The Study Of Molecular Characteristics In Breeder Flocks Of China From 2020 To 2021 And The Prevention And Control Technology Of Salmonella Pullorum

Pullorum disease is a host-specific bacterial infectious disease caused by Salmonella Pullorum,which can be spread horizontally and vertically in breeder flocks.Due to a variety of breeds,diverse breeding patterns and late time to start large-scale breeding in China,Salmonella Pullorum is still prevalent in Chinese breeder flocks,which seriously endangers and restricts the development o f breeder industry.With the implementation of the policy of reducing the use of antibiotics for poultry in 2020,how to prevent and control Pullorum disease has become a major problem nationwide.Therefore,it is crucial to study the epidemiological situation,molecular epidemiological characteristics and control measures of Salmonella Pullorum in breeder flocks.Based on the above background,114 Salmonella Pullorum strains from different chicken breeder farms in 11 provinces of China from 2020 to 2021 were selected to study molecular epidemiological characterization and the correlation analysis between drug resistance genes and drug resistance phenotypes by using next-generation sequencing.In addition,a new highly virulent arthritis-causing Salmonella Pullorum strain was isolated and identified,and its pathogenicity and molecular characteristics were investigated.To expand the control measures of Salmonella Pullorum,this study innovatively evaluated the effectiveness of live Salmonella Enteritidis vaccine and phage against Salmonella Pullorum infection,and provided technical support to assist the Salmonella Pullorum decontamination in breeder flocks of China.1.Molecular epidemiology and drug resistance characteristics of Salmonella Pullorum in breeder flocksIn the present study,114 Salmonella Pullorum strains from different breeder farms in 11provinces were sequenced.The results of MLST showed that the 114 strains belonged to three ST,and the main prevalent ST was ST92（n=104）,accounting for 91.2%of the total.The other two variants ST2151 and ST3717 were only detected from two native chicken breeder flocks in Guangdong and Anhui provinces.Antibiotic resistant phenotypic analysis showed that the proportion of multi-drug resistant isolates in 2021 was significantly higher than in 2020（41.8%vs.27.7%）.114 isolates were mainly resistant to the ampicillin（52.2%-93.6%）and streptomycin（66.0%-76.1%）,and the proportion of drug-resistant strains from fecal and commercial generation dead embryos was significantly higher than that of parental generation dead embryos.Among the 11 provinces,the proportion of multi-resistant strains was as high as 82.8%（24/29）in Jiangsu Province,followed by Anhui Province with 53.8%.The detection results of drug resistant genes showed that theβ-lactamase resistant gene bla_TEM-1B and the sulfonamide resistant gene sul2 had the highest detection rate,and both genes were detected in isolates from different provinces,different sample types and different breeder species.Tetracycline resistant gene tet A,disinfectant resistant gene qac E,sulfonamide resistance gene sul1,the class I integron drf A17 and the resistant gene aad A5were detected in 33%of isolates in 2021.These five resistant genes were simultaneously present in the form of integrons in 22 isolates from Jiangsu province,and the detection rate was 76%（22/29）,and 21 strains were from parental generation dead embryo of breeding hen and one isolate was from broiler waterline swab,which indicated that the most widespread multi-drug resistance gene carriage rate was found in parental dead embryos of Salmonella Pullorum isolates in 2020-2021.In addition,the Ser83 or Ala87 sites of DNA prorotase Gyr A were mutated in all 113 isolates,indicating that Salmonella Pullorum may be resistant to nalidixic acid in current breeding farms.Further analysis revealed that the resistant genotypes of the isolates were significantly correlated with the resistant phenotypes.96.2%（76）of the 79 isolates carried the bla_TEM-1B,and 34 isolates resistant to the sulfonamide cotrimoxazole carried both dfr A17,aad A5 and sul1,or only the sul2 gene.Only 24 of the 82 streptomycin resistant strains carried aad A5,and the main mechanism mediating the significant increase of streptomycin resistant in Salmonella Pullorum may be related to the mutation of gen rsm G.A minimal spanning tree based on geographic information showed that the isolates from the same province had obvious clustering relationships,and the isolates from Jiangsu province being closely related to isolates from the Shandong province,while isolates from Anhui province were more closely related to those from the neighboring Jiangxi,Fujian,Yunnan and Guangdong provinces in the south of China,indicating that the isolates prevalent in different provinces had some geographic relationships.2.The isolation and identification,pathogenic and genome characteristics of highly pathogenic arthritis-causing Salmonella PullorumIn recent years,the incidence of arthritis in chicks caused by Salmonella Pullorum infection is increasing.In this study,four Salmonella Pullorum strains were identified from the liver,spleen and joint fluid of Qingjiaoma chicken breeders with arthritis symptom,and the drug-resistant phenotype and biofilm-forming ability were determined.Chick arthritis model was successfully established using SP206-2 isolated from joint fluid,and the effects of arthritis-causing SP206-2 and diarrhea-causing Salmonella