Fatal equine gas gangrene associated with Clostridium septicum in the Brazilian Amazon: clinicopathological and molecular characterization of a 20-case series

Fatal equine gas gangrene associated with Clostridium septicum in the Brazilian Amazon: clinicopathological and molecular characterization of a 20-case series | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Case Report Fatal equine gas gangrene associated with Clostridium septicum in the Brazilian Amazon: clinicopathological and molecular characterization of a 20-case series Loise Araújo de Sousa, Laura Rosa Corrêa, Hanna Gabriela da Silva Oliveira, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9031945/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 6 You are reading this latest preprint version Abstract Clostridial gas gangrene represents an acute necrotizing infection of skeletal muscle characterized by rapid tissue destruction and severe systemic toxemia in horses, however, systematically documented case series from tropical settings remain scarce. Here, we describe a clinicopathological and molecularly confirmed series of 20 fatal equine gas gangrene cases diagnosed in the eastern Brazilian Amazon (Pará State) between 2018 and 2026. Horses presented with abrupt onset of severe lameness, rapidly progressive swelling of a limb or muscle group, and palpable subcutaneous crepitus, frequently following penetrating trauma, intramuscular injections, or other breaches of tissue integrity. Despite antimicrobial and anti-inflammatory treatment, all animals died or were euthanized due to rapid deterioration and poor prognosis. Necropsy consistently revealed extensive hemorrhagic myonecrosis with serosanguineous exudation and intramuscular/subcutaneous gas accumulation. Histology showed widespread coagulative to floccular myonecrosis, marked interstitial edema and hemorrhage, frequent vascular thrombosis, and abundant intralesional Gram-positive bacilli. A multiplex PCR assay targeting major histotoxic clostridia detected Clostridium septicum DNA in affected skeletal muscle from all cases, with no amplification of other targets. This case series provides clinically relevant documentation of fatal C. septicum –associated myonecrosis in horses raised under tropical conditions and reinforces the importance of early recognition, strict aseptic practices during intramuscular procedures, and integration of pathology with molecular diagnostics for definitive etiological confirmation. equine gas gangrene clostridial myonecrosis multiplex PCR Amazon biome Clostridium septicum Figures Figure 1 Figure 2 Figure 3 1. Introduction Brazil hosts one of the largest equine populations worldwide, and horses remain central to multiple production and service chains, making severe infectious syndromes with high lethality particularly relevant to veterinary practice and animal health management (MAPA 2021). Among these syndromes, clostridial myonecrosis (“gas gangrene”) stands out as a peracute condition marked by extensive muscle necrosis, toxemia, and frequent death, demanding rapid clinical suspicion and prompt etiological confirmation. In horses, myonecrotic infections are classically linked to histotoxic clostridia that reach deep tissues through penetrating trauma or iatrogenic breaches in asepsis, including intramuscular injections and surgical procedures (Uzal et al. 2022 ; Junior et al. 2020 ). Although different agents have been implicated, fulminant courses and high case fatality are consistently reported, reinforcing the diagnostic urgency of this entity in equine medicine (Uzal et al. 2022 ). Published evidence from tropical regions, particularly the Amazon biome, remains limited, despite ecological and management features that may facilitate exposure to clostridial spores and create opportunities for anaerobic proliferation in damaged tissues. The Amazon region is characterized by wide geographic dispersion of farms, heterogeneous veterinary coverage, and logistical constraints that may delay diagnosis and laboratory confirmation, thereby restricting case documentation in the peer-reviewed literature (Salvarani et al. 2025 ). In addition, preventive options for equine clostridial myonecrosis are not standardized; while vaccination strategies exist for other clostridial diseases, commercially available products specifically designed to protect horses against the main histotoxic species involved in gas gangrene are limited, emphasizing the role of basic biosecurity, injection hygiene, and wound management (Uzal et al. 2022 ; Junior et al. 2020 ). Here, we report a clinicopathological and molecularly confirmed case series of 20 horses with gas gangrene from the eastern Brazilian Amazon. The objective is to document the clinical presentation, gross and histological lesions, and multiplex PCR findings, providing region-specific diagnostic evidence and practical insights for prevention focused on management-related risk points. Molecular confirmation is particularly relevant in this context because culture-based diagnosis may be compromised by post-mortem change or prior antimicrobial exposure, and histopathology alone is not agent-specific (Uzal et al. 2022 ). This series also complements previous regional reports of acute equine myonecrosis linked to histotoxic clostridia in Brazil (Farias et al. 2014 ) and supports improved awareness of fatal clostridial myonecrosis in tropical equine systems. Despite the recognized severity of clostridial myonecrosis in horses, systematic clinicopathological documentation from tropical production systems remains limited. Most available reports consist of isolated case descriptions or small outbreaks reported in temperate regions, and information regarding the occurrence, etiological agents, and pathological features of equine gas gangrene in the Amazon biome is scarce. Therefore, documenting well-characterized case series from this region is relevant to improve diagnostic awareness and to identify management practices that may predispose horses to this rapidly fatal infection. 2. Case series description Case identification and inclusion criteria A series of 20 horses with clostridial gas gangrene was identified between January 2018 and January 2026 in the eastern Amazon region of Brazil. Cases were retrieved from veterinary diagnostic laboratory records, clinical reports from field veterinarians, and necropsy submissions originating from several municipalities in the state of Pará. Only cases with compatible clinical history, characteristic gross lesions, histopathological findings consistent with clostridial myonecrosis, and positive multiplex polymerase chain reaction (PCR) detection of histotoxic clostridia were included in the final series. To ensure diagnostic consistency, cases were included only when clinical, pathological, and molecular criteria were concurrently fulfilled. Clinically, horses presented with sudden onset of severe localized swelling, marked pain on palpation, and palpable subcutaneous crepitus, frequently accompanied by systemic manifestations compatible with toxemia such as fever, lethargy, or anorexia. Gross pathological findings obtained during necropsy or surgical exploration consistently revealed extensive hemorrhagic