Effect of formalin percentage, incubation time and temperature on Clostridium chauvoei culture inactivation and immunogenicity.

OBJECTIVES: In order to find the optimal inactivation conditions for Clostridium chauvoei culture, different factors were investigated and the immunogenicity of inactivated cultures was studied. METHODS: C. chauvoei was cultured with different formalin percentages (0.3, 0.5 or 0.7% V/V), inactivation temperatures (37 °C or room temperature) and incubation times (one or two weeks). Sterility tests were performed and residual formaldehyde and pH were measured. Rabbits were immunized twice with inactivated cultures and sera were used for detection of immune response. RESULTS: In the one-week experiment, 0.5 and 0.7% formalin inactivated the bacteria after one week, and the percentage of 0.3 inactivated after three weeks. The residual formaldehyde at weeks 1 and 8 was not significantly different. In the two-week experiment, cultures treated with 0.3 and 0.5% formalin were inactivated after four weeks, and those with 0.7% formalin were inactivated after three weeks. Residual formaldehyde at week 8 differed significantly from that of week 1. Residual formaldehyde was affected by incubation temperature since it was lower at 37 °C than in room temperature. Also, a significant effect was observed for formalin on pH, as higher formalin contents led to lower pH values of the cultures. ELISA showed the lowest antibody titer achieved by 0.7% formalin group. Antibody titer was not different between 0.3 and 0.5% formalin. CONCLUSIONS: The best condition for inactivation of C. chauvoei was considered as one-week incubation with 0.5% formalin at 37 °C, leading to a high antibody response.

Vegetative forms of Clostridium chauvoei trigger apoptotic and inflammatory responses on macrophages.

BACKGROUND: Clostridium chauvoei is a gram-positive, spore-forming, strictly anaerobic bacterium that causes blackleg, a disease that affects cattle by inducing fulminant myonecrosis, thereby leading to high and constant losses of cattle. Macrophages (Mɸs) are depleted in tissues infected with the vegetative form of C. chauvoei, but the mechanism remains partially known. Consequently, Mɸs may be a critical target in the pathogenicity of C. chauvoei. AIM: The objective of this work was to study the mechanism of death of mouse-primary Mɸs infected in vitro for 24 h with the vegetative form of C. chauvoei. METHODS: Mouse peritoneal Mɸs were infected in vitro with different multiplicities of infection (MOIs) of C. chauvoei (i.e., 5:1, 20:1, and 100:1). After 24 h post-infection, cell viability (MTT reduction assay), apoptosis (apoptotic bodies, DNA ladder, and Annexin V assays), and inflammatory cell response (iNOS and TNF-α expression) were assessed. RESULTS: All the MOIs investigated decreased cell viability. An MOI of 20:1 caused the highest production of apoptotic bodies and an electrophoretic DNA-ladder pattern typical of an apoptosis cell death process. These results were corroborated using the Annexin V-flow cytometry assay. Concurrently with apoptotic cell death, Mφs expressed iNOS and TNF-α. CONCLUSION: Inflammation-mediated apoptosis of Mφs can be a potential mechanism of evasion of the immune response used by C. chauvoei in tissues for depleting phagocytic cells at the site of infection.

Clostridial panophthalmitis - An unusual case of localised malignant oedema in Merino lambs associated with Clostridium chauvoei.

The authors report an unusual case of Clostridium chauvoei causing severe panophthalmitis in Merino lambs. More than half of the lambs affected survived, which is unusual for clostridial disease; however, there have been reports in humans that the mortality risk for ocular gas gangrene is lower than when other body parts are affected. A combination of factors in this case included environmental contamination (specific to a particular paddock), genetics predisposing to entropion (lambs born of maiden 2-year-old ewes with some inbreeding), the practice of manually everting eyelids for the entropion and vaccination strategies. C. chauvoei was cultured in pure growth from the eye of a lamb affected by severe panophthalmitis. Histopathology was consistent with severe acute infection, and microscopic sections showed Gram-positive organisms associated with the inflammatory response in the eye. In the acutely affected animal examined there were no signs that the lesions were long-standing. Animals that recovered had one or both eyes destroyed. This report describes that malignant oedema in sheep due to C. chauvoei can manifest as acute and severe panophthalmitis. The case fatality rate of lambs with panophthalmitis was less than 50%, lower than normally occurs for clostridial diseases. Clostridial vaccination of the ewes may have provided a low level of protection in reducing the case fatality rate in the affected lambs as well localisation of the infection from the blood-ocular barrier.

