained for all 57 isolates using The D2 Lineage II specific primers.

**4.2 Serogroup identification by Polymerase Chain Reaction (PCR)** 

gave an amplicon bigger than the expected PCR product (Fig 2 lane 4).

90 minutes and visualized on a UV transilluminator.

**4. Results** 

**3.4 Typing by repetitive element sequence – based PCR** 

using the Dice coefficient. A band matching tolerance of 1.0% was chosen.

**4.1 Isolation and identification of** *L. monocytogenes*

Fig. 1. Genomic DNA from *Listeria monocytogenes* strains subjected to multiplex PCR with primer pairs for D1 and D2. Showing the 214bp PCR product (lanes 1-7). Lane M, MassRuler TM SM0403 (Fermentas).

Fig. 2. Genomic DNA from *Listeria monocytogenes* strains subjected to PCR with GLT primers. Lane 2, 3 and 7 (ATCC 19115 strain) depicting isolates with the expected 483bp PCR product. Lane M, ZipRuler TM SM1378 (Fermentas).

isolates belonging to serogroup 4b, 4d and 4e, the isolates which did not give a PCR product with GLT primers were again subjected to PCR with primers specific to ORF2110. Seventeen

of the 57 isolates gave a 597bp amplicon when the PCR product was resolve on a 1.5% agarose gel. In certain instances unspecific priming was evident (Fig 3 lanes 4, 5 and 6).

Fig. 3*.* Genomic DNA from *Listeria* monocytogenes strains subjected to PCR with primers specific to ORF 2110. Lane 2, 3, 4, 5 6 and 7(ATCC19115 strain) depicting isolates with the expected 596bp PCR product. Lane M, GeneRuler TM SM1148 (Fermentas).

Fig. 4. Genomic DNA from *Listeria monocytogenes* strains subjected to PCR with MAMA-C primers. Lanes 2 to 6 show the expected 268bp PCR product. Lane M, GeneRulerTM SM 1148.

of the 57 isolates gave a 597bp amplicon when the PCR product was resolve on a 1.5% agarose gel. In certain instances unspecific priming was evident (Fig 3 lanes 4, 5 and 6).

Fig. 3*.* Genomic DNA from *Listeria* monocytogenes strains subjected to PCR with primers specific to ORF 2110. Lane 2, 3, 4, 5 6 and 7(ATCC19115 strain) depicting isolates with the

Fig. 4. Genomic DNA from *Listeria monocytogenes* strains subjected to PCR with MAMA-C primers. Lanes 2 to 6 show the expected 268bp PCR product. Lane M, GeneRulerTM SM 1148.

expected 596bp PCR product. Lane M, GeneRuler TM SM1148 (Fermentas).

Furthermore the isolates that were GLT negative were subjected to PCR using MAMA-C primers to identify isolates belonging to division III. From the 51 isolates that were negative for GLT primers, 45 isolates gave a 268bp product with MAMA-C specific primers, indicating suggesting that they belonged to Division III. Included in the 45 positive isolates that were positive for PCR with MAMA-primers were the 17 isolates that had proved of positive for ORF2110 specific primers. The rhamnose fermentation test was positive for all 45 isolates suggesting that they belonged to division III.

Serogroup identification using PCR found that most isolates (49%) belonged to serogroups 4a and 4c. These isolates were found mostly in cabbage and salads. This was followed by isolates in serogroups 4b, 4d and 4e which comprised 30% of the isolates. Isolates within this group appeared in all food types except salads and cheese. Isolates with serogroups 1/2b and 3b were rare with four isolates appearing in salads and one isolate being picked up in cheese and milk respectively. Isolates belonging to division II were not detected at all because no isolates were positive for PCR using D2 specific primers. Four isolates in salads (S10, S31, S200, S258) and two (V208, V225) in cabbage were found to belong to division I, but could not be characterized further into serogroups because they proved to be negative for PCR serogroup identification using GLT and ORF2110 specific primers.


