Clostridium Novyi Research Articles

Large Clostridial Toxins: A Brief Review and Insights into Antigen Design for Veterinary Vaccine Development.

The group of large clostridial toxins (LCTs) includes toxins A (TcdA) and B (TcdB) from Clostridioides difficile, hemorrhagic and lethal toxins from Paeniclostridium sordellii, alpha toxin from Clostridium novyi (TcnA), and cytotoxin from Clostridium perfringens. These toxins are associated with severe pathologies in livestock, including gas gangrene (P. sordellii and C. novyi), infectious necrotic hepatitis (C. novyi), avian necrotic enteritis (C. perfringens), and enterocolitis (C. difficile). Immunoprophylaxis is crucial for controlling these diseases, but traditional vaccines face production challenges, such as labor-intensive processes, and often exhibit low immunogenicity. This has led to increased interest in recombinant vaccines. While TcdA and TcdB are well-studied for human immunization, other LCTs remain poorly characterized and require further investigation. Therefore, this study emphasizes the importance of understanding lesser-explored toxins and proposes using immunoinformatics to identify their immunodominant regions. By mapping these regions using silico tools and considering their homology with TcdA and TcdB, the study aims to guide future research in veterinary vaccinology. It also explores alternatives to overcome the limitations of conventional and recombinant vaccines, offering guidelines for developing more effective vaccination strategies against severe infections in animals.

In silico vaccine design: Targeting highly epitopic regions of Clostridium perfringens type D epsilon toxin and Clostridium novyi type B alpha toxin for optimal immunogenicity

Livestock infections caused by highly toxic bacteria, such as Clostridium perfringens type D and Clostridium novyi type B, present significant challenges in veterinary medicine. Such infections often require complex and elusive treatment regimens. Developing effective vaccines tailored to combat these specific pathogens remains a pressing need within the field. These bacteria are notorious for their extreme toxicity and the difficulty in culturing them for vaccine production. To address this challenge, we engineered a new potential vaccine candidate capable of neutralizing the virulence of both bacterial strains. Leveraging computational techniques, we identified epitopic regions within C. perfringens Epsilon Toxin (ETX) and C. novyi Alpha Toxin (ATX). Through fusion gene design, we integrated these epitopic regions alongside the PADRE-peptide sequence. The PADRE-peptide serves as a universal adjuvant to induce an immune response. The culmination of our efforts materialized in a Recombinant Fusion Protein D (rFPD), a novel vaccine construct designed to elicit robust and specific immune defenses against both bacterial species. By combining in-silico design and molecular engineering, our study represents a promising stride toward combating the impact of these pathogenic bacteria in livestock.

Metagenomic sequencing sheds light on microbes putatively associated with pneumonia-related fatalities of white-tailed deer (Odocoileus virginianus).

With emerging infectious disease outbreaks in human, domestic and wild animal populations on the rise, improvements in pathogen characterization and surveillance are paramount for the protection of human and animal health, as well as the conservation of ecologically and economically important wildlife. Genomics offers a range of suitable tools to meet these goals, with metagenomic sequencing facilitating the characterization of whole microbial communities associated with emerging and endemic disease outbreaks. Here, we use metagenomic sequencing in a case-control study to identify microbes in lung tissue associated with newly observed pneumonia-related fatalities in 34 white-tailed deer (Odocoileus virginianus) in Wisconsin, USA. We identified 20 bacterial species that occurred in more than a single individual. Of these, only Clostridium novyi was found to substantially differ (in number of detections) between case and control sample groups; however, this difference was not statistically significant. We also detected several bacterial species associated with pneumonia and/or other diseases in ruminants (Mycoplasma ovipneumoniae, Trueperella pyogenes, Pasteurella multocida, Anaplasma phagocytophilum, Fusobacterium necrophorum); however, these species did not substantially differ between case and control sample groups. On average, we detected a larger number of bacterial species in case samples than controls, supporting the potential role of polymicrobial infections in this system. Importantly, we did not detect DNA of viruses or fungi, suggesting that they are not significantly associated with pneumonia in this system. Together, these results highlight the utility of metagenomic sequencing for identifying disease-associated microbes. This preliminary list of microbes will help inform future research on pneumonia-associated fatalities of white-tailed deer.

