“ In vivo” properties of lytic bacteriophages isolates in Salmonella Enteritidis infected and uninfected SPF chickens.

1. “In vivo” properties of lytic bacteriophages isolates in Salmonella Enteritidis infected and uninfected SPF chickens. L. Fiorentin, N.D. Vieira and W. Barioni Jr. Marcas da Embrapa em Inglês Marcas do Ministério em Inglês Brazilian Agricultural Research Enterprise Embrapa Swine & Poultry - BR 153, km 110, Vila Tamanduá, Caixa Postal 21 89700-000, Concórdia -SC, Brazil. INTRODUCTION RESULTS • Bacteriophages have recently been studied as a new approach for reducing Salmonella infection in chickens and therefore lowering the risks of contamination of poultry products (Berchieri Jr et al, 1991; Sklar et al., 2001). A perfect understanding of phage biology within the poultry alimentary tract, however, will be necessary for a successful use of phages as therapeutic agents, as well as to protect their longevity in biocontrol. • We have isolated and characterized several bacteriophages lytic to Salmonella Enteritidis and Salmonella Typhimurium (Fiorentin et al., 2002). Three of these phages showing different RAPD profiles were selected for analyses of their invasiveness and rate of excretion in SPF chickens. Cloacal swabs of control birds (Group 3) were negative for both bacteriophages and Salmonella throughout the experiment. Swabs collected from internal organs at necropsy of control birds were also negative for both bacteriophages and Salmonella. SE PT4 uninfected-phage administered birds (Group 2) showed only 1/7 cloacal swabs positive for bacteriophages one day after phage administration and were negative throughout the experiment. Swabs of SE PT4 infected-phage administered chickens (group 1) were 7/7 bacteriophage positive two days after administration and ranging from 4/7 to 7/7 thereafter (Figure 1). All birds in Group 1 were positive at necropsy for both Salmonella and bacteriophages in cecal contents while none was bacteriophage positive for spleen and liver samples, even though they were Salmonella positive in all three samples. Cecal content collected at necropsy showed 87.5 ± 23.7 x 108 CFU/mL. All 35 isolates of Salmonella obtained from cecal contents at necropsy of birds from Group 1 remained sensitive to lysis by all three phages. MATERIAL AND METHODS Bacteriophages were isolated from feces of free range chickens and captive songbirds using a field isolate of Salmonella Enteritidis PT4 (kindly provided by Dr. Paul Barrow, ARFC Institute for Animal Health, Houghton Laboratory, Cambridge, England) or Salmonella Typhimurium ATCC 14028as phage targets. Partial molecular characterization of phage isolates has been obtained and described elsewhere (Fiorentin et al., 2002). The three phages denominated CNPSA1, CNPSA3 and CNPSA4 selected for this study showed icosahedrical head, dsDNA as nucleic acids and had different RAPD profiles. Three groups of 7 SPF chickens (SPAFAS) were housed in isolator cabinets under filtered air from birth till 16 days of age. Group 1 was orally infected at the third day of age with 103 CFU of SE PT4 per bird followed by oral inoculation two days later with a pool of 105 PFU each phage per bird. Group 2 was only administered phage and Group 3 was kept as uninfected control (Table 1). Cloacal swabs were collected daily for SE PT4 and phage isolation. Salmonella was isolated by incubating swabs at 42ºC in peptonated Rappaport-Vasiliadis broth overnight followed by iniculation in Brilliant Green (BG) Agar at 37ºC and identification of Salmonella by colony morphology and serum agglutination with polivalent anti-O reference serum. Bacteriophages were isolated by incubating swabs at 37ºC in one milliliter of a log phase SE PT4 culture, followed by a treatment with 5% chloroform and inoculation of 5µL of supernatant over a loan of SE PT4 grown in nutrient broth solidified with 1% agarose. At the 16th day of life all birds were necropsied and attempts to isolate SE PT4 and bacteriophages were made from cecal content, spleen and liver of every bird. Five isolates of Salmonella obtained from cecal content of each bird in Group 1 were tested for their sensitivity to the phages administered to the birds. Five microliters of each phage was applied over a loan of the Salmonella isolates grown in nutrient broth solidified with 1% agarose. Cecal contents were logarithmically diluted tenfold and inoculated on BG with Novobiocin (40 µg/mL) to obtain CFU/mL. Table 1. Experimental design. Group Birds SE PT4 per bird1 Phage per bird2 1 7 103 CFU at the 3rd day 105 PFU at the 5th day 2 7 None 105 PFU at the 5th day 3 7 None None 1 Field isolate. 2 PFU of each of three phages. CONCLUSIONS We suggest that bacteriophages CNPSA1, CNPSA3 and CNPSA4 do not remain in Salmonella-free birds longer then one day while multiplying in Salmonella-infected birds for longer periods. Selection for phage resistant SE PT4 seemed not to occur within the secretion period studied. BIBLIOGRAPHY • Berchieri Jr., A., Lovell, M. A., Barrow, P.A. The activity in the chicken alimentary tract of bacteriophages lytic for Salmonella typhimurium. Res. Microbiol., 142: 541-549. 1991). • Fiorentin, L., Vieira, N.D.; Barros, S. Bacteriófagos líticos para Salmonella Enteritidis e Salmonella Typhimurium isolados de fezes de aves. In: CONGRESSO BRASILEIRO DE MEDICINA VETERINÁRIA, 29., 2002, Gramado, RS, Anais. Gramado: Sociedade Brasileira de Medicina Veterinária, 2002. CD-ROM. Abstract PAN 1372. • Sklar, B., Joerger, R.D. Attempts to utilize bacteriophages to combat Salmonella Enterica serovar Enteritidis infection in chickens. J. Food Safety, 21: 15-29. 2001.