Whole-genome sequence analysis of Clostridium chauvoei isolated from clinical case of black quarter (BQ) from India.

Black quarter (BQ) is an infectious disease affecting cattle and small ruminants worldwide caused by Gram-positive anaerobic bacterium Clostridium chauvoei. In this study, a draft genome sequence of C. chauvoei NIVEDIBQ1 strain isolated from clinical case of black quarter was analyzed. Sequence analysis indicated that genome had 2653 predicted coding DNA sequences, harbored numerous genes, mobile genetic elements for pathogenesis, and virulence factors. Computational analysis revealed that strain contained 30 virulence-associated genes. An intact genomic region highly similar to the Clostridium phage was present in the genome. Presence of CRISPR systems and the transposon components likely contribute to the genome plasticity. Strain encode diverse spectrum of degradative carbohydrate-active enzymes (CAZymes). Comparative SNP analysis revealed that the genomes of the C. chauvoei strains analyzed were highly conserved. Phylogenetic analysis of strains and available genome (n = 21) based on whole-genome multi-locus sequence typing (wgMLST) and core orthologous genes showed the clustering of strains into two different clusters suggesting geographical links.

Immunomodulatory effect of short-term supplementation with Bacillus toyonensis BCT-7112T and Saccharomyces boulardii CNCM I-745 in sheep vaccinated with Clostridium chauvoei.

The bacterium Clostridium chauvoei is the causative agent of blackleg in livestock, and vaccination is the most effective means of prevention. The aim of this study was to assess the effect of short-term supplementation with Bacillus toyonensis and Saccharomyces boulardii on the immune response to a C. chauvoei vaccine in sheep. Sheep were vaccinated subcutaneously on day 0 and received a booster dose on day 21, with 2 mL of a commercial vaccine formulated with inactivated C. chauvoei bacterin adsorbed on aluminum hydroxide. Probiotics were orally administered B. toyonensis (3 × 108 cfu) and S. boulardii (3 × 108 cfu) over five days prior to the first and second doses of the vaccine. Sheep supplemented with B. toyonensis and S. boulardii showed significantly higher specific IgG, IgG1, and IgG2 titers (P<0.05), with approximately 24- and 14-fold increases in total IgG levels, respectively, than the nonsupplemented group. Peripheral blood mononuclear cells from the supplemented group had increased mRNA transcription levels of the IFN-γ, IL2, and Bcl6 genes. These results demonstrate an adjuvant effect of short-term supplementation with B. toyonensis and S. boulardii on the immune response against the C. chauvoei vaccine in sheep.

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.

Blackleg without skeletal muscle involvement as a cause of sudden death in unvaccinated calves.

Nine, 5-month-old unvaccinated black Angus calves died in less than 48 hours of each other. The calves expressed minimal or no clinical signs prior to death. On-farm post-mortem examinations were carried out on 3 of the deceased calves, and findings included: necrotizing and fibrinosuppurative myositis of the diaphragm muscle, and fibrinosuppurative pleuritis and epicarditis. Fluorescent antibody testing for pathogenic clostridia identified Clostridium chauvoei and a diagnosis of blackleg was made. The remainder of the herd were then vaccinated.

