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Myonecrosis caused by Clostridium septicum in a horse from Southern Brazil

Mionecrose causada por Clostridium septicum em um cavalo do sul do Brasil

ABSTRACT:

Clostridial myonecrosis is a highly fatal infectious disease of the muscle that is caused by pathogenic clostridia. When it occurs in equine muscle, the infection can cause a variety of clinical signs. The present report described a case of acute myonecrosis caused by Clostridium septicum in a horse, which had a favorable clinical evolution. A 6-year-old mare was admitted to a veterinary hospital due to increased volume and lameness in the left pelvic limb after an intramuscular injection of an anti-inflammatory drug. The diagnosis of myonecrosis caused by C. septicum was based on clinical and laboratory findings. The animal showed significant improvement after 5 months of treatment and was discharged from the hospital. There is very limited information regarding myonecrosis caused by C. septicum in horses in Brazil; our study showed that early diagnosis and prompt appropriate treatment can ensure significant improvement and recovery of affected animals.

Key words:
horse; myonecrosis; Clostridium septicum; infectious disease; intramuscular injection

RESUMO:

Mionecroses clostridiais são doenças altamente fatais, causadas por uma ou mais espécies de Clostridium sp. patogênicos. Entretanto, no músculo equino, a infecção pode produzir uma variedade de lesões e sinais clínicos. O presente relato descreve um caso de mionecrose aguda em um cavalo causada por Clostridium septicum, que apresentou uma evolução clínica favorável. Uma égua de seis anos foi internada em um hospital veterinário devido a um aumento de volume e claudicação no membro pélvico esquerdo, observado após injeção intramuscular de anti-inflamatório. O diagnóstico de mionecrose por C. septicum foi baseado nos achados clínicos e laboratoriais. Houve uma melhora significativa do animal após cinco meses de tratamento, permitindo que o mesmo fosse liberado do hospital veterinário. Considerando que as informações sobre C. septicum causadores de mionecroses em cavalos no Brasil são muito limitadas, este relato demonstra que o diagnóstico precoce e a implementação adequada do tratamento podem garantir uma melhora significativa e a recuperação dos animais afetados.

Palavras-chave:
cavalo; mionecrose; Clostridium septicum; doença infecciosa; injeções intramusculares

Clostridial myonecrosis, or gas gangrene, is a necrotizing soft tissue infection caused by various pathogenic Clostridium species, including Clostridium septicum, C. chauvoei, C. novyi type A, C. perfringens type A, and C. sordellii (PEEK et al., 2003PEEK, S. F. et al. Clostridial myonecrosis in horses (37 cases 1985-2000). Equine Veterinary Journal, v.35, p.86-92, 2003. Available from: <Available from: https://beva.onlinelibrary.wiley.com/doi/abs/10.2746/042516403775467513 >. Accessed: Sep. 8, 2021. doi: 10.2746/042516403775467513.
https://beva.onlinelibrary.wiley.com/doi...
; SCOTT & MILLER, 2003SCOTT, D. W.; MILLER, W. H. Bacterial Skin Diseases. In: SCOTT, D.W.; MILLER, W.H. Equine Dermatology. W. B. Saunders, 2003. p. 206-260.). These species belong to a group of histotoxic clostridia, and the infection may lead to toxemia with the worsening of the lesions (ASSIS et al, 2010ASSIS, R. A. et al. Mionecroses clostridiais bovinas. Arquivos do Instituto Biológico, v.77, p.331-334, 2010. Available from: <Available from: https://www.scielo.br/j/aib/a/L9cPX53S6GsKdLNbdCYnqgp/?lang=pt >. Accessed: Oct. 8, 2021. doi: 10.1590/1808-1657v77p3312010.
https://www.scielo.br/j/aib/a/L9cPX53S6G...
).