New threats in the recovery of large carnivores inhabiting human-modified landscapes: the case of the Cantabrian brown bear (Ursus arctos)

Understanding mortality causes is important for the conservation of endangered species, especially in small and isolated populations inhabiting anthropized landscapes where both natural and human-caused mortality may hinder the conservation of these species. We investigated the mortality causes of 53 free-ranging brown bears (Ursus arctos) found dead between 1998 and 2023 in the Cantabrian Mountains (northwestern Spain), a highly human-modified region where bears are currently recovering after being critically threatened in the last century. We detected natural traumatic injuries in 52.63% and infectious diseases in 39.47% of the 38 bears for which the mortality causes were registered, with 21.05% of these cases presenting signs of both infectious diseases and traumas. More specifically, almost 30% of the bears died during or after intraspecific fights, including sexually selected infanticide (10.53%). In addition, primary infectious diseases such as infectious canine hepatitis, distemper, clostridiosis and colibacillosis caused the death of 15.79% of the bears. The number of direct human-caused deaths (i.e., shooting, poisoning, snare) decreased over the study period. This study also reveals three new mortality causes triggered by pathogens, two of which—Clostridium novyi and verotoxigenic Escherichia coli—not previously described in ursids, and the other one, canine distemper virus, never reported in brown bears as cause of death. New management strategies for the conservation of Cantabrian bears, which are urgently needed due to the rapid expansion of the population, should consider the mortality causes described in this study and must promote further research to elucidate how the high prevalence of infectious diseases may threaten the current recovery of the population.

Metagenomic analysis and biodiversity of bacteria in traditional fermented fish or Budu from West Sumatera, Indonesia.

This research aims to investigate the microbial diversity of Budu prepared from fresh and frozen fish from the Pariaman and Pasaman districts in West Sumatra Province, Indonesia, as well as provide basic information about Budu quality. To obtain the bacterial microbial composition, deoxyribonucleic acid extraction was carried out using amplicon-sequencing of the 16S-rRNA gene in the V3-V4 region from two types of Budu and carried out in duplicate. Budu prepared with fresh (Pariaman) or frozen (Pasaman) fish was dominated by Firmicutes (78.455%-92.37%) and Proteobacteria (6.477%-7.23%) phyla. The total microbial species in Budu from Pariaman were higher (227 species) than in Pasaman (153 species). The bacterial species found are Lentibacillus kimchi (1.878%-2.21%), Staphylococcus cohnii (0.597%-0.70%), Peptostreptococcus russeli (0.00%-0.002%), Clostridium disporicum (0.073%-0.09%), Clostridium novyi (0.00%-0.01%), Nioella sediminis (0.00%-0.001%), and Shewanella baltica (0.00%-0.003%). Lentibacillus kimchi, S. cohnii, and C. disporicum are found in both Budu. Nioella sediminis and S. baltica are found in Budu Pariaman. Peptostreptococcus russeli and C. novyi were found in Budu Pasaman. Metagenomic analysis of Budu from different fish, Pariaman (fresh fish) and Pasaman (frozen fish) showed that the biodiversity of bacteria was barely different. Both Budu found lactic acid bacteria from the Enterococcaceae family, genus Vagococcus, and pathogenic bacteria, such as S. cohnii, P. russeli, C. disporicum, and S. baltica. The discovery of various species of pathogenic bacteria indicates that development is still needed in the Budu production process to improve Budu quality.

The first isolation of Clostridium massiliodielmoense from a dead beef cow in Japan and pitfalls to be aware of in identifying the Clostridium species.

Clostridium sp. was detected in the organs of a cow with black watery diarrhea in Japan. Results identifying this species were inconsistent; Clostridium novyi type A infection was suggested by PCR assay targeting Clostridium fliC region (fliC-multiplex PCR), while 16S rRNA gene sequencing identified the isolated bacteria as Clostridium massiliodielmoense. Sequencing of fliC-multiplex PCR products from the isolates revealed the presence of fliC region in C. massiliodielmoense, which had 92.7% nucleotide similarity to that of C. novyi type A JCM 1406T, leading to the false positive detection of C. novyi by the PCR. This is the first C. massiliodielmoense isolation from clinical specimens, suggesting the need for further research on its pathogenicity and improvement in fliC-multiplex PCR.

An intravenous pancreatic cancer therapeutic: Characterization of CRISPR/Cas9n-modified Clostridium novyi-Non Toxic.

