**4. Detection methods**

188 Salmonella – A Dangerous Foodborne Pathogen

and its adaptation to the host, *Salmonellae* may cause severe disease, or go unnoticed and remain in the host for months or years. In this case, the host will be a reservoir of the

The most common symptoms include diarrhea, abdominal pain, vomit and nausea, and may occur together with prostration, muscle pain, drowsiness and fever. Although symptoms generally disappear after 5 days, the microorganisms may be excreted in the feces for many weeks (Jay, 1992). Children, mainly those younger than 1 year of age, elderly and immunocompromised patients are much more susceptible to the disease, and may present more severe infections, such as sepsis, which may lead to death (Gomez & Cleary, 1998;

Salmonellosis is not limited to intestinal infection and gastroenterocolitis. The microorganism may infect other organs; as *Salmonellae* are able to reach the circulation, they may cause diffuse extraintestinal infections, such as meningitis, osteomyelitis, arthritis, pneumonia, cholecystitis, peritonitis, pyelonephritis, cystitis, endocarditis, pericarditis,

*Salmonellae* cross the intestinal epithelium, and reach the *lamina propria* (the layer where epithelial cells are anchored), where they multiply. They are phagocyted by macrophages and monocytes, causing an inflammatory response as a consequence of the hyperactivity of the reticuloendothelial system. Different from what happens in typhoid fever, penetration of *Salmonella* spp. is limited to the *lamina propria* in cases of enterocolitis. In these cases, sepsis or systemic infection are rarely observed, and infection is restricted to the intestinal mucous membrane. Inflammatory response is also related to the release of prostraglandins, which stimulate adenylate cyclase, leading to increased secretion of water and electrolytes and

From 1980 on, human outbreaks caused by *Salmonella* Enteritidis, showed common sources in the US, Great Britain and other European countries (CDC, 2005). Epidemiological surveys from the CDC identified the consumption of eggs or egg-based foods as responsible for most of the outbreaks involving specific phagotypes (PT) of *Salmonella* Enteritidis; PT-4 in European countries, and PT-8 and PT-13a in the US (Perales & Audicana, 1989). The predominant serotypes involved in foodborne diseases changed, in the past decades, from *Salmonella A*gona, *Salmonella* Hadar and *Salmonella* Typhimurium to *Salmonella* Enteritidis, which is the predominant cause of salmonellosis in several countries (Suresh et al., 2006). Changes in the predominance of serotypes reflect changes in animal raising practices and dissemination of new serotypes due to increased international trade. Nowadays, the main concern is the emergence of *Salmonella* serotypes

Cases of disease caused by four serovars of subspecies *enterica* are subject to mandatory reporting, according to regulation number 207 of the Brazilian Agency of Agricultural Defense [SDA; *Secretaria da Defesa Agropecuária*] , reviewed in July 30th, 1995. These serovars are part of the list B of the World Organization for Animal Health (OIE), of diseases that cause regional economic losses. Among them, *Salmonella* Pullorum, Gallinarum, Typhimurium and Enteritidis. About 90 serovars of *Salmonella* spp. are more frequent in

The typification of serovars is important to track the source of infection. For example, *Salmonella* Agona affected humans in the US, in European countries and in Brazil (Synnott et al., 1998; CDC, 2007). According to Clark et al. (1973), human outbreaks in the US and Europe that occurred around 1970 were caused by poultry meat. Animals were infected by

bacteria for susceptible animals.

vasculitis and other disorders (Gelli, 1995).

aqueous diarrhea (Franco & Landgraf, 2004).

that are resistant to multiple antibiotics (Huang, 1999).

cases of human and animal infection (Berchieri Jr. & Freitas Neto, 2009).

Pinto et al., 2004)

*Salmonellae* are short Gram-negative bacilli, about 0.7-1.5 x 2-5 μm, readily stained, and nonsporulating. Most of them move using peritrichial flagella, although serotypes such as *Salmonella* Pullorum and *Salmonella* Gallinarum are nonmotile. They are either aerobic or facultative anaerobic, and grow between 5 and 45°C. Optimum growth occurs at 37°C. Ideal pH for multiplication is 7, but *Salmonella* survives in pH values between 4 and 9. They grow in culture medium for enterobacteria and in blood agar. Colonies are 2-4 mm in diameter, with smooth and round edges. They are slightly raised in medium containing carbon and nitrogen. Colonies may remain viable for a long time when stored in peptone (Holt et al., 1994; Gast, 1997).

