Studies On Multiresistance And Virulence Factors Of Pathogenic Escherichia Coli From Post-weaning Diarrhea And Efficacy Of Specific Egg Yolk Antibody

Post-weaning diarrhea (PWD) is an important widespread disease in porcine industry worldwide. The main symptoms include decreased weight gain and diarrhea. In severe cases, the pigs could die of dehydration and acidosis. Although multiple factors are involved in PWD, Escherichia coli plays a crucial role in the complex biological process. The virulence factors differ geographically and could change with time. Currently, there are few studies in China on the epidemiology of multi-resistance, virulence factors, of the pathogenic Escherichia coli from PWD, as well as the development of PWD model and specific egg yolk antibody evaluations and its optimization of clinical application regimens in the control of the disease.A total of 75 pathogenic Escherichia coli isolates were obtained from rectum swabs of weaned piglets from intensive pig farms of ZheJiang Province. The isolations were identified by routine microbiological techniques and virulence tests in mice and PCR identification of the specific E.coli Eco and UspA genes. In addition, the O serotyping tests showed that the pathogenic Escherichai coli covers at least 11 O serotypes and the predominant serotypes are 0149,0139 and 08.Susceptibility testing by Kirby-Bauer method against 21 antimicrobial agents were carried out. Escherichia coli ATCC25922 was used as reference strain for quality control and clinical categorization of isolates as Susceptible or Resistant was based on the National Committee for Clinical Laboratory Standards (NCCLS/CLSI). The data were analyzed by WHONET. The results showed that none of the 66 isolates were susceptible to all antimicrobial agents tested and most strains (89.4%,59/66) were multi-resistant. The isolates exhibited severe resistance to Chloramphenicol (100.0%), sulfarmethoxazole/Trimethoprim (86.7%), Doxycycline (82.3%) and streptomycin (81.8%). Resistance to Carbenicillin, Polymyxin B and Gentamicin, Ciprofloxacin and Norfloxacin were also quite common, with the resistance rates as high as 69.6%. 66.7%,63.6%,56.4% and 61.5%, respectively. The resistance varies from districts and farms. Of the 66 resistance isolates, there are 36 different resistance patterns. Furthermore, there is one isolate even could resist 13 antimicrobials.64 out of 66 isolates are resistant to more than 2 antimicrobials. Among the multiresistant isolates, resistance to 5 antimicrobial agents was the most prevalent (18.18%), followed by multi-resistance to 6 antibiotics (15.15%) and multi-resistance to 9 antibiotics. In addition, there are cross-resistance between streptomycin and other antimicrobials. In order to investigate the molecular bases for antibacterial resistance. PCR and sequencing techniques were applied to analyse the integrase of classⅠand classⅡintegron and the integron gene cassettes. The results shwoed that, among 75 clinical isolates,66 (88.0%) harbored class . integrase whereas 13(17.3%) harbored classⅡintegrase. All the positive isolates showed resistance to two or more antimicrobial agents(phenotype). Furthermore,46.7% (35/75) of the isolates that were classⅠintegrase positive harbored gene cassettes. Based on analysis of randomly selected 18 isolates, the gene cassettes have fragments size of 250bp,1.1Kb,1.6Kb,1.7Kb and 2.0Kb, respectively, containing aadA and dfrA gene which encodes resistance to the sulfonamides and streptomycin, respectively. In addition,84.6% (11/13) of the classⅡintegrase positive isolates were found also positive for classⅠintegrase, indicating that one isolate could take more than one type of integron. However, no gene cassettes of classⅡintegron were detected. The results indicate that multi-resistance of pathogenic Escherichia coli from PWD piglets is widespread in Zhejiang province; integron plays an important role in the development and dissemination of resistance.Multiplex PCR was used to analyse the virulence factors of F4 and F5, F6, F41, 987P and Fl 8, LT, STa, STb, Stxl, Stx2, SLT-2e. Agglutination test was also applied to test the F4 and F18 pili by respective mono-specific antiserum. The results showed that ETEC accounts for 