Clostridium novyi has demonstrated selective efficacy against solid tumors largely due to the microenvironment contained within dense tumor cores. The core of a solid tumor is typically hypoxic, acidic, and necrotic-impeding the penetration of current therapeutics. C. novyi is attracted to the tumor microenvironment and once there, can both lyse and proliferate while simultaneously re-activating the suppressed immune system. C. novyi systemic toxicity is easily mitigated by knocking out the phage DNA plasmid encoded alpha toxin resulting in C. novyi-NT; but, after intravenous injection spores are quickly cleared by phagocytosis before accomplishing significant tumor localization. C. novyi-NT could be designed to accomplish intravenous delivery with the potential to target all solid tumors and their metastases in a single dose. This study characterizes CRISPR/Cas9 modified C. novyi-NT to insert the gene for RGD, a tumor targeting peptide, expressed within the promoter region of a spore coat protein. Expression of the RGD peptide on the outer spore coat of C. novyi-NT indicates an increased capacity for tumor localization of C. novyi upon intravenous introduction based on the natural binding of RGD with the αvβ3 integrin commonly overexpressed on the epithelial tissue surrounding a tumor, and lead to immune stimulation.

Therapeutic Potential of Clostridium novyi NT in Cancer: Current Knowledge and Future Perspectives.

Resistance to conventional antitumour therapies and Hypoxia in patients with advanced solid tumours are two major reasons for the failure of conventional anti-tumour therapies. Therefore, it is important to find a new therapeutic method that can overcome these problems. An attenuated anaerobic bacterium, Clostridium novyi- NT, could target Hypoxic and Necrotic areas of tumours causing tumour lysis and stimulating a host anti-tumour immune response. To the best of our knowledge, the combination of bacterial anti-tumour therapy, chemotherapy, radiotherapy and immunotherapy may promote tumour regression, inhibit metastasis and develop a new strategy for the treatment of solid tumours. However, the possible molecular mechanisms of the combined therapies are still the biggest challenge. This review provides an overview of the history of bacterial cancer therapy and the development of a non-lethal strain of Clostridium novyi. Below is a precise definition of Hypoxic conditions in solid tumour tissue. To understand the anticancer effect of Clostridium novyi- NT spores, possible cell death mechanisms were summarised by the enzyme phospholipase C (nt01cx0979), which is secreted by Clostridium novyi- NT spores after germination in tumour tissue. The function of Clostridium novyi- NT spores in stimulating the host immune system to elicit anti-tumour responses was reviewed. Then, the results of anti-tumour combination therapies based on Clostridium novyi- NT spores were compiled. Identifying the molecular mechanisms of Clostridium novyi- NT in treating tumours and inducing cell death in invasive cancer cells, ultimately leading to tumour regression, may develop promising clinical strategies in the combined treatment of solid tumours.

Molecular basis and engineering of miniature Cas12f with C-rich PAM specificity.

CRISPR-Cas12f nucleases are currently one of the smallest genome editors, exhibiting advantages for efficient delivery via cargo-size-limited adeno-associated virus delivery vehicles. Most characterized Cas12f nucleases recognize similar T-rich protospacer adjacent motifs (PAMs) for DNA targeting, substantially restricting their targeting scope. Here we report the cryogenic electron microscopy structure and engineering of a miniature Clostridium novyi Cas12f1 nuclease (CnCas12f1, 497 amino acids) with rare C-rich PAM specificity. Structural characterizations revealed detailed PAM recognition, asymmetric homodimer formation and single guide RNA (sgRNA) association mechanisms. sgRNA engineering transformed CRISPR-CnCas12f1, which initially was incapable of genome targeting in bacteria, into an effective genome editor in human cells. Our results facilitate further understanding of CRISPR-Cas12f1 working mechanism and expand the mini-CRISPR toolbox.

