Typing Toxigenic Clostridium perfringens Strains from the Ruminants of Yazd Province by Multiplex Polymerase Chain Reaction

Background: Anaerobic bacterial infections, especially enterotoxemia, are common ruminant disorders. Objectives: The purpose of this study was to identify the toxigenic isolates of Clostridium perfringens in the ruminants of Yazd province. Materials and Methods: In total, 485 fecal and intestinal samples were obtained and analyzed for typing C. perfringens toxovars by multiplex polymerase chain reaction (PCR). Only 179 bacterial strains passed the biochemical tests and 87 C. perfringens strains were confirmed by multiplex PCR. Results: Interestingly, the predominant C. perfringens toxovar was type A (89.7%), while type D (9.2%) was also identified as a pathogen in the ruminants of Yazd province. Conclusions: Detection of toxigenic C. perfringens isolates with multiplex PCR was performed for the first time in this field. The multiplex PCR used in this study provides a useful and reliable tool for C. perfringens genotyping in routine veterinary diagnostics, andepidemiological studies of the prevalent types of C. perfringens in Iran are possible by this technique. Genotyping of C. perfringens is recommended before starting vaccination programs. *Corresponding Author: Mehrdad Shamsaddini Bafti, Tel: +98-9133980638; Fax: +98-34 32233051; E-mail: m.shamsaddini@rvsri.ac.ir Int J Enteric Pathog. 2016 August;4(3):e35249 http://enterpathog.com Copyright © 2016, Alborz University of Medical Sciences. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (http://creativecommons.org/licenses/by-nc/4.0/) which permits copy and redistribute the material just in noncommercial usages, provided the original work is properly cited.

previously. 8ference Strains Standard C. perfringens reference strains (ATCC 13124 , CN 228 , CN 301 , and CN 409 ) were utilized as positive C. perfringens type controls (types A, B, C and D, respectively) as well as C. septicum strain CN 913 as the negative control.
Polymerase Chain Reaction Amplification and Assay DNA extraction from the isolated and reference C. perfringens strains was performed as previously described by the authors. 10C. perfringens typing was performed with the total reaction volume of 50 μL and the following reagents: 5 μL of the extracted DNA with 25 μL of readyto-use PCR master mix (PR901638, Sinaclon, Iran), 1.25 μL (20 pmole/μL) of each primer, and dH 2 O to reach a volume of 50 μL.
For the PCR amplification of 16S rRNA gene and α, β, ε, and ι toxin genes, specific primers (Sinaclon, Iran) were used (Table 1).Amplicons were obtained with 35 cycles following an initial denaturating step at 95ºC for 5 minutes.Each cycle involved denaturation at 94ºC for 1 min-ute, annealing at 53ºC for 1 minute, synthesis at 72ºC for 90 seconds, and final extension step at 72ºC for 5 minutes.
Amplicons were evaluated by electrophoresis on 1.7% agarose gel.The 100 bp DNA ladder (PR911653 and PR901644, Sinaclon, Iran) was used as the molecular marker to indicate the size of the amplicons.The amplified bands were visualized and photographed under UV illumination.

Results
Out of 485 investigated samples, 849 bacterial isolates were morphologically selected for microbiological examination.From these isolates, only 179 strains passed the biochemical tests and were examined by PCR.In this study, we isolated and confirmed 87 C. perfringens strains by multiplex PCR (Table 2).Seventy-eight strains were classified as type A (possessing cpa gene), one strain belonged to type C (possessing cpa and cpb genes), and eight strains belonged to type D (possessing cpa and etx genes) (Figure 1).Other types of C. perfringens (B and E) were not detected.

Discussion
C. perfringens infection in ruminants and other species is generically called enterotoxemia, 4 in which C. perfringens proliferates in large numbers and produces several toxins. 12These toxins are responsible for enterotoxaemia in almost all the ruminants. 13There are geographical differences in the prevalent types of the these bacteria and the type could be different depending on the animal species in the area. 7oxigenic typing of C. perfringens isolates could be performed by different conventional and modern methods.8][19][20][21][22] The multiplex PCR presented here included all the important toxin genes in C. perfringens mediated enterotoxaemia. 8,21Compared to the conventional techniques, PCR method was shown to be much more rapid, provided results in a few hours, and was much more reliable. 23n the present study, typing of C. perfringens isolates by multiplex PCR revealed that types A and D were the most important types of C. perfringens in the ruminants of Yazd province.Type A was the most frequent type of the bacteria which was isolated from the environment and digestive tract of the animals and humans. 1 Similar results were obtained by Greco et al, 17 who reported that, while C. perfringens types A and D were detected at the rates of 84% and 16% by PCR, respectively, type A was reported as the dominant type of C. perfringens in lambs and sheep worldwide. 7n conclusion, the detection of toxigenic C. perfringens isolates circulating in the ruminant fields of Yazd province is very important and was performed for the first time in this field.In contrast to other protocols, in this study, the used multiplex PCR was specific for C. perfringens, since we applied a specific primer (16S rRNA) for the diagnosis of C. perfringens species.This multiplex PCR provided a useful and reliable tool for C. perfringens genotyping in veterinary diagnostics and the type A of C. perfringens was more than other types in small ruminants.

Table 1 .
Sequence of Specific Primers Used for C. perfringens Typing in This Study

Table 2 .
Genotyping of C. perfringens Isolates From the Ruminants of Yazd Province