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Botulism in the Horse
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TOXICOLOGY
BOTULISM IN THE HORSE Francis D. Galey, DVM, PhD
Botulism is a disease consisting of progressive flaccid paralysis. It can occur in all species of animals. 20 The disease is caused by exotoxins from Clostridium botulinum. C. botulinum is an obligate anaerobic, sporeforming, gram-positive rod. 211 The bacterium is ubiquitous and is found in soils and organic matter worldwide. Eight different botulinum toxins are produced and are designated as types A, B, C, C2, D, E, F, and G. 19 · 20 Exposure of the horse to botulism may occur from ingesting preformed toxin in contaminated feed (most commonly) or may result from enteric or wound infection with C. botulinum and subsequent production of toxins in vivo. Clinical paralysis and death may result within hours from exposure to a high toxin level or, as in many cases, may develop and progress over a period of 2 to 3 weeks or more. The toxicity and mechanism of action, pathophysiologic findings, clinical effects, and diagnosis of botulism in horses are discussed. In addition, an unusual case report of type A botulism in the horse is presented. TOXICITY AND MECHANISM OF ACTION OF BOTULISM
Growth of the C. botulinum bacilli and toxin production occur under anaerobic conditions and at pH levels above 6. Growth and toxin production are not encouraged under acidic conditions such as those found in properly prepared haylage or silage. If the ensiling process is incomplete or circumvented, however, botulinum production is possible. The bacterial spores are resistant to heat, requiring temperatures of greater
From the Depa rtment of Ve terinary Sciences and Wyoming State Veterinary Laboratory, University of Wyoming, Lara mie, Wyoming
VETERINARY CLINICS OF NORTH AMERICA: EQUINE PRACTIC E VOLUME 17 • NUMBER 3 • DECEMBER 2001
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than 120°C for destruction.' The botulism toxin itself is a heat-labile protein that is destroyed if heated to sufficiently high temperatures (>80oC).24 Most of the botulism toxins belong to a family of paralysis-inducing proteins that prevent the release of acetylcholine from presynaptic terminals of cholinergic neurons. The one exception is type C2 toxin, which causes changes in membrane permeability. For the neurologic toxins, paralysis occurs at the neuromuscular junction, parasympathetic end plates, and cholinergic ganglia of the sympathetic nervous system as well as within the adrenal glands. 20 As mentioned previously, several subtypes of botulism toxin exist. The toxins are designated as types A, B, C 1, C 2, C, D, E, F, and G. At least some of the toxins (C and D) are encoded in bacteriophages.1 Some evidence suggests that the toxic factors may be transferred between different clostridia.13 In addition, toxin genes may not be expressed in all infected bacteria. Even though C. botulinum is nearly ubiquitous in the environment and in the flora of herbivores, it may not always be toxic, perhaps helping to explain the sporadic nature of outbreaks. The neurotoxic botulinum toxin is a 150-kd protein that has been cleaved into an active dimer. The dimer consists of a 100-kd heavy chain and a 50-kd light chain. The proteins remain linked through a highly conserved disulfide bond. 17 The mechanism of action of the neurologic toxins results from the specific binding of the H chain to the presynaptic membrane of cholinergic nerves.1• 17 This binding is specific and involves protein and cell surface sialogangliosides. Once bound, different Hchain components facilitate internalization within absorption vesicles. Acidification in that microenvironment subsequently facilitates movement of the catalytic L chain into the cytosol. The L chain is a zinc endopeptidase. 1• 17 This proteolytic enzyme cleaves proteins critical to the fusion and release of synaptic vesicles containing acetylcholine. Interference with the release of acetylcholine on nerve stimulation leads to the paralysis. Interestingly, tetanus toxin (Clostridium tetani exotoxin) has the same mechanism as botulinum and apparently differs only in the specificity of binding of the H chain. The H chain of tetanus prefers inhibitory interneurons, explaining the tetanic effect of that toxin. 17 The type C 2 botulinum has adenosine diphosph ate ribosyl transferase activity and clinically can cause increased fluid movement across membranes and subsequent colic and diarrhea. 20 Botulism is one of the most potent toxic diseases known. An approximate median lethal dose for neurotoxic botulinum in mice may be as low as 0.005 µg / kg of body weight." For comparison purposes, the median lethal dose for VX (a very toxic nerve gas) in the mouse is 22 µg / kg by parenteral injection. 15 Putting that information into hypothetical perspective using adult cows as an example, 1 g of pure toxin (< 0.5 tsp), if properly mixed, could kill 400,000 adult animals assuming the toxicity was similar to that of mice on a microgram per kilogram body weight basis.9 Horses are among the most sensitive animals to botulism.
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As a result, the use of traditional mouse bioassay of exposed animals can be frustrating because of a lack of sensitivity for that assay.