+ (positive), - (negative), ND (Not Determined)

Table 1. Serotyping of *L. monocytogenes* by PCR.

#### **4.3 Typing by repetitive element sequence based PCR**

From the 57 *L. monocytogenes* strains that were isolated, 41 were selected and typed using REP-PCR. DNA fingerprints obtained for all isolates had a maximum of five bands ranging from 200bp to 300bp (See figure 5. A, B, C and D). The DNA fingerprints were analyzed using a cluster analysis computer program to derive a dendrogram. A total of 5 clusters could be clearly distinguished at similarity level of more than 40% (Fig. 6) which are designated I, II, III, IV and V. The REP-PCR profiles of the isolates seemed to cluster according to food types, with most of the isolates from salad and cabbage falling into group I and V respectively. The three *L. monocytogenes* isolated from milk fell into group II while all five cheese isolates selected for REP-PCR analysis fell into group III. Group IV was heterogenous group with a balanced mixture of isolates from salad and cabbages. Two pairs of isolates from the two food types had identical REP-PCR profiles (S7 was similar to V131, while S10 was similar to V157). Cabbage had the most diverse REP-PCR profile types that appeared in 4 of the 5 REP-PCR profile groups. Only REP-PCR profile group I had isolates from one commodity (salads). There was no correlation between the REP-PCR clusters and serogroup or the three *L. monocytogenes* genetic lineages.

Fig. 5. REP – PCR fingerprints of *Listeria monocytogenes* isolates (lane 1-7). Lane M, ZipRuler TM SM1378 (Fermentas). A and B shows the diverse profiles of isolates obtained from cabbage and salads. C and D shows isolates with similar REP-PCR profiles obtained from cabbage and salads respectively.

while S10 was similar to V157). Cabbage had the most diverse REP-PCR profile types that appeared in 4 of the 5 REP-PCR profile groups. Only REP-PCR profile group I had isolates from one commodity (salads). There was no correlation between the REP-PCR clusters and

Fig. 5. REP – PCR fingerprints of *Listeria monocytogenes* isolates (lane 1-7). Lane M, ZipRuler TM SM1378 (Fermentas). A and B shows the diverse profiles of isolates obtained from cabbage and salads. C and D shows isolates with similar REP-PCR profiles obtained from

cabbage and salads respectively.

serogroup or the three *L. monocytogenes* genetic lineages.

Fig. 6. REP-PCR Dendrogram representing similarity between *L. monocytogenes* isolates.

#### **5. Discussion**

In this study serogrouping by PCR suggests serotypes belonging to phylogenetic lineage of division I and III, which comprise serogroups 1/2b, 3b, 4a, 4b, 4c, 4d and 4e. Isolates within this group appeared in all food types except salads. It was found that isolates with 1/2b, and 3b were rare with only four isolates appearing in salad and one isolate being picked up in cheese and milk respectively. This is in contrast to most studies that have found serotypes 1/2a and 1/2b were to be the most common serotypes in food (Aarnisalo *et al*., 2003), a finding also supported by Gilot *et al.,* (1996) in a study of foods in Belgium. A study by Wallace *et al.,* (2003) also found serovar 1/2a in 90% of all the *Listeria monocytogenes* isolates tested in food samples. The results obtained from the current study indicated a correlation between certain serotypes and specific food products, serogroups 1/2b and 3b was absent from Cabbage, a food type that had more *L. monocytogenes* isolates than other food type. This observation is in line with results obtained by Vitas and Garcia-Jalon (2004) in a study of fresh and processed foods in Navarra, Spain. In the present study serogroup 4b, 4d and 4e was present in 30% isolates retrieved from the food. This is significant because among the *Listeria monocytogenes* serotypes, serotype 4b has been the number one serotype associated with human listeriosis (Zhang *et al.,* 2007).

