RESUMO
Botulism in cattle is a rare but serious disease. In Germany there is no obligation to report botulism in animals and therefore a precise morbidity rate is not available. In this manuscript we describe an outbreak of Clostridium (C.) botulinum neurotoxin (BoNT) intoxication in a Saxony-Anhalt dairy cow stock of 286 Holstein-Friesian cows and offspring in spring/summer 2009 and its diagnostic approach. 122 animals showed clinical signs of BoNT intoxication. 115 of the affected animals (40.2% of the herd) independent of age died or had to be euthanized. Therapeutic attempts failed in almost all diseased cows, only four calves and three heifers recovered. Diagnostic samples of several animals (n = 4) (liver, ruminal and intestinal contents) and feed (n = 6) were tested for BoNT genes by polymerase chain reaction (PCR). BoNT gene type D was found in several (n = 8) organ samples. The PCR results allowed a preselection of samples for BoNT that were then tested by the mouse bioassay. Thus, the number of mice being inoculated in the mouse bioassay could be reduced. The mouse bioassay turned out positive (wasp-waist) in three preselected organ samples and the neutralization test of one sample with type-specific antitoxin confirmed the presence of BoNT type D. We succeeded in isolating a C. botulinum strain from a liver sample which was typed as a D/C mosaic strain by sequence analysis of the toxin gene. However, the source of the BoNT intoxication could not be traced back.
Assuntos
Botulismo/veterinária , Doenças dos Bovinos/microbiologia , Clostridium botulinum tipo D/classificação , Surtos de Doenças/veterinária , Animais , Botulismo/diagnóstico , Botulismo/epidemiologia , Botulismo/microbiologia , Bovinos , Doenças dos Bovinos/diagnóstico , Doenças dos Bovinos/epidemiologia , Clostridium botulinum tipo D/genética , Clostridium botulinum tipo D/isolamento & purificação , Clostridium botulinum tipo D/metabolismo , Indústria de Laticínios , Feminino , Alemanha/epidemiologia , Camundongos , Toxina Tetânica/genética , Toxina Tetânica/metabolismoRESUMO
A nontoxigenic strain isolated from a fatal human case of bacterial sepsis was identified as a Clostridium strain from Clostridium botulinum group III, based on the phenotypic characters and 16S rRNA gene sequence, and was found to be related to the mosaic C. botulinum D/C strain according to a multilocus sequence analysis of 5 housekeeping genes.
Assuntos
Sangue/microbiologia , Clostridium botulinum tipo C/isolamento & purificação , Clostridium botulinum tipo D/isolamento & purificação , Sepse/diagnóstico , Sepse/microbiologia , Idoso de 80 Anos ou mais , Proteínas de Bactérias/genética , Clostridium botulinum tipo C/classificação , Clostridium botulinum tipo C/genética , Clostridium botulinum tipo D/classificação , Clostridium botulinum tipo D/genética , Análise por Conglomerados , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Evolução Fatal , Feminino , Humanos , Dados de Sequência Molecular , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
Two real-time PCR arrays based on the GeneDisc(®) cycler platform (Pall-GeneDisc Technologies) were evaluated in a multicenter collaborative trial for their capacity to specifically detect and discriminate Clostridium botulinum types C, D and their mosaic variants C-D and D-C that are associated with avian and mammalian botulism. The GeneDisc(®) arrays developed as part of the DG Home funded European project 'AnibioThreat' were highly sensitive and specific when tested on pure isolates and naturally contaminated samples (mostly clinical specimen from avian origin). Results of the multicenter collaborative trial involving eight laboratories in five European Countries (two laboratories in France, Italy and The Netherlands, one laboratory in Denmark and Sweden), using DNA extracts issued from 33 pure isolates and 48 naturally contaminated samples associated with animal botulism cases, demonstrated the robustness of these tests. Results showed a concordance among the eight laboratories of 99.4%-100% for both arrays. The reproducibility of the tests was high with a relative standard deviation ranging from 1.1% to 7.1%. Considering the high level of agreement achieved between the laboratories these PCR arrays constitute robust and suitable tools for rapid detection of C. botulinum types C, D and mosaic types C-D and D-C. These are the first tests for C. botulinum C and D that have been evaluated in a European multicenter collaborative trial.
Assuntos
Botulismo/diagnóstico , Botulismo/microbiologia , Clostridium botulinum tipo C/classificação , Clostridium botulinum tipo C/genética , Clostridium botulinum tipo D/classificação , Clostridium botulinum tipo D/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Animais , Clostridium botulinum tipo C/isolamento & purificação , Clostridium botulinum tipo D/isolamento & purificação , Europa (Continente) , Humanos , Reprodutibilidade dos TestesRESUMO
Diagnosis of botulism in cows is obtained by detecting the neurotoxin and/or Clostridium botulinum in the suspected animal. The standard method for detecting the toxin is the mouse bioassay. However, in recent years, the use of mice has become very costly and inconvenient in some facilities, and public pressure has been increasing to find alternatives to live animal bioassays. In this manuscript, we describe the use of the polymerase chain reaction (PCR) procedures in the diagnosis field cases of bovine type D botulism. Bovine samples from clinical cases diagnosed as C. botulinum type D according by clinical symptoms and bioassay resulted in expected PCR product ( approximately 497 bp) similar to the C. botulinum type D NCTC 8265 strain while the gene product was confirmed by sequence data.
