Gas gangrene in cattle caused by Clostridium novyi - case reportABSTRACT
Gas gangrene is characterized by a severe necrotic condition of subcutaneous tissue and muscle, mainly affecting ruminants and horses, and is caused by species of the genus Clostridium. It can be fatal in animals; pigs, dogs, cats and other species may also be affected by this condition. These agents are responsible for myonecrosis, and they belong to the group of histotoxic clostridial diseases that trigger toxemia and worsen the lesions. While presumptive diagnosis of gas gangrene relies on clinical history and pathological findings, definitive confirmation necessitates precise pathogen identification, particularly when fastidious anaerobes are suspected. Here we describe a classic gas gangrene episode in a 30-month-old-Nelore cattle caused by Clostridium novyi.
Keywords:
Myonecrosis; PCR
RESUMO
Gangrena gasosa é caracterizada por uma condição necrótica severa do tecido subcutâneo e do músculo que afeta principalmente ruminantes e cavalos, causada por espécies do gênero Clostridium, podendo ser fatal em animais; suínos, cães e gatos são outras espécies que podem ser afetadas por essa condição. Esses agentes são responsáveis pela mionecrose e pertencem ao grupo das doenças histotóxicas causadas pelos clostrídios, que desencadeiam a toxemia e pioram as lesões. O diagnóstico presuntivo da gangrena gasosa pode ser estabelecido com base no histórico, em sinais clínicos e em alterações micro e macroscópicas; entretanto, a identificação do organismo é necessária para o diagnóstico confirmatório. Este relato descreve um episódio típico de gangrena gasosa em um Nelore, com 30 meses de idade, causado por Clostridium novyi.
Palavras-chave:
Mionecrose; PCR
INTRODUCTION
The group of infections caused by anaerobic bacteria of the genus Clostridium is called clostridial diseases, which are true infections mediated mostly by toxins, and are highly lethal. When exposed to adverse conditions, these microorganisms transform into a resistant form called spores, remaining potentially infectious in the soil for long periods (Lobato et al., 1998).
Myonecrosis is one of the histotoxic conditions that clostridia can cause, characterized by necrotizing soft-tissue infections caused by one or more pathogenic clostridia. Myonecrosis can affect several animal species and can be divided into two diseases: blackleg and gas gangrene (previously referred to as malignant edema), depending on the etiological agent and pathogenesis. Blackleg is caused exclusively by C. chauvoei, generally affecting cattle aged 6 to 24 months in excellent body condition, and is of endogenous origin. The susceptible animal ingests the spores, which gain access to the bloodstream via the gastrointestinal tract and enter the muscle. Germination is activated when blunt trauma causes low oxygenation (Miyashiro et al., 2007; Nassar et al., 2012; Underwood et al., 2015).
Gas gangrene is an opportunist infection, affecting animals of any age and may have both an exogenous or traumatic origin (Pires et al., 2017). Exogenous infection is characterized when spores enter the muscles via a wound caused, for example, in headbutting, vaccination, castration, dehorning, dystocia, and others (Pires et al., 2017; Underwood et al., 2015). Gas gangrene can be caused by a single Clostridium species or by a combination of several, with the main species involved being Clostridium septicum, C. chauvoei, C. novyi type A, C. perfringens, and Paraclostridium sordellii (Lobato et al., 1998; Underwood et al., 2015). The organisms are ubiquitous in the environment, and they can also be found in the intestinal contents of animals, surviving in the soil for years. It is a fatal disease in which the affected area appears hot and has accumulated gas. Palpation around the infected area reveals crepitus of the subcutaneous tissue, and the skin surrounding this area may have a bluish coloration characteristic of gangrene. Regional lymphadenopathy and fever may occur. Death usually occurs within 12 hours to 2 days due to toxemia triggered by these agents. At autopsy, the necrotic tissue and accumulation of hemorrhagic fluids, crepitant and scattered lesions suggest gas gangrene. Serosal and subendocardial hemorrhages, as well as congestion and edema of regional lymph nodes, kidneys, lungs, and liver, may be observed due to severe toxemia (Oliveira Junior et al., 2020; Underwood et al., 2015).
The C. novyi species is classified into four genotypes (A-D) based on the production of two lethal toxins, alpha (TcnA) and beta, as well as other non-lethal toxins. C. novyi type A produces only alpha toxin (TcnA), causing gas gangrene, while C. novyi type B also produces beta toxin, causing bighead and black disease. C. novyi type C does not encode either of these toxins and is non-toxinogenic. C. novyi type D, which produces only beta toxin, is commonly known as Clostridium haemolyticum and causes bacillary hemoglobinuria. (Navarro and Uzal, 2020), (Table 1).