myonecrosis with intramuscular gas formation, serosanguineous exudation, and a characteristic foul odor. Microbiological confirmation was achieved through multiplex PCR targeting major histotoxic clostridia ( Clostridium chauvoei , C. septicum, C. novyi type A , C. perfringens type A , and C. sordellii ), following the protocol described by Ribeiro et al. ( 2012 ). Clinical presentation and epidemiological context The affected horses originated from five municipalities in the state of Pará, including Castanhal, Novo Progresso, Santo Antônio do Tauá, São Félix do Xingú, and Soure (Marajó Island). Animals ranged from 4 months to 12 years of age, with a predominance of adult individuals. Most cases involved male horses. Clinical evolution was characterized by rapidly progressive swelling of affected limbs or muscle groups, severe lameness, intense pain, and palpable subcutaneous emphysema. Systemic deterioration occurred rapidly, with tachycardia, tachypnea, and signs compatible with systemic toxemia frequently observed during clinical examination, performed according to standard equine semiological guidelines (Feitosa 2020 ). In all animals, a recent breach in tissue integrity was reported prior to the onset of clinical signs. These events included penetrating traumatic injuries, intramuscular injections, minor surgical procedures, or skin lesions associated with environmental hazards such as barbed wire or wooden structures. In several farms, management practices potentially favoring contamination were documented, including reuse of needles and inadequate sterilization of instruments. None of the affected horses had a documented history of vaccination against clostridial diseases. Despite administration of antimicrobial therapy and anti-inflammatory treatment, clinical deterioration progressed rapidly in all animals. The case fatality rate reached 100%, with horses dying or being euthanized due to poor prognosis within a short period after the onset of clinical signs. Representative clinical and gross pathological findings are illustrated in Fig. 1 , which demonstrates marked enlargement of the affected limb with extensive subcutaneous edema and hemorrhage, and Fig. 2 , showing diffuse muscular necrosis with serosanguineous fluid accumulation and tissue liquefaction. Pathological examination Necropsy examinations consistently revealed lesions typical of clostridial myonecrosis. Affected musculature displayed extensive hemorrhagic necrosis with interstitial edema and accumulation of serosanguineous fluid. Gas bubbles were frequently present within muscle fascicles and subcutaneous tissues, producing a characteristic crepitant texture. Histopathological evaluation was performed on representative tissue samples collected during necropsy or surgical intervention. Samples were fixed in buffered formalin, routinely processed, embedded in paraffin, and stained with hematoxylin and eosin following standard histological procedures (Amorim et al. 2011 ). Microscopically, lesions were characterized by severe coagulative to floccular necrosis of skeletal muscle fibers, associated with extensive interstitial edema and hemorrhage. Muscle fibers frequently exhibited fragmentation and loss of cross-striations, often replaced by eosinophilic amorphous debris. Inflammatory infiltrates composed predominantly of neutrophils were present in necrotic areas. Vascular thrombosis was observed in several cases. Gram staining revealed abundant Gram-positive bacilli arranged in clusters or short chains within necrotic muscle tissue, a finding consistent with histotoxic clostridial infection. The principal microscopic findings observed in the case series are summarized in Table 1 . Table 1 Main histopathological findings observed in horses with clostridial gas gangrene in the Brazilian Amazon. Histopathological finding Number of cases (n = 20) Frequency (%) Severe coagulative and/or floccular myonecrosis 20 100 Intramuscular gas vacuoles 20 100 Diffuse interstitial edema 20 100 Intramuscular hemorrhage 20 100 Neutrophil-predominant inflammatory infiltrate 20 100 Intralesional Gram-positive bacilli 20 100 Vascular thrombosis 16 80 Muscle fiber fragmentation and loss of cross-striations 20 100 Centrilobular hepatocellular vacuolization 6 30 Representative histopathological lesions are shown in Fig. 3 , illustrating extensive degeneration and necrosis of muscle fibers associated with inflammatory infiltrates, edema, and hemorrhage. 3. Discussion and conclusions Necrotizing clostridial infections are among the most aggressive bacterial diseases affecting equine skeletal muscle (Vengust et al. 2003 ). The present case series provides clinicopathological and molecular documentation of 20 fatal cases of equine gas gangrene associated with C. septicum diagnosed in the eastern Brazilian Amazon. The most notable observation was the consistent molecular detection of C. septicum in all examined animals, combined with a remarkably uniform clinical and pathological presentation. Although several histotoxic clostridia have been implicated in equine gas gangrene, including C. perfringens, C. novyi, C. chauvoei, and C. sordellii , infections attributed specifically to C. septicum remain comparatively less frequently documented in horses (Uzal et al. 2022 ; Junior et al. 2020 ; Peek et al. 2003 ). Consequently, the present series contributes valuable documentation of the involvement of this species in fatal clostridial myonecrosis affecting horses raised under tropical production conditions. The consistent identification of C. septicum in all analyzed cases suggests that this species may play a more relevant role in equine clostridial myonecrosis in tropical environments than currently recognized, although broader epidemiological studies would be required to confirm regional pathogen distribution. The clinical evolution observed in the affected animals closely corresponds to classical descriptions of clostridial gas gangrene. The abrupt onset of severe lameness, rapidly progressive swelling, and palpable subcutaneous crepitation reflects the extensive tissue destruction and gas production generated during anaerobic bacterial proliferation in damaged muscle tissue (Uzal et al. 2022 ). The uniformly fatal outcome documented in this series further highlights the fulminant nature of this condition. Previous clinical studies have reported mortality rates ranging from approximately 50% to nearly 100% in equine clostridial myonecrosis, depending on the time of diagnosis and the possibility of aggressive surgical intervention (Peek et al. 2003 ; Junior et al. 2020 ). In the present cases, despite the administration of antimicrobial and anti-inflammatory therapy, disease progression was extremely rapid, emphasizing the narrow therapeutic window that characterizes this infection. The gross and microscopic lesions observed in the affected horses were consistent with the well-recognized pathological features of clostridial myonecrosis. Extensive hemorrhagic necrosis of skeletal muscle, diffuse edema, intramuscular gas accumulation, and abundant intralesional Gram-positive bacilli are considered hallmarks of this disease (Amorim et al. 2011 ; Uzal et al. 2022 ). Histopathological uniformity across all