Alpha toxin specific PCR for detection of toxigenic strains of Clostridium perfringens in Poultry

Aim : Isolation of Clostridium perfringens from necrotic enteritis cases in poultry and confirmation by alpha toxin specific PCR Materials and methods: Robertson cooked meat medium with Brain Heart Infusion broth was used for isolation of C. perfringens from intestinal contents of necrotic enteritis suspected birds. Positive cultures from perfringens agar were further confirmed by biochemical tests and subjected to alpha toxin specific PCR. Results and Discussion: Twenty Clostridium perfringens isolates were isolated from intestinal contents of thirty five NE suspected birds. Out of the twenty isolates, 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. Frequency of isolation of C. perfringens was more with Robertson cooked meat medium with BHI broth than thioglycollate broth alone. 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. The isolates produced rough and black colonies with sulphate reduction on perfringens agar, double zone haemolysis on sheep blood agar, stormy clot fermentation on milk medium and opalescence on egg yolk medium. The isolates were found negative for oxidase, catalase, liquefied gelatin, fermented glucose, maltose, lactose and sucrose except mannitol. All the fourteen isolates obtained from commercial broilers proved the alpha toxin producing strains of C. perfringens when they were subjected to alpha toxin specific PCR. Conclusion : This study revealed alpha toxin specific PCR is highly useful for detection of toxigenic strains of Clostridium perfringens in poultry


Introduction
epsilon and iota [4].C. perfringens type A strains produce the chromosomal encoded toxin, while C. Clostridium perfringens type A and to lesser perfringens type C strains produce toxin together with extent type C causes necrotic enteritits (NE) in poultry.toxin [5].Normally, the number of C. perfringens in the intestine However, the chromosomal encoded alpha toxin 4 is low (about 10 cfu/g of digesta).The disease occurs is considered as the main virulence factor for NE in when high numbers of bacteria coincide with a poultry because birds are about 200 times more damaged intestinal mucousa [1].The disturbances in susceptible to alpha toxin than to beta or epsilon toxin normal intestinal microfolora may cause rapid [6].proliferation of C. perfringens, increasing bacterial The alpha toxin has been implicated inseveral 7 9 numbers the range from 10 to 10 colony forming diseases including NE in chickens [7].The toxin units (cfu)/g of digesta resulting in toxin production destruction of mucosal tissue manifests as macroscopic [2].So far, over 800 serotypes of C. perfringens are lesions that are usually seen in jejunum and ileum but known and 17 different toxic fractions have been can also appear in duodenum [8].It is a potent toxin isolated [3].The C. perfringens strains were classified with haemolytic, lethal, dermonecrotic, vascular into five toxinotypes (A, B, C, D and E) based on the permeabilization and platelet aggregating properties production of four major toxins viz., alpha, beta, [9] and it has direct effects on host metabolism including inhibition of neutrophil chemotaxis, vaso-2004, was added to a 50 ul reaction mixture with the constriction, haemolysis of erythrocytes and necrosis following reagents 1.25 U Taq DNA polymerase, 50 of other body cells and modulation of cell metabolism mM Pottassium chloride, 30 mM Tris-Hcl, 1.5 mM preceded by incubation for 2min 30 seconds at 95 C. In India, NE was first reported by Chakraborty, Six microlitre of the amplicons was separated on 1.5% et.al., [15].Now, the NE is emerged as a worldwide agarose gel according to standard procedure.problem [16] and it is a common disease found in all

Results and Discussion
poultry growing areas of the world.
Aim of the study was Isolation of clostridium Inoculation of processed intestinal contents in perfirngens from necrotic enteritis cases in poultry and thioglycollate broth produced turbidity and saccharolytic confirmation by alpha toxin specific PCR.
reaction in Robertson cooked meat medium with brain heart infusion broth.Appreciable luxuriant growths on

Materials and Methods
the clostridial agar were obtained on the initial streak For isolation of the organism causing necrotic from the culture.The selective streaking of these colonies enteritis, thirty five birds suspected for NE were on perfringens agar with supplements revealed rough collected from poultry diagnostic and research centers and black colonies with sulphate reduction. of M/s.Suguna poultry farm, M/s.Venkateshwara The isolates produced double zone haemolysis Hatcheries Limited, Palladam, M/s.Pioneer Hatcheries, on sheep blood agar, stormy clot fermentation on milk Namakkal.Apart from that, ten commercial farms in medium and opalescence on egg yolk medium.The and around Namakkal and Udumalpet area from both isolates were found negative for oxidase, catalase, broiler and layer farms, where, NE cases were reported.liquefied gelatin, fermented glucose, maltose, lactose For isolation of the organism causing necrotic and sucrose except mannitol.enteritis, sterile saline (v/v) was added to the collected Based on the results obtained from the above specimens consisting of intestinal contents and said tests, and in consultation with Bergey's Manual of o scrapings then heated at 80 C for 20 min in water bath.determinative bacteriology [19], the isolates were The processed Intestinal contents were inoculated into identified as C. perfringens.Thus twenty Cl. thioglycollate broth, Robertson cooked meat medium perfringens isolates were obtained from the intestinal with brain heart infusion broth and sterile liquid content of thirty five NE suspected broilers.Out of the paraffin was poured to make a layer over the medium.twenty isolates, fourteen were isolated from o Inoculated medium was incubated at 37 C for 24 hr.commercial broilers of 2 to 6 wk of age, six from The presence of C. perfringens in the inoculated commercial layers of 9 to 15 wk of age.These findings sample is indicated by turbidity in both of the media.correlate with the reports of detection of C. perfringens The positive cultures were streaked on to clostridial in 2-6 wk broiler chickens [20] and isolation of C. agar and perfringens agar with supplements.The perfringens from 7 to 16 wk commercial layer birds o plates were incubated in the anaerobic jar at 37 C for [21].48h.
The primer combination used in this study was The bacteria isolated anaerobically from NE reliable and very specific in amplifying 900 bp specimens showed the characteristic colony types of fragment of the alpha toxin gene-cpa of C. perfringens C. perfringens, were gram stained and confirmed to be but not other genes cpb, etx, iap, cpe and cpb2, C. perfringens by standard biochemical tests as encoding the β, ε, ι, entero and β2 toxins of C. described by Barrow and Feltham [17].
perfringens as proved by Baums, et.al., [18].All the twenty isolates produced the predicted amplification

Identification of toxigenic strains by Polymerase
size of 900 bp, with the gene coding for alpha toxin Chain Reaction (PCR): To design the PCR, alpha production (Fig .1) hence all the isolates are proved as toxin specific primers (CP -F-AGT CTA CGC TTG the alpha toxin producing strains of C. perfringens.GGA TGG AA and CP -R-TTT CCT GGG TTG TCC Similar to present study, Engstrom et al. [12] analysed ATT TC), which flanked 900 base pair DNA sequence, 53 isolates of C. perfringens from NE affected poultry according to Baums et al., [18] were used.
from different parts of Sweden by PCR for toxin To perform the PCR, 2 ul template DNA, typing.They reported that all the isolates were prepared by the heat lysis method of Baums et al.,

2++ by activating the
arachidonic acid cascade and protein Mg , 200 µM of each dNTP and 50 picomoles of each kinase C [10].The polymerase chain reaction assay primer.The thermocycling (incubations for 1 min at o o o (PCR) was used for detection of alpha toxigenic 95 C, 55 C and 72 C respectively was 35 times) was o strains of C.perfringens [11-14].