Charbon symptomatique sans implication des muscles squelettiques comme cause de mort subite chez des veaux non-vaccinés. Neuf veaux non-vaccinés de race Angus âgés de 5 mois sont morts en moins de 48 heures l'un de l'autre. Les veaux ne présentèrent que peu ou pas de signes cliniques avant la mort. Des examens post-mortem réalisés à la ferme ont été effectués sur trois des veaux décédés, et les trouvailles incluaient: myosite nécrosante et suppurative du muscle diaphragmatique, et pleurésie fibrino-suppurative et épicardite. La recherche de clostridies pathogènes à l'aide d'anticorps fluorescents permis d'identifier Clostridium chauvoei et un diagnostic de charbon symptomatique fut posé. Le restant du troupeau fut alors vacciné.(Traduit par Dr Serge Messier).

Marker-less deletion of cctA gene of Clostridium chauvoei.

Clostridium chauvoei causes blackleg disease in domestic animals, especially cattle and sheep. The pathogen produces several toxins including CctA - a hemolysin and protective antigen. Molecular pathogenesis of the disease is poorly understood, possibly due to lack of genetic manipulation tools for C. chauvoei. In the present study, we report the marker-less deletion of cctA gene using the CRISPR-Cas9 system. The C. chauvoei cctA deletion mutant had negligible hemolytic and significantly reduced cytotoxic activities. To the best of our knowledge, this is the first report of genetic manipulation of C. chauvoei. The method we used in this study can be applied for genetic manipulation of C. chauvoei to better understand the pathogenesis and genetics of the pathogen.

Genomic comparison of Clostridium chauvoei isolates from classical and visceral clinical manifestation.

Clostridium chauvoei is the etiological agent of blackleg, an infectious disease affecting cattle and small ruminants worldwide. This disease can manifest as classical blackleg, a condition in which skeletal muscles are affected and visceral blackleg, which affects the heart, sublingual muscles, and the diaphragm. The pathogenesis of the visceral form of the disease is poorly understood. The objective of this study is to determine and analyze complete genomic sequences of six C. chauvoei strains, five isolates from skeletal muscle and one isolate from a visceral case of blackleg in Brazil, to provide insights into the differences in pathogenic profiles of strains causing the different forms of disease. The full genomes of the six C. chauvoei strains were sequenced and comparative analyses were performed among these genomes and the C. chauvoei reference strain JF4335. The results of this study revealed that the genomes of the C. chauvoei strains analyzed are highly conserved; no particular differences were noted that could be associated with the two different clinical manifestations of the disease.

Financial impact of an outbreak of clinically diagnosed blackleg - a case study from Lao PDR.

Blackleg (BL) is an acute to peracute highly fatal infectious disease of mainly large ruminants characterised by lesions of myonecrosis caused by Clostridium chauvoei, usually presenting as a sudden onset of sporadic mortalities. In Southeast Asia, 'BL' is considered a cause of occasional outbreaks of a subacute febrile illness, although there are few published reports available. Investigation of a major outbreak of clinically diagnosed BL occurring in large ruminants (cattle and buffalo) in three neighbouring villages in central Laos in mid-2017, was conducted to determine the financial impacts of BL on smallholder livelihoods. Owners of BL-infected large ruminants in the three affected villages were interviewed (n = 27) and financial losses including losses due to mortality, morbidity and costs of treatments, were determined. The reports of clinical signs of subcutaneous swelling with palpable crepitus in febrile animals were considered consistent with a diagnosis of BL. The outbreak occurred in 47 of a total 449 households with large ruminants across the three villages, affecting 147 of a total population of 3505 'at risk' large ruminants with 71 deaths reported. At the household level, the mean morbidity and mortality rates were 5 ± 4(95% CI: 3-6) and 3 ± 2(95% CI: 1-4) heads per household, respectively. The estimated financial losses due to BL per affected household was USD 822 ± 692(95% CI: 518-1125), being 122% of their annual household income from large ruminant sales. The comparison between the estimated losses due to BL per village and cost of annual BL vaccination programmes, indicated a potential economic benefit of USD3.09 and USD12.37 for every dollar invested in the vaccination programme, if BL outbreaks occur every 20 and 5 years, respectively. This study indicates that clinically diagnosed BL can cause significant losses to smallholder households, and requires Lao animal health authorities to consider vaccination interventions to prevent losses from re-emergence of BL in the known endemically affected areas.

