First Comparative Analysis of Clostridium septicum Genomes Provides Insights Into the Taxonomy, Species Genetic Diversity, and Virulence Related to Gas Gangrene

Clostridium septicum is a Gram-positive, toxin-producing, and spore-forming bacterium that is recognized, together with C. perfringens, as the most important etiologic agent of progressive gas gangrene. Clostridium septicum infections are almost always fatal in humans and animals. Despite its clinical and agricultural relevance, there is currently limited knowledge of the diversity and genome structure of C. septicum. This study presents the complete genome sequence of C. septicum DSM 7534T type strain as well as the first comparative analysis of five C. septicum genomes. The taxonomy of C. septicum, as revealed by 16S rRNA analysis as well as by genomic wide indices such as protein-based phylogeny, average nucleotide identity, and digital DNA–DNA hybridization indicates a stable clade. The composition and presence of prophages, CRISPR elements and accessory genetic material was variable in the investigated genomes. This is in contrast to the limited genetic variability described for the phylogenetically and phenotypically related species Clostridium chauvoei. The restriction-modification (RM) systems between two C. septicum genomes were heterogeneous for the RM types they encoded. C. septicum has an open pangenome with 2,311 genes representing the core genes and 1,429 accessory genes. The core genome SNP divergence between genome pairs varied up to 4,886 pairwise SNPs. A vast arsenal of potential virulence genes was detected in the genomes studied. Sequence analysis of these genes revealed that sialidase, hemolysin, and collagenase genes are conserved compared to the α-toxin and hyaluronidase genes. In addition, a conserved gene found in all C. septicum genomes was predicted to encode a leucocidin homolog (beta-channel forming cytolysin) similar (71.10% protein identity) to Clostridium chauvoei toxin A (CctA), which is a potent toxin. In conclusion, our results provide first, valuable insights into strain relatedness and genomic plasticity of C. septicum and contribute to our understanding of the virulence mechanisms of this important human and animal pathogen.

Epidemiology of Blackleg Disease in Ethiopia

Blackleg is an infectious bacterial disease of cattle and rarely of other ruminants. This bacteria is caused by Clostridium chauvoei which is an anaerobic, gram positive, motile, rod-shaped bacillus bacterium and persists in the soil as resistant spores. Blackleg is an acute or subacute but chronic disease may occur. It occurs most frequently in animals 6-24 months of age and the disease mainly affects none vaccinated as well as animals in good nutritional condition. It produces persistent spores when conditions are not ideal and spores are highly resistant to environmental factors and disinfectants. Infected ruminants do not directly transmit the disease to other animals. The bacteria enter the body through the alimentary mucosa after ingestion of contaminated feed. Secretion of cytolytic toxins that cause necrosis of vascular endothelia .The toxins are absorbed into the animal’s bloodstream which makes the animal acutely sick and causes rapid death. Economic importance due to blackleg is loss of animals, milk production and draft oxen, and cost for treatment and vaccination. Fatality rate of blackleg in fully susceptible populations approaches 100%. Clinical Signs include lethargy anorexia, reluctance to move lameness and recumbence. When superficial muscles are affected, swelling and crepitus are evident. Cattle found dead of blackleg are lying on the side with the affected hind limb stands out stiffly, bloating and putrefaction occur quickly and bloodstained from exudates, nostrils and anus. The disease can be diagnosed using laboratory diagnosis, Immune Fluorescent, Cell Culture and PCR. Control and prevention relies mainly on vaccination.

Diagnosis of blackleg from cattle tissue impregnated in common filter paper

ABSTRACT: Blackleg, an acute myonecrosis caused by Clostridium chauvoei, is usually underdiagnosed since the rapid transport of adequate samples for laboratory testing is difficult. This study tested a direct polymerase chain reaction (PCR) technique using common filter paper impregnated with cattle tissue samples obtained from animals suspected with blackleg. Twenty-five samples, belonging to eleven animals from Rio Grande do Sul State, Brazil, were analyzed. The direct PCR technique identified eight positive animals corroborating with results from microbiological culture. Skeletal muscle was the most common tissue type used in this study and when the animal was positive the pathogen was always detected in this tissue. Storage time of the impregnated filter paper at room temperature did not prove to be a limiting factor for the quality of the results indicating that this procedure can be carried out in the field and samples be sent in regular mail. Our results suggested that direct PCR of common filter paper impregnated with cattle tissue is a practical and economical alternative for the diagnosis of blackleg.

Blackleg in inadequately immunized calves and their recovery following antibiotic therapy

Introduction: There is consensus regarding the importance of blackleg vaccination as a preventive measure, and proper immunization protocols are available. However, few studies have evaluated the effectiveness of vaccine protection against Clostridium chauvoei and the treatment of the disease in calves exhibiting early or advanced clinical courses. This study describes twelve blackleg cases in unvaccinated calves and in calves that received a single dose of the vaccine. It also reports the recovery of some calves after antibiotic therapy. Methodology: Two necropsies of cattle dead from blackleg were performed. Fragments of skeletal muscle from these two cattle were immersed in paraffin for multiplex polymerase chain reaction (PCR) analysis. Results: Twelve calves up to nine months of age developed signs of blackleg and eight died. Ten of those 9-month-old calves had received only the first dose of a blackleg vaccine at 4 months of age, but no booster. The last two affected calves belonged to a herd that had never been vaccinated. Four out of five calves treated with penicillin for 6-7 days recovered from the disease. The diagnosis of blackleg was based on necropsy, histopathological findings and detection of C. chauvoei in skeletal muscle samples of two necropsied calves using PCR. Conclusions: The occurrence of cases only in calves that did not receive a booster dose or were not vaccinated indicated that the vaccine used was effective when performed as recommended by the manufacturer. However, neglecting the booster resulted in casualties due to blackleg.

Prevalence of cardiac lesions in cases of bovine clostridial myositis

ObjectiveThe aim of this retrospective study was to determine the prevalence of cardiac lesions in cases of clostridial myositis. Clostridial myositis (blackleg) is a common cause of death in cattle and is caused by the bacterium Clostridium chauvoei. The characteristic lesions include hemorrhage, necrosis, edema, and emphysema within skeletal muscle and, less commonly, the heart.ResultsOf the 37 cases of blackleg identified, 26 animals (70.3%) had cardiac lesions, including 4 (10.8%) which had only cardiac involvement without skeletal muscle lesions. Cardiac lesions were characterized as necrotizing myocarditis (n=21), fibrinous to fibrinosuppurative pericarditis, epicarditis, or endocarditis (n=24), or both (n=19). These data demonstrate that, in cases of bovine clostridial myositis, cardiac lesions are common, while heart involvement in the absence of skeletal muscle lesions is uncommon.