MOLECULAR EPIDEMIOLOGICAL STUDY ON SHEDDING PATTERN OF E.COLI O157:H7 IN BOVINES WITH RESPECT TO BREED, AGE AND SEASON
| dc.contributor.advisor | Dr. N. KRISHNAIAH | |
| dc.contributor.author | PRADEEP KUMAR | |
| dc.date.accessioned | 2016-12-28T12:33:58Z | |
| dc.date.available | 2016-12-28T12:33:58Z | |
| dc.date.issued | 2016-03-29 | |
| dc.description.sponsorship | Escherichia coli O157:H7 is an emerging zoonotic pathogen. Bovines are thought to be primary reservoir of this pathogen. Animal based foods contaminated with the faeces of bovines would cause foodborne infections in humans with this pathogen emphasising its public health importance. Conventional culture based method for the identification of E. coli O157:H7 is time consuming, needs a lot of media and supplement components and there is a possibility to get false results. And research work on epidemiology of shedding pattern of E. coli 0157:H7 with respect to age, breed, sex and seasons, genetic and physiological factors of animals and environmental factors is very meagre. Hence, the present study was undertaken to standardize multiplex PCR technique for the identification of this pathogen by targeting its six virulence genes; fliCh7, rfbE, eaeA, hlyA, stx1, and stx2 genes to study the epidemiology of shedding pattern of E. coli 0157:H7 with respect to age, breed, sex and seasons, genetic and physiological factors of animals and environmental factors. The standardized multiplex PCR produced amplicons of the size, 625 bp, 397bp, 296 bp, 166 bp, 210 bp and 484 bp, respectively for the fliCh7, eaeA, rfbE, hly, stx1, and stx2 virulent genes. The best DNA bands were obtained at MgCl2 concentrations of 3mM, denaturation at 94˚C for 45 sec and annealing temperature of 60°C and agarose concentration of 1.5%. The threshold sensitivity of standardized multiplex PCR was found to be 0.1 CFU/ml for reference culture of E. coli O157:H7, 1 CFU/g for spiked faecal samples and 0.1CFU/g for spiked faecal samples with one step enrichment. High absolute specificity of the standardized multiplex PCR was observed as none of the six DNA bands was found in the PCR product of negative control cultures. A total of 932 (n) fecal samples were collected from cattle and buffalo farms in different parts of Karnataka from July 2014 to January 2016 and analyzed for the identification and molecular characterization of E. coli O157:H7 by using cultural identification, cultural isolation with biochemical characterization and multiplex PCR. When compared to latex agglutination test, the epidemiological sensitivity and specificity of cultural identification were 100% and 83.35%, respectively, of cultural isolation with biochemical characterization were 100% and 92.51%, respectively, of multiplex PCR were 95.60% and100%, respectively when faecal samples were directly analysed and of multiplex PCR were 100% and 98.69%, respectively when faecal samples with one step selective enrichment were analyzed. The prevalence of shedding of E. coli O157:H7 in bovines was found to be10.94%. The percent of cattle and buffaloes shedding E. coli O157:H7 was 12.10% and 5.55%, respectively. In cattle, the percent of exotic breeds, crossbreds and indigenous breeds shedding E. coli O157:H7 was 18.56%, 13.88% and 4.41%, respectively. Age wise, the percent of shedding of E. coli O157:H7 in calves, heifers and adult animals was 14.80%, 12.12% and 8.60%, respectively. Sex wise, the percent of male and female animals that showed shedding of E. coli O157:H7 was 15.17% and 9.95%, respectively. The percent of bovines shedding E. coli O157:H7 in winter, monsoon and summer seasons was 14.75%, 10.85% and 7.86%, respectively. Maximum percentage of shedding was observed in the month of January and least in the month of May. Depending on environmental temperature, highest percentage of bovines shedding E. coli O157:H7 in faeces was in the temperature range of 20.1-25.0°C and at particular temperature of 22.1-23.0°C indicating that biologically favourable environmental temperature supports the shedding of this pathogen. The percent of healthy and diarrhoeic animals shedding E. coli O157:H7 was 12.06% and 2.68%, respectively, indicating that healthy bovines are more prone for shedding of this pathogen. There was no significant difference in shedding of E. coli O157:H7 at P value of <0.05 among lactating and pregnant (10.66%), lactating and non pregnant (8.54%), and dry pregnant (9.68%) animals. In contrast to these three groups, the shedding of E. coli O157:H7 was significantly very low in non lactating and non pregnant animals (only 1.45%). Apart from 102 E. coli O157:H7 positive samples, the percent of animals that showed shedding of E. coli O157 in the faeces was 13.41% and the percent of animals that showed shedding of STEC was 16.52 %. Highest percent of E. coli O157:H7 positive samples were resistant for Nitrofurantoin (100%) followed by Carbenicillin (92.16%), Co-Trimazine (87.25%), Tetracycline (75.49%), Kanamycin (69. 61%), Amikacin (46.08%), Streptomycin (23.53%) and Ciprofloxacin (only 2.94%). The E. coli O157 positive samples showed highest resistance against Nitrofurantoin (100%) followed by Carbenicillin (88.80%), Co-Trimazine (83.20%), Tetracycline (65.60%), Kanamycin (60.00%), Amikacin (46.4%), Streptomycin (20.8%) and Ciprofloxacin (only 2.4%) and STEC positive samples showed highest resistance against Nitrofurantoin (99.35%) followed by Carbenicillin (88.31%), Co-Trimazine (73.38%), Tetracycline (69.48%), Kanamycin (52.60%), Amikacin (40.91%), Streptomycin (20.78%) and Ciprofloxacin (only 3.25%). The pattern is similar to that of E. coli O157 except the difference in the percentages. The results found in the present study, give enough indication about epidemiological factors influencing the shedding pattern of E. coli O157:H7 in bovines. The association of these factors suggests strategies to reduce its shedding by applying the findings in to the principles of hazard analysis critical control points (HACCP) to strategise pre-harvest preventive and control measures so that the incidence of foodborne outbreaks of this foodborne pathogen in humans could be reduced to great extent. | en_US |
| dc.identifier.uri | http://krishikosh.egranth.ac.in/handle/1/93463 | |
| dc.publisher | PVNR TVU | en_US |
| dc.relation.ispartofseries | D;462 | |
| dc.sub | Veterinary Public Health and Epidemiology | en_US |
| dc.these.type | Ph.D | |
| dc.title | MOLECULAR EPIDEMIOLOGICAL STUDY ON SHEDDING PATTERN OF E.COLI O157:H7 IN BOVINES WITH RESPECT TO BREED, AGE AND SEASON | en_US |
| dc.type | Thesis | en_US |