Production of neutralizing antibodies against the secreted Clostridium chauvoei toxin A (CctA) upon blackleg vaccination.

Clostridium chauvoei is the etiologic agent of blackleg in cattle, inducing fever, severe myonecrosis, oedemic lesions and ultimately death of infected animals. The pathogen often results in such rapid death that antibiotic therapy is futile and thus vaccination is the only efficient strategy in order to control the disease. The ß-barrel pore forming leucocidin Clostridium chauvoei toxin A (CctA) is one of the best characterised toxins of C. chauvoei and has been shown to be an important virulence factor. It has been reported to induce protective immunity and is conserved across C. chauvoei strains collected from diverse geographical locations for more than 50 years. The aim of this study was to identify the location of the CctA toxin during liquid culture fermentation and to use CctA to develop an in vitro assay to replace the current guinea pig challenge assay for vaccine potency in standard batch release procedures. We report that CctA is fully secreted in C. chauvoei culture and show that it is found abundantly in the supernatant of liquid cultures. Sera from cattle vaccinated with a commercial blackleg vaccine revealed strong haemolysin-neutralizing activity against recombinant CctA which reached titres of 1000 times 28 days post-vaccination. Similarly, guinea pig sera from an official potency control test reached titres of 600 times 14 days post-vaccination. In contrast, ELISA was not able to specifically measure anti-CctA antibodies in cattle serum due to strong cross-reactions with antibodies against other proteins present pre-vaccination. We conclude that haemolysin-neutralizing antibodies are a valuable measurement for protective immunity against blackleg and have the potential to be a suitable replacement of the guinea pig challenge potency test, which would forego the unnecessary challenge of laboratory animals.

Pathological and clinical investigations of an outbreak of Blackleg disease due to C. chauvoei in cattle in Punjab, Pakistan.

INTRODUCTION: Clostridium chauvoei (C. chauvoei) is an anaerobic, histotoxic Gram-positive, bacterium causing fatal myonecrosis in livestock with high mortalities. The disease is common in dairy animals, but little is known about the pathophysiology of the disease in exotic (non-native) animals kept under local conditions in Pakistan. METHODOLOGY: Diagnosis of blackleg was made based on hematological and serum biochemical analysis, PCR, necropsy and histopathology. RESULTS: Clinically sick animals exhibited fever, lameness, subcutaneous gaseous swelling and edema particularly in hindquarter and front legs. Hematological analysis showed increases in erythrocyte sedimentation rate and reduces in number of red blood cells, packed cell volume, leukocytes and differential leukocyte count. Serum aspartate aminotransferase, lactate dehydrogenase, alkaline phosphates, alanine aminotransferase, urea, creatinine, creatine kinase, and creatinine phosphokinase were significantly (P < 0.05) higher in the infected animals. At necropsy, swelling areas contained straw-colored fluid with gas bubbles. The muscles were swollen, dark to black and exhibited crepitation sounds at the time of incisions with a rancid odor. Severe pulmonary edema, myocarditis along with petechial hemorrhages, as well as enlargement and congestion of liver and spleen have been observed. Microscopic examination revealed severe inflammatory reaction, edema, and disruption of the myofibrils. Examination of heart, spleen, liver, kidneys, intestine, and lungs showed congestion, severe inflammatory changes with neutrophilic infiltration and necrosis accompanied by dissociation of the normal tissue structure. PCR confirmed C. chauvoei in exudates and different samples of muscles. CONCLUSION: The pathophysiology should be considered in diagnosis of blackleg. The disease is exist in the non-native cattle farms and biosecurity measures have to be elevated.

