1. Salik Nazki, Shakil A. Wani, Rafia Parveen, Zahoor A. Dar, Zahid A
Salik Nazki, Shakil A. Wani, Rafia Parveen, Zahoor A. Dar, Zahid A. Kashoo and Pervaiz A. Dar
Bacteriology laboratory, Division of Veterinary Microbiology and Immunology,
Faculty of Veterinary Sciences and Animal Husbandry, SKUAST-Kashmir, Shuhama, Srinagar , India
Multiplex-PCR based virulence gene profile of Clostridium perfringens isolates from Kashmir
Background
Introduction
Clostridium perfringens (C. perfringens) is a Gram positive spore forming anaerobic bacterium. Mostly clostridial species are saprophytic but some species live as commensals in the intestinal tract of humans and animals. In humans it causes gangrene and gastrointestinal diseases like food poisoning and necrotic enteritis whereas in animals, gastrointestinal and enterotoxaemic diseases occur more frequently. It is one of the most common causes of foodborne illness in the United States. It is estimated that it causes nearly a million cases of foodborne illness each year. Pathovar identification is required for a precise diagnosis of associated pathologies and to define vaccine requirements.
C. perfringens strains are implicated in several human and animal diseases resulting in enormous health and economic consequences. The characterization of toxins released by C. perfringens, so called toxinotyping, is used to determine the pathotype (A to E) involved in a specific disease.
Table 1. Toxins produced by the different toxin types of C. perfringens
Toxinotype
Toxins
Alpha
Beta
Epsilon
Iota A + - B C D E
Material & Methods
In the present study, we used a multiplex PCR assay based on alpha (α), beta (β), beta 2 (β2), epsilon (ε), iota (ι), and enterotoxin (cpe) gene detection for characterization of sheep and goat isolates of C. perfringens.
Fig 2: Postmortem hemorrhagic lesions in sheep abomasum
Fig 3: Isolated colonies of C. perfringens on SPS agar
Fig 4: Gram positive rods of C. perfringens.
Fig 1: Gas Gangrene in Humans
Results & Discussion
A total of 170 C. perfringens strains were isolated from fecal material of healthy and diarrhoeic animals or morbid material including intestinal contents and kidney of animals suspected to have died of enterotoxaemia. Out of 170 isolates 116 (68.24%) were type A and the remaining 54 (31.76%) were type D. None of the samples was positive for C. perfringens type B, type C or type E.
Among C. perfringens type A, two (1.72%) isolates carried β2-gene and one (0.86%) possessed cpe-gene in addition to the species specific α-gene.
Whereas among C. perfringens type D isolates, 14 (25%) were positive for β2-gene and 10 (18.51%) harbored cpe-gene in addition to the α- and ε-genes.
These result revealed that multiplex-PCR is a rapid and effective method of characterizing C. perfringens isolates, and can find utility in efficient management and control of C. perfringens infections in human and animals. M
1000 bp
500 bp
100 bp M
1000 bp
500 bp
100 bp
481 bp
548 bp
485 bp
376 bp
324 bp
Fig 6: Multiplex PCR showing different toxinotypes and virulence genes of C. perfringens.
Lane M: 100bp ladder, Lane 1: C. perfringens Type D with cpa (324 bp) and etx (376 bp) gene amplification (positive control), Lane 2: Negative control, Lane 3: C. perfringens Type D, Lane 4: C. perfringens Type D with cpa, etx and beta2 (548 bp) gene amplification, Lane 5: C. perfringens Type D with cpa, etx and cpe (485 bp) gene amplification, Lane 6: C. perfringens Type A with cpa gene amplification.
Fig 5: Agarose gel electrophoresis of uniplex PCR of 16S rRNA gene.
Lane M: 100bp ladder; Lane 1: Positive control; Lane 2: Negative control; Lane 3 & 4: 481 bp product corresponding to 16S rRNA gene
C. perfringens Type A (116)
Two –1.72% β2-gene
One – 0.84% cpe-gene
C. perfringens Type D (56)
Fourteen – 25% β2-gene
Ten – 1.52% cpe-gene
Conclusions
Incidence of C. perfringens in sheep & goat
C. perfringens Type A and Type D are mostly prevalent in faecal samples and morbid material of sheep and goats in Kashmir.
The multiplex PCR can be efficiently used to detect toxin genes in C. perfringens.
Multiplex PCR based toxinotyping can be used for diagnosis and control of disease in humans and animals.