Using spore-forming bacteria to treat cancer: recent advancements in clostridial-based therapies

The use of bacteria in cancer therapy has emerged as a promising approach, offering unique advantages for targeted treatment and immunotherapy. This review paper explores some recent advances in the application of bacteria, particularly Clostridium species, in cancer therapy. For instance, the Clostridial-Directed Enzyme Prodrug Therapy (CDEPT) utilises non-pathogenic strains of Clostridium as carriers for targeted delivery of anti-cancer drugs to solid tumour cells. This strategy aims to minimise systemic side effects associated with traditional chemotherapy. Additionally, Clostridium novyi and Clostridium sporogenes present oncolytic properties and have shown potential for tumour regression in preclinical models. The engineering of these bacteria to produce cytokines, such as interleukin-12 (IL-12) and interleukin-2 (IL-2), further enhances their therapeutic potential by activating the immune system to target cancer cells. Clinical trials in humans have demonstrated the feasibility and safety of C. novyi-based therapies, and early results indicate potential efficacy in tumour regression. Overall, this review provides valuable insights into the multifaceted roles of bacteria, particularly Clostridium species, in cancer therapy, emphasizing their potential as targeted therapeutics and immunomodulators for improved cancer treatment outcomes.

The Aqueous Leaf Extract of Moringa Oleifera had Immunomodulatory Effects on Sheep that had Been Infected by Fasciola Gigantica, Clostridium Novyi Naturally and Impact to Fasciola gigantica Non-Embryonated

The Aqueous Leaf Extract of Moringa Oleifera had Immunomodulatory Effects on Sheep that had Been Infected by Fasciola Gigantica, Clostridium Novyi Naturally and Impact to Fasciola gigantica Non-Embryonated

A review of clostridial diseases in rhinoceroses

AbstractAlthough rhinoceros conservation efforts are focused on anti‐poaching and combating wildlife trade, infectious diseases are an increasing threat to both free‐ranging and captive rhinoceros populations. Among these, clostridial infections with Clostridium perfringens, Paeniclostridium sordellii, and Clostridium novyi have caused acute disease in black and white rhinoceroses, characterized by a high mortality rate within very brief time spans. The acute nature, as well as limited knowledge of epidemiology, diagnosis, and management, make clostridial diseases an important threat to rhinoceros conservation. This article reviews historical cases of clostridial disease in rhinoceros with the aim of formulating a clearer understanding of the disease's epidemiology and using this information to make recommendations for its management in free‐ranging rhinoceroses. Acute colic and sudden death in rhinoceroses, together with necropsy findings of segmental hemorrhagic enteritis, should lead towards a tentative diagnosis of enteric clostridial disease, although detection and identification of specific toxins are required for a definitive diagnosis. Our findings indicate the need for further research into clostridial disease dynamics in wildlife species and provide the basis for this research in rhinoceroses.

Fist description of bacillary hemoglobinuria in a cattle in Minas Gerais, Brazil

Bacillary hemoglobinuria (BH) is a histotoxic infection caused by Clostridium haemolyticum that affects mostly cattle parasitized with trematodes. The aim of present study was to describe an atypical case of BH in a cow without access to flooded areas and without parasitism by Fasciola hepatica. After eight days of sternal decubitus and apathy, a Nellore cow was euthanized and necropsied. During postmortem examination, mild jaundice, black urine and multifocal lesions in the liver were observed. Histopathology revealed multifocal coagulation foci of necrosis in liver and basophilic bacillary structures, which were confirmed as C. haemolyticum by bacterial isolation, PCR and sequencing techniques. This is the first description of BH in cattle in Minas Gerais state, Brazil and highlight the need for of inclusion of BH as differential diagnosis even in animals not parasitized by trematodes.

Necrotizing hepatitis caused by Clostridium novyi type B in a dog with no predisposing liver lesions: a case report

BackgroundInfectious necrotic hepatitis (INH) is typically a disease of ruminants caused by Clostridium novyi type B. Growth of the causative agent is supported by development of an anaerobic environment within the liver. In dogs, C. novyi is rare and has only been previously reported as a post-mortem diagnosis. In one case, infection was secondary to metastatic pancreatic adenocarcinoma and the other was presumptively diagnosed on histopathology of a hepatic lesion in a dog initially presented for acute collapse.Case presentationAn 8-year-old spayed, female mixed breed dog was presented for acute onset of hyporexia and vomiting. Serum biochemistry revealed elevated hepatocellular injury and cholestatic liver enzymes. Ultrasound revealed peritoneal fluid accumulation and multiple hepatic masses. Cytologic examination of liver aspirates and peritoneal fluid revealed frequent 4 × 1 μm bacilli with a terminal endospore. Anaerobic bacterial growth isolated from the fluid sample could not be identified using typical laboratory identification techniques. Long-read, whole genome sequencing was performed, and the organism was identified as Clostridium novyi type B. Antimicrobial and hepatic support treatment were initiated. The patient re-presented 27 days later, and the follow up liver aspirate with cytology revealed no appreciable bacteria and anaerobic culture was negative. The patient was presented four months later and a large hepatic mass and peritoneal fluid were again identified on abdominal ultrasound. Cytologic examination of the peritoneal fluid revealed bacilli similar to those identified on initial presentation. The patient was euthanized. The most significant finding on necropsy was necrotizing hepatitis with intralesional endospore-forming bacilli compatible with recurrence of Clostridium novyi type B. There was no identifiable cause of an anaerobic insult to the liver.ConclusionsThis case demonstrates the diagnostic utility of using cytology as part of the initial diagnostic work up for infectious hepatitis. The cytologic findings coupled with whole genome sequencing and anaerobic culture were crucial for the identification and classification of the organism identified on fine needle aspirate. Clostridium novyi type B should be considered when bacilli organisms containing a terminal endospore are identified on liver aspirates collected from canine patients.