Blackleg in a free-range brown brocket deer (Mazama gouazoubira) / Carbúnculo sintomático em veado virá de vida livre (Mazama gouazoubira)

A case of blackleg in a brown brocket deer (Mazama gouazoubira) associated with trauma from being hit by a car in southern Rio Grande do Sul is reported. The clinical signs included fever, dehydration and lethargy that worsened progressively until 36 hours after the accident, when the animal died. In the fore right limb, there was a comminuted closed fracture of the radius and ulna but no skin wounds were observed. Grossly, the musculature of the pelvic limbs presented hemorrhage, edema and emphysema. Microscopically, the muscles of both rear legs had necrosis, edema, hemorrhage and mild inflammatory infiltration of neutrophils. Clostridium chauvoei was cultured from affected skeletal muscles, and it was also detected by immunohistochemistry, confirming a diagnosis of blackleg. The overlapping habitat of cattle and brown brocket deer is proposed as a predisposing factor in this case and alerts to spillover cases maybe happening in this region. In addition, blackleg should be included as differential diagnoses of deer with post-traumatic myositis.(AU)

Descreve-se um caso de carbúnculo sintomático em um veado-virá (Mazama gouazoubira), macho, jovem, resgatado após atropelamento em uma rodovia na região sul do Rio Grande do Sul. O cervídeo apresentou febre, desidratação e letargia, progredindo para a morte em 36 horas. No membro torácico direito foi observado fratura cominutiva fechada de rádio e ulna sem a presença de feridas perfurantes. Na necropsia foi observada hemorragia, edema e enfisema na musculatura dos membros pélvicos. Microscopicamente, os músculos dos membros pélvicos apresentaram necrose, edema, hemorragia e discreto infiltrado inflamatório neutrofílico. Houve o isolamento de Clostridium chauvoei e marcação positiva na técnica de IHQ com anticorpo monoclonal anti-C. chauvoei, confirmando o diagnóstico de carbúnculo sintomático. A sobreposição de habitat entre bovinos domésticos e cervídeos pode ser um fator de risco para esta doença e chama a atenção para casos de "spillover" que podem estar ocorrendo na região. Adicionalmente, sugere-se que o carbúnculo sintomático seja incluído nos diagnósticos diferenciais de cervídeos que apresentam miosite pós-traumática.(AU)

Pathology of blackleg in cattle in California, 1991-2015.

Blackleg is an infectious disease of cattle and rarely other ruminants, produced by Clostridium chauvoei and characterized by necrotizing myositis. In most cases of blackleg, the large muscles of the pectoral and pelvic girdles are affected, with other skeletal muscles and the heart involved less frequently. We studied 29 blackleg cases selected from the archives of the California Animal Health and Food Safety Laboratory, 1991-2015. Immunohistochemistry was also evaluated to detect C. chauvoei in formalin-fixed, paraffin-embedded (FFPE) tissues of cattle. Nineteen animals had gross and/or microscopic lesions in both skeletal muscle and heart, 9 had lesions in the skeletal musculature alone, and 1 in the heart alone. Gross lesions in the skeletal musculature involved the following muscle groups: hindquarters ( n = 8), forequarters ( n = 5), neck ( n = 5), lumbar area ( n = 3), brisket ( n = 2), diaphragm ( n = 2), abdominal wall ( n = 1), thoracic wall ( n = 1), and tongue ( n = 1). Of the 20 animals that had lesions in the heart, 11 had pericarditis and myocarditis; 7 had pericarditis, myocarditis, and endocarditis; and 1 each had pericarditis and myocarditis. Immunohistochemistry was 100% sensitive to detect C. chauvoei in FFPE skeletal muscle and/or heart of cattle with blackleg. Simultaneous lesions in skeletal musculature and heart were relatively common in blackleg cases in California; the most affected skeletal muscles were those of the hindlimbs.

Characterization of quorum sensing system in Clostridium chauvoei.