Pullorum strain CVCC526 on growth performance of chicks were compared The genomic characteristics of four arthritis-causing isolates were analyzed by whole-genome sequencing and comparative genomics.All isolates were multi-drug resistant,with 100%resistant to ampicillin,amoxicillin,erythromycin and nalidixic acid.The biofilm assay result showed that the biofilm forming ability of all isolates was not significantly different from that of CVCC526.The virulence assay showed that SP206-2 was a highly pathogenic strain with LD₅₀ of 1.3×10⁶ CFU and acute mortality in chicks within 24 h after infection.In order to compare the effect of SP206-2 and CVCC526 on growth performance,3-day-old Qingjiaoma chicks were divided into control,CVCC526group and SP206-2 group,and mortality,incidence of arthritis and growth performance were determined within 3 weeks after infection.The results showed that the average body weight,daily feed intake and average body weight gain of the SP206-2 group were significantly lower than those of the CVCC526 group（P<0.001）.This indicated that the isolate SP206-2was able to cause more severe damage to growth performance than CVCC526.Arthritis symptoms were observed in chicks 10 days after artificial infection with SP206-2,and the incidence of arthritis was 27.78%.Whole genome sequencing of SP206-2 strain showed that the genome was 4,730,579 bp,52.16%GC content and 5,007 genes,including 4,729 protein-coding regions.The genomic analysis of four arthritis-causing isolates and three diarrhea-causing isolates showed that the genome of arthritis-causing isolates was subject to nonsynonymous mutations,shift mutations and gene copy deletions.Based SNP phylogenetic tree analysis showed that arthritis-causing isolates were in a different evolutionary branch from the diarrhea-causing isolates.Further differential gene analysis showed that the genome of arthritis-causing isolates had missense mutations in genes related to substance metabolism and substance transport as a result of adaptive evolution.3.Study on the effectiveness of live Salmonella Enteritidis vaccine and phage in control Salmonella Pullorum infectionIn view of the severe drug resistant of Salmonella Pullorum,the use of antibiotics for prevention and control of Salmonella Pullorum is gradually being phased out,and the prevention and control of Pullorum disease mainly relies on integrated biosecurity measures to achieve the decontamination of Salmonella Pullorum,and there is no available commercial vaccine for Salmonella Pullorum in China.The effectiveness of the live attenuated Salmonella Enteritidis vaccine against Salmonella Pullorum early infection was evaluated by artificially infecting 1-day-old chicks with Salmonella Pullorum standard strain CVCC526and Salmonella Enteritidis standard strain CVCC3377,respectively.The anti-infection results showed that a single vaccination against better protection agains Salmonella Enteritidis early infection than heterologous serotypes.Live Salmonella Enteritidis vaccine is widely used in China,mainly for the prevention and control of Salmonella Enteritidis,and the complete immunization program needs to be administered in three times at 1 day of age,6 weeks of age,and 16 weeks of age.In this study,157-day-old breeder chickens immunized three times with live Salmonella Enteritidis vaccine were artificially infected with Salmonella Pullorum and the excretion of the vaccine strain was monitored from the brooding period to the egg-laying period.The results showed that compared to the unimmunized+Salmonella Pullorum infection group,vaccine immunization significantly reduced liver index（P<0.01）,spleen bacteria load（P<0.01）and liver bacteria load by 0.36-fold（P>0.05）,and had no significant effect on ovarian and oviduct bacteria load（P>0.05）.It showed that live Salmonella Enteritidis vaccine could alleviate the hepatomegaly caused by Salmonella Pullorum infection and reduce the number of bacterial colonization in the spleen.To evaluate the effectiveness of Salmonella Pullorum phage in control Salmonella Pullorum infection,the study treated chicks with phage after infected with the arthritis-causing Salmonella Pullorum isolate SP206-2.The results showed that phage treatment significantly increased the average body weight of infected chickens（P<0.01）and effectively alleviated the liver lesions caused by the infection,with no significant effect on the bacterial load of liver and spleen（P>0.05）.Analysis of the cecum microbiota revealed that phage treatment was able to reshape the intestinal flora disturbed by Salmonella Pullorum infection and promote intestinal flora homeostasis.In summary,this paper found that ST92 remained Salmonella Pullorum mainly type in some breeder flocks in 11 provinces of China from 2020 to 2021,and the proportion of antibiotic resistant to penicillin and streptomycin was significantly increased.The isolates mainly carriedβ-lactamase resistance gene bla_TEM-1B and sulfonamide resistance gene sul2,and its prevalence had some correlation with geographical distribution.In addition,the impact of highly virulent arthritis-causing Salmonella Pullorum on chick growth performance was more severe than that of the diarrhea-causing isolate,and had become a new feature of the current epidemic of Salmonella Pullorum in breeder farms of China.This study confirmed that the live Salmonella Enteritidis vaccine and phage treatment can be an important tool to assist in the decontamination of Pullorum dieease in breeder flocks.