**2. General aspects**

*Salmonellae* are widely distributed in nature. The main reservoir of these bacteria is the intestinal tract of men and warm-and cold-blooded animals (Jakabi et al., 1999), except for fish, mollusks and crustaceans, which may get contaminated after being fished. Among warm-blooded animals, chickens, geese, turkeys and ducks are the most important reservoirs. Domestic animals, such as dogs, cats, turtles and birds may be carriers, and pose great risk, mainly to kids (Franco & Landgraf, 1996).

The natural habitat of *Salmonella* may be divided into three categories based on the specificity of the host and clinical pattern of the disease: highly adapted to men: *Salmonella* Typhi and *Salmonella* Paratyphi A, B and C, agents of typhoid fever; highly adapted to animals: *Salmonella* Dublin (bovines), *Salmonella* Choleraesuis and *Salmonella* Typhisuis (swine), *Salmonella* Pullorum and *Salmonella* Gallinarum (birds), responsible for animal paratyphoid. The third category includes most of the serovars that affect men and animals, called zoonotic *Salmonella*, responsible for worldwide-distributed foodborne diseases, and detected in most species of animals used for human consumption, wild and domestic animals (Gantois et al., 2009).

*Salmonellae* are short bacilli, 0.7-1.5 x 2.5 μm, Gram-negative, aerobic or facultative anaerobic, positive catalase, negative oxidase; they ferment sugars with gas production, produce H2S, are nonsporogenic, and are normally motile with peritricheal flagella, except for *Salmonella* Pullorum and *Salmonella* Gallinarum, which are nonmotile (Forshell & Wierup, 2006).

Important Aspects of Salmonella in the Poultry Industry and in Public Health 183

houtenae (73); *Salmonella* enterica subsp. indica (13); *Salmonella* bongori (23); the newly proposed species *Salmonella* subterranea was not recognized, and is considered a serovar

Serovars may be further subdivided into biotypes and phagotypes. Biotyping uses different sugar fermentation patterns and assimilation of amino acids among strains of the same serovar, whereas phagotyping is based on the difference in strain susceptibility to a series of

As for their antigenic profile, *Salmonella* has an antigen common to all species in the Enterobacteriaceae family, called Kunin antigen. The presence of this antigen is not routinely analyzed, once it is not a relevant criterion for the differentiation between genus

Some serovars produce a superficial polysaccharide, or capsular antigen, called "Vi". It is found outside the cell wall, and prevents detection of the somatic antigen. It is usually found in strains of *Salmonella* Typhi, *Salmonella* Paratyphi C and *Salmonella* Dublin. Vi antigens are thermolabile, and may be destroyed by heating at 100oC for 10-15 minutes. The somatic antigen, or "O" (Ohne), on the other hand, is specific. It is a lipopolysaccharide, and is resistant to heat and alcohol. It is made up of three parts: a lipid portion, responsible for toxicity and pyrogenic characteristics; a core portion; and the polysaccharide, which confers stability to smooth (S) variants. The "O" antigen is made up of repetitive chains with a definite spatial arrangement. The specificity of "O" antigen is given by this definite nature and the type of bond. The synthesis of this antigen is encoded by about 20 genes (locus rfb). Many somatic antigen factors (67) are recognized and used in the serological identification of *Salmonella*. Although these factors are intimately related, they are not always antigenically identical, and can only be characterized when strains are in the smooth phase. In this phase, colonies show homogenous, shiny surfaces, with regular borders, indicative of the complete "O" antigen. Mutations that affect the core portion of the antigen, or the synthesis of its chain, lead to loss of specificity. In this case, strains are called Rough (R), colonies have irregular borders and surfaces, and it is impossible to recover or recognize their original characteristics. They agglutinate in saline solution, are easily phagocyted, and are sensitive to the action of the complement system. Agglutination of bacterial cells (somatic or "O" antigen) using polyclonal (±7) and specific (65) antisera, which is the laboratory procedure for antigen confirmation, is slow and may form fine granules that are not dissociable by stirring. This occurs because the reaction is based on an interrelationship between the walls

Flagellar antigens, or "H" (Hauch) antigens, are made of a protein called flagellin. Antigenic differences are related to variations in the primary structure or amino acid content of different flagellin molecules. The "H" antigen is thermolabile, may be destroyed at 100oC for 10 minutes, and by slow action of alcohol 50%; but it is resistant to formaldehyde 0.5%. Agglutination of flagellar antigen forms large clumps that are quickly dissociated by stirring. Compared with somatic agglutination, it occurs faster due to the large number of

