Molecular Evaluation of the Enterotoxigenicity of <i>Clostridium difficile</i> and <i>Clostridium perfringens</i> Swine Isolates by PCR Assays — Oak Academic Publishing
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Molecular Evaluation of the Enterotoxigenicity of <i>Clostridium difficile</i> and <i>Clostridium perfringens</i> Swine Isolates by PCR Assays
Department of Veterinary Medical Science—Unit of Infectious Diseases and Microbiology, University of Parma, Parma, Italy
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1 Department of Veterinary Medical Science—Unit of Infectious Diseases and Microbiology, University of Parma, Parma, Italy
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 tcd A / tcd B and cdt A / cdt B 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 tcd A / tcd B and cdt A / cdt B - negative by PCRs and toxins A/B-negative by immunological tests. All C . perfringens strains were type A; eight were also cpb 2-positive. In the sample (diarrhoeic), with double infection, C . difficile tested tcd A / tcd B and cdt A / cdt B -positive by PCRs and toxins A/B-positive by immunoassays; C . perfringens resulted cpb 2-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.
X. Song, J. G. Bartlett, K. Speck, A. Naegeli, K. Carroll and T. M. Perl, “Rising Economic Impact of Clostridium difficile-Associated Disease in Adult Hospitalized Patient Population,” Infection Control and Hospital Epidemiology, Vol. 29, No. 9, 2008, pp. 823-828. doi:10.1086/588756
M. K. Keel and J. G. Songer, “The Comparative Pathology of Clostridium difficile-Associated Disease,” Veterinary Pathology, Vol. 43, No. 3, 2006, pp. 225-240. doi:10.1354/vp.43-3-225
J. G. Songer, “Clostridial Enteric Diseases of Domestic Animals,” Clinical Microbiology Reviews, Vol. 9, No. 2, 1996, pp. 216-234.
B. E. Waggett, B. C. McGorum, U. Wernery, D. J. Shaw and R. S. Pirie, “Prevalence of Clostridium perfringens in Faeces and Ileal Contents from Grass Sickness Affected Horses: Comparisons with 3 Control Populations,” Equine Veterinary Journal, Vol. 42, No. 6, 2010, pp. 494-499. doi:10.1111/j.2042-3306.2010.00105.x
A. A. Baker, E. Davis, T. Rehberger and D. Rosener, “Prevalence and Diversity of Toxigenic Clostridium perfringens and Clostridium difficile among Swine Herds in the Midwest,” Applied and Environmental Microbiology, Vol. 76, No. 9, 2010, pp. 2961-2967. doi:10.1128/AEM.02459-09
J. G. Songer, K. W. Post, D. J Larson., B. H. Jost and R. D. Glock, “Infection of Neonatal Swine with Clostridium difficile,” Swine Health and Production, Vol. 8, No. 4, 2000, pp. 185-189.
M. C. Hammitt, D. M., Bueschel M. K. Keel, R. D. Glock, P. Cuneo, D. W. DeYoung, C. Reggiardo, H. T. Trinh and J. G. Songer, “A Possible Role for Clostridium difficile in the Etiology of Calf Enteritis,” Veterinary Microbiology, Vol. 127, No. 3-4, 2008, pp. 343-352. doi:10.1016/j.vetmic.2007.09.002
A. Rodriguez-Palacios, H. R. Stampfli, T. Duffield, A. S. Peregrine, L. A. Trotz-Williams, L. G. Arroyo, J. S. Brazier and J. S. Weese, “Clostridium difficile PCR Ribotypes in Calves, Canada,” Emerging Infectious Diseases, Vol. 12, No. 11, 2006, pp. 1730-1736. doi:10.3201/eid1211.051581
F. Van Immerseel, J. de Buck, F. Pasmans, G. Huyghebaert, F. Haesebrouck and R. Ducatelle, “Clostridium perfringens in Poultry: An Emerging Threat for Animal and Public Health,” Avian Pathology, Vol. 33, No. 6, 2004, pp. 537-549. doi:10.1080/03079450400013162
L. Petit, M. Gilbert and M. R. Popoff, “Clostridium perfringens: Toxinotype and Genotype,” Trends in Microbiology, Vol. 7, No. 3, 1999, pp. 104-110. doi:10.1016/S0966-842X(98)01430-9
D. Slavic, P. Boerlin, M. Fabri, K. C. Klotins, J. K. Zoethout, P. E. Weir and D. Bateman, “Antimicrobial Susceptibility of Clostridium perfringens Isolates of Bovine, Chicken, Porcine, and Turkey Origin from Ontario,” Canadian Journal of Veterinary Research, Vol. 75, No. 2, 2011, pp. 89-97.