animals examined strengthens the diagnostic consistency of the case definition and provides biological support for the molecular results obtained in the present study. The frequent observation of vascular thrombosis and widespread muscle fiber degeneration further reflects the combined effects of bacterial toxins, tissue ischemia, and host inflammatory responses, which together contribute to the rapid clinical deterioration typical of clostridial infections. An important epidemiological observation in this series was the frequent association of cases with recent tissue trauma or intramuscular injections, conditions widely recognized as facilitating the introduction of clostridial spores into muscle tissue. Several classical reports have documented similar iatrogenic or traumatic entry points in equine gas gangrene outbreaks, particularly following intramuscular injections or minor surgical procedures (Peek et al. 2003 ). In the present cases, additional management practices potentially contributing to infection risk were identified, including reuse of needles and inadequate sterilization of instruments. Although causal relationships cannot be definitively established in an observational case series, these findings highlight important opportunities for preventive intervention through improved aseptic practices and veterinary supervision. The exclusive molecular detection of C. septicum in all animals is noteworthy. This species has been recognized as an important cause of malignant edema and spontaneous myonecrosis in several animal species (Raymundo et al. 2010 ; Junior et al. 2020 ; Uzal et al. 2022 ). In horses, however, reports of C. septicum –associated gas gangrene remains limited compared with those involving C. perfringens type A or other histotoxic clostridia (Kennedy et al. 2009 ; Peek et al. 2003 ; Sacco et al. 2020 ). While the consistent identification of C. septicum in this series suggests that this pathogen may play a relevant role in equine clostridial infections within the studied region, caution is required in interpreting this observation. The retrospective nature of the study, the limited number of cases, and the targeted PCR approach preclude definitive conclusions regarding pathogen predominance in the Amazon biome. Nevertheless, the geographic distribution of cases across several municipalities supports the epidemiological relevance of this agent in the investigated context. Environmental factors characteristic of tropical ecosystems may also influence the epidemiology of clostridial infections. High temperatures, elevated humidity, and the persistence of bacterial spores in soil have long been recognized as conditions favoring the environmental survival of clostridia (Junior et al. 2020 ). The Amazon biome is characterized by climatic and ecological conditions that may facilitate the maintenance of these organisms in the environment, although the present study did not include environmental sampling capable of confirming this hypothesis. Future investigations incorporating soil analysis and molecular epidemiology would be valuable to clarify the environmental reservoirs and transmission pathways of histotoxic clostridia in tropical equine systems. The absence of vaccination against clostridial diseases in all affected animals deserves consideration, although its interpretation must remain cautious. Vaccination strategies targeting several clostridial toxins have been developed for livestock species and experimental vaccine approaches have been explored in horses (Zaragoza et al. 2019 ; Freitas et al. 2021 ). However, protective protocols specifically designed to prevent equine gas gangrene remain limited, and the effectiveness of available vaccines against multiple histotoxic clostridia in horses is still incompletely defined (Uzal et al. 2022 ). Consequently, improvements in management practices, particularly strict aseptic techniques during injections and prompt treatment of traumatic wounds remain the most practical preventive strategies currently available. From a diagnostic perspective, the integration of clinical evaluation, pathological examination, and molecular detection proved particularly valuable in confirming the etiological diagnosis. Culture-based identification of clostridia may be compromised in field conditions due to post-mortem autolysis or prior antimicrobial therapy, which frequently occur in severe cases of myonecrosis (Ribeiro et al. 2012 ; Uzal et al. 2022 ). In this context, multiplex PCR represents a useful complementary diagnostic tool capable of detecting specific clostridial species directly in affected tissues. Some limitations inherent to this study should be acknowledged. The retrospective design restricts the availability of complete exposure data, and the relatively small number of cases prevents robust epidemiological inference. In addition, toxin characterization and bacterial isolation were not consistently achieved in all animals. Nevertheless, the strong concordance between clinical signs, pathological findings, and molecular detection provides reliable evidence supporting the diagnosis of clostridial gas gangrene associated with C. septicum . From a practical perspective, these observations emphasize that even minor breaches in tissue integrity may create conditions for clostridial proliferation in equine muscle, particularly in environments where spores are likely to persist in soil and organic matter. However, to the best of our knowledge, this is one of the largest documented case series of equine gas gangrene associated with Clostridium septicum reported in South America. In summary, this case series documents 20 fatal cases of equine gas gangrene associated with C. septicum in the Brazilian Amazon and provides clinicopathological and molecular evidence supporting the involvement of this pathogen in equine clostridial myonecrosis under tropical conditions. The findings reinforce the fulminant nature of the disease, highlight the value of molecular diagnostics for etiological confirmation, and underscore the importance of strict aseptic practices and early clinical recognition in equine management systems. Declarations Acknowledgments The authors are grateful to CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico), Anaerobic Laboratory of the Federal University of Minas Gerais; Biological Institute of São Paulo and UFPA animal pathology laboratory. Funding This work was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Brazil (Finance Code 001). Competing Interests The authors declare no conflicts of interest. Ethics approval and Informed consent statement All procedures involving animals followed internationally accepted principles of animal welfare. The cases described in this study involved naturally occurring disease and diagnostic investigations requested by animal owners and attending veterinarians; therefore, no experimental procedures were performed. Author Contributions Loise Araújo de Sousa, Laura Rosa Corrêa and Felipe Masiero Salvarani contributed to the study conception and design. Material preparation, data collection and analysis were performed by Loise Araújo de Sousa, Laura Rosa Corrêa, Hanna Gabriela da Silva Oliveira and Felipe Masiero Salvarani. The