Spatial arrangement and intrinsic characteristics of the genus lead to the production of two different types of flagella. In a bacterial population of *Salmonella* spp. strains that produce two different types of flagella, the rate of cell variation among those that present one of the two types or phases is about 104. In most *Salmonella* isolates, two genes encode flagellar antigens: fliC (>50 different alleles), with highly conserved terminal sequences in the genus and which encodes phase 1 antigens; and fljB (±30 alleles), also conserved in the genus,

flagella in the cell, and because bacterial cells bind to each other.

bacteriophages (Ward et al., 1987; Grimont et al., 2000; Dunkley et al., 2009).

of the bongori species (Rodrigues, 2011).

and species.

of the bacterial cells.

Optimal pH for multiplication is around 7.0; pH values above 9.0 or below 4.0 are bactericidal. Ideal temperature is between 35 to 37°C, with minimum of 5°C and maximum of 47°C. As for salt concentration, *Salmonellae* do not survive concentrations over 9% (Franco & Landgraf, 1996).

The first bacteria in the genus *Salmonella* were identified towards the end of the 19th century. *Salmonella* Typhy, the first to be recognized as a pathogen, was found in spleen and lymph nodes of humans in 1880. However, isolation and morphological description were only carried out by Gaffky, in 1884.

In 1885, Salmon and Smith isolated a bacillus from diseased pigs, and called it *Bacterium* Suipestifer. They wrongly considered it the agent of swine fever. This bacterium was later on called *Salmonella C*holerasuis. In 1888, there was a report on *Salmonella* Enteritidis by Gaetner; in 1889, Klein identified fowl typhoid in adult birds in England, and in 1892, Loefer isolated *Salmonella* Typhimurium. In 1899, Rettger described pulorosis and differentiated it from the disease that affected pigs. In 1913, Jones used an agglutination test to identify carriers of *Salmonella* Pullorum (Correa & Correa, 1992).

The genus *Salmonella* started to be classified in 1925, with the use of serological methods. S*almonella* Typhimurium, created by Loeffler (1892), and S*almonella* Paratyphi, created by Schottimuller (1899), were included in the genus. Later on, several S*almonella* serotypes were described, and classified according to White (1829) (Correa & Correa, 1992). Popoff et al. (1996) presented a proposal for the reclassification of the genus *Salmonella*, which would have two species: *Salmonella enterica* and *Salmonella bongori.* 

In the current classification of the Bergey's manual, all *Salmonella* serotypes belong to one of two species: *Salmonella bongori*, which has at least 10 extremely rare serotypes; and *Salmonella enterica,* which is phenotypically and genotypically divided into six subspecies *enterica, salamae, arizonae, diarizonae, houtenae* and *indica*, differentiated by their biochemical behavior, mainly in terms of sugar and amino acid metabolism (Forshell & Wierup, 2006)*.* 

In the current nomenclature, the name of the serovar begins with an uppercase letter, but it is never written in italics. For example in subspecies enterica: *Salmonella enterica* subespecies *enterica* serovar Typhimurium. The short form would be *Salmonella* ser. Typhimurium or *Salmonella* Typhimurium. Other subspecies are designated by the name of the serovar, followed by its antigenic formula, explained below.

Typification of *Salmonella* spp. serovars is based on the antigens found in bacterial cells, somatic (O), flagellar (H) and capsular (Vi) (Selander et al., 1996). Vi antigen is associated with virulence, and is only expressed by serovars Typhi, Paratyphi C and Dublin (Rycroft, 2000; Grimont et al., 2000). H antigen is thermolabile, whereas O and Vi are thermoresistant, and not destroyed by heating at 100°C for two hours (Franco & Landgraf, 1996). The combination of the antigens O, H1 (flagellar, phase 1) and H2 (flagellar, phase 2) determine the antigenic formula of a serovar. O antigens receive Arabic numerals, whereas H1 antigens are identified by lowercase letters, and H2 antigens by Arabic numerals. For example, *Salmonella* enterica subsp salamae ser. 50: z : e,n,x, or *Salmonella* serotype II 50: z : e,n,x.

