Prevalence Of Virulence Genes In Escherichia. Coli Associated With Newborn Piglets Diarrhea And Immune Prevention For Newborn Piglets Diarrhea

Diarrhea is a common and important disease in industrial pig farms and Escherichia coli is the main cause of morbidity and mortality in piglets. The preliminary diagnoses on this disease are mainly by clinical symptom and detailed body dissection. To further establish a rapid diagnosis method for porcine diarrhea, four set of primers were designed according to the sequences of the enterotoxin ST1, ST2, LT1 and high pathogenicity island (HPI) genes that published in the GenBank. All the PCR methods were tested for their specificities by using reference E. coli strains. When multiplex PCR was performed to detect the genes of ST1, ST2 and LT-1, the expected size (183 bp for ST1, 360 bp for ST2 and 282 bp for LT1) were seen in the Enterotoxigenic Escherichia coli (ETEC) strains respectively. When PCR was performed to detect the HPI gene, the expected products of 280 bp were only detected in the HPI-positive bacterial strains. A total of 151 feces samples were obtained from live diarrheic piglets from the north region of Jiangsu province. Following by first cultivating in LB broth at 37°C for 4~6 h, all the samples were detected by the PCR methods, and the data showed that 95 case (62%) were infected with HPI-positive E. coli, 14 case (9.27%) were co-infected with HPI-positive E. coli and ETEC, and 24 case (15.18%) were infected with ETEC. In addition, 54 feces samples from healthy piglets were also detected using established PCR methods, the results showed that 28 case (51.8%) were infected with HPI-positive E. coli, and 6 case (11%) were infected with ETEC. the high prevalence of HPI in healthy piglets indicated these E.colis probably are opportunistic pathogen and its pathogenicity is highly related to the host immunity.To determine the distribution of seropathotypes and identification of vaccine candidate with dominant O serotype and prevalent virulence gene against pathogenic Escherichia coli. Identified virulence gene positive fecal samples submitted for bacteriological isolation and culture,biochemical identification and PCR. A total of 89 pathogenic Escherichia coli were isolated and identified. Among the 89 isolates serological typed, 69 isolates belonged to 6 different serogroups with O138 as the dominant serogroup(44%), Followed by O65 11 strains(12%).Serotyping of E.coli isolates showed that the ETEC were more frequently detected in O138(13%),O65(2%),O21(2%);and that the HPI~+ E.coli were more frequently detected in O138(21%),O65(8%),O21(6%),O141(3%),O9(2%),O159(2%);ETEC with HPI~+ E.coli were more frequently in O138(9%),O65(2%),O9(2%),O139(1%),O55(1%);Virulence gene detection indicated that the HPI was highly related to ETEC, we found 39% HPI~+ in ETEC isolates. Among the adhesion gene detected, K88 and 987P were mainly adhesion gene in 89 isolates. The seropathotypes O138:HPI:K88:LT1 was identified as a vaccine candidate against pathogenic E.coli by PCR detection ,O serotyping and fimbriae in vitro expression test.To develop a vaccine against Escherichia coli-induced pre-weaning diarrhea, insights in the induction of the protective immune response following infection with these pathogenic E.coli is needed .Therefore, multivalent inactivated vaccine expressing K88,987P fimbriae in vitro and containing HPI virulence gene against colibacillus diarrhea of newborn piglet was developed and the immune efficacy by vaccination pregnant sow was determined.At the same time, commercial trivalent inactivated vaccine and phosphate-buffered saline(PBS) taking aluminum adjuvant were administrated as control. On days 1,2,3,7,14 and 21 after parturition, the dynamic changes of K88,987P and FyuA whey antibody titers of the puerperal sow were analyzed, The results showed that the multivalent inactivated vaccine produced by this research could significantly enhance whey antibody titers .Compared with control group ,the K88 whey antibody titers and 987P whey antibody titers was significantly difference(P<0.05 or P<0.01), but not a significant difference on FyuA whey antibody titers. Both K88 and 987P whey antibodies reached summit at 1 day post parturition and declined during the lactation period, especially for K88 whey antibody titers and even no titer production at one week after parturition, while 987P whey antibody titers could last for about 21 days after parturition.These findings confirmed multivalent inactivated vaccine produced by this research could provide protection against piglet colibacillosis and would be used as effective vaccine.To determine the adhesive inhibition effect of whey antibodies against Escherichia coli. adhesive inhibition tests were performed using multivalent whey antibodies against K88 and 987P fimbriae. The results showed that both anti-K88 and anti-987P whey antibodies from immunized group and control group at 1 day post parturition were able to inhibit adhesion of K88~+ and 987P ~+ E.coli to porcine enterocytes, Further investigation indicated that anti-K88 whey antibodies from immunized group and control group at 7 day post parturition were uable to inhibit adhesion of K88~+ E. coli to porcine enterocytes. Whereas anti-987P whey antibody from immunized group at 7 day post parturition were able to inhibit adhesion of 987P~+ E. coli to porcine enterocytes.