Salmonella infection in poultry

Magazine da SPM 214 (3) 8.31c Salmonella infection in poultry Lurdes Clemente*, Ivone Correia, Patrícia Themudo Instituto Nacional de Investigação Agrária e Veterinária, Unidade Estratégica de Investigação, Produção e Saúde Animal Laboratório de Bacteriologia e Micologia, Estrada de Benfica, 71, 1549-11, Lisboa *correspondência: lurdes.clemente@iniav.pt Salmonella are pathogenic gram-negative bacilli of the Enterobacteriaceae family, being the second most frequent cause of human gastrointestinal infection in the European Union. The great majority of infections are associated with the consumption of products such as: meat mainly from poultry, eggs, milk, seafood, fruits and vegetables contaminated with Salmonella. Outline Salmonella are a member of the Enterobacteriaceae family, containing over 25 serotypes. Based on phylogenetic studies by 16SrRNA sequence analysis, two species were separated: Salmonella enterica and Salmonella bongori (Grimond and Weill, 27). Most subspecies of S. enterica are only commonly isolated from reptiles and are not associated with disease, whereas subspecies S. enterica subsp. enterica has far more serotypes, is generally isolated from mammals and birds and is important to veterinary and human medicine (Barrow et al., 212). Some serotypes such as Typhi in humans, Pullorum and Gallinarum in poultry, Typhissuis in pigs and Abortusequi in equine are relatively host-specific, whereas Typhimurium has a wide host range. The habitat of member of the genus Salmonella seems to be limited to the digestive tract of humans and animals (ileum, cecum, colon and mesenteric lymph nodes), where macrophages form a barrier to further invasion (Quinn et al., 22). If macrophages are not able to limit the infection, it may spread causing a septicaemia, like typhoid fever, by serovars: Typhi in humans, Gallinarum in poultry, Typhissuis in swine and Typhimurium in mice (Baumler et al., 2). Its presence in other habitats like water, food and natural environment is explained by faecal contamination (Quinn et al., 22). Public Health Importance Although Salmonella may colonize the intestine without causing disease, infections with Salmonella enterica are one of the most important causes of human food-borne diseases worldwide and the second most frequent cause of gastrointestinal infection in the European Union (EU), with a total of 9134 confirmed cases in 212 (EFSA, 214). A statistically significant decreasing trend in the European Union was observed over the period 28-212 (EFSA, 214). Human infections usually result in a selflimiting gastroenteritis; however, young children, elderly and immune-compromised people may experience enteric fever or, an invasive form of the disease requiring antimicrobial treatment (Pui et al., 211). The great majority of infections are associated with the consumption of products such as: meat mainly from poultry, eggs, milk, seafood, fruits and vegetables (Pui et al., 211). The continued increasing of poultry products per capita consumption also potentiates human exposure to Salmonella via the food chain (http://www.thepoultrysite.com). Salmonella Enteritidis and Salmonella Typhimurium were the most frequently reported serotypes in the EU, accounting for 41.3% and 22.1% of all human isolates, respectively (EFSA, 214). In Poultry In poultry, several factors may affect its susceptibility to intestinal colonization, such as: host age, genetic background, intrinsic 1

Magazine da SPM 214 (3) 8.31c serotype competences, initial dose level, environmental stress, antimicrobial or antiinflammatory treatments and competition with the enteric microbiota (Foley et al., 28). Salmonella can be introduced at all stages of the production cycle, though breeding flocks and hatcheries, which are critical sources and responsible for the quick spread of the infection (Foley et al., 28). Infections caused by serotypes Enteritidis and Typhimurium are rarely responsible for clinical severe forms of illness and animals frequently become asymptomatic carriers, excepting young chicks and poults, where acute outbreaks exhibiting clinical disease accompanied by high mortality rates may occur (Padron, 199; Foley et al., 28). These