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Molecular Detection of Pasteurella Multocida Type B Causing Haemorrhagic Septicemia in Cattle and Buffaloes of Bangladesh

MS Ara
Department of Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

MT Rahman
Department of Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

M Akhtar
Department of Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

M Rahman
Department of Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

KHMNH Nazir
Department of Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

S Ahmed
Department of Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

ML Hossen
Department of Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

MFR Khan
Department of Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

MB Rahman
Department of Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Abstract/ Executive Summary:

Hemorrhagic septicemia (HS) is an acute septicemic disease that primarily affects cattle and buffaloes. The disease is caused by Pasteurella multocida sero types B:2 and E:2. The objective of this study was to isolate P. multocida from clinical cases and to confirm its identity using polymerase chain reaction (PCR) based approach. Clinical samples of two suspected cases of haemorrhagic septicemia of cattle and buffalo from Mymensingh and Rajshahi districts respectively were collected. Two isolates were isolated from these suspected cases and primarily identified as P. multocida based on morphological study, staining properties, and cultural and biochemical characteristics. The isolates were confirmed initially as P. multocida at genus level by PCR using genus specific primers. Later, the isolates were confirmed as P. multocida type B, the causal agent of haemorrhagic septicemia, by PCR with primers specific for P. multocida type B. These isolated organisms can be used as vaccine candidate for the production of effective vaccine against haemorrhagic septicemia.

Keywords: Haemorrhagic Septicemia, Cattle, Buffalo, Pasteurella multocida type B, PCR

Location: Bacteriology laboratory of the Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh.

Start Date:

End Date:

Program Area: Animal Health and Management

Commodity: Buffalo, Cattle

Objectives:

To isolate and identify accurately P. multocida from suspected cases of HS by molecular technique i.e., PCR, so that the isolates could later be used for the development of effective vaccine to control the diseases in Bangladesh.

Methodology:

Isolation and identification of Pasteurella- For the isolation of P. multocida edematous fluid was collected from the throat of buffaloes of Rajshahi (N=1) and cattle of Mymensingh (N=1) regions that showed characteristics signs of HS. The samples were immediately transported to the Bacteriology laboratory of the Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh. Isolation and identification of P. multocida was carried out based on morphology, staining, cultural and biochemical characteristic, as described by Cheesbrough (2006). Polymerase Chain Reaction (PCR) for Pasteurella multocida type B- Conformation of the isolated organisms as P. multocida type B, the causal agent of haemorrhagic septicemia in buffaloes and cattle were done based on PCR as described by Townsend et al. (1998), Panna et al. (2015) and Akhtar et al. (2016). Initially PCR was carried out to confirm the isolate as Pasteurella spp. using the primer KMT1T7 5′-ATC- CGC-TAT-TTA-CCC-AGT-GG-3′ and KMT1SP6 5′-GCT-GTA-AAC-GAA-CTC-GCC-AC-3′. This was then followed by conformation of the isolates as P. multocida type B using the specific primers pairs KTT72 5′-AGG-CTC-GTT-TGG-ATT-ATG-AAG- 3′ and KTSP61 5′-ATC-CGC-TAA-CAC-ACT- CTC-3′. Briefly, for PCR bacterial DNA was first extracted using Wizard genomic DNA Purification Kit (Promega, USA) according to the instruction of the manufacturers to use as PCR template. Extraction of DNA and its quality was checked by running 5 μL suspension of the extracted DNA in a 1% (w/v) agarose gel. All the PCR was done in a final 25 μL volume containing 12.5 μL PCR mastermix (Promega, USA) , 1 μL of each primer (10 pmol), PCR grade water 8.5 μL and DNA template 2 μL. The thermal profile used for the Pasteurella genus specific PCR was performed as follows with slight modification from Townsend et al. (1998): initial denaturation at 94°C for 5 min, followed by 30 cycles of denaturant at 94°C for 1 min, annealing at 49°C for 1 min, and elongation at 72°C for 1 min and finally a final extension at 72°C for 9 min. The thermal profile used for the P. multocida type B specific PCR was also done as per protocol described by Townsend et al. (1998) with slight modification: initial denaturation at 95°C for 5 min, followed by 30 cycles of denaturant at 95°C for 1 min, annealing at 49°C for 1 min, and elongation at 72°C for 7 min and finally a final extension at 72°C for 9 min. Following the completion of PCR, 5 μL PCR products were loaded into 2% agarose gel (w/v) along with 1 μL 6X loading dye for electrophoresis in 1X TBE buffer at 100 V for 35 min. A standard 100 bp DNA ladder (Promega, USA) was also loaded in the same gel to check the size of the amplified PCR products. Prior to casting the gel, ethidium bromide (0.5 μg/mL) was added to the gel. The PCR products were visualized under UV light in an image documentation system (Bio Rad, USA).

Source of Information: Progressive Agriculture 27 (2): 175-179, 2016

Article Link (Online Journal):

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

P. multocida has been isolated from the filed cases of haemorrhagic septicemia of cattle and buffalo in Bangladesh. Besides, molecular identification of the isolated organism as P. multocida type B is confirmed by PCR. The isolate can be used in effective vaccine development.

Knowledge Management: Journal