first draft of the manuscript was written by Loise Araújo de Sousa, Laura Rosa Corrêa and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Data Availability Statement Data are contained within the article. References Amorim RM, Rino AS, Dal-Pai-Silva M, Borges AS, Oliveira Filho JP, Freitas NPP, Maia L, Rezende LAL (2011) Morphological aspects of muscle biopsies in horses affected by myopathies. Pesq Vet Bras 31:479–485. https://doi.org/10.1590/S0100-736X2011000700006 Breauhaus BA, Brown CM, Scott EA, Ainsworth DM, Taylor RF (1983) Clostridial muscle infections following intramuscular injections in the horse. J Equine Vet Sci 3:42–46. https://doi.org/10.1016/S0737-0806(83)80003-3 Farias LD, Azevedo MDS, Trost ME, De La Côrte FD, Irigoyen LF, Vargas AC (2014) Acute myonecrosis in horse caused by Clostridium novyi type A . Braz J Microbiol 45:221–224. https://doi.org/10.1590/S1517-83822014005000023 Feitosa FLF (2020) Semiologia: a arte do diagnóstico, 4th edn. Roca, São Paulo Freitas NFQR, Otaka DY, Galvão CC, Almeida DM, Ferreira MR, Conceição FR, Salvarani FM (2021) Humoral immune response evaluation in horses vaccinated with recombinant Clostridium perfringens toxoids alpha and beta for 12 months. Toxins 13:566. https://doi.org/10.3390/toxins13080566 Junior CAO, Silva ROS, Lobato FCF, Navarro MA, Uzal FA (2020) Gas gangrene in mammals: a review. J Vet Diagn Invest 32:175–183. https://doi.org/10.1177/1040638720905830 MAPA – Ministério da Agricultura, Pecuária e Abastecimento (2021) Agropecuária brasileira em números. Available at: https://www.gov.br/agricultura/pt-br/assuntos/politica-agricola/todas-pulicacoes-de-politica-agricola/agropecuaria-brasileira-em-numeros/abn-02-2021.pdf Kennedy CL, Lyras D, Cheung JK, Hiscox TJ, Emmins JJ, Rood JI (2009) Cross-complementation of Clostridium perfringens PLC and Clostridium septicum alpha-toxin mutants reveals PLC is sufficient to mediate gas gangrene. Microbes Infect 11:413–418. https://doi.org/10.1016/j.micinf.2009.01.002 Peek SF, Semrad SD, Perkins GA (2003) Clostridial myonecrosis in horses (37 cases 1985–2000). Equine Vet J 35:86–92. https://doi.org/10.2746/042516403775467513 Raymundo DL, Pavarini SP, Bezerra Junior PS, Antoniassi NAB, Bandarra PM, Bercht BS, Gomes MJP, Driemeier D (2010) Acute myonecrosis caused by Clostridium septicum in horses. Pesq Vet Bras 30:637–640. https://doi.org/10.1590/S0100-736X2010000800005 Ribeiro MG, Silva ROS, Pires PS, Martinho APV, Lucas TM, Teixeira AIP, Paes AC, Barros CB, Lobato FCF (2012) Myonecrosis by Clostridium septicum in a dog diagnosed by multiplex PCR. Anaerobe 18:504–507. https://doi.org/10.1016/j.anaerobe.2012.09.003 Sacco SC, Ortega J, Navarro MA, Fresneda KC, Anderson M, Woods LW, Moore J, Uzal FA (2020) Clostridium sordellii -associated gas gangrene in horses. J Vet Diagn Invest 32:246–251. https://doi.org/10.1177/1040638719877844 Salvarani FM, Oliveira HGS, Correa LYS, Soares AAL, Ferreira BC (2025) The importance of studying infectious and parasitic diseases of wild animals in the Amazon biome with a focus on One Health. Vet Sci 12:100. https://doi.org/10.3390/vetsci12020100 Uzal FA, Navarro MA, Asin J, Henderson EE (2022) Clostridial diseases of horses: a review. Vaccines 10:318. https://doi.org/10.3390/vaccines10020318 Vengust M, Arroyo LG, Weese JS, Baird JD (2003) Preliminary evidence for dormant clostridial spores in equine skeletal muscle. Equine Vet J 35:514–516. https://doi.org/10.2746/042516403775600569 Zaragoza NE, Orellana CA, Moonen GA, Moutafis G, Marcellin E (2019) Vaccine production to protect animals against pathogenic clostridia. Toxins 11:525. https://doi.org/10.3390/toxins11090525 Additional Declarations No competing interests reported. Supplementary Files GA.jpg Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 02 Apr, 2026 Reviewers agreed at journal 15 Mar, 2026 Reviewers invited by journal 15 Mar, 2026 Editor assigned by journal 10 Mar, 2026 Submission checks completed at journal 10 Mar, 2026 First submitted to journal 04 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9031945","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Case Report","associatedPublications":[],"authors":[{"id":606551446,"identity":"3599f0dc-686c-4e45-bf1b-f1555281c42a","order_by":0,"name":"Loise Araújo de Sousa","email":"","orcid":"","institution":"Federal University of Para","correspondingAuthor":false,"prefix":"","firstName":"Loise","middleName":"Araújo","lastName":"de Sousa","suffix":""},{"id":606551448,"identity":"b32388bc-37b3-4f44-8de2-f4052a5c9326","order_by":1,"name":"Laura Rosa Corrêa","email":"","orcid":"","institution":"Federal University of Para","correspondingAuthor":false,"prefix":"","firstName":"Laura","middleName":"Rosa","lastName":"Corrêa","suffix":""},{"id":606551450,"identity":"0ba5543f-8ef6-4a40-970f-0c183d67d30a","order_by":2,"name":"Hanna Gabriela da Silva Oliveira","email":"","orcid":"","institution":"Federal University of Para","correspondingAuthor":false,"prefix":"","firstName":"Hanna","middleName":"Gabriela da Silva","lastName":"Oliveira","suffix":""},{"id":606551452,"identity":"a193cc59-d347-4f11-82c9-cdfed7e99bfe","order_by":3,"name":"Felipe Masiero Salvarani","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyUlEQVRIiWNgGAWjYBAC9gbmhgMgBj8D4wMgdYCwFp4DjBAtkg3MBsRrATMMDhCthb2x8XBBxTZ74xvJbA9+MNzJJ6yF52DD4Rlnbiduu5HMbtjD8MyygZAWe4nEhsO8bbcTzG7kH5PgYThsQNgW+YdALf9u2xvPSGaT/EOUFglGoJaG24wbJJLZpImzhQfosBnHbifOOPOY3VjG4BkRWtgPH/5cUHPbnr89me3hm4o7hLWAADOUZgPGDlEaULSMglEwCkbBKMACANQcQOyxFLXgAAAAAElFTkSuQmCC","orcid":"","institution":"Federal University of Para","correspondingAuthor":true,"prefix":"","firstName":"Felipe","middleName":"Masiero","lastName":"Salvarani","suffix":""}],"badges":[],"createdAt":"2026-03-04 15:26:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9031945/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9031945/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104878770,"identity":"7ee2ff89-508b-457a-b13d-3bfe1e12d26e","added_by":"auto","created_at":"2026-03-18 08:58:21","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3094698,"visible":true,"origin":"","legend":"\u003cp\u003eEquine with gas gangrene: (\u003cstrong\u003ea\u003c/strong\u003e) increased volume of the left hind limb; (\u003cstrong\u003eb\u003c/strong\u003e) hemorrhage and subcutaneous edema, with extravasation of serosanguineous fluid, liquefaction, and tissue necrosis (postmortem).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9031945/v1/1744c93954aa53fac05bfc8c.png"},{"id":104878865,"identity":"5fe0ed6f-e009-417d-aa69-47a027f81dc0","added_by":"auto","created_at":"2026-03-18 08:58:50","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2140292,"visible":true,"origin":"","legend":"\u003cp\u003eEquine with gas gangrene (postmortem): (\u003cstrong\u003ea\u003c/strong\u003e) hemorrhage and subcutaneous edema, with extravasation of serosanguineous fluid, liquefaction, and tissue necrosis in the pectoral region; (\u003cstrong\u003eb\u003c/strong\u003e) ventral subcutaneous edema.