Z. B. Muktha*
Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh
S. M. L. Kabir
Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh
M. T. Rahman
Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh
Isolation, Identification, Bacterial flora
Department of Microbiology & Hygiene, Bangladesh Agricultural University (BAU), Mymensingh-2202
Animal Health and Management
Horse, Diseases
The experimental work was divided into three steps. The first step included selection of sources, collection of samples, isolation, identification and characterization of respiratory bacterial flora on the basis of their colony morphology, staining property, biochemical and PCR characteristics. The second step included determination of prevalence of isolated bacterial flora. The third step included the current status of drug sensitivity and resistance pattern of isolates of respiratory bacterial flora from nasal swab of healthy horses. The research work was conducted in the Department of Microbiology & Hygiene, Bangladesh Agricultural University (BAU), Mymensingh-2202,during the period of July 2014 to December 2014. A total of 18 apparently healthy horses from different area of Mymensingh were selected for collection of nasal swab sample. All samples were collected directly from the respiratory system (nasal swab) of healthy horse into nutrient broth aseptically. The samples were carried to the laboratory in an ice box contained ice and processed for the isolation and characterization of bacteria subsequently. Bacteriological media for culture Commercially available synthetic media used in this experiment for bacteriological analysis were Blood agar (BA), Nutrient agar (NA), Mannitol salt agar (MS), Eosin Methylene Blue agar (EMB), Salmonella Shigella agar (SSA) etc. The following liquid media used for the study were Nutrient broth (NB), MethylRed and Voges –Proskauer broth (MR-VP broth) and different Sugar Media (dextrose, maltose, lactose, sucrose and mannitol). Sugars and reagents for biochemical tests Dextrose (LOBA Chemic Pvt. Ltd., India), Sucrose (Wako, Japan), Lactose (Merc, England), Maltose (Techno Pharma., India), Mannitol (Beximco Pharma., Germany), Methyl Red and Voges-Proskauer broth (MR-VP broth) (Difco, USA), Peptone water and Phosphate buffer solution were used. A commercial PCR master mix (2X master mix, Promega, U.S.A) was used to detect E. coli molecularly. Antibiotic sensitivity disc To perform this test, two types of bacteria isolated in this study were tested individually against 5 antibiotics. (ciprofloxacin, gentamycin, erythromycin, amoxycilin, ampicilin) based on common usefulness for the therapeutic purpose in the field and on the spectrum of their activity to determine the degree of antibiotic sensitivity to each of bacteria. Gram’s staining: Gram’s staining was done as per the methods described by Merchant and Packer (1967) to determine the morphological features of bacteria. Biochemical test: The carbohydrate fermentation test was carried out using suspected two isolates by inoculating a loop full of nutrient broth culture of the organisms into the tubes containing five basic sugars e.g., galactose,maltose, sucrose, mannitol,and glucose and incubated at 37°C for 24 hrs. Acid production was indicated by change of color from reddish to yellow in the medium. Indole test Two ml of peptone water was inoculated with five ml of bacterial culture and incubated at 37°C for 48 hrs.Kovac's reagent 0.5 ml was added, mixed well and examined after one min. Positive test was indicated by the development of red color. Methyl-Red and Voges-Proskauer (MR-VR) test: These two tests were carried out following the routine procedures as described by others. Catalase test: One ml of 3% H2O2 was run down the slope and examined immediately and after 5 mins for the evaluation of gas production. Positive test indicated by the production of bubbles. Coagulase test: For the coagulase test, 0.5 ml of rabbit plasma was diluted with sterile physiological saline (1:5) separately in two different test tubes containing an equal volume of 24 hours old isolated cultured broth and incubated at 37°C for 4 hours. The tubes were examined after 2-4 hours for detecting the presence of clots of plasma. The negative tubes were left at room temperature for over night and then re-examined. A simple slide coagulase test was also performed by mixing an equal volume of freshly cultured broth with rabbit plasma on a glass slide. A positive result was indicated by macroscopically clumping of the bacterial cells within five seconds because fibrinolysin enzyme lysis the rabbit plasma. Pathogenic Staphylococci showed coagulase-positive reaction whereas nonpathogenic Staphylococci showed negative result. In coagulase test, fibrinogen convert to fibrin and formation of clotting occur. DNA extraction A pure bacterial colony of E. coli from EMB agar was mixed with 200 µl of deionized water which were boiled for 10 minutes then immediately kept on ice for cold shock for 10 minutes. Finally centrifugation was done at 10000 rpm for 10 minutes. The supernatant were collected and used as DNA template for PCR. Molecular detection of E. coli by PCR-- Preparation of a PCR mixture PCR mixture (25 µl) was prepared as follows: i) Nuclease free water =5.5 µl ii) 2X PCR master mixture (Promega, USA) = 12.5 µl iii) Forward primer = 1 µl iv) Reverse primer =1 µl v) DNA template =5 µl
J. Bangladesh Agril. Univ. 13(2): 239–246, 2015
Journal