M. Lebrun, J. G. Mainil and A. Linden, “Cattle Enterotoxaemia and Clostridium perfringens: Description, Diagnosis and Prophylaxis,” Veterinary Record, Vol. 167, No. 1, 2010, pp. 13-22. doi:10.1136/vr.167.1.12
B. Kreft, K. Dalhoff and K. Sack, “Necrotizing Enterocolitis: A Historical and Current Review,” Medizinische Klinik (Munich), Vol. 95, No. 8, 2000, pp. 435-441. doi:10.1007/s000630050003
U. Schotte, U. Truyen and H. Neubauer, “Significance of β2-Toxigenic Clostridium perfringens Infections in Animals and Their Predisposing Factors—A Review,” Journal of Veterinary Medicine, Vol. 51, No. 10, 2004, pp. 423-426. doi:10.1111/j.1439-0450.2004.00802.x
P. Lahti, A. Heikinheimo, T. Johansson and H. Korkeala, “Clostridium perfringens Type A Strains Carrying a Plasmid-Borne Enterotoxin Gene (Genotype IS1151-cpe or IS1470-like-cpe) as a Common Cause of Food Poisoning,” Journal of Clinical Microbiology, Vol. 46, No. 1, 2008, pp. 371-373. doi:10.1128/JCM.01650-07
A. G. Buchanan, “Clinical Laboratory Evaluation of a Reverse CAMP Test for Presumptive Identification of Clostridium perfringens,” Journal of Clinical Microbiology, Vol. 16, No. 4, 1982, pp. 761-762.
P. Spigaglia and P. Mastrantonio, “Molecular Analysis of the Pathogenicity Locus and Polymorphism in the Putative Negative Regulator of Toxin Production (TcdC) among Clostridium difficile Clinical Isolates,” Journal of Clinical Microbiology, Vol. 40, No. 9, 2002, pp. 3470-3475. doi:10.1128/JCM.40.9.3470-3475.2002
S. Stubbs, M. Rupnik, M. Gilbert, J. Brazier, B. Duerden and M. Popoff, “Production of Actin-Specific ADP-Ribosyltransferase (Binary Toxin) by Strains of Clostridium difficile,” FEMS Microbiology Letters, Vol. 186, No. 2, 2000, pp. 307-312. doi:10.1111/j.1574-6968.2000.tb09122.x
P. Fach and M. R. Popoff, “Detection of Enterotoxigenic Clostridium perfringens in Food and Faecal Samples with a Duplex PCR and the Slide Latex Agglutination Test,” Applied and Environmental Microbiology, Vol. 63, No. 11, 1997, pp. 4232-4236.
