Isolation And Identification Of Salmonella In Embryo Source Of Barred Chicken And Analysis Of Genes Related To Drug Resistance And Virulence

Salmonella is a clinically common food-borne Gram-negative pathogen with multiple serotypes.Chickens infected with Salmonella can cause a series of symptoms such as decreased egg production and diarrhea.Among them,chickens infected with Salmonella Pullorum usually discharge white paste feces,which can cause death if they are dehydrated too much due to diarrhea.Salmonella Enteritidis is a zoonotic pathogen,which can cause fever,vomiting,diarrhea and other symptoms.At present,salmonellosis is one of the main threats to the healthy development of China's breeding industry.In the previous study found that when the chickens are infected with Salmonella,Salmonella in a sample of dead embryos carrying the highest rate.In order to evaluate the infection status of Salmonella in Barred chickens,the isolation and identification of Salmonella were completed by using the dead embryos of 18 days old,and the drug resistance and virulence genes were analyzed.In April 2019,Salmonella was isolated and identified from 90 dead embryos of chickens that did not come out of the shell in the Barred chicken farm.After the chicken embryo samples were bacterially enriched with BPW and TTB,the bacteria were inoculated in the XLT4 medium using a three-line method.Salmonella was isolated and identified by colony morphology and PCR;Serotyping the isolated strain with Salmonella diagnostic sera;Determine the Salmonella genotype by Multilocus sequence typing?MLST?,and understand the taxonomic characteristics of the isolates and their genetic relationships;Detection of 13common virulence genes in isolated strains using PCR technology;The Kirby-Bauer method was used to detect the sensitivity of 14 antibiotics to Salmonella isolates;At last,PCR technology was used to detect the class I integron and 21 common resistance genes.The results showed that 77 strains of Salmonella were isolated from 90 chicken embryo samples,and the isolation rate was 85.6%?77/90?.77 strains of Salmonella were divided into two serotypes,including 71 strains of Salmonella Pullorum and 6 strains of Salmonella Enteritidis.Two types of ST were detected by MLST.ST92?71/77,92.2%?was the main type,followed by ST11?6/77,7.8%?Thirteen virulence genes were tested,and all of the isolates?77/77,100%?carried invA,hilA,sefA,sipA,sipC,sopB,sopE,spvC,ssaR,ssrA,and stnP1.The positive rates of the two other virulence factors pefA and rck tested were low,both being 7.8%?6/77?.According to virulence gene statistics,it was found that Salmonella Pullorum did not carry pefA and rck virulence genes,but Salmonella Enteritidis carried pefA and rck virulence genes.The isolated strain has the highest resistance to erythromycin,up to 97.4%?75/77?.Followed by streptomycin reaching 26%?20/77?All strains were sensitive to ofloxacin,gentamicin,florfenicol,polymyxin B,doxycycline,gatifloxacin,with a drug resistance rate of0%?0/77?.The multidrug-resistant strains accounted for 14.3%?11/77?,with the most resistant phenotype of STR-EM-FOX?5.2%,4/77?,followed by STR-EM-CTA?2.6%,2/77?.Six different kinds of resistance genes??-lactams,quinolones,tetracyclines,aminoglycosides,sulfonamides,chloramphenicols?showed that 71.4%?55/77?carried resistance genes,19.5%?15/77?of the strains carried more than three resistance genes.A total of 10 drug resistance gene combinations were found in 77 Salmonella strains.The carrying rate of bla_TEM?49/77,63.6%?was the most prevalent,followed by aaC3?16/77,20.8%?and aac?6'?-ib-cr?15/77,19.5%?,None of the 77 Salmonella strains carry bla_SHV?bla_OXA?bla _CTX-M?qnrB?qnrA?qnrC?qnrD?oqxA?aaC1?aaC2?aaC4?tetA?tetB?sul3?cmlA resistance genes;Four of the 77 Salmonella strains carried class I integrons?4/77,5.2%?,and there were three types of gene cassettes:aadA2?2.6%,2/77?,aadA2-intl1-linF?1.3%,1/77?and intl1-aadA2-sul1?1.3%,1/77?.In this study,the prevalence,serotyping,MLST typing,sensitivity of antibiotics,class I integron,resistance genes and virulence genes carrier rate of Salmonella in Barred chickens were studied.The positive rate of Salmonella infection was 85.6%?77/90?,Salmonella Pullorum was the dominant serotype?92.2%?,and all of them are ST92,indicating that the source of Salmonella Pullorum in this group is single.The Salmonella isolated from Barred chicken has no drug resistance to 6 kinds of common drugs such as gentamicin,florfenicol and polymyxin B,which indicates that the chicken farm is relatively safe in the use of drugs.However,the carrier rates of 11 virulence genes such as invA,hilA,and sefA are 100%.The results of this study supplement the epidemiological data of salmonellosis in Barred chicken.It will provide a reference for the prevention and control program of Salmonella Pullorum of Barred chicken.It provides data support for the purification of salmonellosis in self breeding local chicken.