Detection, Recombination And Expression Of Virulence-related Genes Of Major Pathogenic Bacteria Causing Calf Diarrhea

Calf diarrhea is the most serious diseases affecting calf early growth. E. coli,Salmonella spp. and Clostridium perfringens are the major causes of calf diarrhea.Rapid detection of the three bacterial pathogens and investigating popular virulencegenes are especially essential for control of calf diarrhoea. This study developed threemulti-PCR detecting systems for the pathogenic bacteria above and detected the fecalsamples from calves with clinical diarrhoea , from which possible bacterial pathogenswere isolated by experimental animals inoculation and direct-isolation.entero-toxigenic Escherichia coli (ETEC) is the most important cause of calf diarrhea.Two of the more prominent virulence factors identified for ETEC strains are fimbrialantigens and enterotoxins (ST and LT). This study focused on cloning and expressionof st and ltB carried by ETEC. The st and ltB fusion protein expressed in E. colielicited experimental mice to produce high level of serum antibody reaction andprotection against ETEC.In order to detect the major pathogenic bacteria causing calf diarrhea andinvestigate the prevalent virulence genes, three sets of multi-PCR assays weredeveloped: stx2-k99-f41-stx1 (for detecting ETEC and Shiga toxin-producing E. coli),st-invA-eae-lt (for detecting ETEC, Salmonella spp. and Shiga toxin-producing E. coli)and cpb-plc-etx (for detecting Clostridium perfringens).stx2-k99-f41-stx1: Fimbrial genes k99 and f41 carried by ETEC, stx2 and stx1carried by Shiga toxin-producing E. coli (STEC) were used as target genes forMulti-PCR detection. Multi-PCR was developed by optimizing reaction condition.The Cmin of ETEC C83922, EHEC O157 EDL933 and the mixture to be detected bythe PCR were 1×104CFU/ml, 5×103CFU/ml and 2×104CFU/ml, respectively.st-invA-eae-lt: Virulence gene invA carried by Salmonella spp., st and lt carried byETEC, eae carried by enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli(EHEC) were used as target genes for Multi-PCR detection. Multi-PCR wasdeveloped by optimizing reaction condition. The Cmin of C83922, EDL933,Salmonella Dublin, ETEC EWD299 and the mixture to be detected by the PCR were3×103CFU/ml, 5×103CFU/ml, 3×103CFU/ml, 1×104CFU/ml and 2×104CFU/ml,respectively.cpb-plc-etx: Virulence genes cpb, plc and etx carried by Clostridium perfringenswere used as target genes for Multi-PCR detection. Multi-PCR was developed by optimizing reaction condition. The Cmin of Clostridium perfringens to be detected bythe PCR was 5×103CFU/ml.Fifty-nine fecal samples from calves with diarrhoea were detected by the threesets of multi-PCR above. The results showed that pathogenic Escherichia coli are themajor causes of calf diarrhea. Escherichia coli carrying stx1, eae were isolated fromsamples T1, T2 and XL1. Escherichia coli carrying stx1, eae, stx2 and Clostridiumperfringens carrying plc were isolated from sample T3. Escherichia coli carrying ltwere isolated from samples X5 and X1. Escherichia coli carrying eae were isolatedfrom samples X2 and W1. Escherichia coli carrying st, eae, stx1 and stx2, andClostridium perfringens carrying plc were isolated from sample X4. Escherichia colicarrying stx2 were isolated from sample Q3 and Q4.Diarrhoea in calves is commonly caused by ETEC which carried two majorvirulence factors: ST and LT. The sequence of st-ltB was amplified by overlappingPCR, with pulpy polypeptide as linker. The st-ltB fusion gene was inserted intopMD18-T vector, and then the fusion gene was subcloned to pMal-p2x expressionvector and transformed into expression-strain Tb1. The supernatant of soluble proteinswas analyzed by SDS-PAGE with negative control (Tb1 pMal-p2x). The maximalexpression quantity of target protein was 13.4% of TSP (total soluble protein) thatwas got in the optimized condition: final concentration of 0.41mmol/L IPTG,induction of 8h, at 31℃. The Interest fusion protein was purified by affinitychromatograph with amylose column. Mice were immunized 3 times with the st-ltBfusion protein in dosage of 100μg per mouse by intraperitoneal injection at a intervalof two weeks. Serum were collected from immunized mice on the 7th, 14th, 21th,28th, 35th, 42th and 49th day after the first immunization. The antibody titer of miceimmunized was up to 215, measured by indirect-ELISA with polypeptide of ST-LTBas coat antigen. The suckling mice were gavaged by the cultural supernatants of J13neutralized by positive serum, and the result was negative. At the 7th day after thethird immunization with st-ltB fusion protein, 8 of 10 mice were survived afterchallenged with three times the LD50 dose of ETEC J13. Results showed therecombinant antigen may have potential for use as subunit vaccine against ETEC.