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IJAVMS, Vol. 6, Issue 4, 2012: 241-249 doi: 10.5455/ijavms.126
Necrotic Enteritis in Broiler and Layer Farms in Tamil Nadu, India
Malmarugan, S., Boobalan, A and Dorairajan, N
Department of Veterinary Microbiology, Veterinary College and Research Institute, TamilNadu
Veterinary and Animal Sciences University Namakkal – 637 002, Tamil Nadu, India
Corresponding Author’s email: ([email protected])
Rec.Date: Aug 12, 2011 09:04
Accept Date: Dec 24, 2011 19:02
Abstract
Necrotic enteritis caused by Clostridium perfringens is an acute disease chickens,
producing high mortality. Twenty Clostridium perfringens isolates were isolated from intestinal
contents of thirty five NE suspected birds from commercial poultry farms in India. Out of the
twenty, fourteen were isolated from commercial broilers of 2 to 6 wk of age and six from
commercial layers of 9 to 15 wk of age. Robertson cooked meat medium with Brain Heart
Infusion broth was found to be effective for isolation and the isolates produced appreciable
luxuriant growths on clostridial agar and rough and black colonies with sulphate reduction on
perfringens agar with supplements. The isolates were identified based on haemolysis on sheep
blood agar, stormy clot fermentation on milk medium, opalescence on egg yolk medium and
fermentation of sugars such as glucose, maltose, lactose and sucrose. Alpha toxin specific PCR
revealed that all the isolates were alpha toxin producing strains of C. perfringens. Antibiogram
pattern of isolates revealed cent per cent sensitivity to lincomycin and bacitracin
Key Words: Poultry -Necrotic enteritis – Isolation – Identification- Alpha toxin specific PCR
Antibiogram.
Introduction
Necrotic enteritis (NE) is an enterotoxaemia, caused by types A and C of the enteric
gram-positive bacterium Clostridium perfringens 1. It is characterized by death or necrosis of the
intestinal lining predominantly of the middle and lower small intestine which may be
accompanied by necrosis in caeca and liver in some cases 2. NE usually occurs in 2-6 wk old
broiler chickens raised on litter 3,4,5
. However, an outbreak of NE was recorded in 7 to 16 wk old
cage reared commercial layer pullets 4, 6, 7
or even up to 6 months 8.
Normally, the number of C. perfringens in the intestine is low (about 104 cfu/g of
digesta). The disease occurs when high numbers of bacteria coincide with a damaged intestinal
mucosa. The disturbances in normal intestinal microflora caused by co-factors such as coccidial
infection, removal of coccidiostats or antibiotic growth promoters (AGPs) from poultry feed,
environmental and managemental conditions, physiological stress, immunosuppression and
Malmarugan et al., IJAVMS, Vol. 6, Issue 4, 2012: 241-249
doi: 10.5455/ijavms.126
RESEARCH ARTICLE
NECROTIC ENTERITIS IN BROILER AND LAYER FARMS IN TAMIL NADU
nature of diet may cause rapid proliferation of C. perfringens, increasing bacterial numbers the
range from 107 to 10
9 cfu /g of digesta resulting in toxin production
9,10. Alpha toxin, a
phopholipase C, produced by both type A & C strains of C. perfringens, is considered to be a
major contributing factor towards the development of intestinal necrosis, the characteristic lesion
of NE in poultry8. Now, the NE is emerged as a worldwide problem
11 and it is a common disease
found in all poultry growing areas of the world.
In India the poultry production has played a pivotal role in increasing source of
income and employment generation for the educated unemployed youth. To achieve high levels
of economic efficiency, poultry are raised under intensive system in densely populated flock. Due
to the intensification, the birds face lots of stress, which lead to imbalance in the intestinal
microflora and lowering of body defense mechanisms, making them vulnerable to many diseases.
Among the prevailing pathogens, intestinal pathogens are a major cause of death, disease and
poor performance in poultry. The present research work is mainly focused on isolation,
identification and antibiogram pattern of C. perfringens toxigenic strains in necrotic enteritis
cases in poultry farms in Tamil Nadu, India.
