Clostridium Chauvoei Research Articles

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.

Clostridial disease in cattle

Clostridial disease affects cattle across the UK throughout the year but particularly in the grazing season. All clostridial disease occurs when conditions in the muscles or organs become anaerobic allowing for bacterial proliferation and toxin production. The most common pathogenic clostridium is Clostridium chauvoei which causes blackleg and has both skeletal and cardiac forms. Clostridium perfringens is the second most commonly identified clostridia with type A causing enterotoxaemia and type D causing pulpy kidney. Clostridium novyi is the third most common clostridia — type B causes black disease and type D causes bacillary haemoglobinuria. Cattle are most at risk during the grazing season meaning that control and prevention of disease should be particularly targeted at this time of year. The cost of vaccination is so low compared with the cost of losing even one animal that an appropriate protocol should be implemented on all farms.

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.

Cloning and Expression of Twin-Arginine Translocation D Family Deoxyribonuclease of Clostridium Chauvoei

Clostridium chauvoei, an anaerobic bacterium reported worldwide, is responsible for Black Quarter, a dreadful disease of ruminants. This bacterium produces many toxins responsible for the pathogenesis of the disease. Except for the well-studied virulence factors such as cctA, flagellin andsialidase genes, the exact role of other toxins of C. chauvoei remains unknown. This necessitates studies on the activities of the C. chauvoei toxins and virulence. In the present study, Twin-Arginine Translocation D (TatD) family deoxyribonuclease of the bacterium was selected. The tatD gene C. chauvoei was amplified by PCR and cloned into p-Rham-N-His-SUMO-Kan expression vector, followed by transformation into the E.cloni 10G competent cells. Clones obtained were confirmed by colony PCR. These tatDclones were sequenced and analysed phylogenetically, which revealed the close relationship of C. chauvoei strain to C. isatidis, C. saccharobutylicum, C. botulinum and C. taeniosporum based on tatD sequence analysis. Upon induction of the clones with L-rhamnose,the protein expression was obtained at 42.3 kDa and the same was further confirmed by Western blotting.

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

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. Diagnosis of blackleg was made based on hematological and serum biochemical analysis, PCR, necropsy and histopathology. 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. 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.

Vaccine Production to Protect Animals Against Pathogenic Clostridia.

Clostridium is a broad genus of anaerobic, spore-forming, rod-shaped, Gram-positive bacteria that can be found in different environments all around the world. The genus includes human and animal pathogens that produce potent exotoxins that cause rapid and potentially fatal diseases responsible for countless human casualties and billion-dollar annual loss to the agricultural sector. Diseases include botulism, tetanus, enterotoxemia, gas gangrene, necrotic enteritis, pseudomembranous colitis, blackleg, and black disease, which are caused by pathogenic Clostridium. Due to their ability to sporulate, they cannot be eradicated from the environment. As such, immunization with toxoid or bacterin-toxoid vaccines is the only protective method against infection. Toxins recovered from Clostridium cultures are inactivated to form toxoids, which are then formulated into multivalent vaccines. This review discusses the toxins, diseases, and toxoid production processes of the most common pathogenic Clostridium species, including Clostridium botulinum, Clostridium tetani, Clostridium perfringens, Clostridium chauvoei, Clostridium septicum, Clostridium novyi and Clostridium hemolyticum.

Protective efficacy of commercial vaccines against a virulent field strain of Clostridium chauvoei

Blackleg is an acute and frequently fatal infection that mainly affects cattle and is caused by Clostridium chauvoei. Formalin-killed, whole-cell vaccines are commonly used to control blackleg. The aim of this study was to verify the protective efficacy of two commercial vaccines against the infection of guinea pigs with two strains of C. chauvoei, a virulent field strain (SBP 07/09) and the reference strain used in official tests (Manguinhos-Teixeira or MT). The strains used in the challenge were characterized by whole genome sequencing, and the minimal inhibitory concentrations of 15 antimicrobials were determined. To assess the protective efficacy, guinea pigs were vaccinated and subsequently challenged with C. chauvoei. All four vaccinated and challenged groups seroconverted after vaccination, while the control group remained seronegative, as determined by indirect ELISA. The identical performance of the two C. chauvoei strains in terms of virulence after challenge and their inability to infect vaccinated animals was correlated with their high genetic homology. Both commercial vaccines showed good protective efficacy against both the reference and field strains. Although C. chauvoei vaccination failures have been reported, the results from our study and others reported high similarity among C. chauvoei strains from all over the world, which suggests that the vaccine failures are not due to antigenic variability but inadequate vaccine management.

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 β-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.

Expression of Amino-terminal Fragment of Clostridium chauvoei toxin A

Clostridium chauvoei, the causative of black quarter in ruminants, is an anaerobic spore forming organism, produces different types of exotoxins. Clostridium chauvoei toxin A (cctA) is an important toxin possessing hemolytic and cytotoxic property and also claimed to be a protective antigen. Characterization of the different regions of the toxin has not been carried out yet. Hence, the regions responsible for different activities of the toxin are not clear. As an initial step, amino-terminal region (34-75aa) of cctA gene was cloned and expressed in pRham-N-His-Sumo-Kan vector. Expressed protein was confirmed by SDS-PAGE analysis.