Clostridium chauvoei causes fatal black quarter infection in cattle and buffaloes. The quorum sensing (QS) system, a bacterial cell to cell communication process, of the pathogen was characterized in the current study. The results indicated that C. chauvoei lacked luxS (autoinducer-2) based quorum sensing as detected by the sensor strain Vibrio harveyi BB170. This was supported by absence of luxS gene in C. chauvoei genome. However, the genomic analysis indicated the presence of agrBD system in all three genomes of C. chauvoei available at the NCBI database. The AgrD, which synthesizes QS messenger auto-inducing peptide, was a 44 amino acid protein which shared 59% identity and 75% similarity with AgrD of C. perfringens strain 13 and 56% identity (20% coverage) with Staphylococcus aureus N315. The functional cysteine amino acid was conserved in all the strains. The genomic organisation further suggests the presence of diguanylate cyclase, a gene responsible for synthesis of secondary messenger cyclic di-GMP, at 3' immediate downstream of agrD gene. The real time expression analysis for agrD gene indicated that expression was better at 37 °C (1.9-3.7 fold increase) compared to a higher temperature of 40 °C. However, stable expression was observed at different growth stages (log and early stationary phase) with 0.8-1.4 fold changes in expression pattern. The results indicate the presence of a constitutively expressed agrBD quorum sensing system in C. chauvoei.

Blackleg in cattle in the state Mato Grosso do Sul, Brazil: 59 cases / Carbúnculo sintomático em bovinos em Mato Grosso do Sul: 59 casos

This study aimed to review cases of blackleg (Clostridium chauvoei infection) diagnosed in cattle from Midwestern Brazil from 1994 to 2014 considering epidemiological, clinical, necropsy and histopathological findings. Also the following laboratory tests were used for the diagnosis of some cases of blackleg: microbiological culture and identification of the agent, microbiological culture and identification of the agent by the polymerase chain reaction (PCR), and identification of the agent in formalin fixed paraffin embedded tissues (FFPE). Criteria for presumptive diagnosis of blackleg included necrohemorrhagic emphysematous myositis consisting of inflammatory infiltrate, coagulative necrosis of myofiber, interstitial edema, hemorrhage, and gas bubbles between myofibers. Fifty nine cases from 51 outbreaks of blackleg were found, which corresponded to 1.1% of 5,375 cattle deaths investigated. In five of those outbreaks, samples of affected muscles cultures for the identification of pathogenic clostridia were made. Another three samples of similar material were cultured for clostridia with subsequent identification of the isolate by PCR. Twelve samples of FFPE affected muscle fragments were submitted to PCR for identification of the etiological agent. Except for January, cases were observed in each month of the year, with higher numbers in July-October. Most affected cattle were in the age of 7-12 years, but calves younger than 6 month-old and older than 24 months were also observed. Vaccination histories were scarce. In 32 outbreaks some vaccination history was available, but only in two of those vaccination has been carried out properly. In 56 six cases the skeletal muscles were involved. Muscles of the hind limbs were the most affected. In ten cases muscles of the tongue, myocardium and diaphragm were also affected. In three of the cases the visceral form was observed. Deaths occurred after a clinical course of 6-24 hours, but in most cases cattle were found death. Sudden death was the outcome in visceral cases (cardiac) blackleg. Clostridium chauvoei was confirmed to be the cause by culturing in 5 cases, and by PCR and histopatology in 8 cases. Bacterial culture followed by PCR did not demonstrate C. chauvoei. Calculation of the economic impact indicates that blackleg is a frequent disease in the state of Mato Grosso do Sul (MS) that inflicts significant economic loss. The amount of these losses would be reduced through proper vaccination programs against the prevalent strains of C. chauvoei in the region.(AU)

