Introduction: Helicobacter pullorum is one best-known species belonging to the group of urease-negative enterohepatic helicobacter species (EHC). This gram-negative bacterium has been isolated from different sources, namely humans, animals, water, and raw meat, highlighting the precise role of this agent as an emerging foodborne human pathogen. Conventional culture method using membrane filter is one of the most common methods for detecting H.pullorum from poultry cecum. However, the actual contribution of this fastidious microorganism in pathological processes is hindered by difficulty in cultivating and the use of unsuitable culture media. Accordingly, the present study aimed to evaluate a useful protocol for isolation H.pullorum from poultry cecum.
Methods: A total of 15 cecal samples, comprising five broiler chickens, five turkeys, and five laying hens were collected from an abattoir in Semnan city. As for the conventional method, 200 mg of each sample homogenized in 400 µl mixture containing 7.5 g glucose, 25 ml BHI broth, and 75 ml of inactivated horse serum. Then, 300 µl of each sample was placed on Brucella agar containing 5% sheep blood and selective supplement with 0.45 µm cellulose filter membrane. After incubation for 1h at 37 0C under microaerobic atmosphere with H2 , the filter was removed and the plate was incubated under the same conditions. As for the enrichment method, 200 mg of each sample homogenized in 225 ml of Bolton broth with selective supplement and lysed horse blood for selective pre-enrichment. The samples were incubated at 420C for 48h under microaerophilic conditions with hydrogen. Afterwards, 100 µl of the mixture were applied onto a 0.45 µm cellulose filter membrane and placed directly on Brucella agar with 5% sheep blood and selective supplement. This plate was incubated aerobically at room temperature for 15 min; the membrane was then removed and the plate incubated under hydrogen-enriched microaerophilic atmosphere for 48h at 420C. All statistical analyses were performed with spss 22.0 and p<0.05 was considered as statistically significant level.
Results: The suspected colonies were subjected to PCR assay for final confirmation if they were gram-negative, spiral shaped, oxidase and catalase positive, and urease negative. The results of the present study explicitly illustrated that a significantly higher (p<0.05) isolation rate of H.pullorum positives was obtained with enrichment method (15/15, 100%) than with conventional method (9/15, 60%).
Conclusion: In general, our study puts forward a highly beneficial technique for isolation H.pullorum from poultry cecum by the combination of enrichment broth and cellulose filter membrane, preferably 0.45 pore size onto a Brucella containing 5% sheep blood and selective supplement in an atmosphere enriched with H2. This finding emphasizes the need for implementation this method to improve recovery and selectivity rate and also alleviate contamination in isolation of H.pullorum from poultry cecum.