Experimental infection of chickens one day of age with salmonella serotypes isolated from poultry facilities, wild birds and swine

Brazil is currently regarded as the largest exporter of broiler meat in the world and ranks third worldwide in production of this meat (Brazilian Union of Poultry Farmers, UBA, 2009) and fifth in egg production (Organization of the United Nations for Food and Agriculture, FAO, 2010).

To preserve the Brazilian poultry industry and its high production and export rates, measures are required for prevention and control of some agents causing infectious diseases in birds, for example Salmonella spp.

The incidence of food-borne infections in humans due to Salmonella spp. has increased in some parts of the world, despite all technological development in food production and the adoption of better hygienic and sanitary measures. Foods of animal origin are still primarily responsible for human infection, including poultry meat, eggs, and by-products (Gast et al., 2008).

Many serotypes of Salmonella can survive during weeks or months in chicken feces and litter, wild birds manure, equipment, empty sheds (houses, aisles or galleys), their surroundings, dust particles, feeders and food (Davies and Wray, 1996; Berchieri Junior Freitas Neto, 2009).

The aim of this study was to investigate the presence of Salmonella spp. in materials proceeding from four groups of animals and their environments: broilers, wild birds, feces of rodents prowling poultry facilities, and feces of pigs raised in sheds within the same property, and also verify the pathogenesis of these different Salmonella types, through inoculating them to chickens one day of age.

The research was conducted in cooperation with a broiler farm located in the state of São Paulo, Brazil. Samples of the broilers were collected (carcasses of one day of age and adult chicken, chicken transport crates when reaching the farm, food in the silo and in feeders, water at farm supply and at dispensers, feces [from the first day of life until slaughter], litter from the house and beetles [Alphitobius diaperinus] found in the litter), from six chicken batches during the 45-day stay at the farm. Wild birds frequenting the sheds were captured over a period of one year and with authorization of the Brazilian Institute of Environment and Renewable Natural Resources (IBAMA, permit No. 14909-1, IBAMA Register No. 1902993), swabs were taken from the cloaca, organs and intestinal contents of such birds. A total of 36 wild birds were tested, all captured within the broiler house, mainly during the its sanitary emptying. During this period we observed an increase in the number of wild birds within the aisles in search of food. Among these, 14 were smooth-billed ani (Crotophaga ani), 9 ruddy ground dove (talpacoti Columbina), 3 eared doves (Zenaida auriculata), 3 carpenters (Colaptes campestris), 2 guira cuckoo (Guira guira), 2 plumbeous pigeon (Patagioenas plumbea), 2 southern lapwing (Vanellus chilensis) and one great kiskadee (Pitangus sulphuratus). All birds were examined (nutritional status, color of mucous membranes, natural orifices, and presence of ectoparasites) and none showed alteration signs in assessed parameters.

We found and collected feces of various rodent species that frequent the poultry facilities. On the same property there were, additionally to broiler houses, four corridors for pork production in several development phases, from which we collected feces samples.

All samples were placed in individual sterile vials containing 1% peptone water and were incubated for 24 hours at 37° C. From this initial culture we transferred 2 mL to tubes containing 20 mL of selenite/novobiocin (SN) broth and 0.2 mL aliquots to tubes containing 20 ml of Rappaport (RP) broth, which were incubated for 24 hours at 37° C. Then, the broths were cultivated on MacConkey agar media, Brilliant green (VB) and Xylose Lysine Tergitol 4 (XLT-4) and incubated for 24 hours at 37° C. Colonies suspect of belonging to the genus Salmonella were inoculated in presumptive media TSI (triple sugar and iron) and LIA (lysine iron agar) and incubated at 37° C for 24 hours. Fermentation and characterization were performed at Adolfo Lutz Institute of São Paulo, SP, Brazil.

Salmonella serotypes isolated were prepared for resistance to nalidixic acid at a concentration of 50 µg/mL. Subsequently, all Salmonella spp. cultures were prepared in 10 mL Luria-Bertoni (LB)   broth at 37° C for 24 hours under agitation (100 rpm). Then 0.1 mL of the culture was diluted in buffered saline solution with pH 7.4 (PBS) at a 1:10 ratio, for later preparation of decimally solutions up to 10^-6. From each dilution 0.1 mL was transferred to a plate containing VB agar, which was incubated for 24 hours at 37° C. After this time, we proceeded to count the colonies. Cultures contained 1.2 x 10⁸ colony forming unit/mL (CFU/mL).