Clostridium novyi typing according to lethal toxins (alfa and beta) (Navarro and Uzal, 2020)
Bighead disease is a disease observed in sheep characterized by non-gassy, non-hemorrhagic edema of the head and neck. The edema can migrate to ventral regions such as the throat, and most affected animals die within 48 to 72 hours. Black disease, or infectious necrotic hepatitis, is a fatal, peracute disease most common in cattle and sheep, but can also be seen in goats. The clinical course usually lasts 1-2 days in cattle and is somewhat shorter in sheep, and it affects apparently healthy animals. Occasionally, hyperthermia, tachypnea, inability to keep up with other animals, and recumbency shortly before death may be observed (Underwood et al., 2015).
A presumptive diagnosis of gas gangrene can be inferred based on the history, including clinical signs, sudden death, micro- and macroscopic changes. However, laboratory tests are essential for a confirmatory diagnosis by identifying the clostridial species involved. Muscle, tissue, and/or subcutaneous fluid samples from recently deceased animals should be submitted to the laboratory in a sterile, refrigerated or frozen container for anaerobic culture and/or PCR (polymerase chain reaction) (Oliveira Junior et al., 2020).
This present study describes a gas gangrene episode in cattle and laboratory tests for confirmatory diagnosis.
ETHICAL ASPECTS
This research was not submitted to the Ethics Committee on Animal Use.
MATERIAL AND METHODS
In July 2024, there was the sudden death of a 30-month-old-Nelore bovine belonging to a reserve batch of 10 animals from a farm in the Itapira region of São Paulo, whose carcass presented generalized edema and subcutaneous crepitus, hemorrhagic organs, nasal bleeding and necrotic muscles (Figures 1 and 2).
At necropsy, muscle necrosis and presence of gas in the subcutaneous tissue suggested gas gangrene episode
The extensive livestock farm breeds and rears Nelore cattle, with approximately 520 animals, and uses a nutritional management system based on pasture and proteinated mineral salt. The 10 animals in the reserve batch had been vaccinated against clostridial infections with the Polistar (Vallée) vaccine and dewormed 14 days before death.
Necropsy was performed a few hours post-mortem and the carcass showed blood in the nostrils, generalized edema, and rigor mortis. Macroscopic observations during necropsy revealed congestion of all tissues and necrosis, with friable organs, especially the kidneys. Tissues were collected for laboratory testing for histotoxic clostridial agents at the DPDSA (Animal Health Research and Development Division) laboratory of the Instituto Biológico in São Paulo, Brazil.
The tissues received in the laboratory were macerated with sterile 0.85% saline solution, pre-enriched in Tarozzi medium for 24 hours, and then inoculated onto Reinforced Clostridial Medium (Difco®) agar supplemented with 5% sheep blood. Inoculated plates were incubated anaerobically for up to 72 hours.
Muscle samples and pooled liver and kidney samples were processed for DNA extraction using the Reliaprep DNA tissue kit (Promega®). Subsequently, the extracted genetic material was subjected to PCR with the primers described by Baums et al. (2004) for the detection and typing of Clostridium perfringens, Kikuchi et al. (2002) for the detection of Paraclostridium sordellii, and Sasaki et al. (2002) for research on C. chauvoei, C. septicum, C. haemolyticum, C. novyi type A and C. novyi type B.
Specific amplicons of the flagellin gene (fliC) from Clostridium novyi amplified as type A and B, were purified and subjected to Sanger sequencing using specific primers and subsequent analysis on a capillary sequencing platform.
RESULTS
The samples were treated as a pool of organs (liver and kidney), and muscle was treated separately; by PCR, they were positive for both C. novyi type A and C. novyi type B. We were unable to isolate microorganisms using the described microbiological cultivation methodology.
Sanger sequencing of flagellin gene amplicons of Clostridium novyi types B and A revealed identities of 96.83% and 91.79% with Clostridium novyi sequences deposited at NCBI (Accession OQ628259.1 and AB058936.1, respectively). Considering the high variability of this gene among strains, both values are compatible with the detection of C. novyi. However, they do not allow conclusive identification of the subtype or strain. Alignment analyses also demonstrated the conservation of typical flagellin structural motifs, supporting the phylogenetic association with the species.
DISCUSSION
The occurrence of gas gangrene depends on contact between the clostridial species involved and animals, typically following wound contamination after vaccination, calving, branding, venipuncture, and other traumatic interventions. The disease occurs sporadically, but outbreaks have been reported following the injection of contaminated products or the use of contaminated needles (Ferreira Junior et al., 2020; Pinto et al., 2005).
