MOLECULAR DETECTION AND CHARACTERIZATION OF CLOSTRIDIUM PERFRINGENS TOXIN GENES RESPONSIBLE FOR NECROTIC ENTERITIS IN POULTRY OF ANDHRA PRADESH
Loading...
Files
Date
2008-04
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA
Abstract
ABSTRACT:
The present study was carried out to investigate the prevalence of Clostridium
perfringens toxin genes responsible for necrotic enteritis in poultry of Andhra Pradesh.
A total of 691 faecal samples (396 necrotic enteritis suspected and 295 apparently
healthy) comprising of cloacal swabs from live birds and intestinal scrapings from dead
birds were collected from different districts of Andhra Pradesh viz., Chittoor, Guntur,
Nellore, Krishna, East Godavari and West Godavari. Gross pathological studies of
affected birds revealed necrosis of the small intestinal mucosa and submucosa with
fibrin deposition resulting in pseudo membrane formation and turkish towel appearance
was noticed in the small intestine. Microscopically lumen of intestine with
fibrinonecrotic material which forms a visible pseudo membrane composed of cell
debris, necrotic/distorted villi, inflammatory cells and clumps of bacteria were
observed. The samples were inoculated in to fluid thyoglycollate broth and incubated
overnight. DNA extracted from 24 h broth cultures by boiling method were screened by
multiplex PCR for simultaneous detection of α, β and β2 toxin genes. Out of 396
(broiler 282 & layer 114) necrotic enteritis suspected samples 337/396 (85.1%) were
positive for α toxin gene of which 189/337 (56.08%) were β2 toxin gene positive. Out
of 295 (broiler 182 & layer 113) apparently healthy samples 61/295 (20.67%) were
positive for α of which 4/61 (6.55%) were β2 positive. None of the sample showed
amplification of β toxin gene indicating the absence of C. perfringens type C. As some
recent studies focused the involvement of NetB toxin in pathogenesis, therefore, uniplex
PCR amplification of NetB gene was done from alpha toxin gene positive samples (C.
perfringens type A) yielded no positives for NetB toxin gene. From chi square analysis
a significant difference (p<0.01) in the prevalence of toxin genes (cpa & cpb2) between
necrotic enteritis suspected and apparently healthy at 99% level of confidence with an
increased number of positives from necrotic enteritis suspected group. The present
research indicates C. perfringens type A along with β2 toxin gene might be responsible
for causing necrotic enteritis in broilers and layers in Andhra Pradesh.
The multiple sequence analysis of the amplified partial cpa and cpb2 gene
sequences revealed 100% homology between the present isolates, and with selected
published sequences from GenBank were found to be 98-99% and 94-99% homology
respectively. The phylogenetic analysis of the cpa gene of the present C. perfringens
isolate (MG600591) with the selected published sequences of cpa revealed the close
segregation in distinct clade with cpa gene of broiler isolate of C. perfringens
(GU581186) from Iran. The phylogenetic analysis of three cpb2 sequences of present
isolates (MF471364; MF471366; KX001813) segregated into close group of poultry
originated sequences with exception of MF471365 which segregated in distinct clade
with noporcine originated C. perfringens sequence (AY609173) from USA.
Since alpha toxin gene (cpa) is considered as signature toxin gene for C.
perfringens, amplification of cpa by PCR is considered as confirmative diagnosis of C.
perfringens. Hence, in the present study all the PCR positives for cpa (n=398) were
isolated by culturing revealed only 221/398 (55.52%) isolates indicating PCR is more
sensitive in detecting C. perfringens when compared with isolation by culturing.
In the present study culture supernatant of B. subtilis isolated from healthy
intestinal contents of birds successfully inhibited C. perfringens by disk diffusion
method indicating its importance as a probiotic in controlling necrotic enteritis in
poultry.
Description
THESES
Keywords
null