Abstract: The present research was undertaken to isolate and identify the etiological agents responsible for diarrhoea in goats. For this purpose, faecal samples were cultured and subcultured in basic medium named Nutrient agar medium and Blood agar medium to obtain pure cultures of separate genera. From pure cultures, bacterial colonies were picked up and further cultured in different selective medium viz. MacConkey agar medium, Bacto fluid thioglycollate broth, Cooked meat broth, Brain-heart infusion broth, Eosin methylene blue agar medium and Salmonella-Shigella agar medium to observe the cultural characteristics of the isolates. In different selective medium, bacterial genera were identified on the basis of their cultural morphology (in case of solid media) and change of color and gas production (in case of liquid media). By Gram staining, short, single or paired gram positive and gram negative bacteria were observed under the electric microscope in the smears prepared from the pure colonies from basic and selective medium. Finally Biochemical tests viz., Catalase, Voges-Proskauer, oxidase, citrate utilization, Coagulase, Oxidation-fermentation, Carbohydrate fermentation test were performed to confirm the specific bacterial genera. Of the 20 experimented faecal samples, Salmonella sp. (5.0%), Staphylococcus sp. (10.0%), Escherichia coli (25.0%), Bacillus sp. (85.0%) and Clostridium sp. (65.0%) were identified as single or mixed infection. 5.0% diarrhoea were found non-bacterial infection. Therefore the present study provides opportunities for the characterization of such bacteria as well as for the prevention of diarrhoea and thus help in goat farming.
Introduction
Diarrhoeal disease seems to be one of the major community health hazards both for men and animals in most countries of the world. It is resulted from the enteritis, which is the inflammation of the intestinal mucosa, characterized by abdominal pain, loose faeces, increase in stool mass, stool frequency, vomiting tendency, or stool fluidity (dehydration) that contain 70-95% water. More than 14 L of fluid may be lost per day in severe cases of diarrhoea. The chronic form of diarrhoea may last for days or week and may culminate in death (Radostits et al., 1995).
Caprine diarrhoea occurs world wide in goats of any age. In Bangladesh, Diarrhoeal disease remains the most often reported clinical problem in goats. Goat diarrhoea is responsible for poor growth in kids and a significant loss of production both through morbidity and mortality. Some enteropathogens like bacteria, viruses, protozoa and helminthes have been recognized to be associated with diarrhoea (Radostits et al., 2000). This disease has been reported from UK, USA, Canada, Argentina, France, Australia, New Zealand, Papua New Guinea, South Africa, Switzerland, Iran, India, Algeria, Pakistan, China and Japan. Reports on diarrhoea associated with enteropathogens are very much limited from Bangladesh. Here, it may be mentioned that, research work on diarrhea in goat has not been undertaken earlier in Bangladesh. Therefore, an attempt was made to isolate and identify the enterobacteria from goat diarrhea.
Since 1992, goat rearing has become seriously impaired due to high mortality with diarrhoea like symptoms. The marginal and land-less farmers most easily live on rearing of goats in Bangladesh. So, goat is called the poor man’s cow that is the second important livestock in Bangladesh which plays an important role in the rural economy and we can earn substantial amount of foreign currency by exporting skin and other by-products. The estimated goat population in Bangladesh in 1997-98 was 33.50 millions, which was 33.331 millions in 1996-97 and growth rate of goat is about 8.2% yearly whereas cattle growth rate is about 0.80%. The numbers of goat farm at the private sector were 5584 in 1997-98 (Amin, 1998). So, above information indicates the goat population in our country is increasing day by day.
At present, Asian and other under developing countries, agriculture means crop and cereal production and relatively less importance is given to livestock health and production. As a result, deficiencies of proteins become an unsolved problem. Therefore, enhancement on small ruminants production especially goat in the framework of small holder agriculture assume the great significance. So, present study will be helpful toward goat rearing and enhance dynamism of goat farming which not only alleviate the poverty but also boost up the national economy. Considering all above-mentioned points, the present work was designed with the following objectives:
| • | Isolation and identification of the bacterial agents through different selective culture media and biochemical tests. |
| • | To find out the actual bacterial agents responsible for goat/kid diarrhea so that appropriate regulatory actions are taken. |
Materials and Methods
The research work was carried out at Bacteriology Laboratory of Animal Health Research Division (AHRD), Bangladesh Livestock Research Institute (BLRI), Savar, Dhaka, Bangladesh during October, 2005 to January, 2006. Diarrhoeic goats were collected from Bangladesh Livestock Research Institute’s goat farm.
