Blackleg without skeletal muscle involvement as a cause of sudden death in unvaccinated calves

Abstract

Nine, 5-month-old unvaccinated black Angus calves died in less than 48 hours of each other. The calves expressed minimal or no clinical signs prior to death. On-farm post-mortem examinations were carried out on 3 of the deceased calves, and findings included: necrotizing and fibrinosuppurative myositis of the diaphragm muscle, and fibrinosuppurative pleuritis and epicarditis. Fluorescent antibody testing for pathogenic clostridia identified Clostridium chauvoei and a diagnosis of blackleg was made. The remainder of the herd were then vaccinated.

In August 2019, a beef farmer contacted Springer Animal Hospital in Sturgeon Falls, Ontario to report the sudden death of 4 black Angus calves overnight. The calves were approximately 5 mo old and belonged to a cow-calf operation with 35 cows in northeastern Ontario. The herd was managed on rented pasture and water was provided by a spring-fed pond. The calves were born on a home farm and transferred to pasture in early June. All the cows and calves in the herd were unvaccinated. A veterinarian visited the farm that afternoon and upon arrival discovered that 4 more calves had died. The farmer reported that he had not observed any of the calves exhibiting abnormal clinical signs prior to death. Upon initial examination, the deceased calves were in good condition (body condition score of 3/5) and noted by the farmer to be thriving. No ocular, nasal, or oral discharge was noted. Of the calves still alive, only 1 was displaying signs of weakness and lethargy. The entire pasture was walked and examined, and no potential toxin exposure was noted.

On-farm post-mortem examinations were carried out on 3 of the deceased calves (Calves A, B, and C), 2 having died the night before, while the third had died closer to the time the veterinarian arrived on-farm. No signs of outward trauma or swelling were present when the exterior of the calves was examined, and no crepitus was palpable. Pelvic and pectoral skeletal muscles of all 3 calves were grossly unremarkable; however, the diaphragmatic striated muscle of Calf B was diffusely necrotic. Calf A possessed loosely adherent fibrin lining the epicardium, and the cardiac muscle appeared congested. Pulmonary edema and congestion were present in Calf C, with a thin layer of non-adherent and discontinuous fibrin lining the pleural surface (Figure 1). The lungs were floating in excess blood-stained pleural fluid (Figure 1). Samples of heart, liver, lung, hind limb and forelimb skeletal muscle, diaphragm, thymus, and kidney from all 3 calves, as well as the brain from 1 calf, were collected and transported without fixation or fixed in formalin [60 mL 10% buffered formalin (F6000; ACP Chemicals, Montreal, Quebec)] to the Animal Health Laboratory (AHL) at the University of Guelph for histopathology and relevant diagnostic testing.

Figure 1.

Lung of Calf C, showing mild subpleural suppurative interstitial pneumonia. Alveolar septa are mildly expanded and edematous. Thin arrow identifies discontinuous non-adherent layer of fibrin present at pleural surface. Note that the lungs are floating in excess blood-stained pleural fluid (thick arrow).

Prior to initial handling of tissue, an anthrax enzyme-linked immunosorbent assay (ELISA) was carried out for screening purposes and was negative. Histological examination revealed Calf A to have mild fibrinosuppurative epicarditis and Calf B to have severe necrotizing and fibrinosuppurative myositis of the diaphragm as well as mild fibrinosuppurative pleuritis (Figure 2). Calf C was diagnosed with mild subpleural suppurative interstitial pneumonia. Several small clusters of plump bacilli were present among the interstitial debris of the diaphragm muscle, as well as small clumps of cellular debris found in the epicardium of Calf A (Figure 2). These findings prompted the AHL to perform fluorescent antibody testing for pathogenic clostridia (C. chauvoei, C. novyi, C. septicum, and C. sordellii) on the formalin-fixed, paraffin-embedded diaphragm muscle from Calf B as well as the cardiac muscle from Calf A. Along with the compatible histologic lesions, fluorescent antibody testing revealed the presence of 4+ Clostridium chauvoei (the causative agent for blackleg) in both the diaphragm and cardiac muscle samples, confirming the diagnosis of blackleg as the cause of sudden and unexpected death in the calves. The remaining herd, including adults were vaccinated with C. chauvoei, C. haemolyticum, C. novyi Type B, C. perfringens Types B, C & D, C. septicum, and C. tetani, (TASVAX 8; Merck Animal Health, Kirkland, Quebec). Only 1 calf died after vaccination, bringing the total to 9 sudden deaths in less than 48 h due to this C. chauvoei outbreak.