Materials and Methods
Specimens: For isolation of the organism causing necrotic enteritis, thirty five birds suspected for
NE were collected from poultry diagnostic and research centers of M/s. Suguna poultry farm,
M/s. Venkateshwara Hatcheries Limited, Palladam, M/s. Pioneer Hatcheries, Namakkal. Apart
from that, ten commercial farms in and around Namakkal and Udumalpet area from both broiler
and layer farms, where, NE cases were reported.
Necropsy findings: Twenty birds (14 broilers and 6 layers) had lesions of necrotic enteritis. The
findings revealed gas filled, dilated, thin walled, friable intestine with yellowish brown
diphtheritic membrane on the mucosa in middle and distal intestines. The mucosal surface of the
affected area of intestine was covered with a tan orange pseudomembrane. The lesions were of
necrotic enteritis, possibly of Clostridium perfringens etiology. Smears made from intestinal
scrapings revealed high numbers of gram-positive plump rods by Gram’s staining. The
examination of mucosal scrapings also revealed 2-10 oocysts per 10X microscopic field in the
intestines of some chickens. Morphologically, the oocysyts resembled those of Eimeria maxima
and Eimeria acervulina. The mortality in the affected houses was not adjudged to be due to
coccidiosis.
Isolation: For isolation of the organism causing necrotic enteritis, sterile saline was added to the
twenty biomaterials such as intestinal contents and scrapings collected from the birds had NE
lesions and heated at 80o C for 20 min in water bath. Then the processed Intestinal contents were
inoculated into thioglycollate broth, Robertson cooked meat medium with brain heart infusion
broth and sterile liquid paraffin was poured to make a layer over the medium. Inoculated medium
was incubated anaerobically at 37o C for 24h. The presence of C. perfringens in the inoculated
sample is indicated by turbidity in both the media. The positive cultures were streaked on to
clostridial agar and perfringens agar with supplements. The plates were incubated in the
anaerobic jar at 37o C for 48h.
Identification: The following ready-made dehydrated media were prepared as per the
manufacturer’s instruction (M/s. Hi-Media laboratories, Mumbai, India) for identification of the
NECROTIC ENTERITIS IN BROILER AND LAYER FARMS IN TAMIL NADU
C. perfringens, they were viz., Milk medium with reducing agent, Egg yolk agar (EYA) base,
Egg yolk emulsion, Blood agar base and Nutrient broth. Blood agar was prepared with 5%
defibrinated sterile sheep blood.
The positive colonies obtained on clostridial agar and perfringens agar were inoculated
on milk medium with reducing agent, egg yolk agar and sheep blood agar and they were
subjected to biochemical tests for identification. Oxidase discs, phenol red agar base, sugars such
as glucose, maltose, lactose, sucrose and mannitol, materials for catalase, indole and gelatinase
were procured from Hi-Media Laboratories, Mumbai and they were prepared and the biochemical
tests were carried out as per the procedure described by Barrow and Feltham 11
.
The positive colonies obtained on clostridial agar and perfringens agar were inoculated
on milk medium with reducing agent, egg yolk agar and sheep blood agar and they were gram
stained and tested for their ability to ferment glucose, maltose, lactose, sucrose, and mannitol.
The isolates were also subjected to oxidase, catalase, and gelatin liquification tests as described
previously by Barrow and Feltham11
.
Identification of toxigenic strains by Polymerase Chain Reaction (PCR): To design the PCR,
alpha toxin specific primers (CP – F- AGT CTA CGC TTG GGA TGG AA and CP – R- TTT
CCT GGG TTG TCC ATT TC), which flanked 900 base pair DNA sequence, from Baums et al., 12
were used.
To perform the PCR, 2 ul template DNA, prepared by the heat lysis method of Baums et
al., 12
, was added to a 50 ul reaction mixture with the following reagents 1.25 U Taq DNA
polymerase, 50 mM Pottassium chloride, 30 mM Tris-Hcl, 1.5 mM Mg2++
, 200 µM of each
dNTP and 50 picomoles of each primer. The thermocycling (incubations for 1 min at 95oC, 55
oC
and 72oC respectively was 35 times) was preceded by incubation for 2min 30 seconds at 95
oC.
