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R esearch
(Published online:
15-09-2015)
8.
Rapid detection of Mannheimia haemolytica in lung tissues
of sheep and from bacterial culture -
Jyoti Kumar, Shivendra
Kumar Dixit and Rajiv Kumar
Veterinary World, 8(9): 1073-1077
doi:
10.14202/vetworld.2015.1073-1077
Jyoti
Kumar:
Division of Animal Health, Central Sheep and Wool Research
Institute, Avikanagar - 304 501, Rajasthan, India; jyotivet@gmail.com
Shivendra Kumar Dixit:
Division of Animal Health, Central Sheep and Wool Research
Institute, Avikanagar - 304 501, Rajasthan, India; shivendradixit@yahoo.co.in
Rajiv
Kumar: Animal Biotechnology Section, Central Sheep and Wool
Research Institute, Avikanagar - 304 501, Rajasthan , India;
rajivbiotech028@gmail.com
Received: 10-01-2015, Revised: 02-07-2015, Accepted: 09-07-2015,
Published online:15-09-2015
Corresponding author:
Jyoti Kumar, e-mail: jyotivet@gmail.com
Citation:
Kumar J, Dixit SK,
Kumar R (2015) Rapid detection of Mannheimia haemolytica in
lung tissues of sheep and from bacterial culture, Veterinary
World 8(9): 1073-1077.
Abstract
Aim:
This study was aimed to detect
Mannheimia haemolytica in lung
tissues of sheep and from a bacterial culture.
Introduction: M. haemolytica
is one of the most important and
well-established etiological agents of pneumonia in sheep and
other ruminants throughout the world. Accurate diagnosis of
M. haemolytica
primarily relies on bacteriological
examination, biochemical characteristics and, biotyping and
serotyping of the isolates. In an effort to facilitate rapid
M. haemolytica
detection, polymerase chain reaction
assay targeting Pasteurella haemolytica
serotype-1 specific antigens (PHSSA),
Rpt2 and 12S ribosomal RNA (rRNA) genes were used to detect
M. haemolytica
directly from lung tissues and from
bacterial culture.
Materials and Methods: A total of 12 archived lung tissues
from sheep that died of pneumonia on an organized farm were used.
A multiplex polymerase chain reaction (mPCR) based on two-amplicons
targeted PHSSA and Rpt2 genes of M.
haemolytica were used for
identification of M. haemolytica
isolates in culture from the lung
samples. All the 12 lung tissue samples were tested for the
presence M. haemolytica
by PHSSA and Rpt2 genes based PCR and its
confirmation by sequencing of the amplicons.
Results: All the 12 lung tissue samples tested for the
presence of PHSSA and Rpt2 genes of M.
haemolytica by mPCR were found
to be positive. Amplification of 12S rRNA gene fragment as
internal amplification control was obtained with each mPCR
reaction performed from DNA extracted directly from lung tissue
samples. All the M. haemolytica
were also positive for mPCR. No
amplified DNA bands were observed for negative control reactions.
All the three nucleotide sequences were deposited in NCBI GenBank
(Accession No. KJ534629, KJ534630 and KJ534631). Sequencing of the
amplified products revealed the identity of 99-100%, with
published sequence of PHSSA and Rpt2 genes of
M. haemolytica
available in the NCBI database. Sheep
specific mitochondrial 12S rRNA gene sequence also revealed the
identity of 98% with published sequences in the NCBI database.
Conclusion: The present study emphasized the PCR as a valuable
tool for rapid detection of M.
haemolytica in clinical
samples from animals. In addition, it offers the opportunity to
perform large-scale epidemiological studies regarding the role of
M. haemolytica
in clinical cases of pneumonia and other
disease manifestations in sheep and other ruminants, thereby
providing the basis for effective preventive strategies.
Keywords: lung tissues,
Mannheimia haemolytica,
multiplex polymerase chain reaction,
Pasteurella haemolytica
serotype-1 specific antigens, Rpt2, 12S ribosomal RNA, sheep.
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