67.92% (36/53) as the predominant pathogenic Escherichia coli involved in PWD. F4 and F18 were the predominant fimbriae, amounting to 24.53% (13/53) and 28.30% (15/53),respectively; STa, STb, STa+STb, LT and SLT-2e enterotoxins genes were detected as 30.19%(16/53).35.85% (19/53),18.9% (10/53),1.89% (1/53) and 9.43% (5/53), respectively. The main fimbriae are F4 and F18, while the more prevalent toxins were STa, STb, STa+STb. Further analysis of the subtypes of F4 and F18 by PCR and duplex PCR revealed that F4ac (76.9%,10/13) and F18ac (15/15) are more predominant, whereas 23.1% (3/10) belongs to F4ab variant.The IgY were obtained by immunized hens with purified F4 fimbriae emulsified with or without Freund's adjuvant. Antibody titers of egg yolk and serum were detected by indirect ELISA. The results showed that F4 fimbria is a satisfactory immunogen to induce high titer antibodies. The egg yolk antibody titer of hens immunized with F4 fimbriae emulsified with Freund's adjuvant (FA group) reached 4.41g at week 3 after the boost injection. Adjuvant is needed to keep a long period (9 weeks) of high antibody titer. However, the antibody titer of both egg yolk and serum from PBS group is lower than that of FA group and could not stay at high level for long. In addition, the hens injected with 10μg bird-1 of F4 fimbrial protein, induced almost equal antibody titers, compared with the doses of 50μg and 250μg bird-1(p>0.05). It is therefore concluded that the practical immune dose could be as low as 10μg bird-1.Spray drying and other two methods were applied to prepare full egg powder successfully. The acute toxicity test in mice demonstrated that the egg powder prepared was virtually non toxic. Efficacy in vitro showed that, IgY (antibody titer 1:3000) can control the growth of F4+ETEC (104cfu/ml) for 8h at 37℃. Thereafter, the efficacy of the egg powder on the control of PWD was investigated in weaned piglets experimentally challenged orally by local pathogenic Escherichia coli isolate(s). PWD model was developed with triplex PCR method tracking the shedding profiles of the challenged pathogenic Escherichia coli in the fecal. The study was performed in two sets for the in vivo passive protection of piglets. In set 1, piglets were challenged with both local isolates of Escherichia coli harbored F4 and F18 respectively, while in set 2, only F4+ETEC local isolate was used for the challenge. The challenging dose in both sets is 1012cfu/mlx60ml per piglet. Antimicrobial group were included in both sets, however, in set 1, antimicrobial was used with the egg yolk, while in set 2, the antibiotic was applied alone. The results demonstrated that, in both sets, piglets in the positive controls developed severe diarrhea that lasted for 7 days and above, and the fecal shedding of the challenged pathogenic ETEC of the piglets treated with the egg powders stays lower and short time than those of the piglets without specific egg yolk fed; Fecal diarrhea scores of piglets fed with full egg powder (0.43) were much lower (p<0.05) than that of the control group (1.43); In set 2, fecal diarrhea scores of piglets fed with anti-F4 egg yolk antibody (0.86) is much lower than that of positive control (1.57). It is concluded that the PWD model was repeatable, reliable and could be applied in the study of the pathogenesis of PWD and virulence factors related and evaluation of anti-diarrhea drugs. In addition, The triplex PCR is reliable and specific and relatively sensitive, could rapidly tracking the shedding profiles of the challenging pathogenic Escherichia coli. The egg yolk antibodies are promising alternative to antimicrobials in controlling PWD. The recommended egg yolk to the piglets is the combination of both anti-F4 and anti-F18 IgY at the doses of 0.3g/kg.bw each time,3 times a day and for 3-5 days running after weaning, antimicrobials are not suggested to use with the egg yolk, combinations with anti-F18 IgY is recommended.In summary, results from the above investigations have laid good foundation for further exploration of the pathogenesis of pathogenic Escherichia coli and resistance mechanisms and immune control of PWD by egg yolk antibody developed from fimbriae extracted from Pathogenic Escherichia coli.