Somatic (O) and flagellar (H) antigens, determine different serovars in each subspecies, in a total of 2,610 serovars today, as recognized by Kauffman-White scheme (Grimont & Weill, 2007). Although all of them are considered to be potentially pathogenic to men, only 200 are more frequently related with human disease (Baird-Parker, 1990). Distribution according to species and subspecies is as follows: *Salmonella* enterica subsp. enterica (1,547 serovars); *Salmonella enterica* subsp salamae (513); *Salmonella enterica* subsp arizonae (100); *Salmonella enterica* subsp diarizonae (341); *Salmonella enterica* subsp

Optimal pH for multiplication is around 7.0; pH values above 9.0 or below 4.0 are bactericidal. Ideal temperature is between 35 to 37°C, with minimum of 5°C and maximum of 47°C. As for salt concentration, *Salmonellae* do not survive concentrations over 9% (Franco

The first bacteria in the genus *Salmonella* were identified towards the end of the 19th century. *Salmonella* Typhy, the first to be recognized as a pathogen, was found in spleen and lymph nodes of humans in 1880. However, isolation and morphological description were only

In 1885, Salmon and Smith isolated a bacillus from diseased pigs, and called it *Bacterium* Suipestifer. They wrongly considered it the agent of swine fever. This bacterium was later on called *Salmonella C*holerasuis. In 1888, there was a report on *Salmonella* Enteritidis by Gaetner; in 1889, Klein identified fowl typhoid in adult birds in England, and in 1892, Loefer isolated *Salmonella* Typhimurium. In 1899, Rettger described pulorosis and differentiated it from the disease that affected pigs. In 1913, Jones used an agglutination test to identify

The genus *Salmonella* started to be classified in 1925, with the use of serological methods. S*almonella* Typhimurium, created by Loeffler (1892), and S*almonella* Paratyphi, created by Schottimuller (1899), were included in the genus. Later on, several S*almonella* serotypes were described, and classified according to White (1829) (Correa & Correa, 1992). Popoff et al. (1996) presented a proposal for the reclassification of the genus *Salmonella*, which would

In the current classification of the Bergey's manual, all *Salmonella* serotypes belong to one of two species: *Salmonella bongori*, which has at least 10 extremely rare serotypes; and *Salmonella enterica,* which is phenotypically and genotypically divided into six subspecies *enterica, salamae, arizonae, diarizonae, houtenae* and *indica*, differentiated by their biochemical behavior, mainly in terms of sugar and amino acid metabolism (Forshell & Wierup, 2006)*.*  In the current nomenclature, the name of the serovar begins with an uppercase letter, but it is never written in italics. For example in subspecies enterica: *Salmonella enterica* subespecies *enterica* serovar Typhimurium. The short form would be *Salmonella* ser. Typhimurium or *Salmonella* Typhimurium. Other subspecies are designated by the name of the serovar,

Typification of *Salmonella* spp. serovars is based on the antigens found in bacterial cells, somatic (O), flagellar (H) and capsular (Vi) (Selander et al., 1996). Vi antigen is associated with virulence, and is only expressed by serovars Typhi, Paratyphi C and Dublin (Rycroft, 2000; Grimont et al., 2000). H antigen is thermolabile, whereas O and Vi are thermoresistant, and not destroyed by heating at 100°C for two hours (Franco & Landgraf, 1996). The combination of the antigens O, H1 (flagellar, phase 1) and H2 (flagellar, phase 2) determine the antigenic formula of a serovar. O antigens receive Arabic numerals, whereas H1 antigens are identified by lowercase letters, and H2 antigens by Arabic numerals. For example, *Salmonella* enterica

Somatic (O) and flagellar (H) antigens, determine different serovars in each subspecies, in a total of 2,610 serovars today, as recognized by Kauffman-White scheme (Grimont & Weill, 2007). Although all of them are considered to be potentially pathogenic to men, only 200 are more frequently related with human disease (Baird-Parker, 1990). Distribution according to species and subspecies is as follows: *Salmonella* enterica subsp. enterica (1,547 serovars); *Salmonella enterica* subsp salamae (513); *Salmonella enterica* subsp arizonae (100); *Salmonella enterica* subsp diarizonae (341); *Salmonella enterica* subsp

& Landgraf, 1996).

carried out by Gaffky, in 1884.

carriers of *Salmonella* Pullorum (Correa & Correa, 1992).

have two species: *Salmonella enterica* and *Salmonella bongori.* 

followed by its antigenic formula, explained below.

subsp salamae ser. 50: z : e,n,x, or *Salmonella* serotype II 50: z : e,n,x.

houtenae (73); *Salmonella* enterica subsp. indica (13); *Salmonella* bongori (23); the newly proposed species *Salmonella* subterranea was not recognized, and is considered a serovar of the bongori species (Rodrigues, 2011).

