Isolation And Identification Of Avian Pathogenic Escherichia Coli And Preliminary Studies On Efficacy Of Inactivated Vaccine Based On LpxL And LpxM Deleted Mutants With O1,O2 And O78 Serogroups

Avian pathogenic Escherichia coli (APEC) cause avian pericarditis, perihepatitis, airsacculitis, salpingitis, peritonitis that exert varying degrees of mortality and significant economic costs in poultry. Here, the avian Escherichia coli isolates derived from sick birds originated from some regions in Jiangsu Province were isolated and identified, and their pathogenicity and drug resistance were determined. And, then we developed the polyvalent inactivated vaccine based on lpxL and lpxM deleted mutants with O1,O2 and O78 serogroups. Such data provided a theoretical basis for the prevention and control of avian colibacillosis and laid the foundation for the further development of the polyvalent inactivated vaccine against avian colibacillosis.1 Isolation, identification and pathogenicity of avian pathogenic Escherichia coliOne hundred and sixty-two avian Escherichia coli isolates derived from diseased or sick birds originated from some regions in Jiangsu Province were isolated and identified by standard biochemical procedures. Among these E. coli isolates, One hundred and thirty isolates were placed in determined serogroups and 80.25% isolates belonged to these serogroups, thirty two isolates were unserogroupable. These isolates distributed in 34 serogroups and 66.92% of them belonged to seven O serogroups:O78,O88, O65,O161,O1,O24,O13. Therefore they were predominant serogroups. We also examined the One hundred and sixty-two isolates for the prevalence of the virulence-associated genes by PCR, including iss, iroN, tsh, cvaC, iutA, irp2, and the prevalence of them were 83.33%,80.86%,45.06%,48.76%,75.92%,58.02%, respectively, and there were thirty seven strains carrying all of the tested six virulence genes, which accounted for 22.83% of the total isolates. In order to determine the virulence of the isolates,64 of them were selected and evaluated by using a lethality assay in 1-day-old specific pathogen free (SPF) chicks. And the result was that fifty five isolates of the tested strains were high or intermediate pathogenic, accounting for 85.94%. And, fourty isolates of the tested strains were high pathogenic (33 isolates belong to the predominant serogroups); 15 isolates of the tested strains were intermediate pathogenic (8 isolates belong to the predominant serogroups); 9 isolates of the tested strains were low pathogenic. Combined the virulence genes detection result with 1-day-old chicken lethality assay result, we found that at least 80% of the isolates obtained in this study had high pathogenicity.2 The drug resistance of avian pathogenic Escherichia coli isolatesSixty APEC isolates were selected and tested for their sensitivity and resistance to 16 kinds of antibiotics (ampicillin, tetracycline, doxycycline, cephalothin V, sulfamethoxazole, streptomycin, gentamicin, kanamycin, ciprofloxacin, levofloxacin, florfenicol, azithromycin, norfloxacin, neomycin, polymyxin B, ciprofloxacin). The result showed that these isolates were resistant to 16 kinds of antibiotics in varying degrees. For doxycycline, ampicillin, tetracycline, sulfamethoxazole and ciprofloxacin, the drug resistance rate of all tested strains was exceed 80%, and the drug resistance rate of the tested strains was less 50% for azithromycin, gentamicin and polymyxin B. The tested strains had high sensitivity to polymyxin B. All tested isolates presented multidrug resistance.25%(15/60) of the tested strains were co-resistant to 13 kinds of tested antibiotics,63.33%(38/60) of the tested strains were co-resistant to 9 or more kinds of tested antibiotics. The results indicated that the drug resistance of APEC isolates tested in this study was strong.3 The preliminary studies on efficacy of inactivated vaccine based on lpxL and lpxM deleted APEC mutants with O1,O2 and O78 serogroupsIn this study, the O1,O2 and O78 serotype of APEC wild type strains and their lpxL and lpxM mutants constructed in our laboratory were used to develop the polyvalent oil adjuvant inactivated vaccine, and then evaluated the antibody responses and protective efficiency of the prototype vaccine against the parent strain challenge, and the presence of histopathology lesions in organs of the challenged chicken. The results showed that the antibody responses and protective efficiency of the prototype polyvalent oil adjuvant inactivated vaccine developed were better than a licensed vaccine. For the effect of different adjuvants on antibody responses and protective efficiency the polyvalent vaccine emulsified with Montanide TSA 71VG oil adjuvant was prior to that emulsified with mineral oils. The protection to 01,02,078 was 86.67%, 93.33%,100% respectively with Montanide TSA 71VG oil adjuvant vaccine and 80%,86.67%, 86.67% respectively with mineral oils vaccine. For the effect of different immune times on protective efficiency, the efficacy of the prototype vaccine emulsified with Montanide TSA 71 VG oil adjuvant administered once was better than that of the prime-booster vaccination of the commercial vaccine, and it had better immune protection to 01.02.078,80%,93.33%,93.33% respectively. So, the prototype polyvalent Montanide TSA 71VG oil emulsified adjuvant inactivated vaccine exhibited an excellent protection in immunized chicken against parent 01, 02 and 078 strains challenge, even in birds vaccinated once. For a single dose vaccination of developed vaccine, the efficacy was better than the prime-booster vaccination of the tested commercial vaccine.