Molecular Evaluation of the Enterotoxigenicity of Clostridium difficile and Clostridium perfringens Swine Isolates by PCR Assays (original) (raw)
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Advances in Microbiology
Clostridium difficile and C. perfringens are enteric pathogens affecting a variety of mammals. This study evaluated the molecular enterotoxigenicity of Clostridium swine isolates by PCRs. One hundred and ten swine faeces were analyzed by culture assay. The faecal samples were from sixty-seven healthy animals and 43 with gastrointestinal tract disease. C. difficile strains were PCR-screened for the presence of tcdA/tcdB and cdtA/cdtB genes. All C. perfringens isolates were tested for the characterization of the toxinotype. Overall, sixty-five swine resulted positive: 38 for C. difficile and 17 for C. perfringens. One sample tested C. perfringens and C. difficile-positive, at the same time: on the whole, 39 C. difficile strains were isolated. Thirty-eight C. difficile isolates (all from healthy animals) resulted tcdA/tcdB and cdtA/cdtBnegative by PCRs and toxins A/B-negative by immunological tests. All C. perfringens strains were type A; eight were also cpb2-positive. In the sample (diarrhoeic), with double infection, C. difficile tested tcdA/tcdB and cdtA/cdtB-positive by PCRs and toxins A/B-positive by immunoassays; C. perfringens resulted cpb2-positive. The molecular genotypeing/toxinotyping should be applied to establish a final diagnosis and to assess properly the full implications and the epidemiological impact of these findings in particular in samples of healthy animals and aid in the development of effective intervention methods for controlling clostridial disease outbreaks.
Advances in Microbiology, 2012
Clostridium perfringens and C. difficile have been associated with acute and chronic large and small bowel diarrhoea, and acute haemorrhagic diarrhoeal syndrome in dogs. The objective of this study was to investigate by toxin gene profile and PCR-ribotyping the molecular characteristics of 14 C. perfringens and 10 C. difficile isolates from 95 canine faeces (n = 36, diarrhoeic and n = 59, non-diarrhoeic). Concerning C. perfringens, 13 strains (92.9%) were type A, of which 3 (23.1%) also possessed the beta 2 toxin (CPB2)-encoding gene. One isolate (7.1%) was type D and possessed CPB2 gene. On the whole, 4 of the 14 strains (28.6%) tested cpb2-positive. Six C. difficile isolates (60.0%) demonstrated tcdA+/tcdB+ and cdtA+/cdtB+ genotype and tested positive for, in vitro, toxin production by EIA. Eight distinct ribotypes were observed. In conclusion, the PCR assays may provide useful and reliable tools for C. perfringens and C. difficile molecular typing in routine veterinary diagnostics.
Food & Feed Research, 2018
Clostridium perfringens is a Gram-positive, endospore-forming, anaerobic rod, ubiquitous in nature. C. perfringens strains can produce about 17 toxins. Many of them can lead to miscellaneous diseases, among which the enteric ailment may be the most common and is of utmost importance. In the present work 34 strains of C. perfringens isolated from feed and one from a cow suspected to have died of clostridial infection were subjected to molecular analysis. In order to detect the genotypes, the following genes coding for toxins were targetted: cpa, cpb, cpb2, cpe, etx and iap. The multiplex PCR assay revealed that all C. perfringens isolates from animal feed were of type A and 2-toxinogenic type A strains, possessing only the cpa (n=21), or both the cpa and the cpb2 genes (n=13). The importance of C. perfringens toxins and -2 in the pathogenesis of enterotoxemia is discussed and the regulation on the detection of this bacteria in animal feed questioned. The use of PCR in practise could enable the toxin-genotyping of C. perfringens isolates and, thus, provide a real basis for the establishment of maximum acceptable limits of this bacteria in feed.
Anaerobe, 2016
The aim of this study was to report two cases of Clostridium perfringens type A and Clostridium difficile coinfection in adult dogs. Both animals were positive for A/B toxin. Toxigenic C. difficile and C. perfringens type A positive for NetE and NetF-encoding genes were isolated. This report reinforces the necessity of studying a possible synergism of C. difficile and C. perfringens in enteric disorders.
Detection and characterization of Clostridium perfringens in the feces of healthy and diarrheic dogs
Canadian journal of veterinary research = Revue canadienne de recherche vétérinaire, 2012
Clostridium perfringens has been implicated as a cause of diarrhea in dogs. The objectives of this study were to compare 2 culture methods and to evaluate a multiplex polymerase chain reaction (PCR) assay to detect C. perfringens toxin genes alpha (α), beta (β ), beta 2 (β2), epsilon (ɛ), iota (ι), and C. perfringens enterotoxin (cpe) from canine isolates. Fecal samples were collected from clinically normal non-diarrheic (ND) dogs, (n = 105) and diarrheic dogs (DD, n = 54). Clostridium perfringens was isolated by directly inoculating stool onto 5% sheep blood agar (SBA) and enrichment in brain-heart infusion (BHI) broth, followed by inoculation onto SBA. Isolates were tested by multiplex PCR for the presence of α, β, β2, ɛ, ι, and cpe genes. C. perfringens was isolated from 84% of ND samples using direct culture and from 87.6% with enrichment (P = 0.79). In the DD group, corresponding isolation rates were 90.7% and 93.8% (P = 0.65). All isolates possessed the α toxin gene. Beta (β),...