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9031945/v1/0c3832409da97d9a6051fbcb.png"},{"id":104878852,"identity":"3ecf2bfa-4282-4dc7-87bc-013067dd505a","added_by":"auto","created_at":"2026-03-18 08:58:44","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2458583,"visible":true,"origin":"","legend":"\u003cp\u003eEquine gas gangrene: (\u003cstrong\u003ea\u003c/strong\u003e) degeneration and hyaline necrosis of muscle fibers with intense inflammatory infiltrate [H\u0026amp;E obj. 10×]; (\u003cstrong\u003eb\u003c/strong\u003e) area of necrosis associated with edema with hemorrhage [H\u0026amp;E obj. 20×].\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9031945/v1/21b9242ec34854de3eb201db.png"},{"id":104878882,"identity":"24069034-fdc7-4021-a848-c49424a35ab0","added_by":"auto","created_at":"2026-03-18 08:59:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":12534118,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9031945/v1/63944808-5c25-4fa8-b174-e7f5f28ca493.pdf"},{"id":104878813,"identity":"4d5aabdf-af93-482b-bf6e-b572a4a1cfe3","added_by":"auto","created_at":"2026-03-18 08:58:31","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":64535,"visible":true,"origin":"","legend":"","description":"","filename":"GA.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9031945/v1/23064f2f113d8a74971ef6c0.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Fatal equine gas gangrene associated with Clostridium septicum in the Brazilian Amazon: clinicopathological and molecular characterization of a 20-case series","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eBrazil hosts one of the largest equine populations worldwide, and horses remain central to multiple production and service chains, making severe infectious syndromes with high lethality particularly relevant to veterinary practice and animal health management (MAPA 2021). Among these syndromes, clostridial myonecrosis (\u0026ldquo;gas gangrene\u0026rdquo;) stands out as a peracute condition marked by extensive muscle necrosis, toxemia, and frequent death, demanding rapid clinical suspicion and prompt etiological confirmation. In horses, myonecrotic infections are classically linked to histotoxic clostridia that reach deep tissues through penetrating trauma or iatrogenic breaches in asepsis, including intramuscular injections and surgical procedures (Uzal et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Junior et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Although different agents have been implicated, fulminant courses and high case fatality are consistently reported, reinforcing the diagnostic urgency of this entity in equine medicine (Uzal et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePublished evidence from tropical regions, particularly the Amazon biome, remains limited, despite ecological and management features that may facilitate exposure to clostridial spores and create opportunities for anaerobic proliferation in damaged tissues. The Amazon region is characterized by wide geographic dispersion of farms, heterogeneous veterinary coverage, and logistical constraints that may delay diagnosis and laboratory confirmation, thereby restricting case documentation in the peer-reviewed literature (Salvarani et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). In addition, preventive options for equine clostridial myonecrosis are not standardized; while vaccination strategies exist for other clostridial diseases, commercially available products specifically designed to protect horses against the main histotoxic species involved in gas gangrene are limited, emphasizing the role of basic biosecurity, injection hygiene, and wound management (Uzal et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Junior et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHere, we report a clinicopathological and molecularly confirmed case series of 20 horses with gas gangrene from the eastern Brazilian Amazon. The objective is to document the clinical presentation, gross and histological lesions, and multiplex PCR findings, providing region-specific diagnostic evidence and practical insights for prevention focused on management-related risk points. Molecular confirmation is particularly relevant in this context because culture-based diagnosis may be compromised by post-mortem change or prior antimicrobial exposure, and histopathology alone is not agent-specific (Uzal et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This series also complements previous regional reports of acute equine myonecrosis linked to histotoxic clostridia in Brazil (Farias et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) and supports improved awareness of fatal clostridial myonecrosis in tropical equine systems.\u003c/p\u003e \u003cp\u003eDespite the recognized severity of clostridial myonecrosis in horses, systematic clinicopathological documentation from tropical production systems remains limited. Most available reports consist of isolated case descriptions or small outbreaks reported in temperate regions, and information regarding the occurrence, etiological agents, and pathological features of equine gas gangrene in the Amazon biome is scarce. Therefore, documenting well-characterized case series from this region is relevant to improve diagnostic awareness and to identify management practices that may predispose horses to this rapidly fatal infection.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"2. Case series description","content":"\u003cp\u003e \u003cb\u003eCase identification and inclusion criteria\u003c/b\u003e \u003c/p\u003e \u003cp\u003eA series of 20 horses with clostridial gas gangrene was identified between January 2018 and January 2026 in the eastern Amazon region of Brazil. Cases were retrieved from veterinary diagnostic laboratory records, clinical reports from field veterinarians, and necropsy submissions originating from several municipalities in the state of Par\u0026aacute;. Only cases with compatible clinical history, characteristic gross lesions, histopathological findings consistent with clostridial myonecrosis, and positive multiplex polymerase chain reaction (PCR) detection of histotoxic clostridia were included in the final series.\u003c/p\u003e \u003cp\u003eTo ensure diagnostic consistency, cases were included only when clinical, pathological, and molecular criteria were concurrently fulfilled. Clinically, horses presented with sudden onset of severe localized swelling, marked pain on palpation, and palpable subcutaneous crepitus, frequently accompanied by systemic manifestations compatible with toxemia such as fever, lethargy, or anorexia. Gross pathological findings obtained during necropsy or surgical exploration consistently revealed extensive hemorrhagic myonecrosis with intramuscular gas formation, serosanguineous exudation, and a characteristic foul odor. Microbiological confirmation was achieved through multiplex PCR targeting major histotoxic clostridia (\u003cem\u003eClostridium chauvoei\u003c/em\u003e, \u003cem\u003eC. septicum, C. novyi\u003c/em\u003e type \u003cem\u003eA\u003c/em\u003e, \u003cem\u003eC. perfringens\u003c/em\u003e type \u003cem\u003eA\u003c/em\u003e, and \u003cem\u003eC. sordellii\u003c/em\u003e), following the protocol described by Ribeiro et al. (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eClinical presentation and epidemiological context\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe affected horses originated from five municipalities in the state of Par\u0026aacute;, including Castanhal, Novo Progresso, Santo Ant\u0026ocirc;nio do Tau\u0026aacute;, S\u0026atilde;o F\u0026eacute;lix do Xing\u0026uacute;, and Soure (Maraj\u0026oacute; Island). Animals ranged from 4 months to 12 years of age, with a predominance of adult individuals. Most cases involved male horses.