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Research Article
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Molecular Characterization and Toxinotyping of a Clostridium perfringens Isolate from a Case of Necrotic Enteritis in Indian Kadaknath Fowl |
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P. Thomas,
T.R. Arun,
K. Karthik,
P.V. Berin,
M. Asok Kumar,
Neetu Singh,
J. Usharani,
M. Palanivelu,
S.K. Gupta,
K. Dhama
and
K.N. Viswas
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ABSTRACT
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Necrotic enteritis, caused by Clostridium perfringens, is an important
bacterial disease of poultry. A suspected case of necrotic enteritis was presented
for necropsy from an Indian Kadaknath Fowl flock showing diarrhea and progressive
debility. Gross examination revealed necrotic to ulcerative lesions in intestine.
The organism was isolated from the intestinal contents, tissue and liver under
anerobic conditions. The cultural characteristics and Gram staining were suggestive
of C. perfringens. The sequencing of 16s rRNA gene confirmed the isolate
as C. perfringens and which was well differentiated from other clostridia
associated with avian intestinal tract. This study demonstrates that 16s rRNA
gene sequencing can provide rapid and confirmatory identification of C. perfringens.
Further, Multiplex Polymerase Chain Reaction (mPCR) was performed for toxinotyping
and isolate was found to be positive for α toxin (cpa) and β2
toxin (cpb2), a feature of C. perfringens type A isolates. As
some recent studies have highlighted the involvement of NetB toxin in pathogenesis,
therefore, PCR was carried out to find the presence of this toxin, the isolate
was found to be negative for netB gene. This study emphasizes the molecular
characterization and toxinotyping as a rapid tool for detection of C. perfringens
from suspected necrotic enteritis cases. Very few reports regarding molecular
characterization are available from India, hence it adds to the available data
on this important poultry pathogen. Further investigations are required to understand
the exact role of NetB toxin in pathogenesis as various studies including the
current one reports NetB negative strains involved in necrotic enteritis.
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How
to cite this article:
P. Thomas, T.R. Arun, K. Karthik, P.V. Berin, M. Asok Kumar, Neetu Singh, J. Usharani, M. Palanivelu, S.K. Gupta, K. Dhama and K.N. Viswas, 2014. Molecular Characterization and Toxinotyping of a Clostridium perfringens Isolate from a Case of Necrotic Enteritis in Indian Kadaknath Fowl. Asian Journal of Animal and Veterinary Advances, 9: 385-394.
DOI: 10.3923/ajava.2014.385.394
URL: https://scialert.net/abstract/?doi=ajava.2014.385.394
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Received: January 05, 2014;
Accepted: April 17, 2014;
Published: June 11, 2014
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INTRODUCTION
India ranks first in livestock population which contributes 4% to total Gross
Domestic Product (GDP) and 27% to agriculture (DAHDF, 2012).
Reproductive traits, short productive lifespan, production of good quantity
of eggs and worldwide distribution, favour the use of poultry as a major source
of animal protein which maximized the growth of poultry industry in the last
two decades. For the growing human population there is much demand for good
quantity and quality protein (Godfray et al., 2010).
To meet this demand, rearing of poultry by intensive system is practised in
most part of the world but which leads to major disease outbreaks, even in indigenous
breeds of chickens like Kadaknath, well known for its disease resistance ability.
Most alarming among all the diseases is the one which affects the gastrointestinal
tract because of production losses, high mortality and risk of contamination
of poultry products for human consumption (Dahiya et
al., 2006). Necrotic enteritis is a sporadic disease of avian species
which has been reported from most areas of world in which poultry are reared
(Dahiya et al., 2006). Implementation of legislative
restriction on usage of growth promoting antibiotics in animal feed and reduction
of using antimicrobial growth promoters due to concern of spreading antimicrobial
resistance imparted the increased prevalence of diseases such as necrotic enteritis
(Van Immerseel et al., 2009).
Etiological agent of Necrotic enteritis is Clostridium perfringens type
A and to a lesser extent the type C (Cooper and Songer,
2009), an anaerobic gram positive, spore forming, ubiquitous organism found
in the gastrointestinal tract of healthy birds also. Predisposing factors for
this condition includes changes in diet, poor hygienic conditions, stress, mucosal
damage causing parasites such as Coccidia and the presence of other pathogenic
C. perfringens strains. Outbreaks occur most frequently in chickens around
2-6 weeks of age when the maternal antibodies ebbed around 2 weeks before attaining
maturity of the chicken immune system. Occurrence in broilers up to 11 weeks
of age, 3-6 month old commercial layers, 12-16 week replacement pullets have
also been reported (Malmarugan et al., 2012).
