Sample collection
A total of 100 fecal samples were aseptically collected from sick ducks with clinical signs of duck cholera (showing high morbidity and mortality, depression, anorexia, mucoid discharge from the mouth, ruffled feathers, yellow to greenish watery diarrhea, increased respiratory rate, etc.) during suspected duck cholera outbreaks in Kishoreganj district, Bangladesh during the period from January to December, 2009. Freshly defecated fecal samples were collected using sterile cotton swabs in a view to prevent extraneous contamination. The collected samples will then put into sterile test tubes containing nutrient broth. Immediately after collection the samples were transferred to the Laboratory of Microbiology & Hygiene, Bangladesh Agricultural University, Mymensingh, Bangladesh by using ice box. The samples were then preserved in refrigerator before cultivation.
Isolation and Characterization of P. multocida
The collected samples were processed as per the procedure of Cheesbrough (2006). For isolation and identification of P. multocida organisms, the procedures suggested by Cowan (1985) were followed throughout the experiment. Briefly, the processed samples were inoculated into nutrient broth followed by inoculation onto blood agar media from the broth culture. The inoculating media was incubated at 370C in bacteriological incubator for characteristic colony formation. Subsequent subculture was done for getting pure culture. Stock cultures were maintained in both Agar slant and 20% sterile buffered glycerin (Merchant and Packer, 1967).
The isolated organisms were identified based on colonial morphology by Gram’s staining technique, Leishman's staining technique and motility test (Kumar et al., 2004). For biochemical characterization Carbohydrate fermentation tests, catalase test, Methyl Red test, Voges Proskauer test, Indole test, oxidase test, nitrate reduction test, citrate utilization test and H2S production test were performed according to standard laboratory procedures (Bhattacharya, 2005).
Antibiotic sensitivity tests
Antibiotic sensitivity tests were performed using disc diffusion test of the method described by Kirby-Bauer (Bauer et al., 1966). Commercially available antimicrobial discs (Becton, Dickinson and Company, USA) were used for the determination of the drug sensitivity. The concentration of antimicrobial agent per disc was: Azithromycin (15 μg), Tetracyclin (30 μg), Penicillin G (10 μg), Amoxycillin (10 μg), Ciprofloxacin (5 μg), Ampicillln (10 μg), Erythromycin (15 μg) and Gentamycin (10 μg). For this purpose, at first broth cultures were prepared from stock cultures using nutrient broth. Then, 01-02 ml of freshly growing broth cultures were poured on Blood agar plate and spread uniformly. Antibiotic discs were placed apart onto the surface of the inoculated plates aseptically with the help of a sterile forceps and incubated at 37°C for 24 hours. After incubation, the plates were examined and the diameters of the zone of inhibition were measured and were interpreted with the standard diameters of NCCLS, (1999) and recorded as Sensitive (S), Intermediate sensitive (I) and Resistant (R).