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

Carlos Romero, Nuria Nicodemus, Maria Luisa Jarava, David Menoyo, de Blas Carlos


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 days were housed in pairs and fattened until the age of 63 days.  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 days, 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.



Alpha-toxin; Clostridium perfringens; digestive diseases; Epizootic Rabbit Enteropathy; growth depression; toxinotype

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AOAC. 2000. Official Methods of Analysis, 17th Ed. Association of Official Analytical Chemists, Arlington, VA.

Baskerville M., Wood M., Seamer J.H. 1980. Clostridium perfringens type E enterotoxaemia in rabbits. Vet. Rec., 107: 18-19.

Baums C.G., Schotte U., Amtsberg G., Goethe R. 2004. Diagnostic multiplex PCR for toxin genotyping of Clostridium perfringens isolates. Vet. Microbiol., 100: 11-16.

Bio-X Diagnostics BIO K084 (currently BIO K289). Clostridium perfringens Alpha toxin antigen detection kit. Bio-X Diagnostics, Jemelle, Belgium. Available at: Accessed September 2011.

Boucher S., Nouaille L. 2002. Manuel pratique: Maladies des lapins. Éditions France Agricole, Second Edition, France.

Boucher S., Leplat A. 2005. De quoi meurent les lapins d'engraissement? Table Ronde: Effets des conduites post-sevrage sur les performances et la santé des lapereaux. In: 11èmes Journées de la Recherche Cunicole, Paris, France, Available at: Accessed August 2011.

Cocchi M., Agnoletti F., Bacchin C., Bano L., Guolo A., Drigo I. 2007. Tossinotipizzazione di Clostridium perfringens isolato da coniglio. In Proc.: Giornate di Coniglicoltura ASIC, Forlì, Italy, 67-69.

Coudert P., Licois D. 2005. Epizootic rabbit enteropathy. Study of early phenomena with fresh inoculum and attempt at inactivation. World Rabbit Sci., 13: 229-238.

Coudert P., Lebas F., Licois D. 1997. Une nouvelle pathologie ravage les élevages. La profession se mobilise. Cuniculture, 24: 225-229.

Coudert P., Peeters J.E., Rosell J.M., Argüello J.L., Badiola J.I., Blas E., Cuervo L., Drouet-Viard F., Licois D., Provôt F. 2000. Enfermedades del aparato digestivo: causas parasitarias y otras. In: Rosell J.M. (ed), Enfermedades del Conejo. Mundi-Prensa, Spain, 219-263.

Dewrée R., Meulemans L., Lassence C., Desmecht D., Ducatelle R., Mast J., Licois D., Vindevogel H., Marlier D. 2007. Experimentally induced epizootic rabbit enteropathy: clinical, histopathological, ultrastructural, bacteriological and haematological findings. World Rabbit Sci., 15: 91-102.

Duperray J., Boisot P., Guyonvarch A., Richard A. 2003. Persistance de l'efficacité de la bacitracine pour lutter contre l'entéropathie épizootique du lapin (EEL) après quatre années d'utilisation sur le terrain. In Proc.: 10èmes Journées de la Recherche Cunicole, Paris, France, 271-274.

Garrido S., Nicodemus N., García J., Chamorro S., De Blas J.C. 2009. Effect of breeding system and farm hygiene on performances of growing rabbits and lactating does over two reproductive cycles. World Rabbit Sci., 17: 71-78.

Gurjar A.A., Hedge N.V., Love B.C., Jayarao B.M. 2007. Real-time multiplex PCR assay for rapid detection and toxintyping of Clostridium perfringens toxin producing strain in faeces of dairy cattle. Mol. cell. prob., 22: 90-95.

Hale M.L., Stiles B.G. 1999. Detection of Clostridium perfringens alpha toxin using a capture antibody ELISA. Toxicon, 37: 471-484.

Huybens N., Houeix J., Licois D., Mainil J., Marlier D. 2009. Inoculation and bacterial analyses of fractions obtained from the reference inoculum TEC4 which experimentally reproduces epizootic rabbit enteropathy. World Rabbit Sci., 17: 185-193.

ISO - International Organization for Standardization - 1997. Method ISO 7937. Available at: Accessed August 2011.

Kalender H., Ertas H.B., Cetinkaya B., Muz A., Arslan N., Kilic A. 2005. Typing of isolates of Clostridium perfringens from healthy and diseased sheep by multiplex PCR. Vet. Med.-Czech, 50: 439-442.

Licois D. 2004. Domestic rabbit enteropathies. In Proc.: 8th World Rabbit Congress, Puebla, México, 385-403.

Licois D. 2007. Étude in vivo de la fraction surnageante de l'inoculum TEC4, inoculum utilisé pour la reproduction expérimentale de l'Entéropathie Epizootique du Lapin. In Proc.: 12èmes Journées de la Recherche Cunicole, Le Mans, France, 217-220.

Licois D. 2010. Pathologie d'origine bactérienne et parasitaire chez le lapin: apports de la dernière décennie. Cuniculture, 37: 35-49.

Licois D., Wyers M., Coudert P. 2005. Epizootic Rabbit Enteropathy: experimental transmission and clinical characterization. Vet. Res., 36: 601-613.

Licois D., Coudert P., Marlier D. 2006. Epizootic rabbit enteropathy. In: Maertens L., Coudert P. (Eds.), Recent Advances in Rabbit Sciences. Institute for Agricultural and Fisheries Research, Melle, Belgium, 163-170.

Liu Z.L., Blaschek H.P. 1996. Monoclonal antibody-based ELISA for detection of Clostridium perfringens alpha-toxin. J. Food Protect., 59: 621-625.