PATHOPHYSIOLOGIC FINDINGS OF BOTULISM IN THE HORSE
Botulism can occur from exposure to the botulinum toxin and usually takes place according to one of three scenarios. Most commonly, ingestion of preformed toxin associated with carcasses, decayed organic matter (e.g., poorly ensiled small-grain haylage or hay), or coprophagy (in the case of poultry) is the source of exposure. Toxicoinfectious botulism is another source of exposure in which the organism grows in the gut, leading to toxin production. This form of botulism occurs in young animals such as foals 25 and possibly in poultry. In addition, it has been speculated that equine grass sickness, a form of dysautonomia in horses, may result from toxicoinfection. 12 The third form of botulism is wound botulism. 26 Wound botulism results from infection of an anaerobic wound leading to toxin production. For example, the "shaker foal syndrome" results from botulism in ulcers or other necrotic lesions in 2- to 4-week-old foals. 26 Like tetanus, botulism may also result from other external wounds. One horse reportedly developed botulism-type signs after an open castration procedure.2 As mentioned, exposure to preformed toxin is the most common cause of botulism. A common source of preformed toxin is forage that is contaminated with carrion. In general, forage contaminated with carrion tends to have type C or D toxin if botulism is present. For example, horses attended by this author died after ingestion of hay that contained a dead fox. In another widely discussed case, several horses died after ingesting alfalfa cubes that had been widely distributed to several locations.13 Those cubes were found to contain carrion that had high levels of type C botulinum. Horses are sufficiently sensitive to botulism that one report suggests birds may act as a vector, bringing toxic levels of botulism toxin onto a farm from a distant site where carcasses had been previously stored. 23 Another common source of preformed toxin is decayed or improperly preserved forage. For example, forage that has become wet and decayed or improperly ensiled may be a cause of botulism.10• 18· 30 Botulism induced by forage is often caused by type B botulinum. The most common types of clinical botulism in horses result from types B, C, and D. The protein toxins are absorbed mostly in the small intestine in young animals and in the large intestine in adult animals. 4 The absorption is accomplished via endocytosis. Only small amounts of protein escape digestion to be absorbed; however, the extremely high toxicity of botulinum can make even those small amounts extremely hazardous. 4 Once absorbed, either by the gut in cases with toxicoinfectious botulism and preformed toxin exposure or directly in cases with
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wound botulism, the toxin then binds to the neurons and initiates the paralytic process. CLINICAL FINDINGS
Clinically, the paralytic toxin causes progressive paralysis in animals with botulism. Death is a frequent result. Initial signs that may be observed in the horse include intolerance to exercise, weakness and paralysis of the third eyelid, and tremors.13• 20 Some horses have a noticeable twitch in the triceps region of the front legs and carpal buckling. Further signs include ataxia, weakness, paralysis often progressing from the hind limbs forward, labored breathing, anorexia, and increased salivation. Signs are often aggravated by stimulation. Other signs may involve decreased sounds of gastrointestinal borborygmi or constipation.2· 26 The autonomic effects may predominate in cases of grass sickness if this form of equine dysautonomia is truly a result of botulism. 12 Animals may develop a diarrhea from C2 toxin or perhaps from poor or garbage-laden feed.9• n Some instances of botulism may result in d ysphagia in horses; how ever, th at finding is not ubiquitous and affected animals may eat and drink until the end in some cases. 13 Animals with late-stage botulism become recumbent (sternal recumbency at first and then lateral recumbency), and agonal signs may include some paddling. 13 The cause of death is usually from respiratory paralysis and failure along w ith heart failure. 20 The clinical course of the d isease generally lasts from 1 to several days. Animals exposed to lower levels of toxins may survive for up to 2 weeks. Recovery may occur in mildly affected horses, although some muscle wasting may occur, which may take months to resolve. Mortality in livestock may range from as low as 8% to 100% depending on the number of exposed animals. In cattle, 30%, to 45% mortality is most common. 1• 9 • 28 Gross postmortem findings in animals with botulism are often unremarkable. Gelatinous edema has been found in the intramuscular areas of the neck. 13 This edema m ay extend into the ligamentum nuchae in the midlumbar area. Nonspecific petechiation as well as vascular congestion and engorgement may be present in the central nervous system.8 Specific histologic lesions are usually absent. Evidence of a wound may aid diagnosis in cases of wound botulism. DIAGNOSIS OF BOTULISM
Botulism is usually a clinical diagnosis. Definitive diagnosis of botulism in the laboratory is elusive. The diagnostic difficulty results because of several factors. Pathognomonic gross and histologic lesions are absent. 8 Circulating toxin levels are often low. Current analytic methods
