IDENTIFICATION OF BACTERIAL AGENTS FROM THE FAECAL SAMPLES OF DIARRHOEIC SHEEP AND THEIR ANTIBIOTIC SENSITIVITY

Bacteria Diarrhoea Sheep Antibiotic Sensitivity The current study was carried out to identify the bacterial species in the faecal samples of 20 diarrhoeic sheep and to observe their sensitivity to different antibiotics. This investigation was performed by collecting diarrhoeal samples from the sheep (n=20) under goat and sheep research farm of Bangladesh Livestock Research Institute (BLRI), Savar, Dhaka, Bangladesh. The average age and body weight of the animals were 25 days and 2.5 kg respectively. Of the bacteria responsible for diarrhoea in case of sheep, Escherichia coli alone was found in 6 samples (30%), Escherichia coli combined with Proteus mirabilis was found in 12 samples (60%), and no bacteria could be detected in 2 of the 20 samples tested. Both the bacteria were highly sensitive to ceftriaxone, and moderately sensitive to ciprofloxacin and gentamicin. It could be stated that ceftriaxone is the antibiotic of first choice for the treatment of diarrhoea in sheep, where Escherichia coli is suspected to be the principal causal agent of diarrhoea.

The current study was carried out to identify the bacterial species in the faecal samples of 20 diarrhoeic sheep and to observe their sensitivity to different antibiotics.This investigation was performed by collecting diarrhoeal samples from the sheep (n=20) under goat and sheep research farm of Bangladesh Livestock Research Institute (BLRI), Savar, Dhaka, Bangladesh.The average age and body weight of the animals were 25 days and 2.5 kg respectively.Of the bacteria responsible for diarrhoea in case of sheep, Escherichia coli alone was found in 6 samples (30%), Escherichia coli combined with Proteus mirabilis was found in 12 samples (60%), and no bacteria could be detected in 2 of the 20 samples tested.Both the bacteria were highly sensitive to ceftriaxone, and moderately sensitive to ciprofloxacin and gentamicin.It could be stated that ceftriaxone is the antibiotic of first choice for the treatment of diarrhoea in sheep, where Escherichia coli is suspected to be the principal causal agent of diarrhoea.

INTRODUCTION
Diarrhoea characterized by an increased frequency, fluidity, or volume of faecal excretion is a major problem in the farm animals.It is caused by a wide range of microbial, parasitic and environmental factors namely-bacteria, virus, helminths, protozoa, toxic substances, sands, lush pasture, overfeeding, overcrowding, poor sanitation, inadequate intake of colostrum, poor quality colostrum, poor quality milk replacers, feeds difficult to digest, etc.On the basis of literature the bacterial agents causing diarrhoea in sheep are Escherichia coli, Salmonella spp.and Clostridium perfringens, where Proteus spp., and some other species of bacteria may be associated in some cases (Rahaman, 1995).
According to Hindson and Winter (2002), a combination of reduced reabsorption from the lumen of the intestine, and increased fluid loss through damaged mucosa into the intestine is found in many forms of both gastroenteritis and inflammatory change in the lower intestine.Such a condition ranges from bacterial infections such as E. coli and Salmonella to parasitic diseases such as coccidiosis and parasitic gastroenteritis.Diarrhoea or enteritis, to a greater extent, is related with clostridial diseases.These clostridial diseases of sheep have been recognized clinically for over 200 years, but not until the end of the nineteenth century did their bacterial nature start to be unraveled, a process that continued over the next 50 years (Sterne, 1981).Even during the 1990s, new information came to light as the importance of Clostridium sordellii as a cause of abomasitis and enteritis in all ages of sheep was established (Lewis and Naylor, 1998).The haemorrhagic enteritis affecting lambs in the first few days of life is caused by C. perfringens types (either beta or beta 2 toxin) or C, and differs from lamb dysentery only in the gross pathology and being marginally less acute, affecting lambs up to 3 weeks of age (Lewis, 2007).
E. coli is a major cause of diarrhoea in calves, piglets and lambs, and the term 'colibacillosis' is commonly used.It causes huge economic loss in this age group of animals (Radostits et al., 2000).However, selection of suitable antibiotics is a good strategy for successful treatment of bacterial diarrhoea.But all antibiotics are not always useful for the treatment of diarrhoea because of development of antibiotic resistance.So, the present study was carried out to identify the bacterial species in the faecal samples of diarrhoeic sheep and to observe their sensitivity to different antibiotics.