Biochemically, *Salmonella* strains have the ability to metabolize nutrients, and catabolize Dglucose and other carbohydrates, except lactose and sucrose, with production of acid and gas. They are catalase positive and oxidase negative, as are all genera in the *Enterobacteriaceae* family. They do not ferment malonate, do not hydrolyze urea, do not produce indol, use citrate as a sole source of carbon, reduce nitrate to nitrite, and may produce hydrogen sulfide (Quinn et al., 2000).

Conventional culture methods for isolating *Salmonella* spp. in poultry or animal feed or in feed ingredients have been reported in a number of studies, which were summarized by Williams (1981). Although all methods follow the basic strategy of preenrichment followed by selective enrichment, differential plating and biochemical or serological confirmation, there is no single internationally accepted procedure for *Salmonella* spp. detection.

The Food and Drug Administration (FDA), for example, recommends lactose broth for preenrichment (Andrews el al*.* 1998), while Wyatt et al*.* (1993) used buffered peptone water. Cox et al. (1982) reported that preenrichment decreased the recovery of *Salmonella* spp. from artificially contaminated poultry feed when compared with direct enrichment. Suggested protocols also vary with the substrate: Kafel (l981) suggested the use of anaerobic lactose broth, followed by selection in tetrathionate brilliant green broth and plating on brilliant

Important Aspects of Salmonella in the Poultry Industry and in Public Health 191

factors have little or no discriminatory value, and are normally associated because they represent a complex, such as O:12 (121, 122, 123), with O:2, O:4 and O:9. For example, *Salmonella* Paratyphi A (O:1,2,12), *Salmonella* Typhimurium (O:1,4,5,12) and *Salmonella*

Some antigens appear as a consequence of a change in the structure, such as O:1, which is a result of the insertion of galactose in the polysaccharide; O:5 a results of the acetylation of abequose, found in the repetitive units of the polysaccharide responsible for specificity, such

As for the characterization of flagellar antigens, it should be taken into account the fact that some *Salmonella* serovars have only one flagellar phase. They are called monophasic: *Salmonella* Enteritidis (9,12: g,m:-), *Salmonella* Typhi (9,12 [Vi]:d:-); however, most serogroups show two flagellar phases, that is, they are diphasic strains, such as *Salmonella* Typhimurium (1,4,5,12: i: 1,2) and *Salmonella* Hadar (6,8: z10: e,n,x), which express phase 1 (antigens i or z10) and phase 2 antigens (respectively, antigens 1,2 or e,n,x). Nonmotile strains, which have

Microbial resistance is related to strains of microorganisms that are able to multiply in the presence of concentrations of antimicrobial compounds even higher than those given as therapeutic doses to humans. Development of resistance is a natural phenomenon that followed the introduction of antimicrobial agents in clinical practice. The irrational and widespread use of these agents has added to the problem, and resistance rates vary from

One of the major concerns of the poultry industry is maintaining the sanitary status of the herds. In the incubators where birds are born, there is an attempt to reduce contamination to minimum levels in all phases of the process. Lack of contact with natural biota soon after birth interferes with the normal development of bird intestines (Silva, 2000). Generally, antimicrobial substances (antibiotic or chemotherapic agents), called growth promoters, are used in the feed from the first day of life to the moment of slaughter of the birds, respecting the recommended withdrawal period (Mota, 1996). These growth promoters improve performance because they "modulate" intestinal microbiota and improve feed efficiency. Suppliers of growth promoters guarantee that these substances are not absorbed through the intestinal walls and are shed in feces, where they are quickly biodegraded. Thus, they do not leave residues in the animal, and do not pose risks to human health or the environment (Mota, 1996). However, consumers are constantly concerned on the possible risks that

In veterinary medicine, antimicrobial agents are used in therapy, metaphylaxis, prophylaxis, and as growth promoters (Scharwz et al., 2001). The use of subtherapeutic doses of antibiotics as growth promoters is a public health problem, because many resistant microorganisms may transfer resistance to microorganisms found in bird feces. This kind of use may be responsible for selective pressure that generates resistant bacteria, a current, worldwide-spread, public health problem, due to the risk of dissemination of pathogens and transfer of resistance genes, via food chain, to pathogenic and commensal microorganisms

Since antimicrobials started to be widely used by humans at the end of the 1940s, the emergence of resistant strains was observed in most bacterial species, and against all drugs available (Flemming, 2005). The use of antimicrobials, combined with improvements in

of humans, decreasing the treatment options for infections (Medeiros, 2011).

as in serovar *Salmonella* Typhimurium O:4,12 and O:1,4,5,12.

no flagella, have also been recognized (Rodrigues, 2011).

place to place, depending on the local use of antibiotics.

antimicrobial resistance poses to human health.