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(such as bioassay) are of insufficient sensitivity relative to the extreme sensitivity of the horse to screen for clinically relevant levels of the toxin. 9 Source material may have higher and more easily detected toxin levels, but it is often absent or otherwise unavailable for testing. Definitive diagnosis is difficult primarily because of the high toxicity of botulinum toxin. 211 • 22 Diagnosis of botulism begins with recording the appropriate clinical signs and a lack of significant postmortem lesions. Other potential neurologic diseases of the horse must be considered. Examples of diseases that must be ruled out include the various encephalitides and infection with equine herpesvirus I. In addition, exposure to a number of poisons can also be assessed. Examples of toxicants that may cause neurologic disease and weakness in horses include heavy metals like lead, various insecticides, cholinesterase inhibitors, ionophore antibiotics, neurotoxic baits, some mold toxins, and pharmaceutic agents. Occasionally, diagnosis of botulism in the clinic is helped by electrophysiologic techniques that suggest a lower motor neuron deficit. In humans, a small evoked action potential can usually be elicited; however, the decremental response to slow stimulation rates is absent. Postactivation exhaustion is not a major factor with the poisoning. 6 The major definitive test used to identify botulinum uses the mouse protection bioassay. Because of the high toxicity of botulinum, the toxin is frequently not identified in animal-related samples and requires the source of the botulinum. In this standardized test, samples of source materials, serum, liver, and, in some cases, gut contents are assayed. Although performed in many laboratories,1 1 Galey et al9 provide a brief description of a method that can be used. Briefly, material is extracted in buffer and administered via intraperitoneal injection in mice. Mice with botulism develop an abdominal breathing pattern that classically appears as a "wasp waist" within 72 hours of injection. In questionable cases, the samples may be pretreated with trypsin or heat to help differentiate effects. If signs appear, a second batch of mice is injected with the toxic extract after being pretreated with a polyvalent antiserum for botulinum. If the treated mice survive, individual groups are treated with separate antisera specific to the different toxin types to determine the type of botulism that is present. It has been suggested that horses may be anywhere between 1 and 10,000 times more sensitive to botulinum than the bioassay mouse. 13 When toxin is taken up and bound in the cell, it is much less likely to be present in the circulation in appreciable amounts. Diagnosis of botulism using this assay in livestock cases is often limited by the ability of the investigator to find sources of botulinum in the feed or environment. For that reason, diagnoses of many cases of botulism, including some that involve large numbers of animals,9· 28 are presumptive, based on the presence of appropriate clinical signs and ruling out other causes of neurologic disease. More recently, sensitive immunoassay-based assays have been developed for specific types of botulism. For example, an enzyme-linked
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immunosorbent assay (ELISA) test was developed for type C botulinum.21 These assays are useful to help detect one type of botulinum. A polyspecific test that can screen for multiple types in a diagnostic situation has not been developed, however. In addition, the sensitivity of the ELISA test relative to the mouse bioassay is in question. Culture of fecal materials and polymerase chain reaction assays may help to identify the bacterium. Given the ubiquitous nature of C. botulinum in the environment and in gut flora from many animals, however, the usefulness of this testing is questionable for animals ingesting preformed toxin. Identification of the organism, and perhaps genetic testing to identify which organisms are making toxin, may be useful in the future for cases involving toxicoinfectious or wound botulism, where the organism may still be present. 7, 27, 31 For example, an assay has been developed that uses the nested polymerase chain reaction to identify the C 1 toxin gene in sediments from wetlands. 31 Diagnosis of botulism is still a difficult task. A thorough investigation to rule out the many possible causes of weakness and death in livestock is still indicated. The mouse protection bioassay is useful to the diagnostician primarily in cases where a possible source is identified in the feed or environment. New specific ELISA tests are helpful once the type of botulinum is known, but at this time, improved diagnostic methods in terms of sensitivity and specificity are needed, TREATMENT AND PREVENTION
Treatment of botulism largely relies on supportive care and injection of antiserum (polyvalent or monovalent depending on the type of toxin exposure). Injection of antisera, if done early in the course of the disease before recumbency occurs, may be of benefit, at least for type B botulism.29 Horses with mild disease that is slowly progressing may recover without antitoxin therapy. Antitoxin is not effective after the botulinum has entered the cells. 29 Obtaining antitoxin is not always easy, and the practitioner may want to contact a nearby veterinary school or other large program when seeking antitoxin. Supportive care includes use of antimicrobial therapy specific for the gram-positive bacillus. The antibiotics can be used to treat the botulism in cases where bacterial growth is suspected (e.g., wound botulism). Antibiotic therapy may also be indicated to treat complications of paralysis like aspiration pneumonia. Avoid the use of compounds that may impair the neuromuscular system. In addition, avoid the temptation to use neurostimulants on patients. Neurostimulants such as neostigmine deplete the acetylcholine and are likely to aggravate the situation. Whitlock and Buckley29 suggest that mineral oil may be used to combat ileus and constipation. In addition, histamine blockers may be used to prevent gastric ulcers. Supportive care is an important aspect of botulism treatment. Muz-
