Clostridium Chauvoei Research Articles

Protection efficacy and immunogenicity of Clostridium chauvoei proteins as a subunit blackleg vaccine or an adjuvant for Clostridium perfringens epsilon toxoid

Potential application of Clostridium chauvoei proteins was studied as a subunit blackleg vaccine or a biological adjuvant for Clostridium perfringens epsilon toxoid vaccine. Extracellular and cell surface proteins were extracted from C. chauvoei culture, and their protective efficacy was evaluated by potency test in guinea pigs. In order to investigate the effect of cell surface proteins on C. perfringens epsilon toxoid immunogenicity, rabbits were inoculated subcutaneously twice with: C. perfringens type D toxoid supernatant +200 μg C. chauvoei cell surface proteins (PR-200), toxoid supernatant + 400 μg cell surface proteins (PR-400), inactivated C. perfringens type D vaccine (Vac), toxoid supernatant (Tox), or PBS. Isolation of cell surface proteins yielded about 2.5 mg/L culture protein with a sharp band at 43 kDa probably corresponding to flagellin. Potency test demonstrated the protection ability of both cellular and extracellular proteins of C. chauvoei. ELISA showed that the highest antibody titers against epsilon toxoid belonged to PR-400 and Vac groups. The effect of days post immunization on antibody response was not significant. No significant difference was observed between PR-400 and Vac, as well as PR-200 and Tox groups. Clostridium chauvoei cell surface proteins may have the potential for application as a blackleg disease vaccine and an adjuvant for clostridial toxoids.

Production of highly soluble and immuno-reactive recombinant flagellin protein of Clostridium chauvoei

ObjectiveFlagellin protein, an integral component of flagella, provides motility to several bacterial species and also acts as a candidate antigen in diagnostics and subunit vaccines. The bulk production of flagellin with retention of all conformational epitopes using recombinant protein technology is of paramount importance in the development of pathogen-specific immuno-assays and vaccines. We describe the production of highly soluble and immuno-reactive rFliA(C) protein of Clostridium chauvoei, a causative agent of blackleg or black quarter (BQ) affecting cattle and small ruminants worldwide. The bacterium is known to possess peritrichous flagella that provide motility and also act as a virulence factor with high protective antigenicity. MethodsUpon sequence and structural analysis, a partial fliA(C) gene from Clostridium chauvoei was cloned and the recombinant mature protein with N- and C- terminal truncation was over-expressed as a His-tagged fusion protein (∼25 kDa) in Escherichia coli. Subsequently, rFliA(C) protein was purified by single-step affinity chromatography and characterized for its immuno-reactivity in laboratory animals, Western blot, and indirect-ELISA format. ResultsrFliA(C) was highly soluble and was purified in high quantity and quality. rFliA(C) elicited antigen-specific conformational polyclonal antibodies in rabbit and guinea pig models, as well as anti-Clostridium chauvoei-specific antibodies being specifically detected in BQ-vaccinated and convalescent sera of bovines in Western blot and in indirect-ELISA format. Further, no cross reactivity was noted with antibodies against major bovine diseases (e.g., foot-and-mouth disease, IBR, LSDV, hemorrhagic septicaemia, brucellosis, and leptospirosis). ConclusionThe study indicated the production of conformational recombinant flagellin—rFliA(C)—antigen and its potential utility in development of diagnostics for detection of Clostridium chauvoei-specific antibodies in BQ-recovered and/or vaccinated animals.

Blackleg: A Review of the Agent and Management of the Disease in Brazil.

The genus Clostridium is an important group of pathogenic and nonpathogenic Gram-positive anaerobic bacteria with a sporulation capacity and wide distribution in different environments, including the gastrointestinal tracts of healthy and diseased animals and humans. Among the pathogenic species of the genus, Clostridium chauvoei stands out as a histotoxic agent. It causes significant myonecrosis such as blackleg, a disease with high lethality, especially in young cattle, and is responsible for significant livestock losses worldwide. The pathogenicity of the disease is complex and has not yet been fully elucidated. Current hypotheses cover processes from the initial absorption to the transport and deposition of the agent in the affected tissues. The virulence factors of C. chauvoei have been divided into somatic and flagellar antigens and soluble antigens/toxins, which are the main antigens used in vaccines against blackleg in Brazil and worldwide. This review provides important information on the first and current approaches to the agent C. chauvoei and its virulence factors as well as a compilation of data on Brazilian studies related to blackleg.

