Isolation Identification and Characterization of Bacterial pathogens causing Calf Diarrhea with special reference to Escherichia coli.

摘要

Though India is an agricultural country, livestock plays an important role. In bovine, calf mortality is due to many diseases, in that diarrhea or white scour plays a major role. Enteriocolibacillosis is the disease caused by Escherichia coli. About 16 samples were collected and 12 samples were positive for Escherichia coli. Antibiotic susceptibility test were performed and finding out the sensitivity and resistance pattern. In this all the isolate were resistant to oxytetracyclin. DNA was isolated using conventional boiling and rapid cooling method. Polymerase chain reaction was carried out using tet (A) primer to detect the presence of tet (A) plasmid. Amplified product shows approximately 417 bp fragments in agarose gel electrophoresis viewed under UV illuminatation. Introduction The livestock population of India is huge and animals as a whole play an importance role in the agricultural economy even though they often receive inadequate nourishment. Diarrhea in calves can be caused by a variety of pathogens including bacteria, viruses, protozoa and intestinal parasites. Among bacteria, enterotoxigenic Escherichia coli (ETEC) and Salmonella are know to be the most common and economically important agents ( House, 1978), but other bacteria eg.Campylobacter spp. have also been identified as cause of enteric disease and diarrhea in calves. In acute neonatal diarrhea, an important disease of calves, 4 microorganism in particular, are of widespread occurrence and proven enteropathogenicity: rota virus, coronavirus, cryptosporidia and enterotoxigenic Escherichia coli (ETEC) (Acres et al.,1975). Overfeeding, overpopulation, cold temperature, bad hygiene, artificial feeding and colostrums deprivation are all predisposing factor which can be important in the complex etiology of the disease. Among these organism Escherichia coli is the main cause for the calf diarrhea as “white” scour. The disease characteristically affects calves within the first 10-14 days of age, usually within the first week when there is sudden onset of profuse yellow or white diarrhea causing rapid and severe dehydration. The calf quickly becomes recumbent. Accumulation of fluid in the abomasums and intestines gives the abdomen a bloated appearance. These enterotoxigenic Escherichia coli are shed into the environment by infected animals in the herd and are ingested by newborn calves soon after birth. There is some natural immunity to enterotoxigenic Escherichia coli; however, it often fails to protect calves born and raised under modern husbandry conditions. Materials And Methods Collection of sampleSterilized new polypropylene storage vial of 5ml capacity was used for collection of fecal sample from the diarrheic calves.Medium MacConkey agar, Eosin Methylene Blue agar, Brillant Green agar, Xylose-Lysin Deoxycholate, Nutrient agar, Peptone water, MR-VP broth, Nitrate broth, Phenol Red Base broth, Simmon’s citrate agar, Triple Sugar Iron agar, Christensen’s urea agar base, Motility test medium and Muller Hinton agar. It was autoclaved at 121oC for 15 minutes. Antibiotic test Antibiogram of the Escherichia coli isolates were carried out, as per the standard single disc diffusion technique according to Kirby- Bauer method ( Bauer et.al, 1965), modified by National Committee for clinical Laboratory Standard M2A4. The zone of inhibition was measured, recorded and interpreted according to the 3rd International supplement (1991). DNA isolationIsolation of DNA from the Escherichia coli was carried out by conventional boiling and rapid cooling method. A single colony of the test culture was inoculated in 10 ml nutrient broth and incubated at 37oC for 24 hours. The cells were harvested by centrifugation at 5000rpm for 10 minutes. The pellet was washed with phosphate buffer solution by centrifuging at 5000 rpm for 10 mintues. Supernatant was decanted and the pellet was washed with PBS again. Then the pellet is resuspended in 20μl nuclease free water and boiled for 5-10 minu