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: (micromals@rediffmail.com)

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