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

ObjectivesIn order to find the optimal inactivation conditions for Clostridium chauvoei culture, different factors were investigated and the immunogenicity of inactivated cultures was studied. MethodsC. 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. ResultsIn 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. ConclusionsThe 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.

Prevalence of Cardiac Lesions in Cases of Bovine Blackleg in Tennessee (USA), 2004–2018

Blackleg is a common cause of death in cattle, mostly caused by the bacterium Clostridium chauvoei. Cardiac lesions were traditionally considered uncommon in cases of blackleg in cattle until a 2018 study reported otherwise. This study was aimed at determining the prevalence of cardiac disease among cattle that died of blackleg in Tennessee, USA. The outcome of this study would reinforce the importance of assessing cardiac lesions in suspected cases of blackleg in cattle. The University of Tennessee Veterinary Medical Center database searched for cattle with a confirmed diagnosis of blackleg necropsied between 2004 and 2018. Of the 120 necropsy reports, 37 had a diagnosis of blackleg. Histology slides of skeletal muscle (26/37) and the heart (26/37) were reviewed to confirm the presence of supportive lesions. Of the 37 cases of blackleg identified, 26 animals (70.3%) had cardiac lesions, including 4 (10.8%) that had only cardiac involvement without skeletal muscle lesions. Specifically, (5.4%; 2/37) had only necrotizing myocarditis; (13.5%; 5/37) had only fibrinous or fibrinosuppurative pericarditis, epicarditis, or endocarditis; (51.4%; 19/26) had a combination of myocarditis and pericarditis, epicarditis, or endocarditis; and (29.7%; 11/37) had no lesions. Furthermore, of the 26 cases with cardiac lesions, 24 cases had gross lesions, while 2 cases were identified only by microscopic examination. This indicates that gross examination alone is insufficient to identify cardiac involvement in blackleg cases in cattle. Contrary to traditional perceptions, cardiac lesions in cases of bovine blackleg could be as high as 70% and are most often associated with skeletal muscle lesions. The prevalence of cardiac lesions in cases of blackleg in cattle may be higher when the heart is examined microscopically than if it is only evaluated grossly. Pathologists should specifically evaluate the heart for lesions in suspected cases of blackleg in cattle and utilize microscopic examination when gross lesions are absent.

COMPARATIVE EVALUATION OF ANTIBODY TITER AGAINST PASTEURELLA MULTOCIDA IN RABBITS BY USING FOUR DIFFERENT ADJUVANTS IN HS+BQ COMBO VACCINES

Hemorrhagic Septicemia (HS) caused by Pasteurella multocida is an economically significant disease of bovines that instigates high monetary losses due to unexpected mortality of animals in emerging countries like Pakistan. Similarly Black Quarter (BQ) caused by Clostridium chauvoei is also an acute infectious ailment of cattle and buffaloes in which severe inflammation ofskeletal and cardiac muscles, severe systemic toxicity and a high mortality occurs. The only way to prevent losses due to both diseases is an effective and long lasting vaccination programme. Four HS+BQ combo vaccines adjuvanted with four different adjuvants were prepared. Two HS+BQ vaccines were prepared by addition of two oil adjuvants i.e Montanide ISA-50V-2 and Eolane-170 while one HS+BQ vaccine was prepared by addition of gel while fourth HS+BQ vaccine was prepared by addition of alum. The bacterial dry weight of P. multocida was adjusted to 2 mg/0.75 mL while the bacterial dry weight for Cl. chauvoei was set at 2.5 mg/0.75 mL. The dose of all vaccines was adjusted to 3 mL /animal. Four groups of rabbits having five rabbits in each group were selected at Veterinary Research Institute, Lahore. From four groups of rabbits one was inoculated with HS+BQ oil based vaccine containing Montanide ISA-50 V2 as adjuvant while other group was inoculated with HS+BQ oil based vaccine containing Eolane-170 oil as adjuvant. Third group was vaccinated with HS+BQ vaccine adjuvanted with gel while fourth group of rabbit was vaccinated with HS+BQ vaccin adjuvanted with alum. All rabbits were vaccinated at dose rate of 1mL / rabbit. Rabbits were again vaccinated with the same vaccines as booster dose after 27 days. Indirect HemAgglutination test was conducted after 141 days intervals on serum samples of all groups. The results showed that both HS+BQ combo oil based vaccines have higher antibody titers as compared to gel and alum based vaccines against HS disease.