young birds can be infected by vertical transmission through infected parent stock, or by horizontal transmission in hatcheries, in sexing, transport equipment and in poultry farms through bedding material, unclean facilities and vectors, such as humans, birds, insects and rodents (Foley et al., 28). Antimicrobial resistance Besides causing illnesses or death in both humans and poultry, there is a worldwide concern on the persistence of Salmonella serotypes that are resistant or show decreased susceptibility to several antimicrobials, reducing therapeutic options and, more importantly, leading to treatment failure (Newell et al., 21). Fluoroquinolones such as ciprofloxacin and third generation cephalosporins are important antimicrobials in human and veterinary medicine. In animal isolates, the highest occurrence of decreased susceptibility to quinolones has been recorded in Salmonella spp. recovered from live poultry (Gallus gallus) and broiler meat (EFSA, 213). Although the frequency of nonwild type Salmonella isolates to third generation cephalosporins is very low in European countries, this is an issue is of great concern, because the spread of MDR isolates producing extended-spectrum β- lactamases (ESBL) or plasmid-mediated AmpC β-lactamases (PMAβ) may result from co-selection with other resistance determinants, through the use of other antimicrobials (EFSA, 213). The inappropriate use of antimicrobials in veterinary medicine or in feed additives has been linked to the emergence and spread of Salmonella resistant strains with potentially serious effects in food safety (Hao- Van et al., 212). Table 1. Most frequent Salmonella serotypes isolated from breeders, broilers and layers in Portugal (29-211) Serotypes Breeders (n=58) Broilers (n=275) Layers (n=285) S. Enteritidis 36 71 96 S. Typhimurium 11 8 S. Virchow 1 6 1 S. Infantis 1 3 S. Havana 8 77 28 S. Mbandaka 1 66 35 S. I 4,[5],12:i:- 7 3 S. Virchow 1 6 1 S. Tennessee 4 1 9 I 1,3,19:-:-:Rz27. 1 13 Other 6 29 7 Laboratorial Methods National surveillance control programmes in food-producing animals and food of animal origin were established in the EU countries, according to the guidelines of the Commission Decision 27/47/CE. In poultry, all the production sectors (breeders, layers and broilers) are sampled regularly. During the period from 29 to 211, faecal and environmental samples were collected using sterile boots/sock swabs and isolates recovered and identified according to ISO norm 6579: 22 (Anonymous 22). Serotyping was based on the Kauffmann- White scheme (Grimond and Weill, 27) and 2

Magazine da SPM 214 (3) 8.31c antimicrobial susceptibility was tested through the determination of Minimum Inhibitory Concentrations (MICs) using the agar dilution method (CLSI, 213); interpretation of the results was done according with epidemiological cut-off (http://mic.eucast.org) and clinical breakpoints (http://www.eucast.org/ clinicalbreakpoints), established by EUCAST. Epidemiological data - Portugal Salmonella Serovars Among the 618 Salmonella spp. isolates (Table 1) recovered from live poultry (breeders, broilers and layers), three main serotypes were identified: S. Enteritidis (32.8%, 23/618), S. Havana (18.3%, 113/618) and Salmonella Mbandaka (16.5%, 12/618). Salmonella serotypes is influenced by changes in animal husbandry or other practices, such as: surveillance programs, depopulation of infected flocks, bio security measures and vaccination of layer and breeder flocks (Barrow et al., 212). S. Enteritidis, S. Typhimurium, S. Havana and S. Mbandaka are all considered zoonotic or potentially pathogenic serovars for humans (Schiff and Saphra, 1941; Menon et al., 1994; Scheil et al.,1998; Backer et al., 2; Boisrame-Gastrin, et al., 211, EFSA, 214), and were among the most prevalent serotypes recovered from Portuguese broiler flocks. It is likely that, at least for serotypes Havana and Mbandaka, poultry feed containing cereal grain imported from non- European countries may be one of the main sources for these serotypes in live birds; 36% of poultry feed samples received, were contaminated with one of these serotypes (data not shown). Table 2. Antimicrobial susceptibility of Salmonella isolates (n=618) in