| Calf diarrhea was a gastrointestinal infectious disease caused by enterotoxigenic Escherichia coli, manifested as severe diarrhea, dehydration and even death, which had brought serious economic losses to the calf industry. In addition, irregular clinical use of antibiotics, aggravated the development of drug-resistance, and increased seriously. Currently, the main method to prevention and treatment of such disease is making multivalent vaccines by the strengths virulence factors in different regions and drugs. So, the investigation and detection of virulence genes and drug-resistant of calf diarrhea caused by Escherichia coli, have more practical significance to prevention and control this disease. This research mainly includes3aspects:the established of the multiplex PCR method, the detected of virulence genes and the investigated of drug resistance.The established of multiplex PCR detection method. According to the literature synthesis of four pairs of specific primers to amplify the Escherichia coli estA (229bp), estB (480bp), elt-I (605bp) and elt-II (300bp) gene fragments, after optimizing the reaction conditions, specificity, sensitivity of the multiplex PCR method were studied. The results indicated that this method was specificity, high sensitivity, faster and stable, and the detection limit were2.55×101CFU/μL,2×101CFU/μL,2×101CFU/uL and2.47×103CFU/μL, respectively.The detected of virulence genes. We collected131calf diarrhea stool samples from12dairy farms in Harbin, Acheng, Mudanjiang, Bei’an, Zhaodong, Kedong and zhaodong regions, Heilongjiang Province,342strains of Escherichia coli were isolated. Using conventional and multiplex PCR, to detected Escherichia coli adherence factor (K88, K99,987p, F1, F41, F17, F18, saa, CS31A, afaE-8), toxins(estA, estB, elt-I, elt-II, Stxl, Stx2, EAST1, afaD-8), pathogenicity island (eaeA, ler, irp2, hlyA, sepA, ETT2) and0157, H7antigen. The results showed that,121strains of Escherichia coli carrying virulence genes,52.1%(63/121)were positive for at least one fimbrial or non-fimbrial adhesin genes, of which the F17(44isolates) was the most prevalent adhesin, followed by K88(12isolates), CS31A(13isolates), afaE-8(10isolates), saa(2isolates). In contrast, no isolates were found to express K99,987p, F41or F18adhesin genes. For the toxin-associated genes, EAST1and irp2were the most prevalent genes, and the detection rate were33.9%(41isolates) and30.6%(37isolates), respectively. Followed by ler(12isolates,9.9%), afaD-8(10isolates,8.3%), elt-II(8isolates,6.6%), eaeA(6isolates,5.0%), hlyA(5isolates,4.1%), Stxl(5isolates,4.1%), estA(4isolates,3.3%), Stx2(4isolates,3.3%), sepA(1isolates,0.8%).11 isolates were H7antigen-positive Escherichia coli, no O157antigen, elt-1and estB positive strains were detected.The investigated of drug resistance. By the kirby-Bauer agar diffusion method,121pathogenic Escherichia coli isolated were detected the antibiotic sensitive and the results showed that121pathogenic Escherichia coli had different resistance and sensitivity for the14kinds of commonly antibiotic. Based on the results, ampicillin, amikacin and norfloxacin were extremely resistance, the resistant rate were73.55%,71.90%and60.33%, respectively. In addition, amoxicillin, cephradine, cefotaxime and gentamicin were relatively sensitive, the sensitive rate were79.34%,70.25%,71.07%and76.86%, respectively. From the resistance spectrum we can see that the strains multi-drug resistance of Escherichia coli was serious, more than three-resistant strains accounted for65.29%(79/121) of the isolates, of which16isolates were more than ten-resistant.This study established the multiplex PCR method for detection of enterotoxigenic Escherichia coli, grasp the epidemic situation of main virulence genes and antibiotic resistance of Escherichia coli strains isolated from calves with diarrhea in Heilongjiang province, which provides a scientific method and experimental evidence for the rapid diagnosis and prevention of Escherichia coli diarrhea in calf. |
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