Molecular characterization of virulence genes cctA, nanA, and fliC in Clostridium chauvoei from Rio Grande do Sul and São Paulo State, Brazil

ABSTRACT: Clostridium chauvoei toxin A (CctA), neuraminidase (NanA), and flagellin (FliC) proteins contribute to the pathogenicity of Clostridium chauvoei, the causative agent of blackleg in cattle. The aim of this study was to analyze the genetic variability of cctA, nanA, and fliC genes in C. chauvoei isolates from the Rio Grande do Sul and São Paulo state- Brazil, during different sampling periods. The presence of these genes was verified through PCR amplification and partial gene sequencing of 17 strains. Alignment of PCR amplicons combined with bioinformatics analysis was used in an attempt to study the variability across C. chauvoei solates. The similarity among the partial sequences of cctA and nanA genes was 100%. The sequencing of fliC revealed three different paralog alleles of flagellin, and two strains were seen to be polymorphic, with amino acid alterations in the predicted protein. Overall, this study indicates that strains of C. chauvoei isolated in Brazil are highly conserved with respect to the virulence factors evaluated.

Isolation and identification of Clostridium chauvoei from cattle in Mymensingh

Black quarter (BQ), caused by Clostridium chauvoei, is an economically important, highly infectious bacterial disease of cattle, goat and sheep. The present study was conducted to isolate and identify the causal agent of BQ from field cases. A total of 32 samples from necropsied areas of cattle presented to the Teaching Veterinary Hospital, Bangladesh Agricultural University were collected. The samples were subjected for bacteriological culture followed by identification through a series of conventional bacteriological techniques, staining properties and biochemical tests. The bacteria were cultured in anaerobic condition using candle jar. Morphological characters of bacteria were observed after Gram stain. The suspected samples were subjected for several passages in liver infusion broth to get pure culture. On neomycin blood agar, the bacteria showed hemolysis. Based on the cultural and staining characteristics and biochemical tests, 4 (12.5%) out of 32 samples were confirmed as Cl. chauvoei. It is concluded that Cl. chauvoei has been successfully isolated and identified from BQ affected cattle.

Cloning and Expression of Carboxyl-terminal Region of cctA Gene of Clostridium chauvoei

The carboxyl-terminal region of cctA gene of Clostridium chauvoei was cloned and expressed in prokaryotic expression system. Further, expressed recombinant carboxyl-terminal region of cctA protein was purified and the expression was confirmed by SDS-PAGE analysis.

Blackleg in a free-range brown brocket deer (Mazama gouazoubira)

ABSTRACT: 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.

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.

Comparative pathogenesis of enteric clostridial infections in humans and animals

Several enteric clostridial diseases can affect humans and animals. Of these, the enteric infections caused by Clostridium perfringens and Clostridium difficile are amongst the most prevalent and they are reviewed here. C. perfringens type A strains encoding alpha toxin (CPA) are frequently associated with enteric disease of many animal mammalian species, but their role in these diseased mammals remains to be clarified. C. perfringens type B encoding CPA, beta (CPB) and epsilon (ETX) toxins causes necro-hemorrhagic enteritis, mostly in sheep, and these strains have been recently suggested to be involved in multiple sclerosis in humans, although evidence of this involvement is lacking. C. perfringens type C strains encode CPA and CPB and cause necrotizing enteritis in humans and animals, while CPA and ETX producing type D strains of C. perfringens produce enterotoxemia in sheep, goats and cattle, but are not known to cause spontaneous disease in humans. The role of C. perfringens type E in animal or human disease remains poorly defined. The newly revised toxinotype F encodes CPA and enterotoxin (CPE), the latter being responsible for food poisoning in humans, and the less prevalent antibiotic associated and sporadic diarrhea. The role of these strains in animal disease has not been fully described and remains controversial. Another newly created toxinotype, G, encodes CPA and necrotic enteritis toxin B-like (NetB), and is responsible for avian necrotic enteritis, but has not been associated with human disease. C. difficile produces colitis and/or enterocolitis in humans and multiple animal species. The main virulence factors of this microorganism are toxins A, B and an ADP-ribosyltransferase (CDT). Other clostridia causing enteric diseases in humans and/or animals are Clostridium spiroforme, Clostridium piliforme, Clostridium colinum, Clostridium sordellii, Clostridium chauvoei, Clostridium septicum, Clostridium botulinum, Clostridium butyricum and Clostridium neonatale. The zoonotic transmission of some, but not all these clostridsial species, has been demonstrated.

Blackleg in cattle: current understanding and future research needs

ABSTRACT: Blackleg is an endogenous acute infection that principally affects cattle, whose etiologic agent is the anaerobic bacterium Clostridium chauvoei. In recent years, the major virulence factors of C. chauvoei have been discovered and described. However, the pathogenesis of blackleg in cattle, and in particular, the movement of the pathogen from the point of entry to the affected tissues is not yet fully elucidated. Disease control is based on appropriate management and vaccination. This review summarizes the latest research findings that contribute toward the understanding of the disease in cattle, provide a foundation to preventive strategies, and identify future research needs.

RETRACTED: Blackleg in cattle: Current understanding and future research perspectives-A review

Blackleg is an endogenous acute infection that principally affects cattle. The disease is caused by Clostridium chauvoei (C. chauvoei), an anaerobic spore forming bacterium. Control of this disease is based on stringent husbandry measures and scheduled vaccination plan. In recent years, the major virulence factors of C. chauvoei have been discovered and described. However, the pathogenesis of blackleg in cattle and in particular, circulation of the pathogen from point of entry to target tissues is yet not fully elucidated. This review summarizes the latest review of literature that significantly contributed for understanding the disease in cattle, and provides a foundation to preventive strategies.

Blackleg in cattle in the state Mato Grosso do Sul, Brazil: 59 cases

ABSTRACT: 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.