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zling helps to prevent aspiration of bedding in patients that are recumbent and still attempting to eat. Animals that are struggling can be carefully sedated w ith xylazine or diazepam. Ointments may be useful to help protect the eyes. Severely affected and recumbent animals may require assisted ventilation, bladder catheterization, and oral alimentation.29 As with many diseases, the best way to deal with botulism is prevention. Much of prevention involves good husbandry. It is suggested that horse owners be fastidious about how they feed . Examine forage closely as it is being fed for bits of carrion or other foreign matter (including poisonous plants). It is a good general practice not to feed poor-quality hay to horses. Keep surrounding premises clear of rotting carcasses, dead wildlife, and decayed vegetation. Also, provide strict control of mice, birds, and other potential carriers of disease or toxin. Properly care for penetrating wounds, and be aware of the potential for toxicoinfection in some foals. A toxoid vaccine is available for type B botulism. 29 Multiple doses are required for the vaccine to be effective. 14 Annual vaccination of horses in endemic areas is suggested. This booster is suggested in mares at about 4 to 6 weeks prepartum to provide foals with colostral protection.14 Currently, no multivalent vaccine is available. Case Report
An example of h ow botulism may run its course in a herd of horses was provided in a case from a guest ranch located in a mountainous area. Horses (n = 54) are generally brought in from a nearby state every year. In the middle of January, a single horse was found dead. A second horse (a mare) had mild colic with a paralytic ileus and an enlarged bladder. The mare seemed to be drowsy and died that evening. The following day, a 14-year-old mare was noticed having trouble holding her head up. Two more horses subsequently developed similar signs and became recumbent. A review of the history indicated that the horses had been fed a 1-ton bale of alfalfa hay that had been flooded from a nearby creek and was "rotten and partially fermented. " That bale had been fed 2 days before the index animal was found dead. The remaining fermented hay was removed from the horses 5 days after it was initially fed and 3 days after the first horse died. Over a period of approximately 3 weeks after the hay was fed, 15 of the 54 horses became ill, and 14 of the 15 affected horses died. Clinical signs were primarily of weakness. Horses were unable to hold up their heads. Incoordination, fasciculations in the triceps muscles, d ysphagia, drooling, and flaccid paralysis of the cervical muscles were all noticed. Horses retained control of their bladders and bowels. They also were aware of their surroundings and were not blind, although some seemed to be drowsy or lethargic. Two horses were treated using a polyvalent antiserum for C. botulin um type B and C toxins, without effect. Testing of the hay material using the mouse protection bioassay caused death in mice. Mice developed gastrointestinal signs in addition to weakness and death. Subsequent coadministration of extracted hay material with polyvalent antiserum to botulism toxins and the use of anti-A botulinum resulted in survival of some treated mice. Use of antitoxins to other types of botulism toxins was not effective. Filtration of hay extract to remove large molecules such as
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proteins was also protective of mice, as w as use of heat (70°C for 10 minutes, which would also destroy the toxin). It w as concluded that the hay contained type A botulinum toxin and perhaps another toxin that would have caused the gastrointestinal problem (perhaps C 2, an endotoxin, another bacterial toxin, or a mycotoxin). The clinical signs and history s upported the diagn osis of botulism as did the absence of gross and histologic lesion s in the central nervous system and musculoskeletal system.8 Additionally, infectious syndromes and poisonous diseases such as cholinesterase inhibition and metal poisoning were ruled out. The clinical progression of botulism and the challenge of this case are typical of most similar cases in horses. Visibly moldy or decayed alfalfa h ay has been identified as the cause of type B botulism in horses. 30 Decayed grass haylage and bagged fescue hay have caused type B botulism in cattle."'· 32 Pharyngeal paralysis (dysphagia) as rep orted in this case has been reported for horses with type B botulism. 30 This case is somewh at unique in the identification of type A botulism toxin in the source m aterial. As m en tion ed previously, typ es B, C, and D are the most common toxins associated with botulism in the horse. As a result, the need for diagnostic approaches that cover all types of botulism is underscored. Additionally, the lack of response of any animals to antiserum targeted to type B and C botulism is explained by the finding of type A botulinum in this case. The use of sp ecific toxoids or antitoxins is also complicated by the p ossibility that more than one typ e of botulinum may be present in any case of botulism .