Collection of Samples
The isolates of bacteria used in the present study belonged to 20 goats
(Bengal goats, Jamunapari) aged between one month to three years which were
noticed to have diarrhoea. Faecal samples were selected as experimental sample.
Samples were collected randomly from those goats that were noticed Diarrhoeal
sign. Table 1 represents collected samples and their place
and date of collection.
For the purpose of sampling, sampling kit consisting of several sterile plastic bags, a marking pen, spatula and alcohol were taken. For isolation and identification, faecal samples were collected from the diseased goats directly from the rectum by using sterile plastic gloves. About 20 g of faeces were collected, kept in a polythene bag and tagged. At each time of collection hands were sterilized with alcohol (95%) and the bags were partially tilled with material. Then the bags were properly tied and labeled. Special care was always taken to avoid contamination as far as practicable.
Preparation of Culture Media
Commercially available media were used during this study. Different media
were prepared according to the direction of the manufacturers for the culture
and subculture of the organisms for their proper isolation and identification.
| Table 1: | Collected faecal samples and their place, sex, age, size and date of collection |
Parasitological Examination of the Collected Samples by Direct Microscopy
Each of the faecal samples were examined on conventional direct smear method
and followed by sedimentation methods to detect the parasitic eggs which were
identified by their morphological features as described by Samad (2001).
Observation of Bacterial Growth in Different Culture Media
All the collected diarrhoeic faecal samples were examined for isolation
and identification of bacteria. Samples were streaked on different agar medium,
incubated at 37°C in aerobic incubator and in anaerobic incubator (containing
5% CO2) for 24-48 h or sometimes 72 h.
Isolation of Colonies
From each petri dish plate, culture was subcultured in Nutrient agar and
Blood agar medium and kept in aerobic and anaerobic condition respectively.
Subcultures were repeated on both agar plate by streak plate method (Cheesbrough,
1985) until the colonies were considered the pure separately by visually and
also by microscopically.
Final Selection of Isolates
The colonies were selected according to their size, shape, color, height,
smoothness, margin and other main properties. Regarding to the above properties,
colonies that possessing the highest positive signs were selected for further
tests.
Purification of the Selected Isolates
For purification, the selected colonies were picked up by platinum loop
and plating on agar plate (Nutrient agar and Blood agar) by streaking method.
Morphological and Cultural Studies for Identification of the Selected Colonies
The faecal samples were cultured in NA medium in aerobic incubator at 37°C
for 24-48 h. Samples were also cultured in BA medium (5% Sheep blood) in anaerobic
incubator (5% CO2) at 37° for 48 h. The isolates were also cultured
on different agar medium and inoculated in different broth. After incubation,
the colonies showed different characteristics. For observing cultural characteristics,
discrete colonies on the agar surfaces were selected to study their shape, size,
consistency and color.
Preparation for Microscopic Examination
For microscopic examination, many methods are available to prepare the slides
and here fixed smear method was applied.
Preparation of the Smear
A portion of bacterial culture was taken out by a sterilized loop and smeared
on a slide and a very thin film was made which was allowed to dry in air. The
smear was fixed by slightly heating the slide over a sprit lamp.
Staining
For the observation of the presence of the organisms (bacteria) and also
to study their cellular morphology, selected isolates were stained. From the
entire medium, isolates were collected and stained with Gram’s staining
method as per recommendation of Cowan (1985).
Biochemical Studies of the Selected Isolates
According to the procedure described by Buxton and Fraser (1977-78), the
biochemical tests viz., Catalase, Voges-Proskauer, Oxidase, Citrate utilization,
Oxidation-fermentation and Coagulase tests were performed. Carbohydrate fermentation
test was performed with five basic sugars (Dextrose, Sucrose, Lactose, Maltose
and Mannitol) and thereby production of acid or gas or both.
Results and Discussion
Microscopic Studies and Staining Properties of the Bacterial Isolates
Bacteria were identified on the basis of colony shape, arrangements and
their ability to persisting safranine color in gram staining test (Table
2).