Portugal (29-211) Serotypes Breeders (n=58) Broilers (n=275) Layers (n=285) Ampicillin % DS 1.7 8 3.2 Cefotaxime % DS % R Nalidixic acid % DS 51.7 54.2 2.7 Ciprofloxacin % DS % R 53.4 Chloramphenicol % DS 1.1 Florfenicol % DS 1.1 Gentamicin % DS 1.8 1.4 Streptomycin % DS 5.2 5.8 4.9 Sulfamethoxazole % DS 18.5 1.4 Tetracycline % DS 13.5 4.6 Trimethoprim % DS 3.4 12.7.4 Decreased susceptibility (DS); Resistance (R) EUCAST epidemiological breakpoints Ampicillin, >8mg/L; Cefotaxime, >.5mg/L; Nalidixic acid, >16mg/L; Ciprofloxacin, >.6mg/L; Chloramphenicol, >16mg/L; Florfenicol, >16mg/L; Streptomycin, >16mg/L; Gentamicin, >2mg/L; Sulphamethoxazole, >256mg/L; Tetracycline, >8mg/L; Trimethoprim, >2mg/L EUCAST, clinical breakpoints Cefotaxime, >2mg/L; Ciprofloxacin, >1mg/L.7.7 6.7 22.1.7 3

Magazine da SPM 214 (3) 8.31c Table 3. Antimicrobial susceptibility among the most common serotypes of Salmonella spp in Portugal (29-211) S. enteritidis S. havana S. mdanbaka S. thyphimurium Breed. Broil. Lay. Breed. Broil. Lay. Broil. Lay. Broil. Lay. AMP 5.6 2.8 4.2 11.7 2.9 36.4 25 CTX 2.6 NAL 83.3 95.8 41.7 15.6 7.1 92.4 2.9 9.1 12.5 CIP 86.1 95.8 44.8 35.1 1.7 97 9.1 12.5 CHL 1.3 18.2 FL 1.3 1.5 9.1 GEN 1 1.3 1.5 STR 1.4 4.2 6.5 3.6 1.5 27.3 25 SMX 5.6 31 2 16.9 4.5 45.5 25 TE 5.6 32.4 4.2 1.3 1.5 8.6 36.4 25 TRIM 5.6 28.2 1 1.3 4.5 9.1 Decreased susceptibility of AMP, Ampicillin; CTX, Cefotaxime; NAL, Nalidixic acid; CIP, Ciprofloxacin; CHL, Cloramphenicol; FL, Florfenicol; GEN, Gentamicin; STR, Streptomycine; SMX, Sulphamethoxazole; TE, Tetracycline; TRIM, Trimethoprim. Antimicrobial susceptibility phenotype Susceptibility profiles are shown in Tables 2 and 3 and differ according to the serovars and the origin of the isolates, as reported in other studies (Newell et al., 21; EFSA, 212; Clemente et al., 213; Clemente et al., 214). Although resistance to ciprofloxacin was absent or at very low prevalence, the percentage of poultry isolates with decreased susceptibility to quinolones was very consistent mainly in broilers, in contrast to data obtained from laying hens. The decreasing susceptibility to quinolones might be an indicator of emerging resistance (de Jong et al., 29). Although the frequency of resistance to cefotaxime was very low and comparable to most European countries (EFSA, 212), two isolates of S. havana, recovered from broilers, harbored the bla CTX-M-1 gene (Clemente et al., 213), an extended-spectrum β-lactamase (ESBL) encoding gene, which might be transmissible to humans through contact or the food chain. Future perspectives Presently, the Portuguese reference laboratory (INIAV/IP) is responsible for the application of the antimicrobial susceptibility surveillance programmes of zoonotic bacteria, such as, Salmonella and Campylobacter. From 214 onwards and, according to CD 652/213, Monitoring and Reporting of Antimicrobial Resistance in Zoonotic and Commensal Bacteria surveillance, will be extended to other animal species (bovine and swine) and organisms (commensal Escherichia coli, Campylobacter spp., Enterococcus spp and Escherichia coli extended β-lactamases and carbanemase producers), will be monitored. Monitoring antimicrobial susceptibility, alongside the use of epidemiological cut-off values to interpret final results, seems to be the most sensitive way to detect changes in resistance patterns in different animal populations. Alongside, it is intended to continue the collaboration with the National Reference Laboratory of Antimicrobial Resistance of the National Institute of Human Health. Acknowledgements The authors would like to thank Professor Fernando Bernardo from the Faculty of Veterinary Science, Technical University of Lisbon, for the revision of the manuscript. We are grateful to all staff involved in the Salmonella National Control Programme. References Anonymous, 22. International Organization for Standardization (ISO) 6579:22. Microbiology of food and animal feeding stuffs - Horizontal method for the detection of Salmonella spp. Backer, H., Mohle, B., Janet, C., Benson W., Abbott, S., Farrar, J., Vujia, D., 2. High incidence of extra-intestinal infections in a Salmonella Havana outbreak associated with alfalfa sprouts. Public Health Rep. 11: 339-345. Boisrame-Gastrin, S., Tande, D., Munck, Marie-Reine, Gouriou, S., Nordmann, P. Naas, T. 211. Salmonella carriage in adopted children from Mali: 21 8. J. Antimicrob. Chemother. 66: 2271 2276. Baumler, J., Tsolis, R. and Heffron, F. 2. Virulence Mechanisms of Salmonella and their Genetic Basis. In Salmonella in Domestic Animals, CAIB Publishing, Chapter IV, pg 57-72. 4