Figure 2.

a — Healthy striated muscle. b — Photomicrographs of striated diaphragmatic muscle from Calf B. Most myocytes are necrotic and have hypereosinophilic swollen sarcoplasm with fine or coarse vacuolation (v). Some myocytes have hypercontraction bands present. The endomysium is expanded by edema, fibrin (F), and patchy hemorrhage (H) intermingled with loose aggregates of degenerative leukocytes (arrows). Several small clusters of plump bacilli are present among interstitial debris. Photomicrographs courtesy of the Animal Healthy Laboratory, University of Guelph.

Discussion

Clostridium chauvoei is an anaerobic, Gram-positive, histiocytic, spore-forming bacillus and the well-known cause of blackleg disease (clostridial myositis) in cattle (1). The endospores reside in soil and prefer moist conditions; therefore, ruminants housed on pasture are most associated with the disease (1). Outbreaks of blackleg tend to be seasonal, as cattle ingest spores from the environment upon grazing. Contamination of soil persists via fecal shedding of grazing cattle, as spores are not spread from animal to animal by direct contact (1). Blackleg is well-documented for causing sudden death in ruminants (1,2). Cattle affected are primarily between the ages of 6 mo and 2 y and are often considered to be the “best-growing” animals in the herd, fitting with the case described here (2). Affected cattle are often found dead and show no clinical signs before death. If clinical signs are present, they can include lameness, weakness, fever, swelling, or palpable crepitation and heat over the affected area of skeletal muscle (1). If spores germinate within deep skeletal muscles such as the diaphragm, it is common that no clinical signs will be seen before death, as occurred with the calves in this case (1). Cattle exhibiting signs of lameness or fever rarely survive more than 24 to 36 h after onset of symptoms (1).

Despite being one of the oldest known diseases affecting cattle (first described in 1870), the pathogenesis of blackleg is still not completely understood (3). The current agreed upon pathogenesis begins with cattle ingesting spores from the environment, and the organism proliferating rapidly in the intestines of the animal (1). The spores then cross the intestinal mucosa and are distributed to tissues throughout the body, most commonly residing in skeletal muscle (1). It has been recently shown that C. chauvoei spores can remain viable after being taken up and internalized by bovine macrophages (4). Furthermore, the macrophages undergo an anti-inflammatory response while the spores reside in them, which promotes the survival of the organism within the macrophage (4). This finding provides evidence that the transportation of C. chauvoei spores from the intestine to other tissues of the body could be facilitated by macrophages (4).

Blackleg is considered an endogenous infection, as spores can remain latent in the tissues of cattle for extended periods, and it is commonly believed that a traumatic event creating muscle damage is necessary in order for the spores to cause disease (2). However, more recently it has been hypothesized that, in addition to a traumatic event, increased cortisol and catecholamine concentrations associated with stress can also create an opportune anaerobic environment for C. chauvoei spores to germinate (2). Moreover, it has been shown in sheep that hypoxia associated with excessive exercise can also elicit spore germination, multiplication, and exotoxin release (2). In the case described here, the rapid rate at which a large number of calves simultaneously succumbed to the disease, suggests stress or excess exercise induced spore germination and exotoxin release rather than blunt trauma somehow affecting multiple calves at once.

Following germination of the spores, C. chauvoei vegetative cells produce lethal exotoxins (5). Clostridium chauvoei toxin A (CctA) is considered the main cytotoxic and hemolytic agent of this clostridial disease (5). The organism also produces deoxyribonucleases (beta toxin), hyaluronidases (gamma toxin), neuraminidase, and oxygen labile hemolysins (3). Together, these toxins cause necrosis and migration of neutrophils to the affected areas, as well as degeneration and necrosis of vascular endothelium (3).