Clostridial myonecrosis is most common in cattle, sheep, and goats, which are more susceptible than pigs and horses (RAYMUNDO et al., 2010RAYMUNDO, D. L. et al. Mionecrose aguda por Clostridium septicum em equinos. Pesquisa Veterinária Brasileira, v.30, p.637-640, 2010. Available from: <Available from: https://www.scielo.br/j/pvb/a/TQ9TSGJTxS7tkyLBcxtqPXw/?format=pdf⟨=pt >. Accessed: Sep. 9, 2021. doi: 10.1590/1808-1657v77p3312010.
https://www.scielo.br/j/pvb/a/TQ9TSGJTxS...
). Few studies have reported cases of myonecrosis in horses in Brazil, but the animals died of the clostridial infection in all of these cases (MACÊDO et al., 2007MACÊDO, J. T. S. A., et al. Malignant edema caused by Clostridium chauvoei in a horse. Acta Scientiae Veterinariae, v.41, p.1-4, 2007. Available from: <Available from: https://seer.ufrgs.br/ActaScientiaeVeterinariae/article/view/101969 >. Accessed: Sep. 18, 2021. doi: 10.22456/1679-9216.101969.
https://seer.ufrgs.br/ActaScientiaeVeter...
; RAYMUNDO et al., 2010; FARIAS et al., 2014FARIAS, L. D. et al. Acute myonecrosis in horse caused by Clostridium novyi type A. Brazilian Journal of Microbiology, v.45, p.221-224, 2014. Available from: <Available from: https://www.scielo.br/j/bjm/a/z6gkjLFMqv9ZkRLMfJP5nDx/?lang=en >. Accessed: Oct. 6, 2021. doi: 10.1590/S1517-83822014005000023.
https://www.scielo.br/j/bjm/a/z6gkjLFMqv...
; FERREIRA JÚNIOR et al., 2020FERREIRA JÚNIOR, J. A. Malignant edema in horse by Clostridium perfringens type A. Acta Scientiae Veterinariae, v.48, n.1, p.1-4, 2020. Available from: <Available from: https://seer.ufrgs.br/ActaScientiaeVeterinariae/article/view/101969 >. Accessed: Oct. 14, 2021. doi: 10.22456/1679-9216.101969.
https://seer.ufrgs.br/ActaScientiaeVeter...
).

Clostridium spp. are Gram-positive, anaerobic, spore-forming rods that exist as saprophytes in the environment. They produce a wide variety of toxins and cause numerous diseases (SCOOT & MILLER, 2003). C. septicum, C. chauvoei, and C. perfringens produce toxins that are important in the pathogenesis of myonecrosis, including α-toxin, which is a lecithinase that is necrotizing, leukocidal, and hemolytic (QUINN et al., 2005QUINN. P. J. et al. Microbiologia Veterinária e Doenças Infecciosas. São Paulo: Ed. Artmed, 2005.; KENNEDY et al., 2009KENNEDY, C. L. et al. Cross-complementation of Clostridium perfringens PLC and Clostridium septicum a-toxin mutants reveals PLC is sufficient to mediate gas gangrene. Microbes and Infection, v.11, p.413-418, 2009. Available from: <Available from: https://www.sciencedirect.com/science/article/pii/S1286457909000082?via%3Dihub >. Accessed: Sep. 26, 2021. doi: 10.1016/j.micinf.2009.01.002.
https://www.sciencedirect.com/science/ar...
); β-toxin, which is a deoxyribonuclease that is leukotoxic; and γ-toxin, which is a hyaluronidase that promotes spread of the infection (QUINN et al., 2005).