SUMMARY
Botulism should be considered in cases where weakness, p aralysis, or intolerance to exercise might be seen in the horse. Dysphagia may also be present, although it is not a consistent finding. Potential sources include carrion in hay, moldy or otherw ise rotted vegetation or forage, birds carrying material from animal burial or other similar sites, and contaminated carcasses on-site. Horses, especially foals, may also suffer from toxicoinfectious botulism, a condition where the C. botulinum might colonize and produce toxin within the gastrointestinal tract. Wounds also may harbor the organism and otherw ise promote botulism . Diagnosis of botulism is often a clinical diagnosis backed up by elimination of other possible infectious, injurious, or toxic causes of weakness of the horse. Definitive diagnosis and type identification in the laboratory are difficult and usually require a suitable sample of the source material. Treatment often is unrewarding unless a case is identified early and the proper antitoxin is readily available. Prevention involves common sense approaches to feeding and care of the horse and, where possible, judicious use of vaccination in endemic areas. ACKNOWLEDGMENTS The author thanks Ors. Donal O'Toole and Merl Raisbeck (Wyoming State Veterinary Laboratory), Dr. Ken Griggs (practitioner in Wyoming), and Dr. Richard Walker (California Animal H ealth and Food Safety Laborato ry, Davis, CA) for input and collabora tion in p reparing the case report portion of this article.
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References 1. Abbitt B, Murphy MJ, Ray AC, et al: Catastrophic death losses in a dairy herd
attributed to Type D botulism. JAVMA 185:798- 801, 1984 2. Bernard W, Divers TJ, Whitlock RH, et al: Botulism as a sequel to open castration in a horse. JAVMA 191:73-74, 1987 3. Biberstein EL, Hirsh DC: The clostridia. /11 Hirsch DC, Zee YC (eds): Veterinary Microbiology. Malden, MA, Blackwell Science, 1999, pp 233- 245 4. Bonventre PF: Absorption of botulinal toxin from the gastrointestinal tract. Review of Infectious Disease 1:663-667, 1979 5. Chao LP: The mechanism of botulism. Med Hypotheses 19:83-87, 1986 6. Cherington M: Electrophysiologic methods as an aid in diagnosis of botulism. Muscle Nerve 5(suppl):S28-29, 1982 7. Fach P, Gilbert M, Griffais R, et al: Investigation of animal botulism outbreaks by PCR and standard methods. FEMS Imrnunol Med Microbiol 13:279- 285, 1996 8. France MP: Histological exa mination of the central nervous system in the diagnosis of botulism. J Comp Pathol 10:101-108, 1989 9. Galey FD, Terra R, Walker R, et al: lype C botulism in dairy cattle from feed contaminated with a dead cat. J Vet Diagn Invest 12:204-209, 2000 10. Haagsrna J, Haesebrouck F, Devriese L, et al: An outbreak of botulism type B in horses. Vet Rec 127:206, 1990 11. Hathaway CL: Laboratory procedures for cases of suspected infant botulism. Review of Infectious Disease 1:647- 651, 1979 12. Hunter HC, Miller JK, Poxton IR: The association of Clostridiu111 botuli1111111 type C with equine grass sickness: A toxicoinfection? Equine Vet J 31:492-499, 1999 13. Kinde H, Bettey RL, Ardans A, et al: Clostridi11111 botuli11u111 Type-C intoxication associated with consumption of processed alfalfa hay cubes in horses. JAVMA 199:742- 746, 1991 14. Lewis GE, Kulinski S, Fallon EH, et al: Evaluation of a Clostridiu111 botulinwn toxoid, type B, for the prevention of shaker foal syndrome. Proc Arn Assoc Equine Pract 27:233-237, 1981 15. National Institute of Occupational Safety and Health: Phosphonothioic acid, methyl-, S-(2-(diisopropylarnino) ethyl) 0-ethyl ester. Registry of Toxic Effects of Chemical Substances. RTECS Number TB1090000. Cincinnati, 1999 16. Noterrnans S, Dufrenne J, Oosterorn J: Persistence of C/ostridium botulinwn type B on a cattle farm after an outbreak of botulism . Appl Environ Microbiol 41:179-183, 1981 17. Pellizzari R, Rosetto 0, Schaivo G, et al: Tetanus and botulinurn neurotoxins: Mechanism of action and therapeutic uses. Trans R Soc Lond B Biol Sci 354:259-268, 1999 18. Ricketts SW, Greet TRC, Glyn PH, et al: Thirteen cases of botulism in horses fed big bale silage. Equine Vet J 16:515-518, 1984 19. Rings DM: Bacterial meningitis and diseases caused by bacterial toxins. Vet Clin North Arn Equine Pract 3:85- 97, 1987 20. Rocke TE: C/oslridi11111 botuli1111111. In Gyles CL, Thoen CO (eds): Pathogenesis of Bacterial Infections, ed 2. Ames, IA, Iowa State University Press, 1993, pp 86- 96 21. Rocke TE, Smith SR, Nashold SW: Preliminary evaluation of a simple in vitro test for the diagnosis of Type C botulism in wild birds. J Wildlife Dis 34:744-751, 1998 22. Saguchi G: Clostridi11111 bot11/im1111 toxins. Pharrnacol Tuer 19:165-194, 1983 23. Schoenbaurn MA, Hall SM, Glock RD, et al: An outbreak of type C botulism in 12 horses and a mule. JAVMA 217:365-368, 2000 24. Siegel LS: Destruction of botulinum toxins in food and water. In Hauschild AHW, Dodds KL (eds): Clostridiurn Botulinurn, Ecology and Control in Foods. New York, Marcel Dekker, 1993, pp 323- 341 25. Smith L, Holdeman L: Pathogenic Anaerobic Bacteria. Springfield, IL, Charles C Thomas, 1968, p 305 26. Swerzek TW: Toxicoinfectious botulism in foals and adult horses. JAVMA 176:217220, 1980 27. Szabo EA, Pemberton JM, Gibson AM, et al: Application of PCR to a clinical and