Cultural Characteristics
The cultural characteristics of bacterial isolates were observed on the
basis of pigmentation, form, margin and elevation. Cultural colony characteristics
of the bacteria isolated from the diarrhoeic goats are presented in Table
3.
Results of Parasitic Examination
Different microscopic slides were prepared by smearing method from the isolates.
No parasitic egg was found. This may be due to the goats were treated with anthelminthic
drugs just 14 days back.
Haemolytic Activities of the Isolated Organisms
In sheep BA medium, the haemolytic activities of five isolates of each colony
of sample no. 1623, 74, 1390, 1627 and 379 from NA medium were tested for haemolysin
production of which 6 (75.0%), 5 (62.5%), 7 (87.5%), 6 (75.0%) and 7 (87.5%)
isolates were found positive respectively which were to able for haemolysin
production, respectively (Table 4).
| Table 2: | Microscopic studies and staining properties of the bacterial isolates |
| Table 3: | Cultural colony characteristics of the bacteria on different media |
| Circular (Unbroken peripheral edge), Entire (sharply defined, even), Raised (Slightly elevated), Uniform fine turbidity (finely dispersed growth throughout the solution), Flocculent (flaky aggregates dispersed throughout the solution) | |
| Table 4: | Results of haemolytic activities of the isolates on BA medium |
Biochemical Tests
The biochemical characteristics of bacterial isolates were observed on the
basis of broth color, or gas formation, or clot formation. The different isolates
of the organisms showed identical results in different biochemical tests. The
actual causes for which the manifestation of more or less identical result in
biochemical tests by the five groups of known identified isolates were not clear.
It is thought that all isolates in the present study possess some common genetic
materials which are responsible for the manifestation of similar type of biochemical
reaction as reported by Pandey et al. (1979) and Hodna et al. (1982).
Biochemical characteristics of the bacteria isolated from the diarrhoeic goats
are presented in Table 5.
Carbohydrate Tests
The carbohydrate characteristics of the bacteria isolated from the diarrhoeic
goats are presented in Table 6.
Oxidation-fermentation Properties of Bacteria
All isolates of 1623, 74, 1390 and 1627 were found positive for fermentation
whereas isolates of 379 showed both fermentative and oxidative properties (Table
7).
Provisional Identification of Selected Bacteria
Table 8 Contains the provisional identification of selected
isolates obtained from diarrhoeic goat’s faecal samples. The main etiological
agents were Salmonella sp., Staphylococcus sp., E. coli,
Bacillus sp. and Clostridium sp.
The present study includes isolation and identification of bacteria recovered from faecal samples of diarrhoeic goats of Bangladesh Livestock Research Institute’s goat farm. Most of the clinical signs and bacterial isolates that are recorded in the present study are correlated with the study in calves by the authors like Chakrabarty et al. (1980), Chattopadhyay and Harbola (1988), Baldassi et al. (1995) and Uzal et al. (1996).
Of the 20 diarrhoeic goat faecal samples examined, 19 (98%) had enterobacterial infection, of which 1 sample (5.0%) were found positive for Salmonella spp., 2 samples (10.0%) for Staphylococcus sp., 5 samples (25.0%) for E. coli, 17 samples (85.0%) for Bacillus sp., 13 samples (65.0%) for Clostridium sp. and 1 sample (5.0%) were found negative for bacterial infection.
| Table 5: | Results of biochemical characteristics of the selected isolates |
| (+) sign indicates positive reaction, (-) sign indicates negative reaction | |
| Table 6: | Results of Carbohydrate characteristics of the Isolates |
| (+) sign indicates positive reaction, (-) sign indicates negative reaction | |
| Table 7: | Oxidation-fermentation properties of bacteria isolated from diarrhoeic goats |
| (+) Sign indicates positive reaction, (-) Sign indicates negative reaction | |
| Table 8: | Provisional identification of selected isolates |
Conclusion
Bengal goats and Jamunapari are potential and economic livestock of Bangladesh. A large number of goat populations are decreasing due to diarrhoea in every year. This study was undertaken to identify the main causal agents of diarrhoea and it was found that Salmonella sp., Staphylococcus sp., E. coli, Bacillus sp. and Clostridium sp. play a vital role for goat diarrhoea. Results of this study will help to develop an effective treatment method of goat diarrhoea against those bacteria.