There is also a report in older birds (9 month) from India in 1974. Alpha toxin
is considered as the major virulence factor responsible for producing lesions
in necrotic enteritis. But alpha toxin null mutant was also equally able to
produce disease as compared to wild strains (Keyburn et
al., 2008). Recently, a novel pore forming toxin has been described,
Perfringens necrotic enteritis B-like toxin (NetB). The netB gene
is located on an approximately 85 kb plasmid (Lepp et
al., 2010), associated with pathogenesis causing by damage to host cells.
Clinical necrotic enteritis is manifested in two forms, classical acute form
characterised by sudden increase in flock mortality without any predictive signs,
albeit wet litter is an early indicator of disease. The course of disease is
often peracute indicated by death within 1-2 h, sometimes mortality may raise
up to 50% (Helmboldt and Bryant, 1971; Riddell
and Kong, 1992). The subclinical form is more significant than clinical
form due to persistence of disease in flocks without any noticeable clinical
signs and mortality. Chronic intestinal damage produced by this form of disease
results in poor digestion and absorption, reduced weight gain and increased
feed conversion ratio which ultimately leads to poor production performance
(Elwinger et al., 1992; Kaldhusdal
et al., 2001).
Present study reports disease investigation of an outbreak of deadly necrotic enteritis in a poultry farm of Kadaknath fowl, the isolation and identification of the causative agent (Clostridium perfringens), pathology, molecular detection by Polymerase Chain Reaction (PCR) and multiplex PCR, molecular characterization, 16s rRNA gene sequencing, phylogenetic analysis and toxinotyping of the Clostridium perfringens isolate. MATERIALS AND METHODS
Clinical history and necropsy: An adult (8-10 weeks of age) female Kadaknath
chicken carcass, representing the desi breed flock, was brought for necropsy
examination and diagnosis to Division of Pathology, Indian Veterinary Research
Institute, Izatnagar. History revealed that the affected chicken flock from
an organized farm in Bareilly, Uttar Pradesh suffered from coccidiosis a month
ago and were treated accordingly. A few birds in the same flock showed signs
consisting of inappetence, diarrhoea and progressing debility. Necropsy was
carried out for the fowl presented. Samples were collected aseptically which
included intestinal contents, pieces of liver and intestinal scrapings for bacteriological
examination. These samples were also processed for histopathological studies.
Isolation and identification: Isolation and identification of the causative agent of Necrotic Enteritis (NE) was carried out in the laboratory. Sterile saline was added to the clinical materials such as intestinal contents, liver pieces and scrapings collected from the bird with NE suspected lesions. These were heated at 80°C for 20 min in a water bath. Then the processed samples were initially inoculated in Robertson cooked meat medium and were incubated anaerobically at 37°C for 24 h. The positive cultures were streaked on to blood agar with neomycin antibiotic and incubated in the anaerobic jar at 37°C for 48 h. Grams staining was carried out for the recovered bacterial isolates. Again, the positive colonies were streaked in egg yolk agar and were kept in the anaerobic jar at 37°C for 48 h.
DNA isolation from the culture: DNA was extracted from the bacterial
culture obtained as per the protocol of Wilson (1987).
Briefly, bacterial culture of 48 h was transferred to 2 mL collection tube and
centrifuge at 12,000 rpm for 5 min for pelleting of bacteria. The pellet was
suspended in lysis buffer i.e., 200 μL of 1X TE (pH 8.0), 50 μL 10%
SDS and 2 μL proteinase K, vortexed and incubated overnight at 37°C.
Next day, 200 μL of 5 M Nacl, 100 μL of 10% CTAB (Cetyl Trimethyl
Ammonium Bromide) was added and incubated at 68°C for 10 min in water bath.
Then equal amount of chloroform, isoamyl alcohol (24:1) was added, vortexed
and centrifuged at 10,000 rpm for 10 min. The supernatant was transferred to
a new microcentrifuge tube and an equal volume of phenol:chloroform, isoamyl
alcohol (25:24:1) was added, vortexed and centrifuged at 10,000 rpm for 10 min.
Again supernatant was transferred to a new microcentrifuge tube, then 0.6 volumes
of 99% isopropanol was added and incubated at room temperature for 1 h, followed
by centrifugation at 10,000 rpm for 20 min. The pellet was washed with 70% ethanol,
centrifuged at 10,000 rpm for 5 min. The supernatant was discarded and pellet
air dried at 37°C in an incubator for 15-20 min. The pellet was resuspended
in 30 μL of elution buffer and stored at -20°C.