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environmental investigation of a case of equine botulism. J Clin Microbial 32:19861991, 1994 Truman KF, Bock RE, Thomas RJ, et al: Suspected botulism in three intensively managed Australian cattle herds. Vet Rec 130:398-400, 1992 Whitlock RH, Buckley C: Botulism. Vet Clin North Am Equine Pract 13:107-128, 1997 Wichtel J), Whitlock RH: Botulism associated w ith feeding alfalfa hay to horses. JAVMA 199:471-472, 1991 Williamson JL, Rocke TE, Aiken JM: In situ detection of the Clostridii1111 bot11/im1111 type Cl toxin gene in wetland sediments w ith a nested PCR assay. Appl Environ Microbial 65:3240-3243, 1999 Wilson RB, Boley MT, Corwin B: Presumptive botulism in cattle associated with plasticpackaged hay. J Vet Diagn Invest 7:167-169, 1995 Zhou Y, Sugiyamam H, Johnson EA: Transfer of neurotoxigenicity from Clostridium b11tyricum to a nontoxigenic C/ostridi11111 boluli1111111 type E-like strain. Appl Environ Microbiol 59:3825-3831, 1993
Address reprint requests to Francis D. Ga ley, DVM, PhD Wyoming State Veterinary Laboratory 1174 Snowy Range Road Laramie, WY 82070 e-mail: [email protected]
TOXICOLOGY
BOTULISM IN THE HORSE Francis D. Galey, DVM, PhD
Botulism is a disease consisting of progressive flaccid paralysis. It can occur in all species of animals. 20 The disease is caused by exotoxins from Clostridium botulinum. C. botulinum is an obligate anaerobic, sporeforming, gram-positive rod. 211 The bacterium is ubiquitous and is found in soils and organic matter worldwide. Eight different botulinum toxins are produced and are designated as types A, B, C, C2, D, E, F, and G. 19 · 20 Exposure of the horse to botulism may occur from ingesting preformed toxin in contaminated feed (most commonly) or may result from enteric or wound infection with C. botulinum and subsequent production of toxins in vivo. Clinical paralysis and death may result within hours from exposure to a high toxin level or, as in many cases, may develop and progress over a period of 2 to 3 weeks or more. The toxicity and mechanism of action, pathophysiologic findings, clinical effects, and diagnosis of botulism in horses are discussed. In addition, an unusual case report of type A botulism in the horse is presented. TOXICITY AND MECHANISM OF ACTION OF BOTULISM
Growth of the C. botulinum bacilli and toxin production occur under anaerobic conditions and at pH levels above 6. Growth and toxin production are not encouraged under acidic conditions such as those found in properly prepared haylage or silage. If the ensiling process is incomplete or circumvented, however, botulinum production is possible. The bacterial spores are resistant to heat, requiring temperatures of greater
From the Depa rtment of Ve terinary Sciences and Wyoming State Veterinary Laboratory, University of Wyoming, Lara mie, Wyoming
VETERINARY CLINICS OF NORTH AMERICA: EQUINE PRACTIC E VOLUME 17 • NUMBER 3 • DECEMBER 2001
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than 120°C for destruction.' The botulism toxin itself is a heat-labile protein that is destroyed if heated to sufficiently high temperatures (>80oC).24 Most of the botulism toxins belong to a family of paralysis-inducing proteins that prevent the release of acetylcholine from presynaptic terminals of cholinergic neurons. The one exception is type C2 toxin, which causes changes in membrane permeability. For the neurologic toxins, paralysis occurs at the neuromuscular junction, parasympathetic end plates, and cholinergic ganglia of the sympathetic nervous system as well as within the adrenal glands. 20 As mentioned previously, several subtypes of botulism toxin exist. The toxins are designated as types A, B, C 1, C 2, C, D, E, F, and G. At least some of the toxins (C and D) are encoded in bacteriophages.1 Some evidence suggests that the toxic factors may be transferred between different clostridia.13 In addition, toxin genes may not be expressed in all infected bacteria. Even though C. botulinum is nearly ubiquitous in the environment and in the flora of herbivores, it may not always be toxic, perhaps helping to explain the sporadic nature of outbreaks. The neurotoxic botulinum toxin is a 150-kd protein that has been cleaved into an active dimer. The dimer consists of a 100-kd heavy chain and a 50-kd light chain. The proteins remain linked through a highly conserved disulfide bond. 17 The mechanism of action of the neurologic toxins results from the specific binding of the H chain to the presynaptic membrane of cholinergic nerves.1• 17 This binding is specific and involves protein and cell surface sialogangliosides. Once bound, different Hchain components facilitate internalization within absorption vesicles. Acidification in that microenvironment subsequently facilitates movement of the catalytic L chain into the cytosol. The L chain is a zinc endopeptidase. 1• 17 This proteolytic enzyme cleaves proteins critical to the fusion and release of synaptic vesicles containing acetylcholine. Interference with the release of acetylcholine on nerve stimulation leads to the paralysis. Interestingly, tetanus toxin (Clostridium tetani exotoxin) has the same mechanism as botulinum and apparently differs only in the specificity of binding of the H chain. The H chain of tetanus prefers inhibitory interneurons, explaining the tetanic effect of that toxin. 