Outbreak of equine botulism type C associated with consumption of baleage in Brazil

Abstract

An outbreak of botulism occurred in March 2024 among horses at a Quarter Horse stud farm in Central-West Brazil. After ingesting baleage, 22 of 26 (85%) horses housed in stables and fed baleage became ill. The affected horses had dysphagia, muscular weakness, fasciculations, and progressive recumbency; 13 of 22 (59%) died within a few days. The diagnosis of type C botulism was established based on clinical and epidemiologic findings and confirmed by mouse bioassays, which indicated botulinum toxin type C in liver samples and intestinal contents. Furthermore, PCR testing identified toxigenic Clostridium botulinum in the baleage consumed by the horses.

Botulism results from poisoning by neurotoxins produced by Clostridium botulinum (BoNTs) and affects various animal species. The toxins act at neuromuscular junctions and block the release of acetylcholine, resulting in flaccid paralysis. BoNTs are classified as types A to H according to antigenic differences. ¹⁵

Horses are highly susceptible to BoNTs, and cases of botulism resulting from toxins A–D have been described. The prevalence of each type varies according to the geographic region; types B–D are most frequently associated with the disease in horses worldwide. In the United States, type C is uncommon; types B and A are endemic in the mid-Atlantic and western parts of the country, respectively.^8,14,15

In Brazil, there are only 2 reports of botulism in horses, both of which involved type C. The first case occurred in the northeastern region and was attributed to hay contamination by a rat carcass ³ ; the second case occurred in the southern region, and the source was believed to be stagnant water from reservoirs. ²

The main way in which type C botulism occurs is through the ingestion of preformed toxin. In most cases, the feed involved includes hay that has been contaminated with carcasses or forage that has been spoiled, improperly stored, or inadequately ensiled. ⁵

Cases of botulism occurred in March 2024 on a Quarter Horse stud farm located in Central-West Brazil; of 32 Quarter Horses, 6 were kept outdoors and 26 were stabled. All animals received feed and mineral salt, but those housed in stables also received baleage, a form of ensiled forage with 40–60% moisture content that is baled without chopping; 22 of the 26 (85%) stabled horses became ill; 13 of 22 (59%) died, and 9 recovered.

The first case was recorded one day after feeding baleage. This horse had a clinical progression of one day until death; the other affected horses died 3–5 d after the onset of clinical signs. Baleage had been removed immediately after the first animal became ill. All affected horses received treatment with fluids, glucose, dexamethasone, dimethyl sulfoxide (Vetnil), and vitamin B12.

Physical examination of the affected horses indicated that they initially developed muscle fasciculations, mainly in the triceps. They were alert but had dysphagia, as evidenced by increased time to finish chewing and a very weak swallowing reflex, in addition to weak or absent tongue tone. In males, relaxation and partial exposure of the penis were also observed. Clinical progression continued to sternal recumbency, abdominal breathing, and later lateral recumbency, culminating in death of the animals.

Serum biochemistry panel analysis revealed that 4 of 7 affected had moderately elevated creatine kinase activity. However, no other biochemical changes were observed, and the CBC did not reveal any abnormalities. Four of these horses died naturally, 2 of them within 3 d post-exposure, one 5 d after, and another within 7 d, and were autopsied at the Pathological Anatomy Laboratory of the Federal University of Mato Grosso do Sul (UFMS; Campo Grande, MS, Brazil). The postmortem examination did not indicate any macro- or microscopic lesions.

Samples of the spinal cord, cerebellum, thalamus, parietal cortex of the brain, and hippocampus were sent to the official health defense service of Mato Grosso do Sul (IAGRO; Campo Grande, MS, Brazil) for direct immunofluorescence testing, which yielded negative results for rabies virus. Furthermore, liver samples and intestinal contents from the 4 horses autopsied and 3 baleage samples were processed and analyzed for botulinum toxin; 0.5 mL from each sample was inoculated intraperitoneally into 20–25 g mice. ¹² Simultaneously, aliquots of the same samples were subjected to heat treatment at 85°C for 20 min before being inoculated into another group of mice, which were monitored for 7 d. For samples that yielded positive results in the mouse bioassay, seroneutralization tests using botulinum antitoxins type C and D, which were standardized and provided by the Federal Laboratory of Agricultural Defense (LFDA/MG; Pedro Leopoldo, MG, Brazil), were employed to determine the toxin type. ¹²

Additionally, the same 3 baleage samples were cultured in Wright medium and incubated at 37°C for 5 d to allow indirect detection of C. botulinum spores. Subsequently, 0.5 mL of the resulting supernatant was inoculated into mice, which were monitored for 7 d. In parallel, as a complementary approach to the mouse bioassay and seroneutralization, DNA was extracted from the culture supernatants and subjected to PCR testing using specific primers to detect C. botulinum types C and D. ¹⁰

In the mouse bioassay, botulinum toxin was detected in a liver sample from the horse that died 7 d after exposure and in intestinal content from the horse that died 5 d post-exposure. Seroneutralization testing identified the toxin in both samples as type C. Furthermore, all baleage samples tested positive for the presence of C. botulinum types C and D, as confirmed by PCR analysis following enrichment in Wright medium.

