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.

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.

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.

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.

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.

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.

Proteomic analysis and identification of cell surface-associated proteins of Clostridium chauvoei.

Blackleg is a highly fatal disease of cattle and sheep, caused by Clostridium chauvoei, a Gram positive, anaerobic, spore forming bacteria. Cell surface-associated proteins play a major role in inducing the protective immunity. However, the identity of a majority of cell surface-associated proteins of C. chauvoei is not known. In the present investigation, we have used SDS-PAGE, 2D-gel electrophoresis and Western blotting followed by mass spectrometry to identify cell surface-associated proteins of C. chauvoei. Among the identified proteins, which have shown to offer protective antigencity in other bacteria, Enolase, Chaperonin, Ribosomal protein L10, Glycosyl Hydrolase and Flavoprotein were characterized by sequencing and their overexpression in Escherichia coli. In conclusion, cell surface-associated proteins were identified using proteomic approach and the genes for the immunoreactive proteins were expressed, which may prove to be potential diagnostic or vaccine candidates.

Development of a recombinant flagellin based ELISA for the detection of Clostridium chauvoei.

Blackleg, an economically important and highly fatal disease of ruminants, is caused by anaerobic bacillus, Clostridium chauvoei. Identification and differentiation of the causative agent is crucial for implementation of therapeutic and control measures in real time. Most of the diagnostic tests available for blackleg are PCR based, and only a couple of serological tests have been reported. In this study, we targeted flagellin, an important immunogenic protein of C. chauvoei, to develop a sandwich ELISA for detection of C. chauvoei. Sequence analysis of flagellin gene of related Clostridium species showed that central region of flagellin gene is unique to C. chauvoei. Hence, we cloned and expressed central region of flagellin in a prokaryotic expression system. Antiserum against recombinant flagellin was generated in rabbits and chickens. A sandwich ELISA was developed, in which rabbit anti-flagellin antibodies were used as capture antibodies and chicken anti-flagellin antibodies as detecting antibodies. The test was specific and sensitive in detection of up to 10(4) CFU/ml of C. chauvoei. This study shows that assay developed can be used for detection of C. chauvoei in suspected samples.

Patho-genetics of Clostridium chauvoei.

The genomic sequence of Clostridium chauvoei, the etiological agent of blackleg, a severe disease of ruminants with high mortality specified by a myonecrosis reveals a chromosome of 2.8 million base-pairs and a cryptic plasmid of 5.5 kilo base-pairs. The chromosome contains the main pathways like glycolysis/gluconeogenesis, sugar metabolism, purine and pyrimidine metabolisms, but the notable absence of genes of the citric acid cycle and deficient or partially deficient amino acid metabolism for Histidine, Tyrosine, Phenylalanine, and Tryptophan. These essential amino acids might be acquired from host tissue damage caused by various toxins and by protein metabolism that includes 57 genes for peptidases, and several ABC transporters for amino acids import.

The utilization of a commercial soil nucleic acid extraction kit and PCR for the detection of Clostridium tetanus and Clostridium chauvoei on farms after flooding in Taiwan.

Clostridial diseases are zoonoses and are classified as soil-borne diseases. Clostridium chauvoei and Clostridium tetani cause blackleg disease and tetanus, respectively. Since bacteria and spores are re-distributed by floods and then, subsequently, contaminate soils, pastures and water; the case numbers associated with clostridial diseases usually increase after floods. Because Taiwan is often affected by flood damage during the typhoon season, possible threats from these diseases are present. Thus, this study's aim is to apply a combination of a commercial nucleic acid extraction kit and PCR to assess the prevalence of Clostridia spp. in soil and to compare the positivity rates for farms before and after floods. The minimum amounts of Clostridium tetanus and Clostridium chauvoei that could be extracted from soils and detected by PCR were 10 and 50 colony forming units (cfu), respectively. In total, 76 samples were collected from the central and southern regions of Taiwan, which are the areas that are most frequently damaged by typhoons. Noteworthy, the positive rates for Clostridium tetanus and Clostridium chauvoei in Pingtung county after the severe floods caused by a typhoon increased significantly from 13.73 and 7.84% to 53.85 and 50.00%, respectively. This study for the first time provides the evidence from surveillance data that there are changes in the environmental distribution of Clostridium spp. after floods. This study indicates that screening for soil-related zoonotic pathogens is a potential strategy that may help to control these diseases.

Cytotoxin CctA, a major virulence factor of Clostridium chauvoei conferring protective immunity against myonecrosis.