One major finding in this study is that the majority of the isolates found in food samples belonged to lineage III (4a, 4c), which is contrary to results found in previous studies (Gray *et al*., 2004; Ward *et al*., 2004). Isolates in lineage III are known to be more prevalent in animals with clinical Listeriosis (Jeffers *et al*., 2001). What was also significant was that isolates belonging to division II were not detected; no isolates were positive for PCR using D2 specific primers. Division two has serovar 1/2a, a serotype common in food products. Furthermore, four samples belonged to division I, namely; S10, S31, S200 and S258 from salads, V208 and V225 from cabbage, but could not be characterized further into serogroups because they proved to be negative for PCR serogroup identification using GLT and ORF2110 specific primers. This proved to be one of the major limitation of serotype identification by PCR, in that some isolates could not be conclusively be allocated to serogroups. One other shortcoming of serotyping by PCR is that isolates could not definitively be allocated to specific serotypes but only indicated a number of possible serogroups or a division. However, these results are not surprising as the PCR assays used were not based on genes encoding serotype-specific antigens.

Using REP-PCR Typing a significant observation in the results was that most diverse isolates in this study were more common in cabbage and salads than the dairy products. This was to be expected as relatively few *Listeria* isolates were isolated from dairy products. Variations within the sizes of PCR generated fragments using REP-PCR was observed with this studies and two studies carried out by Wojciech *et al.,* (2004). The amplicons obtained by Wojciech *et al.,* (2004) were shorter with sizes ranging between 123 to 735 bp, in comparison to amplicons obtained by Jersek *et al*., (1998) with sizes ranging from 298 to 6100 bp. In this study amplicons obtained ranged from 200bp to 3000 bp. Though the same primers were used, the reason of such differences could be the variation in the DNA polymerase used, PCR machines and protocols used in this study.

In this study, REP-PCR was used as a tool to characterize *L. monocytogenes* strains isolated from food. This method showed great possibilities for the typing of *L. monocytogenes,* as isolated strains showed very similar REP-PCR fingerprints by visual comparison and cluster analysis but managed to show diversity between strains. These data supports previous studies that suggest that REP-PCR can be used as an alternative method for typing *L. monocytogenes* such as automated ribotyping which has proven to be a valuable epidemiological tool in investigations of many infectious diseases.

#### **5.1 Conclusion**

212 Biochemical Testing

In this study serogrouping by PCR suggests serotypes belonging to phylogenetic lineage of division I and III, which comprise serogroups 1/2b, 3b, 4a, 4b, 4c, 4d and 4e. Isolates within this group appeared in all food types except salads. It was found that isolates with 1/2b, and 3b were rare with only four isolates appearing in salad and one isolate being picked up in cheese and milk respectively. This is in contrast to most studies that have found serotypes 1/2a and 1/2b were to be the most common serotypes in food (Aarnisalo *et al*., 2003), a finding also supported by Gilot *et al.,* (1996) in a study of foods in Belgium. A study by Wallace *et al.,* (2003) also found serovar 1/2a in 90% of all the *Listeria monocytogenes* isolates tested in food samples. The results obtained from the current study indicated a correlation between certain serotypes and specific food products, serogroups 1/2b and 3b was absent from Cabbage, a food type that had more *L. monocytogenes* isolates than other food type. This observation is in line with results obtained by Vitas and Garcia-Jalon (2004) in a study of fresh and processed foods in Navarra, Spain. In the present study serogroup 4b, 4d and 4e was present in 30% isolates retrieved from the food. This is significant because among the *Listeria monocytogenes* serotypes, serotype 4b has been the number one serotype associated