\u003c/p\u003e \u003cp\u003eClinical evolution was characterized by rapidly progressive swelling of affected limbs or muscle groups, severe lameness, intense pain, and palpable subcutaneous emphysema. Systemic deterioration occurred rapidly, with tachycardia, tachypnea, and signs compatible with systemic toxemia frequently observed during clinical examination, performed according to standard equine semiological guidelines (Feitosa \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn all animals, a recent breach in tissue integrity was reported prior to the onset of clinical signs. These events included penetrating traumatic injuries, intramuscular injections, minor surgical procedures, or skin lesions associated with environmental hazards such as barbed wire or wooden structures. In several farms, management practices potentially favoring contamination were documented, including reuse of needles and inadequate sterilization of instruments. None of the affected horses had a documented history of vaccination against clostridial diseases.\u003c/p\u003e \u003cp\u003eDespite administration of antimicrobial therapy and anti-inflammatory treatment, clinical deterioration progressed rapidly in all animals. The case fatality rate reached 100%, with horses dying or being euthanized due to poor prognosis within a short period after the onset of clinical signs. Representative clinical and gross pathological findings are illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, which demonstrates marked enlargement of the affected limb with extensive subcutaneous edema and hemorrhage, and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, showing diffuse muscular necrosis with serosanguineous fluid accumulation and tissue liquefaction.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ePathological examination\u003c/b\u003e \u003c/p\u003e \u003cp\u003eNecropsy examinations consistently revealed lesions typical of clostridial myonecrosis. Affected musculature displayed extensive hemorrhagic necrosis with interstitial edema and accumulation of serosanguineous fluid. Gas bubbles were frequently present within muscle fascicles and subcutaneous tissues, producing a characteristic crepitant texture.\u003c/p\u003e \u003cp\u003eHistopathological evaluation was performed on representative tissue samples collected during necropsy or surgical intervention. Samples were fixed in buffered formalin, routinely processed, embedded in paraffin, and stained with hematoxylin and eosin following standard histological procedures (Amorim et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMicroscopically, lesions were characterized by severe coagulative to floccular necrosis of skeletal muscle fibers, associated with extensive interstitial edema and hemorrhage. Muscle fibers frequently exhibited fragmentation and loss of cross-striations, often replaced by eosinophilic amorphous debris. Inflammatory infiltrates composed predominantly of neutrophils were present in necrotic areas. Vascular thrombosis was observed in several cases. Gram staining revealed abundant Gram-positive bacilli arranged in clusters or short chains within necrotic muscle tissue, a finding consistent with histotoxic clostridial infection. The principal microscopic findings observed in the case series are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMain histopathological findings observed in horses with clostridial gas gangrene in the Brazilian Amazon.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHistopathological finding\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNumber of cases (n\u0026thinsp;=\u0026thinsp;20)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFrequency (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSevere coagulative and/or floccular myonecrosis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIntramuscular gas vacuoles\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDiffuse interstitial edema\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIntramuscular hemorrhage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNeutrophil-predominant inflammatory infiltrate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIntralesional Gram-positive bacilli\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVascular thrombosis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e80\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMuscle fiber fragmentation and loss of cross-striations\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCentrilobular hepatocellular vacuolization\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eRepresentative histopathological lesions are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, illustrating extensive degeneration and necrosis of muscle fibers associated with inflammatory infiltrates, edema, and hemorrhage.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"3. Discussion and conclusions","content":"\u003cp\u003eNecrotizing clostridial infections are among the most aggressive bacterial diseases affecting equine skeletal muscle (Vengust et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). The present case series provides clinicopathological and molecular documentation of 20 fatal cases of equine gas gangrene associated with \u003cem\u003eC. septicum\u003c/em\u003e diagnosed in the eastern Brazilian Amazon. The most notable observation was the consistent molecular detection of \u003cem\u003eC. septicum\u003c/em\u003e in all examined animals, combined with a remarkably uniform clinical and pathological presentation. Although several histotoxic clostridia have been implicated in equine gas gangrene, including \u003cem\u003eC. perfringens, C. novyi, C. chauvoei, and C. sordellii\u003c/em\u003e, infections attributed specifically to C. septicum remain comparatively less frequently documented in horses (Uzal et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Junior et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Peek et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Consequently, the present series contributes valuable documentation of the involvement of this species in fatal clostridial myonecrosis affecting horses raised under tropical production conditions. The consistent identification of \u003cem\u003eC. septicum\u003c/em\u003e in all analyzed cases suggests that this species may play a more relevant role in equine clostridial myonecrosis in tropical environments than currently recognized, although broader epidemiological studies would be required to confirm regional pathogen distribution.