To carry out the experimental infection we used one day-old chicken non-vaccinated against Marek's disease, from a commercial strain, from an hatchery located in the state of São Paulo. The experiment was conducted in isolated rooms with temperature, lighting (artificial) and ventilation control, as well as biosafety level two. Birds were kept in metal cages receiving water (sterilized in autoclave) and food ad libitum. The food was made from corn, soy (soybean) and premix, without the addition of antibiotics. Cultures were initially conducted to detect the presence of Salmonella spp. in the food and the floor of the transport crates (meconium) upon arrival of the chickens from the hatchery. Birds were separated in groups of 15 and each chicken received, through probe to the crop, 0.1 mL of culture containing 1.2 x 10⁸ CFU/mL. Animals were observed twice a day, verifying the presence of clinical signs and mortality.

After Salmonella inoculation, fecal samples were taken from each bird cloaca at 24 hours and then at 8, 15 and 21 days, using sterile cotton swabs and afterwards each sample was processed individually. Half of the lot of birds were sacrificed by cervical dislocation at 15 days post-infection (dpi) and the remaining animals at 21 days. Swabs were taken from the liver, spleen and cecal contents from each bird for its incubation in selenite broth added with novobiocin (SN) at 37° C for 24 hours. Following, the broths were cultivated on plates containing VB agar with nalidixic acid (50 µg/mL) and incubated at 37° C for 24 hours, and then make the reading of the plates verifying for the presence of Salmonella in organs and cecal contents. Organs were observed for macroscopic changes, and also slides were prepared for histopathological exam.

With regard to the presence of Salmonella in wild birds, only in one carpenter (Colaptes campestris) S. Heidelberg was isolated in organs and intestinal contents. Several authors have also isolated Salmonella from wild birds (Sousa et al., 2010a, Sousa et al., 2010b). By microscopically analyzing the organs of this positive wild bird, we only observed moderate diffuse peribronchial anthracosis.

Concerning broilers' samples, from the 25 water samples, two were positive for Salmonella spp. (S. Glostrup; S. enterica subspecie enterica 6,8:d:-); from the 36 chicken fecal samples, one of which was a grouped sample or "pool" of 10 fecal swabs, four were positive for Salmonella spp. (S. Heidelberg S. enterica subspecies enterica 6.7:R: -; S. enterica subspecies enterica 4,5,12:R: -; S. Tennessee). Among the 47 litter samples, one of which was a pool of 10 litter swabs, three were positive for Salmonella spp. (S. enterica subspecies enterica 4,5_12:R: -; S. Heidelberg; S. Infantis). From the 25 insect samples, one of which was a pool of 15 grams of insects, one was positive for Salmonella spp. (S. Tennessee).

From the 15 swine fecal samples, one of which was a pool sample of 10 fecal swabs, four were positive for Salmonella spp. From those, two were of the same serotype (S. Panama and . Typhimurium).

From the total of 30 rodents samples analyzed, one of which was a pool of 20 grams of feces, all were negative to Salmonella spp. isolation.

Also, we isolated several serotypes of Salmonella spp. in a research conducted with poultry products, including food, litter and meat meal (Hofer et al. 1998; Andreatti Filho et al., 2001).

The clinical sign observed in chickens during the experimental infection was the presence of loose stools on the cages floor and around the cloaca, which suggests a diarrhea scheme between days 3 and 12 dpi. During the experiment a death occurred in the group of S. enterica subspecies enterica 6.7:R: -. In total, the cloacal swabs taken from the 15 birds in each group on day 1 dpi were positive, except in the group of S. Panama, in which two birds did not excrete Salmonella at the time of the sampling with swabs. Except for a fowl, the group of S. Tennessee did not excrete the agent on day 8 dpi. The cloacal swabs from all birds of the 9 groups of Salmonella were positive on day 15 dpi. Some authors have reported the excretion of Salmonella spp. in the feces for a long time (Pinheiro et al., 2001, Oliveira et al., 2005, Ribeiro et al., 2005).