One major finding in this study is that the majority of the isolates found in food samples belonged to lineage III (4a, 4c), which is contrary to results found in previous studies (Gray *et al*., 2004; Ward *et al*., 2004). Isolates in lineage III are known to be more prevalent in animals with clinical Listeriosis (Jeffers *et al*., 2001). What was also significant was that isolates belonging to division II were not detected; no isolates were positive for PCR using D2 specific primers. Division two has serovar 1/2a, a serotype common in food products. Furthermore, four samples belonged to division I, namely; S10, S31, S200 and S258 from salads, V208 and V225 from cabbage, but could not be characterized further into serogroups because they proved to be negative for PCR serogroup identification using GLT and ORF2110 specific primers. This proved to be one of the major limitation of serotype identification by PCR, in that some isolates could not be conclusively be allocated to serogroups. One other shortcoming of serotyping by PCR is that isolates could not definitively be allocated to specific serotypes but only indicated a number of possible serogroups or a division. However, these results are not surprising as the PCR assays used

Using REP-PCR Typing a significant observation in the results was that most diverse isolates in this study were more common in cabbage and salads than the dairy products. This was to be expected as relatively few *Listeria* isolates were isolated from dairy products. Variations within the sizes of PCR generated fragments using REP-PCR was observed with this studies and two studies carried out by Wojciech *et al.,* (2004). The amplicons obtained by Wojciech *et al.,* (2004) were shorter with sizes ranging between 123 to 735 bp, in comparison to amplicons obtained by Jersek *et al*., (1998) with sizes ranging from 298 to 6100 bp. In this study amplicons obtained ranged from 200bp to 3000 bp. Though the same primers were used, the reason of such differences could be the variation in the DNA polymerase used,

In this study, REP-PCR was used as a tool to characterize *L. monocytogenes* strains isolated from food. This method showed great possibilities for the typing of *L. monocytogenes,* as

**5. Discussion** 

with human listeriosis (Zhang *et al.,* 2007).

were not based on genes encoding serotype-specific antigens.

PCR machines and protocols used in this study.

The findings clearly highlight the possible occurrence of *L. monocytogenes* 1/2b and 4b among foods served by retailers and street vendors in Gaborone. The presence of this human pathogen in ready-to-eat foods should be considered as having significant public health implications, particularly among the immuno-compromised and HIV/AIDS persons who are at greater risk. In order to solve the problems in this study, there is a need for close co-operation between the products suppliers, supermarket management, workers, especially cleaning agents, the staff of cleaning companies, and hygiene specialists from the food industry and health inspectors from the Ministry of health. Street vendors need to be educated about ill health imposed by consumption of food contaminated by *L. monocytogenes,* on the other hand retailers need to be trained and be vigilant of retail assistants on the importance of maintaining the cold chain in order to prevent food borne disease outbreaks. Considering the occurrence of power disruptions that occur now and then and the high ambient temperatures experienced, Supermarket managers should be encouraged to invest in stand-by generators to serve during periods when power cuts occur.

#### **6. Acknowledgements**

The authors wish to convey sincere gratitude and thanks to the University of Botswana, University of South Africa, Walter Sisulu University and National Research Foundation (NRF) South Africa, for providing financial assistance for this research work. We are indebted to Mr Daniel Loeto for the great technical assistance provided during this work. Special thanks go to Dr M. Ditlhogo, Ex-Head of Biological Sciences Department and all the microbiology staff for their unique roles. Finally, appreciation is extended to supermarket managers and street vendors for affording us the opportunity to collect the various samples.

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### *Edited by Jose C. Jimenez-Lopez*

Biochemical testing necessitates the determination of different parameters, and the identification of the main biological chemical compounds, by using molecular and biochemical tools. The purpose of this book is to introduce a variety of methods and tools to isolate and identify unknown bacteria through biochemical and molecular differences, based on characteristic gene sequences. Furthermore, molecular tools involving DNA sequencing, and biochemical tools based in enzymatic reactions and proteins reactivity, will serve to identify genetically modified organisms in agriculture, as well as for food preservation and healthcare, and improvement through natural products utilization, vaccination and prophylactic treatments, and drugs testing in medical trials.

Biochemical Testing

Biochemical Testing

*Edited by Jose C. Jimenez-Lopez*

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