17 The type C 2 botulinum has adenosine diphosph ate ribosyl transferase activity and clinically can cause increased fluid movement across membranes and subsequent colic and diarrhea. 20 Botulism is one of the most potent toxic diseases known. An approximate median lethal dose for neurotoxic botulinum in mice may be as low as 0.005 µg / kg of body weight." For comparison purposes, the median lethal dose for VX (a very toxic nerve gas) in the mouse is 22 µg / kg by parenteral injection. 15 Putting that information into hypothetical perspective using adult cows as an example, 1 g of pure toxin (< 0.5 tsp), if properly mixed, could kill 400,000 adult animals assuming the toxicity was similar to that of mice on a microgram per kilogram body weight basis.9 Horses are among the most sensitive animals to botulism.
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As a result, the use of traditional mouse bioassay of exposed animals can be frustrating because of a lack of sensitivity for that assay.
PATHOPHYSIOLOGIC FINDINGS OF BOTULISM IN THE HORSE
Botulism can occur from exposure to the botulinum toxin and usually takes place according to one of three scenarios. Most commonly, ingestion of preformed toxin associated with carcasses, decayed organic matter (e.g., poorly ensiled small-grain haylage or hay), or coprophagy (in the case of poultry) is the source of exposure. Toxicoinfectious botulism is another source of exposure in which the organism grows in the gut, leading to toxin production. This form of botulism occurs in young animals such as foals 25 and possibly in poultry. In addition, it has been speculated that equine grass sickness, a form of dysautonomia in horses, may result from toxicoinfection. 12 The third form of botulism is wound botulism. 26 Wound botulism results from infection of an anaerobic wound leading to toxin production. For example, the "shaker foal syndrome" results from botulism in ulcers or other necrotic lesions in 2- to 4-week-old foals. 26 Like tetanus, botulism may also result from other external wounds. One horse reportedly developed botulism-type signs after an open castration procedure.2 As mentioned, exposure to preformed toxin is the most common cause of botulism. A common source of preformed toxin is forage that is contaminated with carrion. In general, forage contaminated with carrion tends to have type C or D toxin if botulism is present. For example, horses attended by this author died after ingestion of hay that contained a dead fox. In another widely discussed case, several horses died after ingesting alfalfa cubes that had been widely distributed to several locations.13 Those cubes were found to contain carrion that had high levels of type C botulinum. Horses are sufficiently sensitive to botulism that one report suggests birds may act as a vector, bringing toxic levels of botulism toxin onto a farm from a distant site where carcasses had been previously stored. 23 Another common source of preformed toxin is decayed or improperly preserved forage. For example, forage that has become wet and decayed or improperly ensiled may be a cause of botulism.10• 18· 30 Botulism induced by forage is often caused by type B botulinum. The most common types of clinical botulism in horses result from types B, C, and D. The protein toxins are absorbed mostly in the small intestine in young animals and in the large intestine in adult animals. 4 The absorption is accomplished via endocytosis. Only small amounts of protein escape digestion to be absorbed; however, the extremely high toxicity of botulinum can make even those small amounts extremely hazardous. 4 Once absorbed, either by the gut in cases with toxicoinfectious botulism and preformed toxin exposure or directly in cases with
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wound botulism, the toxin then binds to the neurons and initiates the paralytic process. CLINICAL FINDINGS
Clinically, the paralytic toxin causes progressive paralysis in animals with botulism. Death is a frequent result. Initial signs that may be observed in the horse include intolerance to exercise, weakness and paralysis of the third eyelid, and tremors.13• 20 Some horses have a noticeable twitch in the triceps region of the front legs and carpal buckling. Further signs include ataxia, weakness, paralysis often progressing from the hind limbs forward, labored breathing, anorexia, and increased salivation. Signs are often aggravated by stimulation. Other signs may involve decreased sounds of gastrointestinal borborygmi or constipation.2· 26 The autonomic effects may predominate in cases of grass sickness if this form of equine dysautonomia is truly a result of botulism. 12 Animals may develop a diarrhea from C2 toxin or perhaps from poor or garbage-laden feed.9• n Some instances of botulism may result in d ysphagia in horses; how ever, th at finding is not ubiquitous and affected animals may eat and drink until the end in some cases. 13 Animals with late-stage botulism become recumbent (sternal recumbency at first and then lateral recumbency), and agonal signs may include some paddling. 13 The cause of death is usually from respiratory paralysis and failure along w ith heart failure. 