Clostridial myonecrosis in horses has been reported to occur after infection of wounds in mucous membranes, orchiectomy, and intramuscular injections (MACÊDO et al., 2007MACÊDO, J. T. S. A., et al. Malignant edema caused by Clostridium chauvoei in a horse. Acta Scientiae Veterinariae, v.41, p.1-4, 2007. Available from: <Available from: https://seer.ufrgs.br/ActaScientiaeVeterinariae/article/view/101969 >. Accessed: Sep. 18, 2021. doi: 10.22456/1679-9216.101969.
https://seer.ufrgs.br/ActaScientiaeVeter...
; ANDERSON et al., 2013ANDERSON, F. L. et al. Clostridial myonecrosis, haemolytic anaemia, hepatopathy, osteitis and transient hypertrophic cardiomyopathy after intramuscular injection in a Thoroughbred gelding. Australian Veterinary Journal, v.91, p.204-208, 2013. Available from: <Available from: https://pubmed.ncbi.nlm.nih.gov/23614516/ >. Accessed: Oct. 8, 2021. doi: 1111/avj.12021.
https://pubmed.ncbi.nlm.nih.gov/23614516...
; FARIAS et al., 2014FARIAS, L. D. et al. Acute myonecrosis in horse caused by Clostridium novyi type A. Brazilian Journal of Microbiology, v.45, p.221-224, 2014. Available from: <Available from: https://www.scielo.br/j/bjm/a/z6gkjLFMqv9ZkRLMfJP5nDx/?lang=en >. Accessed: Oct. 6, 2021. doi: 10.1590/S1517-83822014005000023.
https://www.scielo.br/j/bjm/a/z6gkjLFMqv...
). Most cases have been associated with recent intramuscular injection of commonly used therapeutic agents, and horses seem to be more vulnerable to clostridial myonecrosis (also known as gas gangrene) following this type of injection (PEEK & SEMRAD, 2002PEEK, S. F.; SEMRAD, S. D. Clostridial myonecrosis in horses. Equine Veterinary Education, v.14, p.207-215, 2002. Available from: <Available from: https://beva.onlinelibrary.wiley.com/doi/10.1111/j.2042-3292.2002.tb00162.x >. Accessed: Sep. 8, 2021. doi: 10.1111/j.2042-3292.2002.tb00162.x.
https://beva.onlinelibrary.wiley.com/doi...
; PEEK et al., 2003PEEK, S. F. et al. Clostridial myonecrosis in horses (37 cases 1985-2000). Equine Veterinary Journal, v.35, p.86-92, 2003. Available from: <Available from: https://beva.onlinelibrary.wiley.com/doi/abs/10.2746/042516403775467513 >. Accessed: Sep. 8, 2021. doi: 10.2746/042516403775467513.
https://beva.onlinelibrary.wiley.com/doi...
).

Currently, there is limited information regarding myonecrosis-causing C. septicum in horses in Brazil. In addition, the reported survival rate of clostridial myositis is low. Thus, the objective of this study was to report a case of acute myonecrosis in a horse caused by C. septicum triggered by an intramuscular injection of flunixin meglumine in the left hindlimb, with a favorable clinical course. Our findings demonstrated the importance of early diagnosis and appropriate treatment to ensure the recovery of affected animals.

A 6-year-old mare was admitted to a veterinary hospital because of increased volume in the gluteal muscle region and lameness in the left hindlimb after receiving an intramuscular injection of flunixin meglumine 5 days earlier. The mare showed grade V lameness according to the AAEP scale I-V (AAEP, 1991AAEP. Guide for veterinary service and judging of equestrian events. American Association of Equine Practitioners, Lexington, 1991.). Clinical evaluation showed diffuse edema in the limb, with areas of crackling on palpation and percussion. A radiographic study of the hindlimb revealed the accumulation of a large amount of gas in the subcutaneous tissue (Figure 1A) within the patella region. In addition, ultrasonography showed an accumulation of gas in the regions around the main muscles of the pelvic limb.

Figure 1
A) A Cranioproximal-craniodistal oblique radiography of the left hindlimb performed at arrival to the clinic (day 0). Accumulation of gas in the subcutaneous tissue around the patella region; (B) Vertical incisions into the injured muscle to permit drainage and oxygenation of the necrotic tissues (day 0); (C) Wound evolution (day 30); (D) Contraction and epithelialization of the wound (5 months post intervention/hospitalization).

