Ethical approval
The experiment was approved by Institutional Animal Ethics Committee, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh.
Study period
This study was conducted during the period ranging from July to December 2013.
Collection of samples
Sick (n=6) and dead (n=19) birds manifesting the characteristics clinical signs of colibacillosis (watery diarrhea, weakness, anorexia and weight loss, etc.) were collected from five poultry farms in the Mymensingh and Tangail districts. Feces samples of sick birds were collected aseptically in sterile test tubes. Sick birds were euthanized by intracardiac administration of saturated MgSO4 solution. A thorough post-mortem examination of all dead birds was carried out. Liver and spleen samples were collected separately in sterile test tubes. Feces, liver, and spleen samples were used for the bacteriological study. Age (broiler 25-30 days and 31-35 days old and layer 40-45 days and 46-50 days old), chicken line and location of the farms were recorded.
Isolation of bacteria in pure culture
Samples were enriched in nutrient broth at 37°C for 24 h. The overnight bacterial broths were streaked onto eosin methylene blue (EMB) agar and incubated at 37°C for 24 h. A single colony was further sub-cultured until a pure culture was obtained.
Identification of bacteria
Colony characteristics of bacteria such as shape, size, surface texture, edge, elevation and color observed in pure culture, Gram’s-staining and biochemical tests (sugar fermentation, methyl red, Voges Proskauer and Indole production tests) were used for identification of bacteria.
Molecular detection of E. coli by PCR
DNA extraction A pure bacterial colony was mixed with 100 µl of distilled water which was boiled for 10 min then immediately kept on ice for cold shock. Finally, centrifugation was done at 10,000 rpm for 10 min. The supernatant was collected and used as DNA template for PCR.
Primers used for PCR
A genus-specific PCR was performed to amplify 16S rRNA of E. coli using previously published primers.
Antibiotic sensitivity test
Three isolates randomly selected from the samples were tested for antimicrobial drug susceptibility against five commonly used antibiotics such as ampicillin (10 µg/disc), chloramphenicol (30 µg/disc), ciprofloxacin (5 µg/disc), gentamicin (10 µg/disc), and cefalexin (30 µg/disc) by disk diffusion or Kirby-Bauer method. Results of antibiotic sensitivity tests were recorded as sensitive and resistant according to the guidelines of Clinical and Laboratory Standards Institute.
Statistical analysis
The prevalence of colibacillosis in broiler and layer at different age groups of bird and poultry farm compared for statistical significance using Chi-square test (Duncan’s multiple range test, SPSS 11.5, UK). Ap≤0.05 was considered to be statistically significant.