C. G. Baums, U. Shotte, G. Amtsberg and R. Goethe, “Diagnostic Multiplex PCR for Toxin Genotyping of Clostridium perfringens Isolates,” Veterinary Microbiology, Vol. 100, No. 1-2, 2004, pp. 11-16. doi:10.1016/S0378-1135(03)00126-3
M. Rupnik, “Is Clostridium difficile-Associated Infection a Potentially Zoonotic and Foodborne Disease?” Clinical Microbiology and Infection, Vol. 13, No. 5, 2007, pp. 457-459. doi:10.1111/j.1469-0691.2007.01687.x
J. S. Weese, B. P. Avery, J. Rousseau and R. J. Reid-Smith, “Detection and Enumeration of Clostridium difficile Spores in Retail Beef and Pork,” Applied and Environmental Microbiology, Vol. 75, No. 15, 2009, pp. 5009-5011. doi:10.1128/AEM.00480-09
J. Freeman, M. P. Bauer, S. D. Baines, J. Corver, W. N. Fawley, B. Goorhuis, E. J. Kuijper and M. H. Wilcox, “The Changing Epidemiology of Clostridium difficile Infections,” Clinical Microbiology Reviews, Vol. 23, No. 3, 2010, pp. 529-549. doi:10.1128/CMR.00082-09
J. Cloten, S. Kruth, L. Arroyo and J. S. Weese, “Prevalence and Risk Factors for Clostridium difficile Colonization in Dogs and Cats Hospitalized in an Intensive Care Unit,” Veterinary Microbiology, Vol. 129, No. 1-2, 2008, pp. 209-214. doi:10.1016/j.vetmic.2007.11.013
K. W. Post, B. H. Jost and J. G. Songer, “Evaluation of a Test for Clostridium difficile Toxins A and B for the Diagnosis of Neonatal Swine Enteritis,” Journal of Veterinary Diagnostic Investigation, Vol. 14, No. 3, 2002, pp. 258-259. doi:10.1177/104063870201400314
M. K. Keel and J. G. Songer, “The Distribution and Density of Clostridium difficile Toxin Receptors on the Intestinal Mucosa of Neonatal Pigs,” Veterinary Pathology, Vol. 44, No. 6, 2007, pp. 814-822. doi:10.1354/vp.44-6-814
S. L. Marks and E. J. Kather, “Bacterial-Associated Diarrhea in the Dog: A Critical Appraisal,” Veterinary Clinics: Small Animal Practice, Vol. 33, No. 5, 2003, pp. 1029-1060. doi:10.1016/S0195-5616(03)00091-3
M. C. Ferrarezi, T. C. Cardoso and I. S. Dutra, “Genotyping of Clostridium perfringens Isolated from Calves with Neonatal Diarrhea,” Anaerobe, Vol. 14, No. 6, 2008, pp. 328-331. doi:10.1016/j.anaerobe.2008.12.001
L. Ceci, P. Paradies, M. Sasanelli, et al., “Haemorrhagic Bowel Syndrome in Dairy Cattle: Possible Role of Clostridium perfringens Type A in the Disease Complex,” Journal of Veterinary Medicine Series A, Vol. 53, No. 10, 2006, pp. 518-523. doi:10.1111/j.1439-0442.2006.00884.x
C. C. Brown, D. C. Baker and I. K. Barker, “Alimentary System,” In: M. G. Maxie, Ed., Jubb, Kennedy and Palmer’s pathology of domestic animals, 5th Edition, Saunders Elsevier, St. Louis, 2007, pp. 1-296.
W. E. Morris, A. J. Venzano, A. Elizondo, D. A. Vilte, E. C. Mercado and M. E. Fernandez-Miyakawa, “Necrotic Enteritis in Young Calves,” Journal of Veterinary Diagnostic Investigation, Vol. 23, No. 2, 2011, pp. 254-259. doi:10.1177/104063871102300209
Y. S. Sawires and J. G. Songer, “Clostridium perfringens: Insight into Virulence Evolution and Population Structure,” Anaerobe, Vol. 12, No. 1, 2006, pp. 23-43. doi:10.1016/j.anaerobe.2005.10.002
J. G. Songer, R. Jones, M. A. Anderson, A. J. Barbara, K. W. Post and H. T. Trinh, “Prevention of Porcine Clostridium difficile-Associated Disease by Competitive Exclusion with Nontoxigenic Organisms,” Veterinary Microbiology, Vol. 124, No. 3-4, 2007, pp. 358-361. doi:10.1016/j.vetmic.2007.04.019
S. Thiede, R. Goethe and G. Amtsberg, “Prevalence of β2 Toxin Gene of Clostridium perfringens Type A from Diarrhoeic Dogs,” Veterinary Record, Vol. 149, No. 9, 2001, pp. 273-274. doi:10.1136/vr.149.9.273
D. M. Bueschel, B. H. Jost, S. J. Billington, H. T. Trinh and J. G. Songer, “Prevalence of cpb2, Encoding β2 Toxin, in Clostridium perfringens Field Isolates: Correlation of Genotype with Phenotype,” Veterinary Microbiology, Vol. 94, No. 2, 2003, pp. 121-129. doi:10.1016/S0378-1135(03)00081-6