Serovars may be further subdivided into biotypes and phagotypes. Biotyping uses different sugar fermentation patterns and assimilation of amino acids among strains of the same serovar, whereas phagotyping is based on the difference in strain susceptibility to a series of bacteriophages (Ward et al., 1987; Grimont et al., 2000; Dunkley et al., 2009).

As for their antigenic profile, *Salmonella* has an antigen common to all species in the Enterobacteriaceae family, called Kunin antigen. The presence of this antigen is not routinely analyzed, once it is not a relevant criterion for the differentiation between genus and species.

Some serovars produce a superficial polysaccharide, or capsular antigen, called "Vi". It is found outside the cell wall, and prevents detection of the somatic antigen. It is usually found in strains of *Salmonella* Typhi, *Salmonella* Paratyphi C and *Salmonella* Dublin. Vi antigens are thermolabile, and may be destroyed by heating at 100oC for 10-15 minutes.

The somatic antigen, or "O" (Ohne), on the other hand, is specific. It is a lipopolysaccharide, and is resistant to heat and alcohol. It is made up of three parts: a lipid portion, responsible for toxicity and pyrogenic characteristics; a core portion; and the polysaccharide, which confers stability to smooth (S) variants. The "O" antigen is made up of repetitive chains with a definite spatial arrangement. The specificity of "O" antigen is given by this definite nature and the type of bond. The synthesis of this antigen is encoded by about 20 genes (locus rfb).

Many somatic antigen factors (67) are recognized and used in the serological identification of *Salmonella*. Although these factors are intimately related, they are not always antigenically identical, and can only be characterized when strains are in the smooth phase. In this phase, colonies show homogenous, shiny surfaces, with regular borders, indicative of the complete "O" antigen. Mutations that affect the core portion of the antigen, or the synthesis of its chain, lead to loss of specificity. In this case, strains are called Rough (R), colonies have irregular borders and surfaces, and it is impossible to recover or recognize their original characteristics. They agglutinate in saline solution, are easily phagocyted, and are sensitive to the action of the complement system. Agglutination of bacterial cells (somatic or "O" antigen) using polyclonal (±7) and specific (65) antisera, which is the laboratory procedure for antigen confirmation, is slow and may form fine granules that are not dissociable by stirring. This occurs because the reaction is based on an interrelationship between the walls of the bacterial cells.

Flagellar antigens, or "H" (Hauch) antigens, are made of a protein called flagellin. Antigenic differences are related to variations in the primary structure or amino acid content of different flagellin molecules. The "H" antigen is thermolabile, may be destroyed at 100oC for 10 minutes, and by slow action of alcohol 50%; but it is resistant to formaldehyde 0.5%. Agglutination of flagellar antigen forms large clumps that are quickly dissociated by stirring. Compared with somatic agglutination, it occurs faster due to the large number of flagella in the cell, and because bacterial cells bind to each other.

Spatial arrangement and intrinsic characteristics of the genus lead to the production of two different types of flagella. In a bacterial population of *Salmonella* spp. strains that produce two different types of flagella, the rate of cell variation among those that present one of the two types or phases is about 104. In most *Salmonella* isolates, two genes encode flagellar antigens: fliC (>50 different alleles), with highly conserved terminal sequences in the genus and which encodes phase 1 antigens; and fljB (±30 alleles), also conserved in the genus,

Important Aspects of Salmonella in the Poultry Industry and in Public Health 185

According to the World Health Organization (WHO), *Salmonella* is the bacterial agent most frequently involved in cases of foodborne disease all over the world. The agent is normally transmitted to humans by means of foods of animal origin, such as meat, eggs and milk (Nascimento et al., 2003). In the past, the main motivations for controlling *Salmonella* spp. infections in poultry were the losses caused by clinical (pullorum disease and fowl typhoid) and subclinical diseases (paratyphoid infections) (Calnek, 1997). Nowadays, due to the public health implications, prevention of foodborne transmission of *Salmonella* spp. is a

Historically, *Salmonella* Typhimurium was the most common agent of the foodborne disease in humans, although in the past decades *Salmonella* Enteritidis has been most frequently involved in salmonellosis outbreaks (Berchieri Jr. & Freitas Neto 2009; Kottwitz, et al., 2010). There is a growing concern about human infections caused by other serovars, such as