Maertens L., Cornez B., Vereecken M., Van Oye S. 2005. Efficacy study of soluble bacitracin (Bacivet S®) in a chronically infected epizootic rabbit enteropathy environment. World Rabbit Sci., 13: 165-178.

Marlier D., Dewrée R., Lassence C., Licois D., Mainil J., Coudert P., Meulemans L., Ducatelle R., Vindevogel H. 2006. Infectious agents associated with epizootic rabbit enteropathy: Isolation and attempts to reproduce the syndrome. Vet. J., 172: 493-500.

McCullagh P., Nelder J.A. 1989. Generalized Linear Models, Second Edition. Chapman and Hall, New York, USA.

Menoyo D., García C., Jarava M., De Blas C. 2011. Genotyping of Clostridium perfringens strains in rabbits affected by epizootic enteropathy. In Proc.: XIV Jornadas sobre Producción Animal, AIDA-ITEA, Zaragoza, Spain, 863-865.

Mertens D.R. 2002. Gravimetric determination of amylase-treated neutral detergent fibre in feeds with refluxing beakers or crucibles: collaborative study. J. Assoc. Off. Assoc. Chem. Int., 85: 1217-1240.

Möllby R., Holme T., Nord C.E., Smyth C.J., Wadström T. 1976. Production of phospholipase C (alpha-toxin), haemolysins and lethal toxins by Clostridium perfringens types A to D. J. Gen. Microbiol., 96: 137-144.

Patton N.M., Holmes H.T., Riggs R.J., Cheeke P.R. 1978. Enterotoxemia in rabbits. Lab. Anim. Sci., 28: 536-540. PMid:214635

Petit L., Gibert M., Popoff M.R. 1999. Clostridium perfringens: toxinotype and genotype. Trends Microbiol., 7: 104-110.

Richez P., Richard A., Cornez B. 2007. Sensibilité, résistance et profil de bactéricidie de la bacitracine vis-à-vis de souches de Clostridium perfringens isolées lors de cas cliniques d'Entéropathie Epizootique du Lapin. In Proc.: 12èmes Journées de la Recherche Cunicole, Le Mans, France, 209-212.

Romero C., Nicodemus N., Chamorro S., Corujo A., Martínez-Morentin C.G., Margüenda I., de Blas C. 2009a. Efecto de la camada y las condiciones ambientales sobre la mortalidad de gazapos en periodos con distinta incidencia de enteropatía epizoótica del conejo. In Proc.: XIII Jornadas sobre Producción Animal, AIDA-ITEA, Zaragoza, Spain, 191-193.

Romero C., Nicodemus N., García-Rebollar P., García-Ruiz A.I., Ibáñez M.A., de Blas J.C. 2009b. Dietary level of fibre and age at weaning affect the proliferation of Clostridium perfringens in the caecum, the incidence of Epizootic Rabbit Enteropathy and the performance of fattening rabbits. Anim. Feed Sci. Technol., 153: 131-140.

Romero C., Rebollar P.G., Dal Bosco A., Castellini C., Cardinali R. 2010. Efecto de la inclusión de ácidos orgánicos de cadena corta sobre el crecimiento, la mortalidad y el tejido linfoide del intestino en gazapos. In Proc.: XXXV Symposium de Cunicultura, Segovia, Spain, 79-83.

Romero C., Nicodemus N., Rodríguez J.D., García A.I., de Blas C. 2011. Effect of type of grinding of barley and dehydrated alfalfa on performance, digestion, and crude mucin ileal concentration in growing rabbits. J. Anim. Sci., 89: 2472-2484.

Rood J.I. 1998. Virulence genes of Clostridium perfringens. Annu. Rev. Microbiol., 52: 333-360.

Rosell J.M., de la Fuente L.F., Badiola J.I., Fernández de Luco D., Casal J., Saco M. 2009. Study of urgent visits to commercial rabbit farms in Spain and Portugal during 1997-2007. World Rabbit Sci., 17: 127-136.

SAS. 1990. SAS/STAT® User's Guide, Version 6, 4th ed. SAS Inst., Cary, NC, USA.

Songer J.G. 1996. Clostridial Enteric Diseases of Domestic Animals. Clin. Microbiol. Rev., 9: 216-234.

Spanish Royal Decree 1201/2005. 2005. Real Decreto 1201/2005. Sobre protección de los animales utilizados para experimentación y otros fines científicos. Boletín Oficial del Estado, 252: 34367-34391.

Szalo I.M., Lassence C., Licois D., Coudert P., Poulipoulis A., Vindevogel H., Marlier D. 2007. Fractionation of the reference inoculum of epizootic rabbit enteropathy in discontinuous sucrose gradient identifies aetiological agents in high density fractions. Vet. J., 173: 652-657.

Timbermont L., Lanckriet A., Gholamiandehkordi A.R., Pasmans F., Martel A., Haesebrouck F., Ducatelle R., Van Immerseel F. 2009. Origin of Clostridium perfringens isolates determines the ability to induce necrotic enteritis in broilers. Comp. Immun. Microbiol. Infect. Dis., 32: 503-512.

Wasinski B. 2007. Contribution of Clostridium perfringens type A with β2 toxin gene in aetiology of porcine enteric diseases. A case report. B. Vet. I. Pulawy, 51: 509-513.

Zhang G., Darius S., Smith S.R., Ritchie S.J. 2006. In vitro inhibitory effect of hen egg white lysozyme on Clostridium perfringens type A associated with broiler necrotic enteritis and its α-toxin production. Lett. Appl. Microbiol., 42: 138-143.

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