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 sample

Medium

Antibiotic test

DNA isolation

Result and Discussion

Out of 16 fecal samples from the diarrheic calves 12 were positive for bacterial isolation on to media like MacConkey agar, EMB, BGA, XLD. All the 12 isolates produced positive reaction to lactose fermentation on MacConkey agar plate, metallic green sheen colonies on EMB plates, yellowish green colonies on BGA, yellow colonies on XLD. These findings were in correlation with the presentation of Cowan (1993) and John Barnes et al., (2003).

Gram staining were performed for all the isolates and revealed a Gram negative, non-acid fast, uniform staining non-spore forming bacilli. These findings were in correlation with the presentation of John Barnes et al., (2003).

All the 12 isolates evinced positive reaction for indole test by converting the aminoacid tryptophane to indole with help of tryptophanes enzyme by the organism, Methyl Red test by utilization of glucose in the MRVP medium, triple sugar iron agar test, the isolates utilized all three sugars glucose, lactose and sucrose which results in acid slant or acid butt with gas production, carbohydrate fermentation test, utilized all three sugars by change of red colour (indicator phenol red) to yellow with gas production in Durham’s tube in Phenol red base medium, nitrate reduction test by converting the nitrate to nitrite, catalase test by the development of air bubbles on adding 3%H₂O₂ to the isolate in the glass slide.

All the 12 isolates were negative for Voges proskauer test where no acetone production was seen, citrate utilization test where no colour change in Simmon’s citrate medium and also negative for oxidase test.These findings were in correlation with Bettelheim, (1994) and Cowan, (1993).

Figure 1

Antibiotic susceptibility test were done for all the isolates. All the isolates were sensitive to ciprofloxacin(100%) followed by pefloxacin(100%), cephotaxime(100%), chloramphenicol(83.33%), co-trimoxazole(75%) and gentamicin(75%). In the present study the isolates were sensitive to gentamicin was similar to the findings of Jones et al., 1977. It was resistant to oxytetracyclin(100%), chlortetracycline(100%), ampicillin(66.66%) and amoxicillin(58.33%). Palmer et al, (1977) reported that the findings of amoxicillin was effective. Resistant to tetracycline resistant(100%) was smiliar to according to Galland et al.(2001). They found that 38.5 percent isolates were resistant to tetracyline. The variation in the antimicrobial pattern may be due to the use of antibiotic in that region during that period.

Figure 2

PCR amplification were done for two isolates. In the present study the PCR amplified fragments of approximately 417 bp was observed with use of tet(A) specific primers F- CGA GCC ATT CGC GAG AGC and R- GCC TCC TGC GCG ATC TGG in the 1.5% agarose with ethidium bromide stain electrophorsed and viewed under UV illumination. This findings were in agreement with that of Furushita et al., (2003).

Figure 3