Este estudo foi realizado com o objetivo de descrever casos de carbúnculo sintomático (infecção por Clostridium chauvoei) diagnosticados em bovinos do Centro-Oeste brasileiro de 1994-2014, avaliando a epidemiologia, os sinais clínicos, os achados de necropsia e a histopatologia; objetivou-se também avaliar os seguintes testes laboratoriais para o diagnóstico de carbúnculo sintomático: cultura microbiológica e identificação do agente, cultura microbiológica e identificação do agente por reação em cadeia de polimerase (PCR) e identificação do agente em material fixado em formol e incluído em parafina (FFIP). Os critérios para o diagnóstico presuntivo de carbúnculo sintomático incluíram miosite necro-hemorrágica enfisematosa, caracterizada por infiltrado inflamatório, necrose de coagulação de miofribras, edema intersticial, hemorragia e bolhas de gás em meio às miofribras. Cinquenta e nove casos oriundos de 51 surtos foram encontrados, o que corresponde a 1,1% das 5.375 mortes de bovinos investigadas. Em cinco desses casos, amostras do músculo afetado foram cultivadas para clostrídios patogênicos. Amostras semelhantes de outros três animais foram cultivadas para clostrídios e os isolamentos identificados subsequentes por PCR. Doze fragmentos de músculo afetado FFIP foram submetidos a PCR para identificação do agente etiológico. Com exceção de janeiro, os casos de carbúnculo sintomático foram observados em todos os meses do ano com uma maior incidência em junho-outubro. A faixa etária da maioria dos bovinos afetados era de 7-12 anos de idade, mas bovinos mais jovens que 6 meses e mais velhos que 24 meses foram também afetados. Os históricos de vacinação eram escassos nesses surtos. Em 32 surtos havia alguma informação sobre a vacinação, mas em apenas dois casos a vacinação tinha sido realizada adequadamente. Cinquenta e seis casos de carbúnculo sintomático deste estudo eram casos clássicos afetando os músculos esqueléticos. Os músculos mais afetados foram os dos membros pélvicos. Em dez casos os músculos da língua, miocárdio e diafragma estavam também afetados. Apenas três dos casos apresentaram a forma visceral (cardíaca). O curso clínico foi de 6-24 horas, mas na maioria dos casos os bovinos foram encontrados mortos. Em casos da forma visceral ocorria morte súbita. Clostridium chauvoei foi confirmado como o agente causal por cultura em cinco casos e por PCR em amostra FFIP em 8 casos. Cultura bacteriana seguida de PCR do isolado não demonstrou C. chauvoei. Carbúnculo sintomático é uma doença frequente em bovinos no Mato Grosso do Sul podendo provocar importantes prejuízos para os produtores rurais. Esses prejuízos podem ser reduzidos através de um programa de vacinação adequado usando-se vacinas eficazes contra cepas de C. chauvoei prevalentes na região.(AU)

First report of two complete Clostridium chauvoei genome sequences and detailed in silico genome analysis.

Clostridium (C.) chauvoei is a Gram-positive, spore forming, anaerobic bacterium. It causes black leg in ruminants, a typically fatal histotoxic myonecrosis. High quality circular genome sequences were generated for the C. chauvoei type strain DSM 7528T (ATCC 10092T) and a field strain 12S0467 isolated in Germany. The origin of replication (oriC) was comparable to that of Bacillus subtilis in structure with two regions containing DnaA boxes. Similar prophages were identified in the genomes of both C. chauvoei strains which also harbored hemolysin and bacterial spore formation genes. A CRISPR type I-B system with limited variations in the repeat number was identified. Sporulation and germination process related genes were homologous to that of the Clostridia cluster I group but novel variations for regulatory genes were identified indicative for strain specific control of regulatory events. Phylogenomics showed a higher relatedness to C. septicum than to other so far sequenced genomes of species belonging to the genus Clostridium. Comparative genome analysis of three C. chauvoei circular genome sequences revealed the presence of few inversions and translocations in locally collinear blocks (LCBs). The species genome also shows a large number of genes involved in proteolysis, genes for glycosyl hydrolases and metal iron transportation genes which are presumably involved in virulence and survival in the host. Three conserved flagellar genes (fliC) were identified in each of the circular genomes. In conclusion this is the first comparative analysis of circular genomes for the species C. chauvoei, enabling insights into genome composition and virulence factor variation.

Blackleg in cattle: A case report of fetal infection and a literature review.