Genotyping of Clostridium perfringens isolated from calves with neonatal diarrhea
Anaerobe, 2008
Clostridium perfringens is more prevalent type of clostridia genus isolated from the intestinal tract of ostrich (Struthio camelus). Necrotic enteritis (NE) is a potentially fatal gastrointestinal (GI) disease of poultry and other avian species, which produces marked destruction of intestinal lining in digestive tract caused by C. perfringens. Pathogenicity and lesions are correlated with the toxins produced, thus toxin typing of the bacterium has diagnostic and epidemiological significance. The aims of the present study were to determine the biotypes of C. perfringens among ostrich's farms either diseased and healthy ones and to screen the isolates for major toxin genes (cpa, cpb, etx, and iA, cpb2, and cpe). Materials and Methods: Thirty isolates of C. perfringens were obtained from NE-positive and NE-negative ostrich flocks in Khorasan-e-Razavi porvince and analyzed by multiplex PCR assay. Results: All isolates were positive for alpha toxin gene (cpa) and five of those were positive for beta toxin gene (cpb). The presence of cpb2 gene was detected in a high percentage of isolates originating from both healthy (93.3%) and diseased flocks (80%). None of the isolates carried enterotoxin gene (cpe).
Current Science, 2017
Clostridium difficile with its virulence factors A and B toxins cause Pseudomembranous colitis. Bacterium was isolated from 57 dog and 41 pig diarrheic faecal samples in cycloserinecefaxitin fructose agar media and molecular detection was done by amplification of gluD gene (755 bp). Variability of toxin genes in positive isolates was tested by multiplex PCR. Detection of binary toxin genes (cdtA and cdtB) was also done. Results showed 33.67% positivity with 18 and 15 from dog and pig respectively, from which 10 and 5 were toxigenic and 11 pig isolates exhibited binary toxin. PCR-RFLP demonstrated toxinotype 0 in all A + B + isolates.
Toxinotype V Clostridium difficile in Humans and Food Animals
Emerging Infectious Diseases, 2008
Clostridium diffi cile is a recognized pathogen in neonatal pigs and may contribute to enteritis in calves. Toxinotype V strains have been rare causes of human C. diffi cileassociated disease (CDAD). We examined toxinotype V in human disease, the genetic relationship of animal and human toxinotype V strains, and in vitro toxin production of these strains. From 2001 through 2006, 8 (1.3%) of 620 patient isolates were identifi ed as toxinotype V; before 2001, 7 (<0.02%) of ≈6,000 isolates were identifi ed as toxinotype V. Six (46.2%) of 13 case-patients for whom information was available had community-associated CDAD. Molecular characterization showed a high degree of similarity between human and animal toxinotype V isolates; all contained a 39bp tcdC deletion and most produced binary toxin. Further study is needed to understand the epidemiology of CDAD caused by toxinotype V C. diffi cile, including the potential of foodborne transmission to humans.
Clostridium difficile with its virulence factors A and B toxins cause Pseudomembranous colitis. Bacterium was isolated from 57 dog and 41 pig diarrheic faecal samples in cycloserinecefaxitin fructose agar media and molecular detection was done by amplification of gluD gene (755 bp). Variability of toxin genes in positive isolates was tested by multiplex PCR. Detection of binary toxin genes (cdtA and cdtB) was also done. Results showed 33.67% positivity with 18 and 15 from dog and pig respectively, from which 10 and 5 were toxigenic and 11 pig isolates exhibited binary toxin. PCR-RFLP demonstrated toxinotype 0 in all A + B + isolates.
Potential Role Of Clostridium Difficile And Clostridium Perfringens As A Cause Of Diarrhea In Horses
Journal of Applied Veterinary Sciences
Diarrhea is a major clinical problem affecting foals and horses at different ages which can be fatal and cause great economic losses particularly in foals. The aim of this study was to identify Clostridium perfringens and Clostridium difficile involved in enteric infections and the associated virulence factors in diarrheic foals and horses. Thirty fecal samples were examined for identification of C.perfringens and C.difficile (bacteriological culturing, microscopical examination, count and molecular detection). C.perfringens was isolated from young horses less than 6 months age in an incidence of 46.7% with count ranged from 1.3×10 4-5.6×10 8 cfu/g, while from horses of 6 months to one year, it was 20% with count ranged from 1.7×10 4-1.9×10 6 cfu/g, on the other hand, 13.3% with count ranged from 1.4×10 3-2.5×10 4 cfu/g was the incidence of C.perfringens in horse more than one year. C. difficile was not detected in all examined samples. C.perfringens type A was the most predominant type detected in an incidence of 58.3% then type B (4.7%). All C.perfringens isolates were sensitive to penicillin, nitromedazole, fluramfenicole and ceftiofur and all are resistant to oxytetracycline. Based on the high count, eight C.perfringens isolates were chosen to be molecular characterized for the presence of some virulence factor genes which included ; cpa, cpb2, cpe and netF toxin genes. All examined isolates have cpa (alpha) and cpb2 (beta 2) toxin genes, 5/8 isolates were found to have netF gene while only 2 isolates harbored cpe (enterotoxin gene). C. difficile tpi gene was not detected in all examined samples (10). This study may throw the light on the synergistic effect of β2 and net F toxins together with alpha toxin on induction of enterocolitis in horse. ــــــــــــــــــ ـــــــــــــــــــــــ