Combined Molecular and Biochemical Identification of Alpha Toxin in Local Isolated Clostridium novyi from the Sheep Liver.

Clostridium novyi (C. novyi) causes deadly Black disease in sheep and rarely in other animals. Alpha toxin (α-toxin), the most apparent pathogen of this disease, is produced by C. novyi type B. Economic damages of C. novyi include sheep mortality costs, depreciation of affected farms, and health problems with infected carcasses. The identification of C. novyi and isolation of its pathogens by conventional methods is a time-consuming process, necessitating a simple and rapid method for isolating and detecting pathogenic C. novyi. Therefore, this study aimed to molecularly identify α-toxin in local C. novyi isolates from the sheep livers. In this study, 75 livers suspected of Black disease were sampled. The samples of the liver were cultured under anaerobic conditions. Some of the cultured colonies were used in biochemical tests. For molecular confirmation, the DNA of isolates was extracted, and the isolates were confirmed by the polymerase chain reaction (PCR) on the liver tissue and cultured samples using specific α-toxin primers. The PCR on α-toxin produced a band in the range of 609 bp, indicating that the samples belonged to C. novyi. According to the results, of 75 isolates, 18 isolates were confirmed as C. novyi. C. novyitype B was isolated from the liver and confirmed by biochemical and molecular characterization. The PCR assay ensured a sensitive and specific tool for the detection of C. novyi in the samples.

LDLR, LRP1, and Megalin redundantly participate in the uptake of Clostridium novyi alpha-toxin

Clostridium novyi alpha-toxin (Tcnα) is a potent exotoxin that induces severe symptoms including gas gangrene, myositis, necrotic hepatitis, and sepsis. Tcnα binds to sulfated glycosaminoglycans (sGAG) for cell-surface attachment and utilizes low-density lipoprotein receptor (LDLR) for rapid entry. However, it was also shown that Tcnα may use alternative entry receptors other than LDLR. Here, we define that LRP1 and Megalin can also facilitate the cellular entry of Tcnα by employing reconstitutive LDLR family proteins. LDLR, LRP1, and Megalin recognize Tcnα via their ligand-binding domains (also known as LDL receptor type A repeats). Notably, LDLR and LRP1 have contrasting expression levels in many different cells, thus the dominant entry receptor for Tcnα could be cell-type dependent. These findings together increase our knowledge of the Tcnα actions and further help to understand the pathogenesis of C. novyi infection-associated diseases.

Daidzein-directed methionine γ-lyase in enzyme prodrug therapy against breast cancer

Thiosulfinates in situ formed by “pharmacological pair” C115H methionine γ-lyase/S-(allyl/alkyl)-l-cysteine sulfoxides possess cytotoxic activity against various malignant cell lines. To investigate in vivo antitumor activity of thiosulfinates generated directly at the surface of tumor cells, a chemical conjugate between Clostridium novyi C115H methionine γ-lyase (C115H MGL) and isoflavone daidzein was prepared. The binding of conjugate (C115H-Dz) to various breast cancer cell lines was demonstrated, as well as its cytotoxicity in the presence of S-(allyl/alkyl)-l-cysteine sulfoxides. The most promising among thiosulfinates was dipropyl thiosulfinate (IC50 < 0.53 μM). The pharmacokinetic parameters of C115H MGL and C115H-Dz were obtained. Plasma half-lives of the enzyme and conjugated enzyme were 4.4 and 7.2 h, respectively. In vivo antitumor effect of pharmacological pairs on SKBR-3 xenografts was demonstrated. Treatment of tumor-bearing mice with a pair of C115H-Dz/propiin inhibited tumor growth by 85%.