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

BackgroundClostridium 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. AimThe 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. MethodsMouse 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. ResultsAll 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. ConclusionInflammation-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.

A Short Review on Blackleg in Ruminants

Blackleg is an endogenous acute infection that principally affects ruminants. It is Clostridium chauvoei, anaerobic spore-forming bacteria, causes the disease, which manifests as an acute, localized inflammation of muscle tissue produced by development of the blackleg organisms. Although blackleg may occur at any time of year, more cattle are lost during the summer months. In recent years, the major virulence factors of C. chauvoei have been discovered and described. However, the pathogenesis of blackleg in ruminant, and in particular the pathogen’s movement from the place of entrance to the affected tissues is yet unknown. When outbreaks arise, it is prevented by vaccination; in the early stages of the disease, it is treated with antibiotics, most notably penicillin, which is an effective therapy for the condition. Control of this disease is based on strict husbandry methods and a vaccination program. The key virulence factors of C. chauvoei have been found and characterized in recent years. This review covers the most recent research discoveries that contribute to a better understanding of the disease and provides the basis for preventative efforts.

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.

Paralytic-like disease in clostridium chauvoei infected ostriches

This datasheet on paralytic-like disease in clostridium chauvoei infected ostriches covers Identity, Associated Diseases, Pests or Pathogens, Hosts/Species Affected.

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.

Genome Sequence Analysis of Clostridium chauvoei Strains of European Origin and Evaluation of Typing Options for Outbreak Investigations.

Black quarter caused by Clostridium (C.) chauvoei is an important bacterial disease that affects cattle and sheep with high mortality. A comparative genomics analysis of 64 C. chauvoei strains, most of European origin and a few of non-European and unknown origin, was performed. The pangenome analysis showed limited new gene acquisition for the species. The accessory genome involved prophages and genomic islands, with variations in gene composition observed in a few strains. This limited accessory genome may indicate that the species replicates only in the host or that an active CRISPR/Cas system provides immunity to foreign genetic elements. All strains contained a CRISPR type I-B system and it was confirmed that the unique spacer sequences therein can be used to differentiate strains. Homologous recombination events, which may have contributed to the evolution of this pathogen, were less frequent compared to other related species from the genus. Pangenome single nucleotide polymorphism (SNP) based phylogeny and clustering indicate diverse clusters related to geographical origin. Interestingly the identified SNPs were mostly non-synonymous. The study demonstrates the possibility of the existence of polymorphic populations in one host, based on strain variability observed for strains from the same animal and strains from different animals of one outbreak. The study also demonstrates that new outbreak strains are mostly related to earlier outbreak strains from the same farm/region. This indicates the last common ancestor strain from one farm can be crucial to understand the genetic changes and epidemiology occurring at farm level. Known virulence factors for the species were highly conserved among the strains. Genetic elements involved in Nicotinamide adenine dinucleotide (NAD) precursor synthesis (via nadA, nadB, and nadC metabolic pathway) which are known as potential anti-virulence loci are completely absent in C. chauvoei compared to the partial inactivation in C. septicum. A novel core-genome MLST based typing method was compared to sequence typing based on CRISPR spacers to evaluate the usefulness of the methods for outbreak investigations.

Computational Design of a Multi-epitope Vaccine Against Clostridium chauvoei: An Immunoinformatics Approach.