OBJECTIVE: The purpose of this study was to determine the identity of the major toxin of Clostridium chauvoei, an important pathogen of cattle causing black leg and to determine its value as a protective antigen in vaccines against myonecrosis. METHODS: Genomic sequence analysis was used to determine potential virulence genes of C. chauvoei. Subsequently, the putative toxin candidate gene was cloned and expressed to obtain recombinant toxin. This toxin was investigated for its cytotoxic activity, hemolysis and its potential as a protective antigen in the guinea pig potency assay. RESULTS: A novel protein toxin, named Clostridium chauvoei toxin A (CctA) that belongs to the family of ß-barrel pore forming toxins of the leucocidin superfamily of bacterial toxins was discovered by whole genome sequence analysis. The corresponding gene cctA was found in all strains of C. chauvoei analyzed, isolated from various geographical areas over the globe during the last 50 years, but not in other pathogenic Clostridium species. Native CctA and recombinant rCctA produced in Escherichia coli in the form of a rCctA::NusA fusion protein or thrombin processed rCctA were highly cytotoxic for Embryonic Calf Nasal Epithelial (ECaNEp) cells and had high haemolytic activity against sheep erythrocytes in standard haemolysis assays. Polyclonal anti-rCctA rabbit antibodies fully neutralized the cytotoxic and haemolytic activity, not only of rCctA but also of supernatants from cultures of the various C. chauvoei strains, indicating that CctA is the main cytotoxic and haemolytic substance secreted by C. chauvoei. Using a standard vaccine release procedure, we demonstrated that vaccination of guinea pigs with CctA in the form of a fusion protein with the E. coli heat labile toxin B subunit (rCctA::LTB) as a peptide adjuvant protected the animals against challenge with spores of virulent C. chauvoei. CONCLUSIONS: CctA is the major virulence factor of C. chauvoei and the main protective antigen in vaccines against blackleg.

Lethal human neutropenic entercolitis caused by Clostridium chauvoei in the United States: tip of the iceberg?

The patient is a 44-year-old woman with metastatic grade 3 intra-ductal carcinoma of the breast who was started on palliative chemotherapy (docetaxel) 10 days prior to admission and presented to the emergency center complaining of diffuse abdominal pain and generalized weakness. CT abdomen showed diffuse bowel wall thickening from the cecum to the transverse colon with free fluid in the pelvis. The patient was neutropenic on admission (absolute neutrophil count of 600 cells/µl). She received antibiotics for 21 days for neutropenic enterocolitis. Blood culture isolate from admission was sent for 16s rRNA gene sequencing, which identified Clostridium chauvoei. While C. chauvoei has a long history of veterinary importance, this is the first documented case of infection caused by C. chauvoei in a human in the United States. C. chauvoei has a close phylogenetic relationship with C. septicum making the two species difficult to differentiate using conventional microbiologic methods. With increased use of more reliable detection methods the actual prevalence of C. chauvoei causing human disease may be higher than currently recognized.

Genetic and functional characterization of the NanA sialidase from Clostridium chauvoei.

Clostridium chauvoei is the causative agent of blackleg, a wide spread serious infection of cattle and sheep with high mortality. In this study we have analyzed the sialidase activity of the NanA protein of C. chauvoei and cloned the sialidase gene nanA. Sialidase is encoded as a precursor protein of 722 amino acids with a 26 amino acid signal peptide. The mature sialidase has a calculated molecular mass of 81 kDa and contains the carbohydrate binding module 32 (CBM32, or F5/8 type C domain), the sialic acid binding module CBM40 and the enzymatically active sialidase domain found in all pro- and eukaryotic sialidases. Sialidase activity does not require the CBM32 domain. The NanA protein is secreted by C. chauvoei as a dimer. The nanA gene was found to be conserved and sialidase activity was found in C. chauvoei strains isolated over a period of 50 years from various geographical locations. Antiserum directed against a recombinant 40 kDa peptide containing CBM40 and part of the enzymatically active domain of NanA neutralized the secreted sialidase activity of all C. chauvoei strains tested.

Development of a real time PCR Taqman assay based on the TPI gene for simultaneous identification of Clostridium chauvoei and Clostridium septicum.

In the present study, a Taqman allelic discrimination assay based on three SNPs of the TPI gene is described. It was used as a differential diagnostic tool to detect blackleg and malignant edema. Sudden deaths of grazing ruminants, such as cattle, sheep and goats, which show clinical signs related to hyperacute infective processes, encouraged the development of a rapid and precise diagnostic molecular method. Specific primers and probes for Clostridium septicum and Clostridium chauvoei were designed on the basis of the TPI gene sequence. The multiplex PCR was tested on the DNA of a total of 57 strains, including 24 Clostridium chauvoei, 20 Clostridium septicum, 1 Bacillus anthracis and 12 other Clostridium spp. The DNA samples from Clostridium chauvoei and Clostridium septicum strains were amplified. Amplification of other DNA samples was not observed, with the exception of Clostridium tertium, which showed a weak positive signal. To avoid misdiagnosis, a confirmatory assay based on a Sybr green real time PCR was proposed. The authors confirmed the efficacy and the specificity of the test used in this study, which proved to be a useful tool for the diagnosis of clostridiosis that are often diagnosed using only traditional tools.

Development and validation of a multiplex real-time PCR for detection of Clostridium chauvoei and Clostridium septicum.