Six microlitre of the amplicons was separated on 1.5% agarose gel according to standard
procedure.
Antibiogram: Antibiotic sensitivity pattern of the isolates was tested in Muller Hinton Agar
(M/s. Hi-Media laboratories, Mumbai, India) by Kirby-Bauer disc diffusion technique. To
prepare the inocula, growth from an 18-24h culture was used. Inoculated MHA plates were
incubated for 24h in the anaerobic jar. The zones of inhibition surrounding the discs were then
measured and interpreted as per the standard
Results and Discussion
The prominent macroscopic lesions found in the small intestines of NE affected birds
during the postmortem examination were in agreement with the observations of Parish 18
and
Kaldhusdal and Hofshagen 19
. Gas filled, dilated, thin walled, friable intestines with yellowish-
brown diphtheritic membrane found in the current study were also observed by other researchers 5,7
. Mucosal surfaces covered with a grey-brown to yellow-green diphtheritic membrane or
pseudomembrane are denoted as ‘dirty turkish towel’ appearance. Similar findings were also
documented by Immerseel et al. 10
NECROTIC ENTERITIS IN BROILER AND LAYER FARMS IN TAMIL NADU
Fig .1. Large, rough and black colonies of Clostridium perfringens with sulphate reduction on
perfringens agar with supplements.
Fig.2. C. perfringens from a culture: short, Gram-positive plumpy rods with blunt ends. (Gram
stain X 1000).
NECROTIC ENTERITIS IN BROILER AND LAYER FARMS IN TAMIL NADU
Fig.3. Milk medium showing the classical stormy clot reaction of three isolates of C. perfringens.
The tube on the left extreme is uninoculated.
Fig. 4. An agarose gel stained with ethidium bromide with PCR amplification products of C.
perfringens isolates. Lane M: 100bp ladder; Lanes 2 to 6: Isolates
In the present study twenty C. perfringens isolates were obtained from thirty five NE
suspected birds. Out of the twenty isolates, fourteen were isolated from commercial broilers of 2
to 6 wk of age, six from commercial layers of 9 to 15 wk of age. These findings correlate with the
reports of detection of C. perfringens in 2-6 wk broiler chickens 5,15
and isolation of C.
perfringens from 7 to 16 wk commercial layer birds 6,8
.
In the present study, smears made from intestinal scrapings revealed the large numbers of
gram-positive rods by Gram’s staining and coccidial pathogens, such as E.maxima and E.
acervulina oocysts by direct microscopic examination as described by Long et al. 5,7
Broussard et
NECROTIC ENTERITIS IN BROILER AND LAYER FARMS IN TAMIL NADU
al. and Cowen et al.16
. Hence, C. perfringens could be isolated from intestinal contents and
scrapings of intestinal wall from birds affected with NE. These findings are in accordance with
Shane et al.17
. Report on Eimeria species that colonize the small intestine, are known to
predispose to necrotic enteritis by Jackson et al., 20
has given additional support to these findings.
In the present study, Robertson cooked meat medium with BHI broth has served as the
best medium for isolation and it was found that chance of isolation was more with this medium
than thioglycollate broth alone, which might be due to the addition of brain heart infusion broth
as it will increase low redox potential level which provides favorable anaerobic conditions for
growth of C. perfringens.
For further isolation, when positive cultures were streaked on to clostridial agar,
appreciable luxuriant growths were obtained and the selective streaking of these colonies on
perfringens agar with supplements revealed rough and black colonies with sulphate
reduction(Fig.1). Gram stain from these colonies revealed short, Gram-positive plumpy rods with
blunt ends (Fig.2). The isolates developed grey, flat, glistening round colonies surrounded by
inner zone of complete haemolysis and an outer zone of discolouration and incomplete
haemolysis on sheep blood agar after 48 h of incubation at 370C under anaerobic condition. This
feature of the colonial morphology is concurred with earlier observation of Barrow and
Feltham11
, Craven et al.21
and Ficken and Berkhoff, (2004). In milk medium, all the isolates
produced the classical stormy clot or stormy reaction which was indicated as shreds of milk curds
in the sides of the tubes with excessive gas formation (Fig.3). On egg yolk agar, all the isolates
attacked lecithin in the medium and produced opalescence around the streak. These findings are
in accordance with those of Barrow and Feltham 11
. All the isolates were found negative for
oxidase, catalase, liquefied gelatin, fermented glucose, maltose, lactose and sucrose except
mannitol. Barrow and Feltham 11
and Craven et al. 21
reported the similar findings.