Magazine da SPM 214 (3) 8.31c Barrow, P.A., Jones, M.A., Smith, A.L. and Wigley, P. 212. The long view: Salmonella the last forty years. Avian Pathol. 41: 413-42. Clemente, L., Manageiro, V., Ferreira, E., Jones-Dias, D., Correia, I., Themudo, P., Albuquerque, T., Caniça, M. 213. Occurrence of extended-spectrum β-lactamases among isolates of Salmonella enterica subsp. enterica from food-producing animals and food products, in Portugal. International J. Food Microbiol. 167: 221-228. Clemente, L., Correia, I., Themudo, P., Neto, I., Caniça, M. and Bernardo F. 214. Antimicrobial susceptibility of Salmonella enterica isolates from healthy breeder and broiler flocks in Portugal. Vet. J. 2: 276-281 Clinical Laboratory and Standards Institute, 213. Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals; Approved Standard-Third Edition. CLSI document VET1-A 4. Commission Decision (27/47/CE) of 12 June 27 on a harmonised monitoring of antimicrobial resistance in Salmonella in poultry and pigs (notified under document number C 27 2421). de Jong, A., Bywater, R., Butty, P., Derrover, E., Godinho, K., Klein, U., Marion, H., Simjee, S., Smets, K., Thomas, V., Vallé, M., Wheadon, A. 29. A pan-european survey of antimicrobial susceptibility towards human-use antimicrobial drugs among zoonotic and commensal enteric bacteria isolated from healthy food-producing animals. J. Antimicrob. Chemother. 63: 733-744. EFSA-European Food Safety Authority. 213. The European Union Summary Report on antimicrobial resistance in zoonotic and indicator bacteria from humans, animals and food in 211. EFSA Journal 11: 3196. EFSA- European Food Safety Authority. 214. The European Union Summary Report on Trends and Sources of Zoonoses, Zoonotic Agents and Food-borne Outbreaks in 212. European Food Safety Authority European Centre for Disease Prevention and Control. EFSA Journal, 12: 3547. Foley, S.L., Lynne, A.M., Nayak, R. 28. Salmonella challenges: Prevalence in swine and poultry and potential pathogenicity of such isolates. J. Anim Sci. 86: E149-E162. Grimont, P.A., Weill, F.X. 27. Antigenic formulae of the Salmonella serovars (9th edition). WHO Collaborating Centre for Reference and Research on Salmonella. Institute Pasteur, Paris, France. Hao-Van, T., Kha Nguyen, H.N., Smooker, P., Coloe, P. 212. The antibiotic resistance characteristics of non-typhoidal Salmonella enterica isolated from food-producing animals, retail meat and humans in South East Asia. Int. J. Food Microbiol. 154: 98-16. Hoeltzer, K. 211. Animal contact as a source of human non-typhoidal salmonellosis. Vet. Res. 42: 34-61. Menon, P.K., Raguraman, T.S., Chaudhury, M. Kudesia, S., Rao, D.V. 1994. Neonatal Salmonella Havana meningitis. Indian J. Pathol. and Microbiol. 34: 435-438. Newell, D.G., Koopmans, M., Verhoef, L., Duizer, E., Aidara-Kane, A., Sprong, H., Opsteegh, M., Langelaar, M., Threfall, J., Scheutz, F. 21. Food-borne diseases The challenges of 2 years ago still persist while new ones continue to emerge. Int. J. Food Microbiol. 139: S3-S15. Padron, M. 199. Salmonella Typhimurium outbreak in broiler chicken flocks in Mexico. Avian Dis. 34: 221-223. Pui, C.F., Wong, W.C., Chai, LC., Tunung, R., Jeyaletchumi, P., Hidayah, M.S, Ubong, A., Farinazleen, M.G., Cheah, Y., Son, R. 211. Salmonella: A foodborne pathogen. Int. Food Res. J. 18: 465-473. Quinn, P.J., Markey, B.K., Carter, M.E., Donnelly, W.J. and Leonard, F.C. 22. Enterobacteriaceae. In Veterinary Microbiology and Microbial Disease, Chapter 18: 16-123. Schiff, F., Saphra I. 1941. A New Salmonella Type: Salmonella Havana. J. Infect. Dis. 68(2) (March-April), page 12. Scheil, W., Dalton, C., Murray, C., Wilson, D. 1998. A South Australian Salmonella Mbandaka outbreak investigation using a database to select controls. Aust. N. Z. J. Public Health, 22: 536 539. Images from the website: www.google.pt 5