In cases of histotoxic myonecrosis caused by C. perfringens, alpha toxin is the main virulence factor, destroying the phospholipid membranes of erythrocytes, endothelial cells, leukocytes, and muscle fibers, thereby increasing capillary vascular permeability. As the infection expands, inflammatory edema with serosanguineous exudate and gas production occurs (Ferreira Junior et al., 2020; Uzal et al., 2014). Gas gangrene in horses is rare, with few reports in the literature. However, (Ferreira Junior et al., 2020) reported a case of gas gangrene in a horse caused by Clostridium perfringens type A, where the trigger was likely a contaminated needle used during the treatment of colic symptoms. Similarly, Pinto et al. (2005) described a case of malignant edema caused by Clostridium septicum in a female pig from Rio Grande do Sul that died 4 days after birth. At necropsy, the body was in a regular condition, and there was marked subcutaneous edema in the umbilicus and especially in the hind limbs. Upon incision, a large amount of serosanguineous fluid, muscle necrosis, and gas that dissociated the muscle fibers were observed.
C. novyi type A is commonly found in soil and rarely in the microbiome of healthy animals. The microorganism produces alpha-toxin, a large, glycosylating toxin with necrotizing and lethal activity, which is considered the main virulence factor of this agent. C. novyi type A also produces a cytolysin that damages cell membranes (Oliveira Junior et al., 2020). C. novyi type B is also part of the typical soil microbiome and colonizes the intestines of clinically healthy individuals, and spores from dead animals contaminate pastures. In some areas, bacillary hemoglobinuria varies seasonally, associated with fasciolosis, but this condition can also occur in areas free of the parasite Fasciola hepatica, and may be associated with abscesses, hepatocellular fatty changes, toxic plants, liver biopsy and other parasites that create a hypoxic environment favorable to spore germination (Navarro and Uzal, 2020).
In the present case, despite being a rare occurrence, infection likely resulted from wound contamination during vaccination 14 days prior to death, with spores or vegetative forms of histotoxic clostridia, which represent the starting point for most cases of gas gangrene. At least there was no other suspicious traumatic or exogenous episode that could have triggered this gas gangrene occurrence. Subsequently, the low redox potential, decomposing protein metabolism, and acidic pH promoted spore germination and stimulated the proliferation of vegetative forms of clostridia, triggering the production of toxins responsible for the lesions and clinical signs characteristic of gas gangrene, such as edema, hemorrhage, ischemia, and local necrosis. Symptoms as nosebleeds and friable kidneys may indicate systemic toxemia and increased vascular permeability caused by the alpha toxin of Clostridium novyi. (Oliveira Junior et al., 2020).
The diagnosis of gas gangrene is usually based solely on clinical data and/or autopsy findings. However, for effective diagnosis and preventive measures, it is essential to identify the agent(s) involved. The failure to isolate C. novyi via conventional culture underscores the diagnostic challenges posed by its strict anaerobic requirements, rendering molecular tools such as PCR-based detection indispensable for routine surveillance (Pinto et al., 2005; Miyashiro et al., 2007; Nassar et al., 2012; Nyaoke et al., 2018). Muscle, subcutaneous tissue, and/or fluid from grossly affected areas of recently dead animals should be submitted to the laboratory. These samples should be collected in sterile containers and submitted either fresh, refrigerated or frozen for anaerobic culture and/or PCR. Also, air-dried smears of grossly affected tissues should be submitted for an FA test (Oliveira Junior et al., 2020). Thus, samples for microbial analysis must be collected as soon as possible after death, because several clostridial species commonly found in the intestine can invade tissues shortly before or after death, potentially yielding false-positive results. Gas gangrene is usually an acute disease with no time for treatment. In animals that are closely monitored (e.g., horses), treatment can be attempted if the diagnosis is established early. Gregory et al. (2006) reported the treatment and recovery of a blackleg-affected bovine. Treatment consisted of high-dose penicillin and deep cleaning of necrotizing areas.
Clostridial species are widespread, and their spores are highly resistant to environmental conditions; therefore, eradication of the disease is practically impossible (Oliveira Junior et al., 2020). Proper surgical site preparation, instrument sterilization, asepsis of the vaccination site, and post-operative wound care help prevent the disease. Furthermore, multivalent vaccines against clostridial infections should also be routinely administered. The humoral response to toxoids and possibly some somatic antigens result in robust immunity. Vaccines can be administered at any age, but it is generally recommended that the primary vaccination be given at 6 months, followed by a booster 3-4 weeks later. However, the duration of protection is short, so two annual boosters are necessary for high-exposure areas (Underwood et al., 2015; Navarro and Uzal, 2020). Furthermore, all carcasses infected with these clostridial infections should be burned, buried, or removed from the facilities to prevent the spread of C. novyi spores (Navarro; Uzal, 2020).
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