The primer combination CP – F - AGT CTA CGC TTG GGA TGG AA and CP – R –
TTT CCT GGG TTG TCC ATT TC used in this study was reliable and very specific in
amplifying 900 bp fragment of the alpha toxin gene- cpa of C. perfringens but not other genes
cpb, etx, iap, cpe and cpb2, encoding the β, ε, ι, entero and β2 toxins of C. perfringens as proved
by Baums, et.al.,12
. All the twenty isolates produced the predicted amplification size of 900 bp,
with the gene coding for alpha toxin production (Fig .4) hence all the isolates are proved as the
alpha toxin producing strains of C. perfringens. Several authors also noted that isolates from
poultry were alpha toxin producing toxigenic strains of C. perfringens 13,
22
.
Antibiogram pattern of C. perfringens isolates were furnished in Table I. The isolates
revealed cent per cent sensitivity to lincomycin, bacitracin and varying degree of sensitivity to
ampicillin, amoxycillin, cloxacillin, penicillin G, chlortetracycline, oxytetracycline, doxycycline,
virginamycin, chloramphenicol, co-trimoxazole, triple sulpha, gentamicin, ciprofloxacin and
streptomycin.
The variation in antimicrobial pattern of C. perfringens isolates obtained in the present
study was correlated with the reports on antibiogram pattern of C. perfringens in poultry 7. The
variation in the antimicrobial pattern might be due to indiscriminate use of these antibiotics as
feed additive and prophylaxis as well as therapeutic agent in poultry industry in India.
NECROTIC ENTERITIS IN BROILER AND LAYER FARMS IN TAMIL NADU
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NECROTIC ENTERITIS IN BROILER AND LAYER FARMS IN TAMIL NADU
Table .1. Antibiotic sensitivity patterns of Clostridium perfringens isolates
S- Sensitive R- Resistant
*** Drug and Disc Concentration : L- Lincomycin (30 mcg), B- Bacitracin (10 units), A- Ampicillin (25 mcg), Am-Amoxycillin (30
mcg), Cl- Cloxacillin (5 mcg), P-Penicillin G (10 units/disc), Ct- Chlortetracycline (30 mcg), O-Oxytetracycline (30 mcg), D-Doxycycline
(30 mcg), V-Virginamycin (10 mcg), C-Chloramphenicol (30 mcg), Co-Co-trimoxozole (25 mcg), Ts-Triple sulpha (300 mcg), G-
Gentamicin (30 mcg), Cf-Ciprofloxacin (5 mcg), S-Streptomycin (10 mcg).
Isolates
***Drug and Disc concentration
L B A Am Cl P Ct O D V C Co Ts G Cf S
B1 S S S S S S S R R S S R S S R R
B2 S S S S S S S R S S R S R R S R
B3 S S S S S R R S S S S R S R R R
B4 S S S S S S S S R S S R R S R R
B5 S S S R S S S R S S R R S S R R
B6 S S S S S S R S S R S S R S R S
B7 S S S S R S S S R S S R R R S R
B8 S S R S S S R S S S R R R R R S
B9 S S S S S S S S R S R S S S R R
B10 S S S S S S R S S S S R S R R R
B11 S S S S S S S R S S R R R R S R
B12 S S S S S S S S S S S R R R S R
B13 S S S S S S S S S S S R R S R R
B14 S S S S S S R S R S S S R R S S
L1 S S S S S S R S S S S S S R R S
L2 S S S S S S S S S S R R R S S S
L3 S S R S R S S R S S S R R R R R
L4 S S S S S S S R S S S R R R S R
L5 S S S R S S S S S R S R R S S R
L6 S S S S S R S S R S S S R R R S
%
Sensitive
100 100 90 90 90 90 70 70 70 90 70 30 30 40 40 30