Factors Affecting on Production of Clostridium novyi type (B) Alpha Toxin

Factors Affecting on Production of Clostridium novyi type (B) Alpha Toxin

Clostridial Diseases of Horses: A Review.

The clostridial diseases of horses can be divided into three major groups: enteric/enterotoxic, histotoxic, and neurotoxic. The main enteric/enterotoxic diseases include those produced by Clostridium perfringens type C and Clostridioides difficile, both of which are characterized by enterocolitis. The main histotoxic diseases are gas gangrene, Tyzzer disease, and infectious necrotic hepatitis. Gas gangrene is produced by one or more of the following microorganisms: C. perfringens type A, Clostridium septicum, Paeniclostridium sordellii, and Clostridium novyi type A, and it is characterized by necrotizing cellulitis and/or myositis. Tyzzer disease is produced by Clostridium piliforme and is mainly characterized by multifocal necrotizing hepatitis. Infectious necrotic hepatitis is produced by Clostridium novyi type B and is characterized by focal necrotizing hepatitis. The main neurotoxic clostridial diseases are tetanus and botulism, which are produced by Clostridium tetani and Clostridium botulinum, respectively. Tetanus is characterized by spastic paralysis and botulism by flaccid paralysis. Neither disease present with specific gross or microscopic lesions. The pathogenesis of clostridial diseases involves the production of toxins. Confirming a diagnosis of some of the clostridial diseases of horses is sometimes difficult, mainly because some agents can be present in tissues of normal animals. This paper reviews the main clostridial diseases of horses.

Spatial and seasonal analysis and geovisualization of Fasciola hepatica–free bovine bacillary hemoglobinuria outbreaks in eastern Uruguay, 1999–2019

Bovine bacillary hemoglobinuria (BBH) produced by Clostridium novyi type D, is an endemic, highly fatal disease of cattle in the temperate grassland region of eastern Uruguay. A previous study showed that in this region, BBH is not associated with Fasciola hepatica or any other known focal-ischemic liver injury, so the reasons for its high incidence remains undetermined. The objective of this study was to analyze data from 45 Fasciola hepatica-free BBH outbreaks (1999–2019) in order to find common animal, seasonal and/or geographical risk factors, which may explain the occurrence of the epizootics. Fisher’s goodness-of-fit testing showed a significantly higher case proportion of adult cows (N = 368, 80.5%) and lower of calves (N =8, 1.8%), as compared to the expected proportions of the censused population in the study area and historical submissions computed from the laboratory database (Chi-Sq = 346.2 and 174.8, df = 7, P < 0.00). Time series decomposition showed a bi-seasonal pattern, with a larger peak in spring and early summer (October to January) and a smaller increase in autumn (March-May). The lowest seasonal indices were on mid-summer (February) and winter (June-September). A combination of spatial statistics was used to assess the different spatial features of the disease and consistency of the findings. Global spatial autocorrelation showed BBH was significantly clustered (Moran’s I = 0.407, P < 0.001). Both smoothed Anselin’s Local Indicator of Spatial Autocorrelation and Kulldorff’s spatial scan Poisson and Bernoulli models, detected roughly the same high-risk areas in the southeastern part of the Merin Lagoon basin, with the most likely cluster centered in the large wetland biosphere reserve “Eastern Wetlands and Coastal Strip” (RR = 9.12, P < 0.001). Outbreaks were georeferenced (latitude, longitude) and thematic dot-mapping geovisualization in Google Earth™ showed that the results were robust and truly geographic in nature. Most outbreaks (40/45, 88.8%) occurred on wetlands areas and large river valleys, characterized by poorly drained and frequently flooded soils, indicating that moisture-laden soils are the natural habitat of C. novyi type D. Grasslands in these endemic areas support rapid fattening of cattle during spring-summer, and somewhat less in autumn, in almost exact correspondence with BBH peaks, suggesting a close causal association in high-risk areas. Risk is significantly higher in adult cows probably because the spore content in the liver is highest in this category. The altered lipid metabolism and lipotoxicity in the liver may be the precipitating factor for spore germination and epizootic occurrence.