Clostridium chauvoei is the causative agent of blackleg in cattle and sheep. The clinical symptoms of this severe disease are very similar to that of malignant edema (Clostridium septicum), infections of other Clostridium species belonging to the gas edema complex, and anthrax (Bacillus anthracis). C. chauvoei and C. septicum are closely related taxa and share many phenotypic properties hampering diagnosis by using traditional microbiological methods. Thus, there is a need for a fast and reliable identification method for specific detection of both species in clinical samples. The multiplex real-time PCR assay presented here is based on the detection of the spo0A gene and enables the simultaneous identification of C. chauvoei and C. septicum. The assay design includes an amplification control DNA template for the recognition of PCR-inhibitors. Assay validation was performed using a collection of 29 C. chauvoei, 38 C. septicum strains and 26 strains of other Clostridium species. Furthermore, the real-time PCR assay was successfully tested on tissue samples from 19 clinical blackleg cases. The assay allowed the reliable detection of one picogram DNA which represents approximate 239 genome equivalents.

Detection and identification by PCR of Clostridium chauvoei in clinical isolates, bovine faeces and substrates from biogas plant.

BACKGROUND: Clostridium chauvoei causes blackleg, an acute disease associated with high mortality in ruminants. The apparent primary port of entry is oral, during grazing on pasture contaminated by spores. Cases of blackleg can occur year after year on contaminated pastures. A method to determine the prevalence of C. chauvoei spores on pasture would be useful.The standard method for C. chauvoei detection is culture and biochemical identification, which requires a pure culture. In most muscle samples from cattle dead from blackleg the amount of C. chauvoei in samples is high and the bacterium can easily be cultured, although some samples may be contaminated. Detection by PCR would be faster and independent of contaminating flora.Digested residues from biogas plants provide an excellent fertiliser, but it is known that spore-forming baeria such as Clostridium spp. are not reduced by pasteurisation. The use of digested residues as fertiliser may contribute to the spread of C. chauvoei. Soil, manure and substrate from biogas plants are contaminated with other anaerobic bacteria which outgrow C. chauvoei. Therefore, detection by PCR is would be useful. This study applied a PCR-based method to detect of C. chauvoei in 25 muscle and blood samples, 114 manure samples, 84 soil samples and 33 samples from the biogas process. METHODS: Muscle tissues from suspected cases of blackleg were analysed both by the standard culture method followed by biochemical identification and by PCR, with and without preculture. To investigate whether muscle tissue samples are necessary, samples taken by swabs were also investigated. Samples from a biogas plant and manure and soil from farms were analysed by culture followed by PCR. The farms had proven cases of blackleg. For detection of C. chauvoei in the samples, a specific PCR primer pair complementary to the spacer region of the 16S-23S rRNA gene was used. RESULTS: Clostridium chauvoei was detected in 32% of muscle samples analysed by culture with identification by biochemical methods and in 56% of cases by culture in combination with PCR. Clostridium chauvoei was detected in 3 (out of 11) samples from the biogas plants collected before pasteurisation, but samples taken after pasteurisation and after digestion all tested negative. Clostridium chauvoei was not detected in any soil or silage samples and only one manure samples tested positive. CONCLUSION: The diagnostic method used for C. chauvoei was not applicable in estimating the risk of blackleg on particular pastures from manure or soil samples, but found to be highly useful for clinical samples.

Molecular characterization of the diversity of Clostridium chauvoei isolates collected from two bovine slaughterhouses: analysis of cross-contamination.

Clostridium chauvoei is the etiologic agent of blackleg, a high mortality rate disease affecting mainly cattle and sheep. Carcasses of animals affected by the disease are the chief source of soil infection and considered as an ever-present threat to livestock health. A study was undertaken to examine the cross-contamination of C. chauvoei in two different bovine slaughterhouses using restriction endonuclease analysis (REA) and protein analysis. Samples from various sites of two different bovine slaughterhouses were screened and 34 isolates were identified by conventional techniques and 16S rRNA gene (rrs) sequencing. C. chauvoei were isolated from carcass, soil, and sewage from slaughterhouses examined. The isolates were differentiated using REA and whole-cell and excretory protein pattern analysis combined with numerical analysis and cluster formation. The alpha and beta toxins produced by the strains were characterized. Our preliminary results suggest that REA combined with numerical analysis provides additional criteria and characteristic banding patterns for the study of the cross-contamination and characterization of C. chauvoei. The effects of temperature, oxygen tension, and enzymes on C. chauvoei hemolysin activity were also discussed. These microorganisms may be a potential contaminant of carcasses and widespread in soil of abattoir environments. The information of area-specific distribution of C. chauvoei strains and its toxin characteristics may give an efficient program in protecting cattle and other ruminants.

Human fulminant gas gangrene caused by Clostridium chauvoei.

The first human case of fulminant gas gangrene caused by Clostridium chauvoei, a pathogen causing ruminant blackleg, was confirmed for a 58-year-old man suffering from diabetes mellitus. The patient developed conspicuous emphysematous gangrene in the right chest wall as well as intravascular gas entrapments and died 2 h after hospital arrival.