The most frequently discovered post-mortem lesions of clostridial myositis are found within the large muscles of the pectoral and pelvic girdle, and often produce a scent described as rancid butter (1). Classic skeletal muscle lesions are described as dark hemorrhagic zones within necrotized muscle with possible edema and gas production (1). Subcutaneous tissues surrounding the fascia of the lesion are often found to be thick with yellow gelatinous fluid and a hemorrhagic appearance (1). As with this case, examining multiple muscles in great detail is important in sudden death cases, as lesions can often be very difficult to find (1). Lesions within the crura of the diaphragm can be common as with this case, and lesions do not have to be large for animals to succumb to death from toxemia (1).

Although skeletal muscle necrosis is the classic post-mortem finding for blackleg, other common lesions do occur, as demonstrated in the calves that underwent necropsies for this case. Rapid and severe bloating is a common finding in ruminants that have died from blackleg (1). Generalized rapid degeneration of internal organs (including the liver and spleen) can occur, and more specific lesions include fibrinohemorrhagic pleuritis (with fibrin overlying the mediastinum and epicardium), emphysematous myocarditis, and fibrinohemorrhagic pericarditis (1). Pneumonia can be present but is not considered part of the actual lesion (1).

It has been reported that both sheep and cattle possessing no skeletal muscle lesions have been definitively diagnosed with a C. chauvoei infection (6). Instead, myocarditis has been the prominent lesion in these cases (6). The cattle affected all possessed a congested and hemorrhagic myocardium, enlarged spleen, and congested lungs and liver, and blood-stained pericardial fluid, but there was complete absence of skeletal muscle lesions (6). Additionally, none of the cattle were vaccinated against clostridial diseases, and only young animals were affected. Another report described calves being diagnosed with C. chauvoei but only possessing intestinal and lingual lesions (7). As with some of the calves herein, this provides further evidence towards increased stress (e.g., moving from one pasture to another) or excess exercise playing a larger role in inducing C. chauvoei spore germination, and the importance of looking for lesions in less likely areas (7).

Due to the varying types of presentation, C. chauvoei infections can be difficult to diagnose from mere post-mortem lesions (2). Definitive diagnosis of C. chauvoei is achieved by detecting the organism in affected tissues, which can be accomplished by polymerase chain reaction (PCR), culture, immunohistochemistry (IHC), or fluorescent antibody testing (FAT) (2). Whereas other clostridia may be a cause of disease or a post-mortem contaminant, C. chauvoei is not considered a post-mortem contaminant, and therefore identification of C. chauvoei by one of the methods listed is considered diagnostic (1).

Disease due to C. chauvoei exhibits a rapid clinical course, often leaving little, if any, time for treatment. When treatment has been attempted, success has been variable using penicillin antibiotics (7). Success has been reported in cases in which calves showed clinical signs and were treated with an IM injection of long-acting penicillin, or IV injection of sodium benzylpenicillin (7). Clostridium chauvoei is one of the most toxic of the clostridial organisms, which is why treatment is generally difficult (5). Therefore, it is widely accepted that preventative vaccination against clostridial diseases is the most effective way to avoid sudden death in ruminants infected with C. chauvoei (2). Current blackleg vaccines contain bacterin and toxoid culture supernatants and are considered highly effective (5). Flagella, outer membrane proteins, and extracellular proteins have recently been described as important components in inducing protection against blackleg disease (5). Efficacy is considered to be nearly 100% in preventing disease, and 50% to 100% effective even after experimental exposure to C. chauvoei spores, which most likely explains why deaths of the calves stopped once vaccine was administered in the present outbreak (2).

In conclusion, although blackleg has a well-described etiology, epidemiology, and clinical symptoms, pathology can vary, and this case serves to highlight that time and attention to detail are sometimes needed to find more subtle lesions. Moreover, definitive diagnosis of C. chauvoei should be made by identifying the organism in affected tissues. Due to its rapid clinical course, C. chauvoei disease can be difficult to treat, but vaccination is highly effective in preventing illness, as well as potentially reducing the incidence of sudden deaths in a herd already experiencing disease.