Establishment And Application For Determination Of The Chicken Salmonellosis

Chicken salmonellosis caused by one or more Salmonella serotypes,which is the most common infectious disease in chickens.According to the structure it is divided into chicken pullorosis,avian typhoid and poultry paratyphoid.This pathogen poses a serious threat to chicken industry.For that reason,it is urgent to develop a rapid,specific,and effective diagnostic method for the detection of S.Gallinarum,especially the rapid,specific,and sensitive diagnostic tool.The main research work of this topic is as follows:Objective:To understand the prevalence of serotypes and drug resistance of salmonellosis in large chicken farms in northern Xinjiang.This subject aims to use Salmonella specific target gene invA as a diagnostic marker,that based on molecular biology methods and immune colloidal gold technology,established a nested-PCR method,LAMP with LFD,colloidal gold immunochromatography test strip for the detection of S.Gallinarum,and assess the feasibility of these methods.The bacteria of suspected chicken salmonellosis were isolated and identified,Salmonella diagnostic serum for serotype identification and pathological observation.Methods:(1)A plate agglutination test was used to test 750 clinical samples taken from in large chicken farms in northern Xinjiang.Bacteria isolation and identification,serotype identification and pathological anatomy were used to diagnose the case of suspected chickens with salmonellosis.Finally,the PCR method for further validation test,and the resistance of the isolated S.Gallinarum was tested,and the drug sensitivity test was carried out for the isolated Salmonella.(2)According to the Salmonella gene inv A,two pairs of specific primers were designed as outer primers and inner primers.Using the product of the first round of PCR as a template for the second round of PCR reaction,the nested-PCR method of the chicken salmonellosis was established.At the same time,specificity,sensitivity,and clinical samples were tested.(3)According to the highly conserved locality of the invasion protein InvA of Salmonella,a set of LAMP specific primers and probes were designed to optimize the LAMP reaction system and reaction conditions.The LAMP product labeled by biotin was specifically hybridized with the Digoxigenin labeled probe,and LFD was used to complete the detection.The method of bacterial isolation and identification,the conventional PCR method and the established LAMP-LFD method were used to detect the simulated samples and clinical samples of eggs contaminated by Salmonella.(4)The invA gene of Salmonella was cloned,and the prokaryotic expression was carried out.The fusion protein of InvA was purified by Ni-NTA,and the reactivity of the protein was identified by Western blotting and ELISA.The recombinant protein of InvA was used as the envelope antigen to optimize the reaction conditions and the colloid gold test paper was developed.The positive serum and clinical samples of Avian salmonellosis were detected by plate agglutination test and immunochromatography test strip.Results:(1)The isolation rate of S.Gallinarum in this region was 9.2%,and the detection of serotype was D1 type.The isolated S.Gallinarum had the highest resistance to vancomycin and neomycin,which were100.0%and 81.2%,respectively,and ofloxacin.The lowest resistance rate was 4.62%,followed by ceftriaxone,azithromycin,and doxycycline.The drug resistance rates were 5.8%,8.7%,and 13.0%,respectively.(2)The optimal reaction conditions of nested-PCR:The amount of primers in the upper and lower reaches is 0.3?L,the first annealing temperature is 57.4?,and the second annealing temperature is53.5?.The sensitivity of the nested-PCR method to the recombinant plasmid was is 10~2 copies/?L.The results showed that the coincidence of the traditional diagnostic method was 95%.(3)The optimum reaction condition of LMAP-LFD method was 62 C and 50 min could be detected.The sensitivity of this method to invA genome DNA of Salmonella chicken was 1×10~2 CFU/mL,which was 1000 times that of conventional PCR detection method.The minimum initial infection rate of 9 CFU/10 mL was detected Salmonella contamination in eggs.The clinical samples were detected by LAMP-LFD,the result is consistent with that of the bacterial isolation by 95.14%.(4)The pH value of the best colloid gold marker was 7.6,and the antibody concentration was 1.7 mg/mL.The standard positive serum of S.Gallinarum could be detected as 1:256.The 195 chicken serum samples were detected by immunochromatography test strip,and the coincidence rate showed that the coincidence of flat agglutination test was 96.41%.Conclusion:(1)In conclusion,the pathogenicity of S.Gallinarum in this area was isolated,and its serotype type was D1.The drug sensitivity test showed that the resistance of isolate Salmonella was complex.It is suggested that the farms in this area should strengthen health management,purify the breeding hens,reduce the dosage of antibiotics,and ensure the health and safety of food.(2)Using the invA gene of Salmonella as a diagnostic marker,the PCR method and LAMP-LFD method of S.Gallinarum were established.The recombinant protein of InvA was obtained by cloned invA gene and prokaryotic expression,and the immuno colloid gold test strip was developed.These detection methods provide a new technology for the epidemiological screening and purification of the Salmonella infection in chickens.