The hindlimb of the horse was subjected to trichotomy, and a large area of necrosis was observed. To promote aeration and facilitate wound cleaning, the treatment included vertical incisions into the injured muscle to drain the foul exudate and gas (Figure 1B). Blood samples were collected for complete blood count (CBC) and biochemical analysis, and fluid and muscle biopsies were collected for bacteriological examinations (SB 82/20 protocol).

CBC performed on the first day of hospitalization showed only few changes, mainly related to fibrinogen (900 mg/dL), which was above the reference value (200-400 mg/dL). Systemic therapy was initiated on the first day at the clinic, which included intravenous potassium penicillin (40,000 UI/kg QID), phenylbutazone (4.4 mg/kg BID), intravenous fluid therapy, and oral omeprazole (1 mg/kg SID). Penicillin treatment was continued for 23 days. During the first days of hospitalization, wound cleaning and debridement were performed to remove necrotic tissue. Hydrogen peroxide was used to facilitate cleaning, and sugar was added to stimulate granulation tissue (Figure 1C). A full distal limb compressive bandage was placed to avoid distal migration of the edema. After completely removing the necrotic tissue, a 2% chlorhexidine degerming agent and silver sulfadiazine ointment were applied to the wound.

In the laboratory, fluid samples and muscle fragments were plated onto 5% sheep blood agar, MacConkey agar, and semisolid-reinforced clostridial medium (Thermo Scientific™ Oxoid™) and incubated anaerobically at 37 °C. After 48 h, small gray white colonies were observed on blood agar, but no growth was observed on MacConkey agar. Microbiological growth was also verified in the semisolid-reinforced clostridial medium, which showed growth (turbidity) and gas formation. Gram staining of a smear from the culture on blood agar and reinforced medium showed pure large, Gram-positive rods. Antimicrobial testing by the disk diffusion method (CLSI, 2007Clinical and Laboratory Standards Institute (CLSI). Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria, seventh ed., Wayne, Pennsylvania: Clinical and Laboratory Standards Institute (CLSI), 2007.) showed sensitivity of the bacteria to azithromycin, ceftiofur, enrofloxacin, penicillin, and trimethoprim‐sulfamethoxazole and intermediate sensitivity to tetracycline.

The diagnosis was confirmed by polymerase chain reaction (PCR) and molecular analysis. DNA was extracted from the colonies (TAKEUCHI et al., 1997TAKEUCHI, S. et al. Detection of Clostridium septicum hemolysin gene by polymerase chain reaction. The Journal of Veterinary Medical Science, v.59, p.853-855, 1997. Available from: <Available from: https://www.jstage.jst.go.jp/article/jvms/59/9/59_9_853/_article >. Accessed: Sep. 21, 2021. doi: 10.1292/jvms.59.853.
https://www.jstage.jst.go.jp/article/jvm...
) and subjected to multiplex PCR for the identification of Clostridium species (SASAKI et al., 2002SASAKI, Y. et al. Phylogenetic analysis and PCR detection of Clostridium chauvoei, Clostridium haemolyticum, Clostridium novyi types A and B, and Clostridium septicum based on the flagellin gene. Veterinary Microbiology, v.86, p.257-267, 2002. Available from: <Available from: https://www.sciencedirect.com/science/article/pii/S0378113502000020?via%3Dihub >. Accessed: Sep. 19, 2021. doi: 10.1016/s0378-1135(02)00002-0.
https://www.sciencedirect.com/science/ar...
), based on the presence of the flagellin gene. A 294 bp amplicon was observed in isolate SB 82/20, corresponding to amplicon size of C. septicum flagellin gene. The amplicon was sequenced, and the consensus sequence obtained in the Staden package (STADEN, 1996STADEN, R. The Staden sequence analysis package. Molecular Biotechnology, v.5, p.233-241, 1996. Available from: <Available from: https://link.springer.com/article/10.1007%2FBF02900361 >. Accessed: Sep. 19, 2021. doi: 10.1007/BF02900361.
https://link.springer.com/article/10.100...
) was compared with other Clostridium spp. sequences in GenBank using the Basic Local Alignment Search Tool. The sequence was deposited in GenBank (SB 82/20, accession no. MZ508338). The nucleotide sequence of SB 82/20 showed high identity (94.9%-96.9%) with other C. septicum flagellin gene sequences in GenBank (GenBank accession numbers CP034358.1, AB058934.1, HQ650581.1, AB058935.1, CP023671.1, and AB058933.1) and less than 86% identity with those from other Clostridium spp.