Enteritidis (O:1,9,12).

**5. Drug resistance** 

green agar, in the analysis of fish meal. Allen et al. (1991) reported that the sensitivity of Rappaport Vassiliadis medium depended on the substrate in the detection of *Salmonella* spp. in high moisture foods, compared with tetrathionate or selenite cystine broth. Eckner et al. (1992) added novobiocin to tetrathionate selective enrichment and increased the incubation temperature to 42ºC.

The conventional technique for the detection of the microorganism includes the following steps: preenrichment, selective enrichment, isolation and selection, biochemical characterization, serological characterization and final identification. This technique requires at least four days for a negative result and six to seven days for the identification and confirmation of positive samples (Soumet et al*.,* 1997). The presence of *Salmonella* has to be determined in at least 25g or mL of sample.

New methodologies, such as immunological tests, have been proposed as alternatives for direct detection of this pathogen. For example, ELISA (Enzyme-linked Immunosorbent Assay) was used by Loguercio et al. (2002). Immunoenzymatic technology may be combined with other rapid methods in order to decrease total assay time. Luk et al. (1997) combined a digoxigenin-based ELISA with the polymerase chain reaction (PCR) to detect amplified r*fb*S, a lipopolysaccharide gene of *Salmonella* spp.; in this case, preenrichment was no longer than 16 hours.

Other types of assays have also been used: techniques based on molecular biology, such as nucleic acid hybridization or PCR, which was used by Flôres et al. (2003); and tests based on metabolism measurements (impedance and radiometry) (Franco & Landgraf, 1996). Ribotyping is the most recent addition to the automated identification of bacteria.

The RiboPrinterTM Microbial Characterization System is based on the highly conserved nature of the rRNA operon. Ribotyping provides a reproducible method by which rRNA and polymorphic fragments can be compared with a database for identification of genus, species and strain (Grimont & Grimont, 1986). The system is almost completely automated, requiring only picking up the colonies, suspending them in buffer and submitting them to heat treatment in a special carrier. Once heated, the sample is placed in the device, which automatically lyses the bacteria, releasing DNA; digests it with restriction enzymes; transfers the sample to agarose gel; and separates restricted fragments by electrophoresis. DNA fragments separated by size are then transferred to a nylon membrane, which is hybridized with a chemically-labeled and treated DNA antibody/alkaline phosphatase conjugate.

Resulting stained bands are then photographed, and the image is stored in the computer database and compared with other images in it. The database for this system is less comprehensive than that of other automated systems, but it still adequate for *Salmonella*  spp. The system would, however, be invaluable in epidemiological studies related to (HACCP) incidents.

Serotyping is an important epidemiological tool that complements the identification of *Salmonella*, making it possible to determine the prevalence/emergence or to show trends of a given serovar in different geographical regions, as well as to identify outbreaks, and discover sources of infection and routes of transmission. Serotyping is based on the Kauffmann & White classification and involves the identification of somatic and flagellar antigens.

The somatic structure is identified based on the recognition of the serovars, which are represented by uppercase letters. For example, group A (O:2), group B (O:4); group C1 (O:6,7), group C2 (O:6,8,20), group D (O:9), group E1 (O:3,10), group E2 (O:3,15), group E4 (O:1,3,19), etc. Some factors identify the antigenic group, for example, O:4, O:9. Other factors have little or no discriminatory value, and are normally associated because they represent a complex, such as O:12 (121, 122, 123), with O:2, O:4 and O:9. For example, *Salmonella* Paratyphi A (O:1,2,12), *Salmonella* Typhimurium (O:1,4,5,12) and *Salmonella* Enteritidis (O:1,9,12).

Some antigens appear as a consequence of a change in the structure, such as O:1, which is a result of the insertion of galactose in the polysaccharide; O:5 a results of the acetylation of abequose, found in the repetitive units of the polysaccharide responsible for specificity, such as in serovar *Salmonella* Typhimurium O:4,12 and O:1,4,5,12.

As for the characterization of flagellar antigens, it should be taken into account the fact that some *Salmonella* serovars have only one flagellar phase. They are called monophasic: *Salmonella* Enteritidis (9,12: g,m:-), *Salmonella* Typhi (9,12 [Vi]:d:-); however, most serogroups show two flagellar phases, that is, they are diphasic strains, such as *Salmonella* Typhimurium (1,4,5,12: i: 1,2) and *Salmonella* Hadar (6,8: z10: e,n,x), which express phase 1 (antigens i or z10) and phase 2 antigens (respectively, antigens 1,2 or e,n,x). Nonmotile strains, which have no flagella, have also been recognized (Rodrigues, 2011).
