Real-time multiplex PCR assay for rapid detection and toxintyping of Clostridium perfringens toxin producing strains in feces of dairy cattle

Gurjar, Anagha; Hegde, Narasimha V.; Love, Brenda C.; Jayarao, Bhushan M.

doi:10.1016/j.mcp.2007.08.001

Cited by 55 publications

(37 citation statements)

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“…The organism is therefore capable of producing different pathological pictures and of causing numerous different histotoxic and enteric diseases in both humans and other animals (1). Alpha (α), beta (β), epsilon (ε) and iota (ι) comprise the four main C. perfringens toxins which form the basis of categorizing this bacterium into five different types, from A to E (3)(4)(5). These different bacterial types provoke diverse diseases and even one strain may cause the infection in numerous hosts (6,7).…”

Section: Introductionmentioning

confidence: 99%

Clostridium perfringens isolate typing by multiplex PCR

Ahsani¹,

Mohammadabadi²,

Shamsaddini³

2010

J. Venom. Anim. Toxins incl. Trop. Dis

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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...

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Section: Introductionmentioning

confidence: 99%

Clostridium perfringens isolate typing by multiplex PCR

Ahsani¹,

Mohammadabadi²,

Shamsaddini³

2010

J. Venom. Anim. Toxins incl. Trop. Dis

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“…While ELISA has an advantage over plate count methods, in that it can deal with multiple samples, it is not quantitative and cannot detect low bacterial contamination levels (McCourt et al, 2005). In spite of its specificity and rapidity, disadvantages of PCRbased methods include the requirement of a DNA extraction step, the detection of both live and dead cells (Gurjar et al, 2008;Wise and Siragusa, 2005), and reaction interference by certain chemical inhibitors, such as polyphenolic compounds, in complex substrates (Koonjul et al, 1999;Wise and Siragusa, 2005). To overcome these limitations, Ootsubo et al (2003) developed a FISHFC method and showed specific detection and quantification of cultivable Enterobacteriaceae from food and environmental water.…”

Section: Discussionmentioning

confidence: 99%

Rapid Quantitative Detection of Salmonella enterica Using Fluorescence In Situ Hybridization with Filter-cultivation (FISHFC) Method

Shimizu

Aoi

Osanai

et al. 2013

FSTR

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Specific detection and enumeration of Salmonella enterica in food using conventional culture-based methods (CCBM) are time consuming and labor intensive. This study was conducted to develop a rapid S. enterica detection and enumeration method by combining fluorescence in situ hybridization (FISH) with micro-colony formation culture (FISHFC). Specificity tests of the SAL343 probe for S. enterica detection revealed that SAL343-associated fluorescent micro-colonies were observed specifically for S. enterica, but not for any other organisms. This finding suggests that SAL343 is highly specific for detecting S. enterica using FISHFC. For validation, FISHFC with SAL343 was compared to CCBM, with multiple selective agar, using spiked food samples; no significant differences in enumeration were found between FISHFC and CCBM (p > 0.05). The FISHFC method allowed enumeration of S. enterica within 10 h while CCBM allowed enumeration within 5 days. Therefore, the FISHFC method has potential application for more rapid and specific enumeration of S. enterica in food samples compared to other available methods.Keywords: Salmonella enterica, fluorescence in situ hybridization, FISHFC, rapid detection *To whom correspondence should be addressed. E-mail: yamasaki@fish.hokudai.ac.jp IntroductionSalmonella are Gram-negative, facultatively anaerobic, rod-shaped bacteria that exist ubiquitously in the environment and are commonly found in water, animal intestine, reptilian skin and food. Salmonella also causes salmonellosis in humans worldwide due to the consumption of contaminated food such as fresh fruits and vegetables (Matthews, 2006). The genus Salmonella currently consists of two species: S. enterica and S. bongori. S. enterica is comprised of six subspecies: enterica, salamae, arizonae, diarizonae, houtenae and indica, which are further subdivided into more than 2,500 serotypes (Agbaja et al., 2011;Su and Chiu, 2007). Most of the Salmonella responsible for disease in humans and animals are members of S. enterica. S. enterica serotype Enteritidis and Typhimurium are two of the main sources of human food poisoning. Although S. bongori has been reported to infect humans, it is generally associated with cold-blooded animals (Giammanco et al., 2002). Therefore, S. enterica are clinically important pathogens of humans.Routine methods for enumerating Salmonella are traditional microbiological methods based on plating using selective agar and biochemical identification. However, the biochemical characteristics of some isolates may differ from the common phenotypic properties of typical strains. Moreover, it is recommended that multiple selective agars be used for Salmonella detection in food samples (Nye et al., 2002). Thus, the conventional methods for detecting Salmonella are time-consuming and labor-intensive, taking at least several days to obtain the final results (Kim et al., 2007). To overcome technical problems associated with Salmonella detection, a number of molecular-based methods, such as PCR, quantitative PCR (qPCR), enzy...

