Research On Virulence Factors And Pathogenicity Of Salmonella Pullorum In SPF Chickens

The various serotypes of Salmonella can cause disease in humans,livestock,poultry and wildlife.Salmonella-caused diseases spread all over the world.Many serotypes of Salmonella can infect human even cause food poisoning.In recent years,the occurrence of poultry salmonellosis has become increasingly serious,and Pullorum disease is one of them.In addition,this disease has some new features in many aspects such as epidemic,occurrence,clinical manifestations,necropsy changes and drug susceptibility.At present,there is no effective vaccine control against Pullorum disease.In this study,the Salmonella collected from poultry were identified,analyzed for drug resistance and virulence genes,and two strains of S.pullorum were screened.Pathogenicity and immunehistochemical methods were used to investigate their pathogenicity.1?Identification and serotype analysis of Salmonella from poultryIn this experiment,10 strains of Salmonella with clear background were collected and cultured.Gram staining,PCR identification,serotype sequencing analysis and biochemical experiments were performed.Salmonella enteritidis standard strain was used as a control.The results showed that two strains of S.pullorum and one strain of S.choleraesuis came from chickens,six strains of S.typhimurium and one strain of S.enteritidis came from ducks.2?Salmonella resistance gene detection and drug resistance analysis in poultryThe sensitivity test of 14 drugs and the PCR detection of 11 corresponding drug resistance genes were carried out on the 10 Salmonella collected by the K-B method,and E.coli ATCC25922 was used as a control.The results showed that the resistance rate to streptomycin was the highest,reaching 70%;the resistance rate to ampicillin was 30%;the resistance rate to amoxicillin,norfloxacin,tetracycline,and compound sulfamethoxazole were 20%;the resistance rates to cefoxicillin,kanamycin,gentamycin,and chloramphenicol were 10%;All the salmonella were sensitive to cefoperazone,neomycin,ofloxacin,and norfloxacin,four strains showed multi-drug resistance;the resistance gene detection results showed that the detection rate of the drug resistance gene catA1 was 100%;the detection rate of aadA1,aadA2,sulI was more than 50%,The detection rates of qnrB,tetB,blaTEM,tetA and sulII were lower than 35%,and the detection rates of blaPSE,qnrA and tetG were 0.The results of drug resistance and resistance gene of Salmonella showed that the drug resistant phenotype is consistent with the detection rate of drug resistance genes.The drug resistance genes carried by bacteria are correlated with drug resistance.The resistance genes on Salmonella DNA can largely determine their drug resistance.3?Detection of virulence genes of Salmonella from poultryPCR technique was used to detect 15 virulence genes of 10 Salmonella strains and they were known to participate in the adhesion,invasion and survival of Salmonella.The results showed that most Salmonella strains were positive for the selected virulence genes,with a carrying rate of more than 80%,and one strain carried all tested virulence genes;The detection rates of pipD,fimA,mis L,sopA,invA,stn,spvC,spiC,sifA and orf319were 100%;the carrying rate of virK,sipA,spvB,fliC was 90%,80%,60%,and 40%respectively.4?Pathogenicity of S.pullorum in chickenAccording to the clinical symptoms and the virulence genes checking of 10 strains,two strains of S.pullorum were screened for lethal infection in animals,and a LD₅₀ test for a pathogenic strain was screened for the animal's infection test.The antibiotic treatment trials test was conducted according to the test of drug susceptibility and clinical veterinary drug.After intraperitoneal injection,the heart,liver,spleen,lung,caecum and bursa of SPF chickens were stained with HE on the first day,the third day,the fifth day,the seventh day,the tenth day and the fifteenth day;the liver and cecum were selected to measur bacterial load;the location of the virulence protein SpiC was determined by the liver with the most severe injury and the immunohistochemical staining of related liver injury indicators was performed.The peak of death occurred from the third day to the seventh day and was also the most serious period of damage to various organs.At the same time,the expression of SpiC in liver was the most extensive and highest,which was consistent with the distribution of hepatic necrosis,indicating that virulence protein is related to liver injury.Caspase-1 and Caspase-3 were highly expressed during this period,indicating that the liver was damaged.by the bacterial infection caused damage to the organs of the animals,so the host will be ill.