Biological Characteristics Of Pathogenic Escherichia Coli O157 From Pigeon And Evaluation Of Immune Efficacy Of Inactivated Vaccine

In order to identify the causes of diarrhea and death of racing pigeons in Jiangsu Province,the collected disease materials were isolated and identified.The evolutionary relationship of the isolated strains was analyzed by 16S rRNA gene sequencing,and the isolated strains were traced by MLST and Escherichia coli conservative gene sequencing;Further,by detecting the serotype,biofilm phenotype,virulence genes,drug resistance profile of the isolated strains,as well as the susceptibility of chickens to the isolated strains,the biological characteristics of the isolated strains were systematically sorted out.It was confirmed that the isolated strain was pathogenic Escherichia coli O157 from pigeons.The inactivated vaccine was prepared,and the chickens were immunized by oral and subcutaneous injection.The antibody titer in the immunized chickens was detected by ELISA method,and the immune protection effect was evaluated by virus challenge.Research results indicate that:1.The Gram negative bacteria isolated from diseased pigeon tissues,swabs,and feces were initially diagnosed as Escherichia coli O157 in the laboratory;The isolated strains grew white and smooth round colonies on the NA plate,and purplish red colonies on the CT-SMAC plate;The 16S rRNA gene sequence of the isolated strain was more than 98%similar to that of Escherichia coli.The pathogenic bacterium was identified as Escherichia coli by constructing a phylogenetic tree,and was named E.c.86246;Serological tests showed that the isolated strain was Escherichia coli O157:non H7 strain;Sporty;The isolated strains carrying tccp1014,eae A,and hly virulence genes can cause varying degrees of functional damage to the gastrointestinal tract of chickens,even leading to death;Its LD₅₀is 2.0×10⁵CFU/mL,with strong pathogenicity;The isolated strains carry five resistance genes,tetA,tetM,sul2,aadA1,and bla_TEM,with weak biofilm formation ability.They are severely resistant to antibiotics such as tetracycline and aminoglycosides,and are sensitive to amoxicillin,ofloxacin,and norfloxacin.They exhibit high sensitivity to the water extracts of scorpion and fructus aurantii.2.A strain of Enterobacteriac E.coli was isolated from diseased tissues of pigeons,and its biochemical characteristics and morphological identification results were very similar to those of E.c.86246;After PCR detection of specific genes,16S rRNA sequencing,phylogenetic tree construction,and serum diagnosis,the isolated strain was identified as Escherichia coli O157:H7.Through MLST analysis,it was identified as Escherichia coli type ST-636,named XY211007;The specific gene rfbE sequencing analysis showed that the isolated strain XY211007 had a 100%similarity with the deer derived Escherichia coli O157:H7,and a 99%similarity with the pig derived and human derived Escherichia coli O157:H7;The isolated strain XY211007carries three virulence genes:stx2,eaeA,and hly.The artificial infection test found that the infected chickens had poor appetite,depression,diarrhea,or bloody stool,even death.The autopsy found that the chickens had stomach and intestinal bleeding,with a statistical LD₅₀of 5.98×10⁵CFU/mL,indicating that the isolated strain has strong pathogenicity to chickens.The pathogenicity test of Vero cells in vitro found that both the isolated strain and its culture medium can cause Vero cells to shrink,deform,round,die,and shed,indicating that the isolated strain has strong toxicity to Vero cells.The drug property test found that the isolated strain XY211007 carried four drug resistance genes,namely tetM,tetA,sul1 and bla_TEM,was sensitive to 16 kinds of antibacterial drugs,including gentamicin,kanamycin,neomycin and ciprofloxacin,was resistant to seven kinds of drugs,including doxycycline,tetracycline and midecamycin,was highly sensitive to the water extracts of five kinds of Chinese herbal medicines,namely,Polygonum multiflorum Thunb,Fructus Aurantii,Folium Isatidis,Flos Lonicerae Lonicerae,and Cinnamomum cassia,and was moderately sensitive to the water extracts of 19 kinds of Chinese herbal medicines,including Angelica,Siraitia grosvenorii,Atractylodes macrocephala.3.The isolated strain XY211007 was inactivated by heating to prepare an aluminum salt adjuvant inactivated vaccine and immunize chickens.The results showed that oral and subcutaneous inoculation with inactivated vaccine of pathogenic Escherichia coli O157 from pigeons in chickens can both produce high IgG antibody levels in chickens after primary and secondary immunization;After secondary immunization,the IgG antibody titers in the subcutaneous immunization group were significantly different from those in the control group,adjuvant group,and oral immunization group.Three days after the attack,the protection rate of the subcutaneous immunization group was 80%,while the protection rate of the oral immunization group was 90%;After 5 days of challenge with 1672LD50,the protection rates of both subcutaneous and oral immunization groups were 50%..In summary,in this study,two pathogenic Escherichia coli strains with different serotypes were successfully isolated from pigeon sources,including E.c.86246 strain O157:non H7 strain and XY211007 strain O157:H7 strain,both of which are virulent strains with certain drug resistance and can infect chickens.The inactivated vaccine developed with XY211007 has a good protective effect on early infection of chicks under high-dose challenge.This study provides theoretical basis and data reference for the biological characteristics analysis,prevention and control,traceability,precise treatment,and vaccine development of pigeon Escherichia coli O157.