In this report, the diagnosis of myonecrosis caused by C. septicum in a horse was based on clinical signs and biochemical and molecular findings. The applied treatment had significant effects, and the horse showed a considerable improvement in their clinical condition after 5 months of treatment (Figure 1D).

C. septicum is a highly virulent pathogen that is increasingly being recognized as a major contributor to serious clostridial infections (KENNEDY et al., 2005KENNEDY, C. L. et al. The a-toxin of Clostridium septicum is essential for virulence. Molecular Microbiology, v.57, p.1357-1366, 2005. Available from: <Available from: https://onlinelibrary.wiley.com/doi/10.1111/j.1365-2958.2005.04774.x >. Accessed: Sep. 22, 2021. doi: 10.1111/j.1365-2958.2005.04774.x.
https://onlinelibrary.wiley.com/doi/10.1...
). The bacteria can infect the subcutaneous tissues, fascia, or muscle layers, and the infection is characterized by fever, depression, swelling, subcutaneous gas accumulation, rapid tissue necrosis, and extreme pain (KENNEDY et al., 2009KENNEDY, C. L. et al. Cross-complementation of Clostridium perfringens PLC and Clostridium septicum a-toxin mutants reveals PLC is sufficient to mediate gas gangrene. Microbes and Infection, v.11, p.413-418, 2009. Available from: <Available from: https://www.sciencedirect.com/science/article/pii/S1286457909000082?via%3Dihub >. Accessed: Sep. 26, 2021. doi: 10.1016/j.micinf.2009.01.002.
https://www.sciencedirect.com/science/ar...
).

There are several hypotheses about how Clostridium spores are introduced into the skeletal muscles of horses. According to BREUHAUS et al. (1983BREUHAUS, B. A. et al. Clostridial muscle infections following intramuscular injections in the horse. Journal of Equine Veterinary Science, v.3, p.42-46, 1983. Available from: <Available from: https://www.sciencedirect.com/science/article/pii/S0737080683800033?via%3Dihub >. Accessed: Oct. 6, 2021. doi: 10.1016/S0737-0806(83)80003-3.
https://www.sciencedirect.com/science/ar...
), spores can be introduced through medication or by a needle. In our case, the infection was probably induced during an intramuscular injection of flunixin meglumine. Other drugs that have been associated with clostridial infection include ivermectin, antihistamines, phenylbutazone, dipyrone, vitamin B complex, aminopromazine, and synthetic prostaglandin (PEEK et al., 2003PEEK, S. F. et al. Clostridial myonecrosis in horses (37 cases 1985-2000). Equine Veterinary Journal, v.35, p.86-92, 2003. Available from: <Available from: https://beva.onlinelibrary.wiley.com/doi/abs/10.2746/042516403775467513 >. Accessed: Sep. 8, 2021. doi: 10.2746/042516403775467513.
https://beva.onlinelibrary.wiley.com/doi...
; ANDERSON et al., 2013ANDERSON, F. L. et al. Clostridial myonecrosis, haemolytic anaemia, hepatopathy, osteitis and transient hypertrophic cardiomyopathy after intramuscular injection in a Thoroughbred gelding. Australian Veterinary Journal, v.91, p.204-208, 2013. Available from: <Available from: https://pubmed.ncbi.nlm.nih.gov/23614516/ >. Accessed: Oct. 8, 2021. doi: 1111/avj.12021.
https://pubmed.ncbi.nlm.nih.gov/23614516...
). However, it has also been suggested that clostridia, can pass through lesions in the alimentary tract and colonize muscles, remaining inactive until stimulated by certain conditions. This corroborated the hypothesis that tissue damaged by injection of therapeutic agents can create a favorable environment for the activation of resident clostridial spores, resulting in the onset of myonecrosis (PEEK & SEMRAD, 2002; ANDERSON et al., 2013).