\u003c/p\u003e \u003cp\u003eThe clinical evolution observed in the affected animals closely corresponds to classical descriptions of clostridial gas gangrene. The abrupt onset of severe lameness, rapidly progressive swelling, and palpable subcutaneous crepitation reflects the extensive tissue destruction and gas production generated during anaerobic bacterial proliferation in damaged muscle tissue (Uzal et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The uniformly fatal outcome documented in this series further highlights the fulminant nature of this condition. Previous clinical studies have reported mortality rates ranging from approximately 50% to nearly 100% in equine clostridial myonecrosis, depending on the time of diagnosis and the possibility of aggressive surgical intervention (Peek et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Junior et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In the present cases, despite the administration of antimicrobial and anti-inflammatory therapy, disease progression was extremely rapid, emphasizing the narrow therapeutic window that characterizes this infection.\u003c/p\u003e \u003cp\u003eThe gross and microscopic lesions observed in the affected horses were consistent with the well-recognized pathological features of clostridial myonecrosis. Extensive hemorrhagic necrosis of skeletal muscle, diffuse edema, intramuscular gas accumulation, and abundant intralesional Gram-positive bacilli are considered hallmarks of this disease (Amorim et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Uzal et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Histopathological uniformity across all animals examined strengthens the diagnostic consistency of the case definition and provides biological support for the molecular results obtained in the present study. The frequent observation of vascular thrombosis and widespread muscle fiber degeneration further reflects the combined effects of bacterial toxins, tissue ischemia, and host inflammatory responses, which together contribute to the rapid clinical deterioration typical of clostridial infections.\u003c/p\u003e \u003cp\u003eAn important epidemiological observation in this series was the frequent association of cases with recent tissue trauma or intramuscular injections, conditions widely recognized as facilitating the introduction of clostridial spores into muscle tissue. Several classical reports have documented similar iatrogenic or traumatic entry points in equine gas gangrene outbreaks, particularly following intramuscular injections or minor surgical procedures (Peek et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). In the present cases, additional management practices potentially contributing to infection risk were identified, including reuse of needles and inadequate sterilization of instruments. Although causal relationships cannot be definitively established in an observational case series, these findings highlight important opportunities for preventive intervention through improved aseptic practices and veterinary supervision.\u003c/p\u003e \u003cp\u003eThe exclusive molecular detection of \u003cem\u003eC. septicum\u003c/em\u003e in all animals is noteworthy. This species has been recognized as an important cause of malignant edema and spontaneous myonecrosis in several animal species (Raymundo et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Junior et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Uzal et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In horses, however, reports of \u003cem\u003eC. septicum\u003c/em\u003e\u0026ndash;associated gas gangrene remains limited compared with those involving \u003cem\u003eC. perfringens\u003c/em\u003e type \u003cem\u003eA\u003c/em\u003e or other histotoxic clostridia (Kennedy et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Peek et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Sacco et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). While the consistent identification of \u003cem\u003eC. septicum\u003c/em\u003e in this series suggests that this pathogen may play a relevant role in equine clostridial infections within the studied region, caution is required in interpreting this observation. The retrospective nature of the study, the limited number of cases, and the targeted PCR approach preclude definitive conclusions regarding pathogen predominance in the Amazon biome. Nevertheless, the geographic distribution of cases across several municipalities supports the epidemiological relevance of this agent in the investigated context.\u003c/p\u003e \u003cp\u003eEnvironmental factors characteristic of tropical ecosystems may also influence the epidemiology of clostridial infections. High temperatures, elevated humidity, and the persistence of bacterial spores in soil have long been recognized as conditions favoring the environmental survival of clostridia (Junior et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The Amazon biome is characterized by climatic and ecological conditions that may facilitate the maintenance of these organisms in the environment, although the present study did not include environmental sampling capable of confirming this hypothesis. Future investigations incorporating soil analysis and molecular epidemiology would be valuable to clarify the environmental reservoirs and transmission pathways of histotoxic clostridia in tropical equine systems.\u003c/p\u003e \u003cp\u003eThe absence of vaccination against clostridial diseases in all affected animals deserves consideration, although its interpretation must remain cautious. Vaccination strategies targeting several clostridial toxins have been developed for livestock species and experimental vaccine approaches have been explored in horses (Zaragoza et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Freitas et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, protective protocols specifically designed to prevent equine gas gangrene remain limited, and the effectiveness of available vaccines against multiple histotoxic clostridia in horses is still incompletely defined (Uzal et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Consequently, improvements in management practices, particularly strict aseptic techniques during injections and prompt treatment of traumatic wounds remain the most practical preventive strategies currently available.\u003c/p\u003e \u003cp\u003eFrom a diagnostic perspective, the integration of clinical evaluation, pathological examination, and molecular detection proved particularly valuable in confirming the etiological diagnosis. Culture-based identification of clostridia may be compromised in field conditions due to post-mortem autolysis or prior antimicrobial therapy, which frequently occur in severe cases of myonecrosis (Ribeiro et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Uzal et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In this context, multiplex PCR represents a useful complementary diagnostic tool capable of detecting specific clostridial species directly in affected tissues.