Half of the birds were sacrificed at 15 days dpi and the remaining at 21days dpi. During the autopsy samples were taken aseptically with swabs from the liver, spleen and cecal contents to verify from which organs it was possible to isolate Salmonella spp. At 15 dpi Salmonella serotypes were present in the liver and spleen of 27 birds (20%) and in all the cecal contents samples. At 21 dpi, Salmonella serotypes were present in the liver and spleen of 16 birds (11.8%) and remained present in all cecal contents samples. Our results match those of Ribeiro et al. (2005) when they investigated S. Kottbus in one-day chickens.

At necropsy macroscopic lesions were observed: pale liver, spots on the liver, enlargement of the spleen, petechiae on breast and leg muscles, mainly with serotypes S. Tennessee, S. Panama, S. Glostrup and S. Infantis. In regard to microscopic changes in organs of experimentally infected chickens we mainly observed granulocytes in the spleen and Fabricio bursa, and goblet cells in duodenum, ileum and cecum.

By means of this investigation we could isolate Salmonella spp. serotypes from samples taken at broiler and swine farms, as well as from wild birds that frequented poultry facilities, showing the presence of this agent in several analyzed materials. When one-day chickens were inoculated, these serotypes were thrown in the feces for a long time, thus becoming the germ carriers and reservoirs, as well as spreading it to the environment, contaminating water, food and bed.

Andreatti Filho RL, Fernandes SA, Boretti LP, Barros MR, Del Bem SR, Fontana A, Sampaio HM, Savano EN. 2001. Sorovares de Salmonella isolados de materiais avícolas no período de 1994 a 1999. Rev Educ Contin. 4:90-101.

Berchieri Junior A & Freitas Neto OC. 2009. Salmoneloses. pp. 435-454. In: Doenças das aves. Berchieri Junior A, Silva EN, Di Fábio J, Sesti L, Zuanaze MAF. (ed), 2.ed. Campinas: FACTA.

Davies RH & Wray C.1996. Persistence of Salmonella Enteritidis in poultry units and poultry food. Brit Poul Sci 37:589-596.

FAO - Food and Agriculture Organization of the United Nations, 2010. Disponível na internet <http://www.fao.org>. Acesso: 10/12/2010.

Gast RK, Shivaprasad HL, Barrow PA. 2008. Salmonella Infections, pp. 619-674. In: Diseases of Poultry. Saif YM, Fadly AM, Glisson JR, McDougald LR, Nolan LK, Swayne DE. (ed), 12.ed. Blackwell Blackwell Publishing, Athens, Georgia.

Hofer E, Silva Filho SJ, Reis EMF. 1998. Sorovares de Salmonella isolados de matérias-primas e de ração para aves no Brasil. Pesquisa Vet Brás. 18(1):21-27.

Oliveira GH, Berchieri Junior A, Fernandes AC. 2005. Experimental infection of laying hens with Salmonella enterica serovar Gallinarum. Braz J Microbiol. 36:51-56.

Pinheiro LAS, Oliveira GH, Berchieri Junior A. 2001. Experimental Salmonella enterica serovar Pullorum infection in two commercial varieties of laying hens. Avian Pathol. 30:129-133.

Ribeiro SAM, Berchieri Junior A, Orsi MA, Mendonça AO, Ferrati AR. 2005. Experimental infection by Salmonella enterica subsp enterica serovar Kottbus in day-old broiler chickens. Braz J Poult Sci. 7(2):107-112.

Sousa E, Berchieri Junior A, Pinto AA, Machado RZ, Carrasco AOT, Marciano JA, Werther K. 2010a. Prevalence of Salmonella spp. antibodies to Toxoplasma gondii, and Newcastle disease virus in feral pigeons (Columba livia) in the city of Jaboticabal, Brazil. J Zoo Wildlife Med. 41(4):603-607.

Sousa E, Werther K, Berchieri Junior A. 2010b. Assessment of Newcastle and infectious bronchitis pathogens, and Salmonella spp. in wild birds captured near poultry facilities. Arq Bras Med Vet Zoot. 62(1):219-223.

UBA - União Brasileira de Avicultura. Relatório Anual: São Paulo, 2009, 40 p. Disponível na internet <http://www.uba.org.br>. Acesso: 03/01/2011.