Clostridium perfringens isolate typing by multiplex PCR

Abstract

Abstract:Clostridium perfringens is an important pathogen that provokes numerous different diseases. This bacterium is classified into five different types, each of which capable of causing a different disease. There are various methods for the bacterial identification, many are labor-intensive, time-consuming, expensive and also present low sensitivity and specificity. The aim of this research was to identify the different types of C. perfringens using PCR molecular method. In this study, 130 sheep-dung samples were randomly collected from areas around the city of Kerman, southeastern Iran. After processing and culturing of samples, the produced colonies were morphologically studied, gram stain test was also carried out and the genera of these bacteria were identified through biochemical tests. DNA extracted from isolated bacteria for genotyping was tested by multiplex PCR with specific primers. Based on length of synthesized fragments by PCR, toxin types and bacterial strains were detected. C. perfringens isolated types were divided as follows: 17.39% type A, 21.74% type B, 34.78% type C and 26.09% type D. It should be emphasized that, up to the present moment, C. perfringens type A has not been reported in Iran.Key words: Clostridium perfringens, multiplex PCR, biochemical tests, genetic typing. According to Timoney et al. (13), the antitoxin ε was measured in sheep blood sera by ELISA, which proven to be a specific, quick and economical method that may replace the serum neutralization test. ELISA utilizes polyclonal antibodies to identify C. perfringens toxins (14). However, its disadvantage is that the interaction reaction among the produced antibodies works against the toxins, which may difficult the identification of toxin types. Moreover, ELISA falls short of identifying β 2 toxins and in order to identify toxins from spore-forming bacteria, it must be activated by means of special culture methods (15). Original PaPerBiochemical tests are also incapable of distinguishing different types of C. perfringens (16). PCR is the most modern practical technology in diagnosing infectious diseases and compared with classical techniques, it has been shown to be more rapid, with results obtained in a few hours, and also more reliable (12,14). PCR allows a faster bacterial identification directly from clinical samples (17). It should be noted that up to this moment, no research on this subject has been carried out in Iran. MATERIALS AND METHODSIn the present study, sheep dung samples were obtained from 130 Kermani sheep, from nine locations in southeastern Iran. The sheep were from both genders and their ages ranged from 1 to 2.5 years. The samples were collected aseptically in sterile plastic bags and transferred to the laboratory within 1 to 2 hours, after which they were accordingly processed. Samples were diluted in PBS (1:10), the bath temperature was maintained at 80°C for ten minutes in order to eliminate the non-sporeforming bacteria, subsequently they were cultivated on 5% sheep blood agar and anaerobically i...

Molecular Typing of Clostridium perfringens Isolates from Faecal Samples of Healthy and Diarrhoeic Sheep and Goats in Kashmir

“…Various PCR protocols including multiplex-PCR have been developed to detect the toxin genes to type C. perfirngens isolates (Ahsani et al, 2010).Compared to conventional methods, these protocols provide rapid and sensitive detection of the organisms. The multiplex PCR method may provide a more sophisticated approach, enabling a simultaneous and specific detection of all the toxinotypes of C. perfringens.…”

Food Poisoning Caused by Bacteria (Food Toxins)

“…perfringens is an anaerobic bacterium that creates spores that survive in soil, sediments, and areas subject to both human and animal fecal contamination. It is widely distributed in the environment and is frequently found in the human intestine and in several domestic and wild animals' intestines [78].…”

“…The production of toxins in the digestive tract is associated with sporulation. The disease is foodborne; and only one case has implied the possibility of poisoning caused by the preformed toxin [12,78,79].…”

“…Additionally, there is a more severe but less frequent disease caused by eating a food product contaminated with type C strains; this disease is known as necrotic enteritis or pig-bel disease, and it is often fatal. Deaths caused by necrotic enteritis are due to intestinal infection and necrosis, as well as by septicemia, the elderly people being the most affected population [78].…”

“…The disease diagnosis is confirmed by the presence of the toxin in the stools of patients; either by traditional methods (culture from the stools or the food involved) or by molecular methods by looking for the following genes: cpe (CPE toxin), plc (Alpha-toxin), and etx (Epsilon-toxin) [12,78,79].…”