MATERIALS AND METHODS
This investigation was performed in the bacteriological laboratory of Bangladesh Livestock Research Institute (BLRI) by collecting diarrhoeal samples from the sheep (n=20) in BLRI goat and sheep research farm.The average age and body weight of the animals were 25 days and 2.5 kg, respectively.The duration of the experiment was from August, 2010 to May, 2011.

Sample collection
The faecal samples were collected aseptically into stool containers directly from the rectum with the help of small polybags.

Laboratory tests for identification of bacteria
The collected samples were then allowed for culture in bacteriological media (nutrient agar, EMB agar, SS agar, Blood agar, Mac Conkey agar), Gram's staining and biochemical tests (Methyl Red test, Indole test, Catalase test, etc) for isolation and identification of the bacterial species.

Antibiotic sensitivity test
The antibiotic sensitivity test was done by using disc diffusion method (Cowan and Steel, 1965).The commercially available antibiotic discs such as, oxytetracycline, gentamicin, amoxycillin, penicillin G, cloxacillin, sulphamethoxazole, streptomycin, ciprofloxacin and ceftriaxone were used to know the sensitivity of Escherichia coli and Proteus mirabilis to these drugs.

RESULTS AND DISCUSSION
The isolates were identified as Escherichia coli on the basis of morphology (Gram negative rod), cultural characteristics (Green metallic sheen on EMB agar as in fig. 1) and biochemical characteristics and Proteus mirabilis on the basis of morphology (Gram negative rod), cultural characteristics (Swarming growth on Mac Conkey agar as in fig. 2 and Brilliant Green Agar) and biochemical characteristics.Among the different kinds of bacteria responsible for diarrhoea in sheep, only Escherichia coli was found in 6 samples (30%), Escherichia coli alone combined with Proteus mirabilis was found in 12 samples (60%), and no bacteria were found in 2 of the 20 samples tested (Table 1).
Both the bacteria were highly sensitive to ceftriaxone, and moderately sensitive to ciprofloxacin and gentamicin (Table 2).The inhibition zones, in case of ceftriaxone, were 29 mm for Escherichia coli and 27 mm for Proteus mirabilis while these zones were 17 mm for Escherichia coli and 21 mm for Proteus mirabilis in case of ciprofloxacin and 17 mm for both Escherichia coli and Proteus mirabilis in case of gentamicin (Table 2).These bacteria were found resistant to other antibiotics used in the sensitivity test (Table 2).Wang et al. 2014 noticed decreased susceptibility of P. mirabilis in Taiwan to some broad spectrum antibiotics, including 3rd-generation cephalosporins and ciprofloxacin, whereas Kwiecińska-Piróg et al. 2013 found that in most of the tested concentrations, ciprofloxacin was more efficient than ceftazidime against the P. mirabilis biofilm.They became able to prove that the efficiency of antibiotics against P. mirabilis biofilm depends on its maturity and strains' origin.
Bacterial resistance to a particular antibiotic might occur due to use of that antibiotic for a longer period of time.The present study revealed that E. coli isolates were resistant to several antibiotics like penicillin G, amoxycillin, cloxacillin, streptomycin and oxytetracycline.These findings are in support of Ershaduzzaman et al. 2007, Tadesse et al. 2012. But Islam et al. 2007 found streptomycin as a sensitive antibiotic to E. coli which contradicts the present findings.However, this might happen due to strain variation of E. coli.
In this investigation the highest sensitivity was recorded for ceftriaxone because it is a new generation of cephalosporin and has not been used by the physician for long time.The interpretation was done as resistant (≤10 mm), less sensitive (11-14 mm), moderately sensitive (15-18 mm) and highly sensitive (≥19 mm) except penicillin G, where zone of inhibition for resistance range is ≤28 mm according to Kirby-Bauer Method.

CONCLUSION
In conclusion, it might be stated that ceftriaxone is the antibiotic of first choice, and ciprofloxacin and gentamicin are the antibiotics of second choice to be used for the treatment of diarrhoea in sheep, where Escherichia coli is suspected to be principal causal agent of diarrhoea.

Table 1 .
Prevalance of bacteria found in the diarrhoeal samples of sheep

Table 2 .
Inhibition zones produced by antibiotics used against Escherichia coli and Proteus mirabilis in the sensitivity test. N.B.: