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Isolation and Characterization of Bacteria from Keratoconjunctivitis Affected Cattle with Their Pathogenicity and in Vitro Antibiotic Sensitivity

M. Rahamatunnabi
Department of Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

M. R. Ali
Department of Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

M. A. Islam
Department of Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

M. T. Hossain
Department of Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Abstract/ Executive Summary:

Isolation and characterization with antibiotic sensitivity of the bacteria in the eye swab of 34 keratoconjunctivitis affected cattle were carried out on the basis of their morphology, staining, cultural and biochemical properties during the period from October 1999 to March 2000. Staphylococcus spp. (76.5%), Streptowuus spp. (38.2%), E. coli (52.9%), Bacillus spp. (70.6%), unidentified Gram positive cocci (5.9%) and unidentified Gram negative rods (20.6%) were identified as a single or mixed infection. Pathogenicity study of these isolated organisms showed conjunctivitis associated with Staphylococcus spp., Streptococcus spp., Bacillus spp. and E. coli in experimentally inoculated mice whereas unidentified Gram positive cocci did not show any conjunctivitis in mice. The mixed intraocular inoculation of these isolated bacteria produced severe keratoconjunctivitis within 24 hours both in mice and calves. Results of antibiotic sensitivity test showed that all types of bacterial isolates were found highly sensitive to oxytetracyclin (80-100%) and chloramphenicol (70-100%). StaphyloctJccus spp., Streptacoccus spp. and Bacillus spp. were moderately susceptible to streptomycin (69.56%), trisulfa (60%) and trisulfa (80%) respectively. It may be concluded that among the tested antibiotics, oxytetracyclin may have the preference in clinical therapy of keratoconjunctivitis in cattle caused by bacteria in Bangladesh.

Keywords: Isolation, Characterization, Bacteria, Keratoconjunctivitis, Pathogenicity, Antibiotic sensitivity

Location: Central Cattle Breeding Station and Dairy Farm (CCBSDF), Savar, Dhaka; Bangladesh Agricultural University (BAU) Veterinary Clinic,

Start Date: 00-10-1999

End Date: 00-03-2000

Program Area: Animal Health and Management

Commodity: Cattle

Objectives:

This study was undertaken for the isolation and identification of bacterial pathogens from clinical cases of keratoconjunctivitis in cattle and to characterize their pathogenicity in mice and calves and in vitro sensitivity to antibiotics.

Methodology:

Eye swab samples: An eye swab sample from each of 34 randomly selected severely infected different aged groups of cattle was collected by using sterile. cotton and screw capped tube from Central Cattle Breeding Station and Dairy Farm (CCBSDF), Savar, Dhaka; Bangladesh Agricultural University (BAU) Veterinary Clinic, Bangladesh Agricultural Un Culture of swab samples: Each swab sample was divided and inoculated separately in nutrient agar (NA) and blood agar (BA) media to promote the growth of bacteria. The aerobic culture plates were incubated at 37°C for 72 hours and the anaerobic culture plates were incubated at 37°C for 2-7 days. The colonies on primary cultures were repeatedly subcultured by streak plate method (Cheesbrough, 1985) until the pure culture with homogenous colonies were obtained. Media such as nutrient agar (NA), sheep blood agar (SBA), eosin methylene biue (EMB) agar, MacConkey agar (MA) and Chocolate agar (CA) were used for subcultures. Haemolytic patterns of the bacteria were categorized according to the type of haemolysis they produced on SBA (Carter, 1986). Identification of bacterial isolates: The bacterial isolates were identified by their cultural, morphological and biochemical characters. For the cultural characteristics, discrete colonies on the agar surface were observed. Their shape, size, consistency and colour were observed. Gram stained slides of the isolates were examined microscopically to study their cellular morphology. The biochemical tests were performed as fermentation with five basic sugars (dextrose, maltose, sucrose, lactose and mannitol) and thereby production of acid or acid and gas, IMViC utilization test, catalase and coagulase .tests. Individually isolated colonies of the same morphology were selected from appropriate agar plates, cloned and checked for purity of growth prior to characterization of the respective genera and species. Characterization into respective genera and species were done on the basis of morphological, cultural and biochemical reactions. The classification and specification of organisms were based on the scheme presented in Bergey's Manual of Systemic Bacteriology (Halt et al., 1985). Catalase test: The test was performed as described by Cowan (1985). To perform the test a 3.0 ml of 3% hydrogen peroxide solution was poured into a test tube. A confluent growth of test organism was immersed into the solution by mixing with a sterile glass rod. Bobbles of oxygen will be seen by the accumulation on the wall of the glass rods if the organisms are catalase producer. Coagulase test: The test was performed as described by Carter (1986). For this test l-2 drops of diluted rabbit plasma was mixed with an equal volume of freshly cultured broth of a particular organism on a slide and examined under microscope for the occurrence of any coagulation. IMViC utilization test: Motility indole urea medium (MIU), Bacto MR-VP medium, Koser's citrate medium are commonly used for this test following the procedures described by Cowan (1985). The IMViC bacteria must reveal the following characteristics such as indole production from tryptose, methyl red for acid production from glucose, and Voges-Proskauer for the production of acetone from glucose, and ability of the positive bacteria to utilize citrate as the sole source of carbon. Carbohydrate fermentation test: The production of acid or acid and gas was tested for Staphylococcus spp., Streptococcus spp., E. coli, Bacillus spp., unidentified Gram positive cocci and unidentified Gram negative rods using five basic sugars namely dextrose, sucrose, maltose, lactose and mannitol. The carbohydrate test was performed by inoculating a loop-full organisms into the tubes containing different sugar media and incubated for 24 hours at 37°C. Acid production was indicated by the change of the phenol red to yellow colour and accumulation of gas in the inverted Durham's tube indicated gas production. Maintenance of stock culture: The stock culture was maintained following the procedures of Carter (1979). Nutrient agar slants used for the maintenance of stock culture for each of the bacterial isolate. After growth of the organism in the slant, the sterile mineral oil was overlaid and the culture was kept at room temperature for use as seed.

Source of Information: Bangl. J. Vet. Med. (2005). 3 (1): 25-31

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

The results of isolation, identification, biochemical test, frequency distribution, pathogenicity and antibiotic sensitivity of the bacteria isolated from cattle affected with keratoconjunctivitis in the present study indicated that five different types of bacteria are responsible for keratoconjunctivitis in cattle of Bangladesh. This experiment also suggests that there might be some mutual exchange of virulent factors between the organisms which ultimately helps to aggravate the infection in cattle.

Knowledge Management: Journal