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Isolation and Molecular Detection of Pasteurella Multocida Type a from Naturally Infected Chickens, and Their Histopathological Evaluation in Artificially Infected Chickens in Bangladesh

Sayedun Nahar Panna
Department of Microbiology and Hygiene, Bangladesh Agricultural University Mymensingh-2202, Bangladesh.

K. H. M. Nazmul Hussain Nazir
Department of Microbiology and Hygiene, Bangladesh Agricultural University Mymensingh-2202, Bangladesh.

M. Bahanur Rahman
Department of Microbiology and Hygiene, Bangladesh Agricultural University Mymensingh-2202, Bangladesh.

Sultan Ahamed
Department of Microbiology and Hygiene, Bangladesh Agricultural University Mymensingh-2202, Bangladesh.

Md. Golam Saroare
Business Development Officer, Chars Livelihoods Program, Bangladesh

Shovon Chakma
One Health Epidemiology Fellow, Massey University, New Zealand

Tazrin Kamal
Department of Microbiology and Hygiene, Bangladesh Agricultural University Mymensingh-2202, Bangladesh.

Ummay Habiba Majumder
Department of Microbiology and Hygiene, Bangladesh Agricultural University Mymensingh-2202, Bangladesh.

Abstract/ Executive Summary:

Pasteurella multocida type A is the etiologic agent of fowl cholera, a highly contagious and fatal disease of chickens. The present research work was performed for the isolation, identification and molecular detection of P. multocida Type A from chickens. Liver, heart and spleen of suspected dead chicken (n=35) were collected from Gazipur and Pabna districts in Bangladesh. The targeted bacteria from the samples were isolated, identified and characterized based on their morphology, staining, cultural, biochemical characters, pathogenicity test, histopathological study and Polymerase Chain Reaction (PCR). The P. multocida organism was isolated from 11.42% (n=4/35) samples. The organisms were gram negative, non-spore forming rod, non-motile, occurring singly or pairs in Gram staining, whereas in Leishman’s stain, bipolar shaped organisms were observed. All the isolates were found positive for oxidase and catalase tests, produced indole, and fermented glucose, mannitol and sucrose. Necrotic foci in liver and congestion with hemorrhages in heart were found on necropsy. After pathogenicity test, the pathological changes were reconfirmed by histopathology depicting congestion, hemorrhage and lymphocyte infiltration in heart, liver and spleen tissues. In type specific PCR reaction, the organisms were confirmed as P. multocida Type A. In conclusion, P. multocida type A is prevalent among poultry in the studied regions; thus, care must be taken to control of the disease.

Keywords: Chicken, Fowl cholera, Histopathology, Pasteurella multocida type A, PCR

Location: Gazipur and Pabna districts of Bangladesh.

Start Date: 00-00-0000

End Date: 00-00-0000

Program Area: Pest Management

Commodity: Chicken, Chicken

Objectives:

To identification and molecular detection of P. multocida Type A from naturally infected chicken.

Methodology:

This study was conducted in two different areas namely Gazipur and Pabna districts of Bangladesh. The study areas were selected based on the report of FC outbreak during the study period. Clinical specimens such as heart, liver and spleen of FC suspected chickens (n=35) were randomly collected. The samples were collected as per the standard guidelines, with prior permission from the owners. After collection, the samples were transported to Bacteriology Laboratory at the Department of Microbiology and Hygiene, Bangladesh Agricultural University (BAU). P. multocida organisms were cultured according to the standard method described by Cowan. In brief, the samples were inoculated separately into different culture media such as Blood agar (BA), Nutrient agar (NA) and Nutrient broth (NB) for screening of the samples. The inoculated media were incubated at 37^oC for the appearance of characteristic colony. Based on the colony characteristics, subsequent selective subculture was done to obtain pure culture of P. multocida. The isolated pure culture was subjected for Gram staining and Leishman’s staining for morphological identification of the bacteria. For biochemical characteristics, different tests such as Methyl red (MR), Voges-Proskauer (VP), Indole production, catalase, oxidase, and sugar fermentation tests were done. NA slants were used to maintain stock culture. For the maintenance, the P. multocida bacteria were inoculated in slant by streaking and were incubated at 37^oC overnight. Finally, sterile mineral oil was overlaid and kept the slant at room temperature for future use Crude DNA was obtained from the isolates using boiling method mentioned by Queipo-Ortun et al. with slight modification. Briefly, the organisms were cultured onto NA at 37^oC for overnight aerobically. Then, a loopful of bacteria was picked up by swiping and mixed in 200 μL of deionized water. The mixture was then heated in boiling water for 10 min followed by dipping into ice for 10 min and centrifugation was done at 13,000 rpm for 10 min. The supernatant was collected and stored at -20ºC until used. athogenicity was observed by giving challenge infection with virulent P. multocida Type A. The challenge was done by following the guidelines set by the ethics committee of the Department of Microbiology and Hygiene, BAU. Unvaccinated chickens (n=3) were challenged with virulent P. multocida Type A isolate. The challenge dose containing 2.93x108 CFU/0.5 mL was administered through intramuscular route. Isolation of bacteria following challenge was performed according to the procedures suggested. After 24 h of challenge, the chickens were sacrificed, and swab or tissue materials were taken from liver, lungs and spleen. Immediately after collection, the samples were streaked onto BA and NA plates, and were incubated at 37^oC overnight. The positive cases were reconfirmed by re-isolation of P. multocida Type A by following the standard procedures described earlier . The collected samples (heart, liver and spleen) from the artificially infected chickens were collected and were sent to the Department of Pathology, BAU for histopathological study. The formalin-fixed liver, heart and spleen samples from chickens were trimmed, processed, sectioned and stained following the standard hematoxylin and eosin (H&E) staining techniques.

Source of Information: J. Adv. Vet. Anim. Res., 2(3): 338-345.

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

Isolation and identification of Pasteurella multocida was successfully done from naturally infected chickens. The bacteria were subsequently confirmed to be P. multocida Type A, for the first time in Bangladesh. Histological examination of livers and hearts of chickens revealed the changes consistent with this bacterium. Thus, the bacteria are present in chickens in the study areas of Bangladesh, which needs necessary attention for effective control of this disease.

Knowledge Management: Journal