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“…A rabbit with mild diarrhoea, distension of the anterior digestive tract with gas and liquid contents, liquid or impacted caecal contents and without macroscopic signs of inflammation was deemed as an ERE-confirmed case. When clinical signs and lesions of ERE were identified, the toxinotype of C. perfringens (Petit et al, 1999) was determined by PCR using primer sequences of Baums et al (2004) and Gurjar et al (2007). Rabbits were naturally infected.…”

Section: Health Status Evaluationmentioning

confidence: 99%

“…The QIAamp DNA Stool Mini KitTM (Qiagen, CA, USA) was used to isolate bacterial DNA with a previous sample treatment with reinforced Clostridium medium following the method used by Gurjar et al (2007). Three independent duplex PCR reactions were standardised for the detection of Clostridium perfringens genes encoding a-, b-, ε-, ι-, b2-and CPE toxins with available specific primer sequences and conditions published by Baums et al (2004) and Gurjar et al (2007). Detailed information regarding duplex PCR conditions can be found in Menoyo et al (2011).…”

Section: Clostridium Perfringens Genotypingmentioning

confidence: 99%

Characterisation of Clostridium perfringens presence and concentration of its α-toxin in the caecal contents of fattening rabbits suffering from digestive diseases

et al. 2011

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Digestive diseases are the main cause of morbidity and mortality in growing rabbits. Clostridium perfringens is a widely occurring pathogenic bacterium in enteric diseases of domestic animals and its pathogenicity stems from the production of potent exotoxins. This work aimed to quantify the concentration of C. perfringens α-toxin in caecal samples of rabbits with digestive diseases and relate these concentrations to C. perfringens counts. Additionally, C. perfringens strains isolated from rabbits with clinical lesions of Epizootic Rabbit Enteropathy (ERE) were toxinotyped. To conduct this work, a total of 711 rabbits weaned at 35 d were housed in pairs and fattened until the age of 63 d. No experimental infection was performed and no antibiotics were provided in the feed or drinking water. All rabbits displaying symptoms of digestive diseases were slaughtered and necropsied. At 46 d, 88 healthy rabbits were also slaughtered. Caecal contents were sampled from all slaughtered animals. Thirty-seven out of the 69 rabbits with digestive diseases (88.5% of sick animals) showed ERE-confirmed symptoms and lesions. Apart from diarrhoea, the most constant ERE signs were abdominal bloating and borborygmi. At necropsy, the anterior digestive tract was found filled with large amounts of gas and liquid. Twenty-seven rabbits had liquid caecal contents whereas caecal impaction only appeared in 10 rabbits. Live weight was lower by 49.2% (P<0.001) in diseased rabbits as compared with healthy rabbits of the same age. For C. perfringens counts lower than 6.0 log cfu/g, the α-toxin concentration remained below 2.6 μg/mg. However, for bacterial counts above 6.0 log cfu/g the concentration of α-toxin ranged from 0.12 to 60.9 μg/mg. Nevertheless, both caecal concentration of C. perfringens (7.65 vs. 3.09 log cfu/g, P<0.001) and that of its α-toxin (6.02 vs. 0.17 μg/mg, P<0.001) were higher in diseased rabbits than in healthy ones. C. perfringens toxinotype A was found in all ERE-affected rabbits. No other toxinotype was identified and no isolate contained the enterotoxin gene. In conclusion, C. perfringens α-toxin should not be considered a good indicator of the bacterium's presence, as high counts of colonies are not always associated with high toxin concentrations.

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Real-time multiplex PCR assay for rapid detection and toxintyping of Clostridium perfringens toxin producing strains in feces of dairy cattle

Cited by 55 publications

References 20 publications

Clostridium perfringens isolate typing by multiplex PCR

Clostridium perfringens isolate typing by multiplex PCR

Rapid Quantitative Detection of Salmonella enterica Using Fluorescence In Situ Hybridization with Filter-cultivation (FISHFC) Method

Characterisation of Clostridium perfringens presence and concentration of its α-toxin in the caecal contents of fattening rabbits suffering from digestive diseases

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