\u003c/p\u003e \u003cp\u003eSome limitations inherent to this study should be acknowledged. The retrospective design restricts the availability of complete exposure data, and the relatively small number of cases prevents robust epidemiological inference. In addition, toxin characterization and bacterial isolation were not consistently achieved in all animals. Nevertheless, the strong concordance between clinical signs, pathological findings, and molecular detection provides reliable evidence supporting the diagnosis of clostridial gas gangrene associated with \u003cem\u003eC. septicum\u003c/em\u003e. From a practical perspective, these observations emphasize that even minor breaches in tissue integrity may create conditions for clostridial proliferation in equine muscle, particularly in environments where spores are likely to persist in soil and organic matter. However, to the best of our knowledge, this is one of the largest documented case series of equine gas gangrene associated with Clostridium septicum reported in South America.\u003c/p\u003e \u003cp\u003eIn summary, this case series documents 20 fatal cases of equine gas gangrene associated with \u003cem\u003eC. septicum\u003c/em\u003e in the Brazilian Amazon and provides clinicopathological and molecular evidence supporting the involvement of this pathogen in equine clostridial myonecrosis under tropical conditions. The findings reinforce the fulminant nature of the disease, highlight the value of molecular diagnostics for etiological confirmation, and underscore the importance of strict aseptic practices and early clinical recognition in equine management systems.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors are grateful to CNPq (Conselho Nacional de Desenvolvimento Cient\u0026iacute;fico e Tecnol\u0026oacute;gico), Anaerobic Laboratory of the Federal University of Minas Gerais; Biological Institute of S\u0026atilde;o Paulo and UFPA animal pathology laboratory.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior (CAPES), Brazil (Finance Code 001).\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eEthics approval and Informed consent statement \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll procedures involving animals followed internationally accepted principles of animal welfare. The cases described in this study involved naturally occurring disease and diagnostic investigations requested by animal owners and attending veterinarians; therefore, no experimental procedures were performed. \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLoise Ara\u0026uacute;jo de Sousa, Laura Rosa Corr\u0026ecirc;a and Felipe Masiero Salvarani contributed to the study conception and design. Material preparation, data collection and analysis were performed by Loise Ara\u0026uacute;jo de Sousa, Laura Rosa Corr\u0026ecirc;a, Hanna Gabriela da Silva Oliveira and Felipe Masiero Salvarani. The first draft of the manuscript was written by Loise Ara\u0026uacute;jo de Sousa, Laura Rosa Corr\u0026ecirc;a and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eData Availability Statement \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData are contained within the article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAmorim RM, Rino AS, Dal-Pai-Silva M, Borges AS, Oliveira Filho JP, Freitas NPP, Maia L, Rezende LAL (2011) Morphological aspects of muscle biopsies in horses affected by myopathies. 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Toxins 11:525. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/toxins11090525\u003c/span\u003e\u003cspan address=\"10.3390/toxins11090525\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"veterinary-research-communications","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"verc","sideBox":"Learn more about [Veterinary Research Communications](https://www.springer.com/journal/11259)","snPcode":"11259","submissionUrl":"https://submission.nature.com/new-submission/11259/3","title":"Veterinary Research Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"equine gas gangrene, clostridial myonecrosis, multiplex PCR, Amazon biome, Clostridium septicum","lastPublishedDoi":"10.21203/rs.3.rs-9031945/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9031945/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eClostridial gas gangrene represents an acute necrotizing infection of skeletal muscle characterized by rapid tissue destruction and severe systemic toxemia in horses, however, systematically documented case series from tropical settings remain scarce. Here, we describe a clinicopathological and molecularly confirmed series of 20 fatal equine gas gangrene cases diagnosed in the eastern Brazilian Amazon (Par\u0026aacute; State) between 2018 and 2026. Horses presented with abrupt onset of severe lameness, rapidly progressive swelling of a limb or muscle group, and palpable subcutaneous crepitus, frequently following penetrating trauma, intramuscular injections, or other breaches of tissue integrity. Despite antimicrobial and anti-inflammatory treatment, all animals died or were euthanized due to rapid deterioration and poor prognosis. Necropsy consistently revealed extensive hemorrhagic myonecrosis with serosanguineous exudation and intramuscular/subcutaneous gas accumulation. Histology showed widespread coagulative to floccular myonecrosis, marked interstitial edema and hemorrhage, frequent vascular thrombosis, and abundant intralesional Gram-positive bacilli. A multiplex PCR assay targeting major histotoxic clostridia detected \u003cem\u003eClostridium septicum\u003c/em\u003e DNA in affected skeletal muscle from all cases, with no amplification of other targets. This case series provides clinically relevant documentation of fatal \u003cem\u003eC. septicum\u003c/em\u003e\u0026ndash;associated myonecrosis in horses raised under tropical conditions and reinforces the importance of early recognition, strict aseptic practices during intramuscular procedures, and integration of pathology with molecular diagnostics for definitive etiological confirmation.\u003c/p\u003e","manuscriptTitle":"Fatal equine gas gangrene associated with Clostridium septicum in the Brazilian Amazon: clinicopathological and molecular characterization of a 20-case series","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-18 08:56:21","doi":"10.21203/rs.3.rs-9031945/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-04-02T22:38:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"255711167601668655255803215854745656546","date":"2026-03-16T03:23:47+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-15T20:03:59+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-10T22:31:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-10T22:30:17+00:00","index":"","fulltext":""},{"type":"submitted","content":"Veterinary Research Communications","date":"2026-03-04T15:14:32+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"veterinary-research-communications","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"verc","sideBox":"Learn more about [Veterinary Research Communications](https://www.springer.com/journal/11259)","snPcode":"11259","submissionUrl":"https://submission.nature.com/new-submission/11259/3","title":"Veterinary Research Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"1be342ae-51cc-4d54-b33c-98734bc9640a","owner":[],"postedDate":"March 18th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-03-18T08:56:22+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-18 08:56:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9031945","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9031945","identity":"rs-9031945","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}