The horse was treated with antibiotics, cutaneous incisions, wound washing, and debridement. Earlier studies have suggested that bacteria associated with gas gangrene and myositis are typically susceptible to penicillin, provided adequate tissue levels are maintained (PERDRIZET et al., 1897PERDRIZET, J. A. et al. Successful management of malignant oedema caused by Clostridium septicum in a horse. The Cornell Veterinarian, v.77, p.328-338, 1897.). In the present case, penicillin therapy was initiated, and the animal showed significant improvement, emphasizing that the combination of antibiotic therapy with clinical management (skin incisions, fluid drainage, tissue debridement, and topical treatment) was essential to resolve the infection.

The prognosis of clostridial myonecrosis appears to be dependent on the Clostridium spp. involved (PEEK & SEMRAD, 2002PEEK, S. F.; SEMRAD, S. D. Clostridial myonecrosis in horses. Equine Veterinary Education, v.14, p.207-215, 2002. Available from: <Available from: https://beva.onlinelibrary.wiley.com/doi/10.1111/j.2042-3292.2002.tb00162.x >. Accessed: Sep. 8, 2021. doi: 10.1111/j.2042-3292.2002.tb00162.x.
https://beva.onlinelibrary.wiley.com/doi...
). According to PEEK et al. (2003), clostridial myonecrosis due to C. perfringens has the most favorable prognosis, whereas there is a 50% survival rate following infection with C. septicum (PEEK et al., 2003). However, when dual infections occur, the fatality rate is 100% (VALBERG & MCKINNON, 1984VALBERG, S. J.; MCKINNON, A. O. Clostridial cellulitis in the horse: A report of five cases. Canadian Veterinary Journal, v.25; p.67-71, 1984.). Thus, an accurate diagnosis and species identification are essential to improve the chances of recovery from the infection. Moreover, studies have shown that treatment is only effective when performed in the initial stages of the disease (PEEK et al., 2003). Our findings corroborated these results, as the mare received the treatment and showed significant improvement in its recovery from the infection.

In summary, the present study reported the isolation and molecular identification of C. septicum from a case of myonecrosis in a horse and discussed the clinical and therapeutic approaches that were essential for recovery. The trigger specific to the disease may have been an intramuscular injection. While there is a low incidence of C. septicum infection in horses, prophylaxis as well as preventive measures are important to avoid infection.

ACKNOWLEDGEMENTS

We thank Professor Agueda P. Castagna de Vargas for her generous assistance and indispensable support in all activities developed in Laboratório de Bacteriologia from Universidade Federal de Santa Maria. This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001.

REFERENCES

  • CR-2021-0736.R1

Edited by

Editor:

Rudi Weiblen (0000-0002-1737-9817)

Publication Dates

  • Publication in this collection
    27 Apr 2022
  • Date of issue
    2022

History

  • Received
    14 Oct 2021
  • Accepted
    02 Dec 2021
  • Reviewed
    07 Mar 2022
Universidade Federal de Santa Maria Universidade Federal de Santa Maria, Centro de Ciências Rurais , 97105-900 Santa Maria RS Brazil , Tel.: +55 55 3220-8698 , Fax: +55 55 3220-8695 - Santa Maria - RS - Brazil
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