Clostridium chauvoei causes blackleg in cattle. The disease has been reported worldwide, and although it can be prevented by vaccination, sporadic cases and occasional outbreaks still occur. We describe a case of blackleg in a 2-y-old, pregnant Gyr cow with in utero transmission to the fetus. The cow had characteristic gross and microscopic lesions of blackleg including widespread necrohemorrhagic and emphysematous skeletal and myocardial myositis, and fibrinous pericarditis. Her uterus contained a near-term, markedly emphysematous fetus with skeletal muscle and myocardial lesions similar to those seen in the dam. Histopathology of dam and fetal tissues revealed numerous gram-positive bacilli, many of them with sub-terminal spores, in multiple tissues. These bacilli were identified as C. chauvoei by immunohistochemistry. Anaerobic culture and fluorescent antibody tests performed on skeletal muscle from both the dam and fetus were positive for C. chauvoei, confirming a diagnosis of blackleg. Blackleg is a so-called endogenous infection, and the currently accepted pathogenesis involves ingestion of spores that are transported to muscle tissues where they lie dormant until anaerobiosis prompts germination. Germinating bacteria are histotoxic, producing severe, local necrosis and ultimately lethal toxemia. This model, however, has not been confirmed experimentally and also fails to explain some cases of the disease. A presumptive diagnosis of blackleg is based on clinical, gross, and histologic findings. Diagnostic confirmation necessitates the detection of C. chauvoei by culture, PCR, or immunodetection methods.

Spatial-temporal cluster analysis of fatal Clostridium chauvoei cases among cattle in Styria, Austria between 1986 and 2013.

Clostridium chauvoei is a gram positive, spore building bacterium that causes blackleg, a mostly fatal disease in cattle and other ruminants. Although the disease is common, little is known on the epidemiology of blackleg. As infection occurs through the environment, the risk of blackleg might be increased in areas with a specific climate or soil type. Therefore, the objective of the present study was to identify spatial and temporal clusters in the incidence of blackleg in the province of Styria, Austria. Data were collected within the governmentally delivered blackleg control program which includes vaccination of cattle with access to known blackleg pastures and compensation for fallen stock. Between 1986 and 2013, 1448 suspect blackleg cases were reported to official veterinarians; with blackleg confirmed through bacteriology in 266 cases (18%). The number of confirmed blackleg cases was highest in 2011 (25 cases) and lowest in 2004 (2 cases). Mean annual blackleg incidences varied considerably between different municipalities from 0 cases in most of the Southern parts of the province to 584 cases/1,000,000 cattle in some Northwestern municipalities. The spatio-temporal analysis identified one high risk cluster in the Northwest where cattle had 9.56 times the risk to develop blackleg compared to those in Northern and Northeastern parts of the province. Furthermore, a low-risk cluster was identified in the southeastern part of the province, where cattle had a relative risk of 0.015 to die of blackleg. No temporal or spatio-temporal clusters were identified. Results of the present study suggest that blackleg cases are clustered within certain geographic areas which might be due to soil type and water permeability. Results of this study should be used to motivate farmers to vaccinate cattle against Clostridium chauvoei in known areas with high risk.

Intracellular survival of Clostridium chauvoei in bovine macrophages.

Clostridium chauvoei is the etiological agent of blackleg, a severe disease of domestic ruminants, causing myonecrosis and serious toxemia with high mortality. Despite the known importance of this agent, studies evaluating its pathogenesis of blackleg are scarce, and many are based on an unproven hypothesis that states that macrophages are responsible for carrying C. chauvoei spores from the intestines to muscles in the early stages of blackleg. Therefore, the present study aimed to investigate the survival of C. chauvoei vegetative cells or spores after phagocytosis by a murine macrophage cell line (RAW 264.7) and bovine monocyte-derived macrophages and to profile inflammatory and anti-inflammatory cytokine transcripts of bovine macrophages infected with C. chauvoei vegetative cells or spores. Both vegetative cells and spores of C. chauvoei remain viable after internalization by murine and bovine macrophages. Bovine macrophages infected with vegetative cells showed a pro-inflammatory profile, while those infected with spores displayed an anti-inflammatory profile. Together, these results corroborate the classical hypothesis that macrophages may play a role in the early pathogenesis of blackleg. Moreover, this is the first study to evaluate the infection kinetics and cytokine profile of bovine monocyte-derived macrophages infected with a Clostridium species.