Blackleg is an infectious disease of animals that is commonly caused by Clostridium chauvoei and characterized by localized muscle necrosis. In this study, proteome-mining and immunoinformatics approaches were applied to identify novel antigenic proteins and to construct a multi-epitope vaccine against C. chauvoei. All proteins of C. chauvoei strains were retrieved from the NCBI Microbial Genome Database containing both genomic and proteomic data of prokaryotes. The proteins were analyzed to exclude non-redundant sequences and to determine antigenic, virulent, and non-allergenic vaccine candidates through several online tools, resulting in seven protein candidates. Cytotoxic T and B cell epitopes of these proteins were evaluated through the tools present in the immune epitope database and the prioritized antigenic epitopes were then conjugated via appropriate linkers to construct the vaccine candidate. After the evaluation of physicochemical properties of the construct, the tertiary structure was modeled and refined through trRosetta and GalaxyRefine, respectively. The quality of the 3D structure was validated by ERRAT score, z-score, and Ramachandran plot and the construct was then docked with bovine Toll-like receptor 4 (TLR 4) using ClusPro. The docked complex was subjected to Molecular Mechanics/Generalized Born Surface Area in the HawkDock server and normal mode analysis in the iMODS simulation suite to assess the binding energy and stability of the complex, respectively. Overall, the vaccine construct was found stable and energetically feasible for bovine TLR 4 binding. Therefore, it can be used as a multi-epitope vaccine construct in clostridial vaccines to control the blackleg disease.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10989-021-10279-9.

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.

Clinico-hematological, patho-anatomical and molecular based investigation of blackleg disease in Cholistani cattle

Clostridium chauvoei, causing myonecrosis in livestock animals, lives in the feces, surface water and soil. The blackleg due to C. chauvoei is very common in dairy animals. Still, no report is available about the pathophysiology of disease in Cholistani cattle kept under tropical and desert conditions of Cholistan, Pakistan. Therefore, in this study, we report for the first time the pathophysiology of a visceral form of blackleg infection in indigenous cholistani cattle breed reared in desert conditions of Cholistan. Clinically morbid animals exhibited different signs of infection including fever, crepitation sounds, gaseous swelling and edematous lesions. Blood was collected for hematological and serum biochemical investigation. Hematological examination indicated a significant increased erythrocyte sedimentation rate, lower red blood cells, hematocrit, and total white blood cell count. Results on serum biochemistry showed significantly (p<0.02) increased creatine kinase levels, creatinine, urea, alanine aminotransferase, aspartate aminotransferase, alkaline phosphates, creatinine phosphokinase and lactate dehydrogenase in infected cattle. At necropsy level, light pink color fluid under swelling areas of skin, gas bubbles, dark to black in color of affected muscles and crepitation sounds at palpation were observed. Necropsy showed marked myocarditis, petechial hemorrhages, consolidation and severe pulmonary edema. Spleen showed petechial hemorrhages and congestion. Histological analysis of muscular tissues indicated severe inflammatory reaction comprising of cellular infiltration, marked edema, necrosis and disruption of myofibrils. PCR confirmed the presence of C. chauvoei in muscles, heart and exudates of the lungs. This is the first report on molecular detection of Clostridium chauvoei from a visceral form of Blackleg disease in cholistani breed of cattle naturally infected in the Cholistan desert of Punjab, Pakistan.

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.

Vaccination against pathogenic clostridia in animals: a review.

Clostridium is a Gram-positive, rod-shaped, anaerobic, and spore-forming bacterium, which is found in the surrounding environments throughout the world. Clostridium species cause botulism, tetanus, enterotoxaemia, gas gangrene, necrotic enteritis, pseudomembranous colitis, blackleg, and black disease. Clostridium infection causes severe economic losses in livestock and poultry industries. Vaccination seems to be an effective way to control Clostridial diseases. This review discusses the toxins and vaccine development of the most common pathogenic Clostridium species in animals, including Clostridium perfringens, Clostridium novyi, Clostridium chauvoei, and Clostridium septicum. In this comprehensive study, we will review different kinds of clostridial toxins and the vaccines that are experimentally or practically available and will give a short description on each vaccine focusing on its applications, advantages, and disadvantages.

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.

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. 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. 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. 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.

Gas gangrene in mammals: a review.

Gas gangrene is a necrotizing infection of subcutaneous tissue and muscle that affects mainly ruminants and horses, but also other domestic and wild mammals. Clostridium chauvoei, C. septicum, C. novyi type A, C. perfringens type A, and C. sordellii are the etiologic agents of this disease, acting singly or in combination. Although a presumptive diagnosis of gas gangrene can be established based on clinical history, clinical signs, and gross and microscopic changes, identification of the clostridia involved is required for confirmatory diagnosis. Gross and microscopic lesions are, however, highly suggestive of the disease. Although the disease has a worldwide distribution and can cause significant economic losses, the literature is limited mostly to case reports. Thus, we have reviewed the current knowledge of gas gangrene in mammals.