A diagnosis of botulism was initially established based on the horses’ histories and clinical signs, as well as the exclusion of other diseases based on the lack of significant macro- or microscopic lesions. The diagnosis was confirmed by mouse bioassay. The detection of C. botulinum in the baleage suggested a possible source of the toxin; however, the botulinum toxin itself was not directly identified in the sample.

Clinical signs of botulism can appear within a few hours to several weeks after exposure, depending on the dose ingested. Manifestations include difficulty in locomotion, reduced tongue-muscle tone, dysphagia, pelvic limb ataxia, tremors, and lateral recumbency preceding death.^5,8,15 Dysphagia is often a finding that distinguishes the condition from other neuromuscular diseases. ¹³ In our outbreak, the horses had typical clinical signs of botulism, and the disease progressed for 1–6 d from the onset of clinical signs until death. The horses that recovered had mild signs and recovered in 7–9 d.

Given the clinical signs, some differential diagnoses could be considered, including rabies, leukoencephalomalacia, protozoal myeloencephalitis, and ionophore toxicity. Although these conditions have clinical manifestations like those of botulism, histologic lesions are characteristically absent in cases of botulism. No significant hematologic changes were observed, and there were only moderate elevations in muscle enzymes related to prolonged recumbency.

Definitive confirmation of the diagnosis of botulism requires the detection of BoNTs in food samples or in tissues or fluids from affected animals; the mouse bioassay is the gold-standard test. However, the bioassay has low epidemiologic sensitivity because some species, such as horses, are more sensitive to the toxin than are mice, or the toxin may no longer be circulating at the time of collection. Thus, detection of the toxin in 2 of the 4 horses was sufficient to confirm the diagnosis, and its absence in the others did not rule out the disease.^1,8 The fact that only horses fed baleage became ill suggests that the feed was the source of contamination, and the detection of a strain of C. botulinum in the feed reinforces this hypothesis. Although PCR-based methods can detect C. botulinum and support the diagnosis, they identify only the presence of the bacterium or its neurotoxin genes, not the active toxin itself. Therefore, these results must be interpreted in conjunction with clinical signs and epidemiologic data. When associated with compatible clinical signs, the detection of C. botulinum—especially after enrichment—can be considered strong evidence of botulism, even though the organism may be present in the environment without necessarily producing the toxin. ⁸ Thus, PCR testing was applied as a complementary tool to support our diagnostic investigation.

Botulinum toxin production occurs only under conditions of high humidity and neutral or alkaline pH. Hence, the presence of the toxin in well-preserved silage or haylage is highly unlikely, since silage has a very low pH, while haylage has a low moisture content. ⁹ Another factor that may indicate a risk for the occurrence of botulism is haylage with a high ash content (>10%), which suggests that a large amount of soil has been incorporated into the product. In our case, no bromatologic analyses were performed, and no data are available regarding the pH or ash content of the product used.

Botulism types A and B generally result from the proliferation of C. botulinum in decomposing plant material, favored by improper silage storage or damaged packaging. Type C, on the other hand, more frequently results from feed contamination by spores originating from decomposing carcasses. ¹³ In horses, type C botulism has been reported in various contexts: one outbreak involved 12 horses and a mule and was attributed to feed contaminated with bird carcasses ¹¹ ; another case was linked solely to the consumption of processed alfalfa hay cubes, with no evidence of carcass contamination ⁷ ; and a third was associated with the ingestion of contaminated soil accumulated in feeding troughs. ⁶ These reports collectively demonstrate that type C botulism in horses can occur under varied conditions and emphasize the importance of considering multiple sources of exposure during outbreak investigations. In our outbreak, no animal carcasses were observed in the baleage, and there were no signs of poor storage conditions or visible soil in the feeding areas. Nonetheless, the absence of grossly visible contamination does not exclude other potential risk factors for toxin production, such as the presence of small undetected carcasses, localized pockets of poor fermentation, or excessive soil contamination during harvesting. Ash content >10% in forage indicates soil contamination and may increase the risk of C. botulinum proliferation under anaerobic conditions. Therefore, evaluating forage quality before feeding is essential, and, where available, vaccination with type C/D toxoids should be adopted as an additional preventive measure.

Epidemiologic characterization is essential for botulism cases as it directs the investigation of the sources of food or water consumed by the affected animals, which enables confirmation of the presence of toxins in these resources. This confirmation helps to identify risk factors and indicates the need for antitoxins and vaccination. Worldwide, only a few vaccines have been approved for protection of horses against types B, D, and C. ⁴