Amplification of 16s rRNA gene using universal primers: The C. perfringens
suspected DNA was amplified with 16s rRNA universal primer. Briefly, PCR was
carried out in 25 μL reaction mixture containing 10X Dream Taq PCR buffer
(MBI Fermentas), 0.2 mM dNTP mixture, 1.25 units Dream Taq DNA Polymerase (MBI
Fermentas), 10 pmol of each universal primers SR-FWD 50-AGAGTTTGATYMTGGC-30
(positions 4-19) and SR-REV 50-GYTACCTTGTTAC GACTT-30 (positions 1505-1488)
as reported by Davies et al. (1996). Agarose
gel electrophoresis was carried out to see the results and gel extraction using
QIA quick Gel extraction kit was carried out using manufacturers instruction.
The eluted PCR product was submitted for sequencing to Eurofins, Bangalore,
India for gene sequencing.
Phylogenetic analysis of 16s rRNA sequence: The 16s rRNA sequence was
compared with sequences available in the GenBank (NCBI) database using the Basic
Local Alignment Search Tool (BLAST). The sequence was compared with the 16s
rRNA gene sequences of pathogenic avian Clostridia viz., C. colinum,
C. fallax, C. baratti producing enteritis in chicken (Cooper
et al., 2013) and also with commensal Clostridia associated
with avian gastrointestinal tract viz., C. polysaccharolyticum, C.
xylanolyticum, C. aurantibutyricum, C. glycolicum, C.
leptum, C. lituseburense, C. saccharolyticum, C. thermocellum
and C. propionicum (Zhu et al., 2002)
using MEGA6 software (Tamura et al., 2013).
The evolutionary history was inferred using the Neighbor-Joining method (Saitou
and Nei, 1987) and the evolutionary distances were computed using the Jukes-Cantor
method (Jukes and Cantor, 1969).
Toxinotyping of the isolate: Multiplex Polymerase Chain Reaction (mPCR)
targeting the four major toxin genes namely cpa, cpb, etx
and iap, the enterotoxin gene cpe and all variants of cpb2
was carried out for toxinotyping the Clostridium perfringens isolate
obtained using the primers described by Van Asten et
al. (2009). Agarose gel (1.5%) electrophoresis was carried out to visualize
the results after amplification by PCR. Similarly, to know the presence of netB
gene in the isolate, PCR was carried out targeting the netB gene primers
as reported by of Tolooe et al. (2011).
RESULTS On gross examination, the affected birds of the poultry farm under investigation were found weak and emaciated. Systematic necropsy examination revealed gross pathological changes predominantly in the intestinal segments. The intestinal wall appeared thickened, dark and discoloured to bluish tint. Upon opening the lumen, the mucosa showed nearly circumscribed to oval or elongated shaped necrotic to ulcerative lesions with raised and hyperemic borders (Fig. 1a). The lesions were more severe in duodenum, jejunum followed by ileum, cecum and colo-rectum. No pathologically significant gross lesions in other organs were observed. Histopathology revealed thickened serosal and muscular layers. The submucosa showed varying degrees of degenerative to necrotic changes amidst infiltration by mononuclear cells. The mucosa was characterized by superficial epithelial denudation to complete loss of villi in the necrotic areas (Fig. 1b). The duodenum in presented case consistently presented hyperplasia of submucosal glands, mononuclear cell infiltration and mild fibroplasia (Fig. 1c). Although, no significant pathological changes were observed in the liver on gross examination, histopathology revealed foci of hepatocyte necrosis and severe perivascular infiltration predominated by mononuclear lymphocytic cells (Fig. 1d) followed by a few heterophils. Bacteriological and biochemical examination were matching characteristics of C. perfringens. On blood agar, the bacterial colonies were small, dew drop like and had double zone of haemolysis (Fig. 2). In egg yolk agar, opalescence was observed confirming the presence of alpha toxin of C. perfringens. Grams staining revealed gram positive bacterial rods.
Amplification of 16s rRNA gene yielded expected product size of 994 bp (Fig.
3) and its sequencing and phykigenetic analysis revealed that the isolate
belonged to Clostridium perfringens type A and evolutionarily related
to C. baratti and C. fallax among Clostridium species associated
with avian gastrointestinal tract (Fig. 4). Multiplex PCR
for toxin typing of Clostridium perfringens was positive for α toxin
(cpa) and β2 toxin (cpb2), a feature of C. perfringens
type A isolates (Fig. 5). The C. perfringens isolate
was found to lack netB toxin gene as revealed by netB gene specific
PCR testing.
