| Salmonella usually affects chickens of all ages and exists at different stages of production.It is necessary to understand the distribution of Salmonella,in the whole production chain of the breeding farm in order to effectively prevent and control the contamination and transmission of Salmonella.At present,the conventional measure to control salmonellosis is based on the usage of antibiotics,but the emergence of drug resisitance,especially the multidrug resistance increased the difficulties of the measure and may poss the potential threat to the food safety.It is crucial to develop the alternatives of the antibiotics.As a bacterial viruse that infects specific bacterial cells and produce no resistance,phage is a possible alternative to antibiotics.In the study,the prevalence of Salmonella in the whole production chains of five Yellow-chicken breeding companies in Guangxi was carried out to investigate the contamination of Salmonella,drug resistance and the potential critical control points of salmonellosis.The genetic relationship among the Salmonella isolates of the same farm or between the farms was studied by genotyping to evaluate the application value of genotyping techniques.Phages using Salmonella Pullorum as the host were isolated from the feces samples of a breeding farm.The biological characteristics of the phage and the evaluation of the antibacterial effects in vitro were studied to evaluate the potential development value of the phage.A total of 1 325 samples were collected from 7 kinds of samples in 6 links from 5breeding farms,including the eggshell surface of hatching eggs at different phases(pre-disinfection,post-disinfection and piped),dead embryos during late stage of incubation,day-old weak chicks,pullorum disease-fowl typhoid(PD-FT)antibody positive and negative breeder hens.A total of 232 Salmonella strains were isolated and the mean isolation positive was 17.51%,with the highest of 32.08%(85/265)in company DGGX12 and the lowest of11.70%(31/265)in company DGGX11.The Salmonella isolation positive was also different on the samples of different production links,and the PD-FT antibody positive birds had the highest of 48.00%(24/50),followed by dead embryos during late stage of incubation(30.40%,76/250),day-old weak chicks(26.00%,65/250),PD-FT antibody negative birds(8.00%,2/25),shell surfaces of piped eggs(23.20%,58/250),shell surfaces of eggs before disinfection(2.00%,5/250)and shell surfaces of eggs after disinfection(2.00%,5/250).Twenty-four out of the 50 PD-FT antibody positive birds were Salmonella-positive and Salmonella could be detected in the samples of the total 10 different organs/sites tested.The detection rate of the ovary was the highest(37.50%,9/24),followed by oviduct(33.33%,8/24),spleen(29.17%,7/24)and lung(29.17%,7/24),while those of cecum(including the ileocecal junction and the contents)and cloacal swabs were the lowest(4.17%,1/24).The results of the serotyping of all the isolates showed that Salmonella Pullorum(SP)(85.17%)was the most prevalent serovars,followed by Salmonella Give(8.48%),Salmonella Kedougou(2.97%),Salmonella London(1.69%),Salmonella Weltevreden(1.27%)and 1 unserotyped strain.Antimicrobial resistance tests showed that all the isolates had different resistance degrees against the 17 common antibiotics tested.The resistance to SIZ(99.0%),SXT(98.01%),TSU(97.51%),NAL(97.51%),AMC(96.02%)and AMP(94.53%)were the highest,followed by STR(74.13%),CAZ(57.71%),TET(34.83%),CRO(30.85%),CTX(30.35%)and AKN(30.35%),while were sensitive to CIP?KAM?NIT?GEN?FFC.The majority of the isolates(99.00%)showed multidrug resistance patterns.ERIC-PCR was used to classify all the 367 Salmonella strains isolated from different production links in the five breeding farms.Then based on the years,farms and the ERIC-PCR genotyps of the strains,31 representive isolates were further calssified by MLST.The results showed that a total of 24 genotypes,which were classified into 17 clusters were found in the 367 isolates.And the isolates from different farms had their own unique genotype,while a few of genotypes show multiple-site ditributions.All the isolates were classified into the same ST type ST92(5-2-3-7-31-41-11)by the MLST.Two lytic phages of Salmonella Pullorum were isolated from the fecal samples of a breeding farm and named SP11 and SP19,respectively.The results of the study on the biological characteristics of the two phages showed that the lytic plaques of the phages were transparent and with clear edgea,and the diameter were 2.5 mm and 3.5 mm,and the titers were 2.45×10~8PFU/m L and 1.08×10~9PFU/m L,respectively.The two phages were also highly host specific according to the tests of lysis spectrum on a total of 218 strains of bacteria including Salmonella of 13 serotypes,Escherichia coli,E.coli and Staphylococcus aureus,and the phage SP19 had a stronger lytic effect.The further study on SP19 showed that the phage possess an icosahedral head(52 mm in diameter)and an apparent tail under the tramsmission electron microscope.Its optimal MOI is 0.1.The test of step growth curve showed that the latent period was 5 min,cracking period was 45 min and the burst size was 11PFU/cell.Phage SP19 could remain at high activity in the temperatures of 40-70?and the pH values of 3-12.In the evaluation of antibacterial effect in vitro,phage SP19 can effectively inhibit the propigation of the host bateria.In summary,the results of the investigation demonstrated that there were different degrees of Salmonella contamination among the links of the production chain on the breeding farms.Salmonella Pullorum was the dominant serotype and the multi-drug resistance of the isolates was serious.And the isolates from different sites and links show cross distribution.Phage SP19 was host specific and can effectively control the proliferation of host bacteris,so it has potential development value. |
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