Concerns about the presence of *Salmonella* spp. in foodstuffs of poultry origin increased in the 1980s, when *Salmonella* Enteritidis phagotype 4 was responsible for several outbreaks of foodborne disease in England, caused by the ingestion of foods containing poultry ingredients (Colin, 1996; Baxter-Jones, 1996). The vertical transmission of *Salmonella* Enteritidis in commercial poultry was responsible for the increased number of cases of human infection in Europe, North America and other parts of the world (Humphrey et al., 1988; International Commission for Microbiological Safety of Foods (ICMSF), 1998). These species replaced *Salmonella* Typhimurium, which was the most common agent of human

The introduction of *Salmonella* Enteritidis in Brazil probably occurred in the end of the 1980s, by means of breeders acquired from European countries. This may have also facilitated the introduction and dissemination of phatogotype PT-4 beginning in 1993 (Irino et al., 1996),

In the 1990s, there were several reports of foodborne disease outbreaks in humans mainly caused by the ingestion of poultry products (Taunay et al., 1996). Between 1995 and 2011, there were 406 reported outbreaks and 16,304 cases of salmonellosis in Brazil, Chile,

According to the National Health Surveillance Agency in Brazil [ANVISA; *Agência Nacional de Vigilância Sanitária*], among the etiological agents of foodborne diseases identified between 1999 and 2004, *Salmonella* spp. was the most prevalent in Brazil, with the predominance of *Salmonella* Enteritidis between 2001 and august 2005 (Rodrigues, 2005). According to the WHO, *Salmonella* is one of the pathogens that causes the greatest impact on population health, and is associated with outbreaks and with sporadic cases of foodborne disease. According to data of the Brazilian Ministry of Health, 6,602 foodborne disease outbreaks were recorded between 1999 and 2008, and *Salmonella* spp. was associated with

In the European Union, *Salmonella* Enteritidis, *Salmonella* Typhimurium, *Salmonella* Infantis, *Salmonella* Hadar and *Salmonella* Virchow are considered by the European Food Safety Authority the most important serovars in terms of public health (EFSA, 2007). In Japan, between 1999 and 2002, 32% of the cases of foodborne infection were due to *Salmonella*, with Enteritidis, Typhimurium and Infantis as the predominant serovars. In 2005, in the US, the serovars that were most frequently isolated from human sources were *Salmonella* Typhimurium, *Salmonella* Enteritidis, *Salmonella* Newport, *Salmonella* Heidelberg and

Infantis, Agona, Hadar, Heidelberg and Virchow (Freitas Neto et al., 2010).

foodborne infection until the 1980s (Olsen et al., 2003; Jay, 2000).

the predominant phagotype in Europe at this time (Wall & Ward, 1999).

Argentina, Peru, Uruguay, Paraguay and Ecuador (Franco et al., 2003).

43% of the cases in which the etiological agent was identified (Medeiros, 2011).

*Salmonella* Javiana (Centers For Diseases Control and Prevention - CDC, 2007).

priority for the poultry sector (Oliveira & Silva, 2000).

which encodes phase 2 antigens. These genes are expressed by a phase-variation mechanism, with fliC being found in all *Salmonellae*, and having a homologous gene found in *E*. *coli;* whereas fljB is located in a region exclusive to the *Salmonella* genome, and is found in four of the six subspecies. In some cases, triphasic strains may be isolated. Besides the other two genes, it was described that these strains presented the flagellin gene (flpA) in a plasmid. The genes that encode flagellin in *Salmonella* spp. are generally highly conserved in extremities 5' and 3', whereas the central region is highly variable.

In practical conditions, rapid agglutination with polyclonal antisera (12 polivalent and 85 monovalent antisera) may frequently occur in the absence of expression of one of the phases, preventing the identification of the serovar. This may happen in some serovars, when cell subpopulations, each possessing a given antigen or set of antigens associated with their flagella, are able to produce a third or fourth type of flagellum. Identification, in these cases, requires "immobilization" of one of the phases, in order to characterize the unknown phase, a technique called "phase inversion". When the phase is not recognized, the serovar will not be conclusively diagnosed, preventing effective control actions. However, considering the complexity of flagellar antigens, if not all monovalent antisera are used, results on antigenic structure may be incorrect, such as g,m; g,t; g,p; g,q; g,p,s; g,z61; m,t.