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Fig. 1(a-d): |
(a) Necrotic to ulcerative lesions in
the mucosa intestine showing raised and hyperemic borders (arrows), (b)
Mucosal ulcer showing epithelial denudation to complete loss of villi
(stars), (c) Hyperplastic submucosal glands amidst mononuclear lymphoid
infiltration as aggregates (4-point star) in the duodenum and (d) Section
of liver showing infiltration of lymphoid cells in the perivascular space
(4-point star) |
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Fig. 2: |
Blood agar plate showing colonies with double zone of haemolysis |
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Fig. 3: |
16s rRNA PCR amplification: Lane 1: 100
bp ladder (BR biochem life sciences), 2 and 3: Positive sample (replicate) |
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Fig. 4: |
Phylogenetic analysis of Clostridium perfringens isolate |
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Fig. 5: |
Gel electrophoresis results of toxin
typing of Clostridium perfringens by Multiplex PCR: Lane 1: 50
bp ladder (BR biochem life sciences), 2 and 5: Negative control (replicate),
3 and 4: Positive sample (replicate), 6: Positive control |
DISCUSSION
Clostridium perfringens, the causative agent of necrotic enteritis in
poultry, was isolated and identified from a clinical case of 8-10 weeks old
Kadaknath fowl from an organized farm in Bareilly, Uttar Pradesh. Similar findings
that necrotic enteritis can occur between 7-16 weeks of birds were reported
by earlier workers (Frame and Bickford, 1986; Malmarugan
et al., 2012). Grams staining of intestinal scrapping revealed
Gram positive bacterial rods along with oocyst of Eimeria spp., which
was correlating with the previous report (Malmarugan et
al., 2013). The causative agent of necrotic enteritis, C. perfringens,
though is a commensal organism in healthy poultry but can take upper hand when
there is breach in the gut wall and immunity which may be due to infections
like coccidiosis (Williams, 2005). In the present study,
the poultry flock investigated was recently affected with coccidiosis and hence
due to the damage caused by Eimeria, C. perfringens might have
flared up.
Gross pathology revealed gas filled, dilated, thin walled, friable intestine
with yellowish brown diphtheritic membrane which was in accordance with earlier
reports by Malmarugan et al. (2012). Histopathological
changes revealed partial to complete loss of villi in the necrotic areas, there
was hyperplasia of submucosal glands, mononuclear cell infiltration and mild
fibroplasias. Similar histopathological changes were reported by Van
Immerseel et al. (2009). Bacterial collagenases were thought to be
the reason for the abundant damage caused in the intestine (Olkowski
et al., 2008).
Bacterial colonies on the blood agar showed double zone of haemolysis and these
were small, dew drop like which were similar to the findings reported by Craven
et al. (2001). The 16s rRNA sequence analysis showed that the sequence
was similar to Clostridium perfringens type A and the phylogenetic tree
based on the 16s rRNA sequence revealed that the isolate was evolutionarily
related to C. baratti and C. fallax but was well differentiated
from the sequences studied, indicating that 16s rRNA and gene sequencing can
be employed as an alternate tool for confirmative diagnosis of C. perfringens
type A. Multiplex PCR for toxin typing of C. perfringens was positive
for α toxin (cpa) and β2 toxin (cpb2), a feature of
C. perfringens type A isolates.
The isolate obtained was tested for the presence of netB gene by PCR
amplification but the result revealed that the isolate was negative for netB.
Recent studies have indicated that netB gene is a virulence factor for
the causative of necrotic enteritis in chicken (Keyburn et
al., 2008, 2010). NetB toxin producing gene was
found on a large plasmid (Lepp et al., 2010).
Different studies have suggested various opinions regarding the role played
by NetB toxin in necrotic enteritis (Chalmers et al.,
2008; Martin and Smyth, 2009). NetB toxin positive
isolates have been recovered from healthy birds also and similarly NetB negative
isolates also got recovered from diseased birds, indicating that necrotic enteritis
may be a multifactorial condition (Timbermont et al.,
2011). Further studies are required to know the exact role played by NetB
toxin and also to find other toxin(s) that are actually the cause of necrotic
enteritis as in the case of NetB negative isolates.
CONCLUSION The present study reports the isolation and identification of Clostridium perfringens, causing necrotic enteritis which is an important bacterial disease of poultry, from a farm of Kadaknath fowl in India. It is a thorough disease investigation report based on clinical pathology, molecular detection of C. perfringens by PCR and multiplex PCR and its molecular characterization by 16s rRNA gene sequencing, phylogenetic analysis and toxinotyping. The utility of molecular detection, characterization and toxinotyping needs to be explored for rapid detection and confirmation of C. perfringens from suspected necrotic enteritis cases. Further studies are also suggested to find out the role of toxins in the development of necrotic enteritis in poultry.
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