20 The clinical course of the d isease generally lasts from 1 to several days. Animals exposed to lower levels of toxins may survive for up to 2 weeks. Recovery may occur in mildly affected horses, although some muscle wasting may occur, which may take months to resolve. Mortality in livestock may range from as low as 8% to 100% depending on the number of exposed animals. In cattle, 30%, to 45% mortality is most common. 1• 9 • 28 Gross postmortem findings in animals with botulism are often unremarkable. Gelatinous edema has been found in the intramuscular areas of the neck. 13 This edema m ay extend into the ligamentum nuchae in the midlumbar area. Nonspecific petechiation as well as vascular congestion and engorgement may be present in the central nervous system.8 Specific histologic lesions are usually absent. Evidence of a wound may aid diagnosis in cases of wound botulism. DIAGNOSIS OF BOTULISM
Botulism is usually a clinical diagnosis. Definitive diagnosis of botulism in the laboratory is elusive. The diagnostic difficulty results because of several factors. Pathognomonic gross and histologic lesions are absent. 8 Circulating toxin levels are often low. Current analytic methods
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(such as bioassay) are of insufficient sensitivity relative to the extreme sensitivity of the horse to screen for clinically relevant levels of the toxin. 9 Source material may have higher and more easily detected toxin levels, but it is often absent or otherwise unavailable for testing. Definitive diagnosis is difficult primarily because of the high toxicity of botulinum toxin. 211 • 22 Diagnosis of botulism begins with recording the appropriate clinical signs and a lack of significant postmortem lesions. Other potential neurologic diseases of the horse must be considered. Examples of diseases that must be ruled out include the various encephalitides and infection with equine herpesvirus I. In addition, exposure to a number of poisons can also be assessed. Examples of toxicants that may cause neurologic disease and weakness in horses include heavy metals like lead, various insecticides, cholinesterase inhibitors, ionophore antibiotics, neurotoxic baits, some mold toxins, and pharmaceutic agents. Occasionally, diagnosis of botulism in the clinic is helped by electrophysiologic techniques that suggest a lower motor neuron deficit. In humans, a small evoked action potential can usually be elicited; however, the decremental response to slow stimulation rates is absent. Postactivation exhaustion is not a major factor with the poisoning. 6 The major definitive test used to identify botulinum uses the mouse protection bioassay. Because of the high toxicity of botulinum, the toxin is frequently not identified in animal-related samples and requires the source of the botulinum. In this standardized test, samples of source materials, serum, liver, and, in some cases, gut contents are assayed. Although performed in many laboratories,1 1 Galey et al9 provide a brief description of a method that can be used. Briefly, material is extracted in buffer and administered via intraperitoneal injection in mice. Mice with botulism develop an abdominal breathing pattern that classically appears as a "wasp waist" within 72 hours of injection. In questionable cases, the samples may be pretreated with trypsin or heat to help differentiate effects. If signs appear, a second batch of mice is injected with the toxic extract after being pretreated with a polyvalent antiserum for botulinum. If the treated mice survive, individual groups are treated with separate antisera specific to the different toxin types to determine the type of botulism that is present. It has been suggested that horses may be anywhere between 1 and 10,000 times more sensitive to botulinum than the bioassay mouse. 13 When toxin is taken up and bound in the cell, it is much less likely to be present in the circulation in appreciable amounts. Diagnosis of botulism using this assay in livestock cases is often limited by the ability of the investigator to find sources of botulinum in the feed or environment. For that reason, diagnoses of many cases of botulism, including some that involve large numbers of animals,9· 28 are presumptive, based on the presence of appropriate clinical signs and ruling out other causes of neurologic disease. More recently, sensitive immunoassay-based assays have been developed for specific types of botulism. For example, an enzyme-linked
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immunosorbent assay (ELISA) test was developed for type C botulinum.21 These assays are useful to help detect one type of botulinum. A polyspecific test that can screen for multiple types in a diagnostic situation has not been developed, however. In addition, the sensitivity of the ELISA test relative to the mouse bioassay is in question. Culture of fecal materials and polymerase chain reaction assays may help to identify the bacterium. Given the ubiquitous nature of C. botulinum in the environment and in gut flora from many animals, however, the usefulness of this testing is questionable for animals ingesting preformed toxin. Identification of the organism, and perhaps genetic testing to identify which organisms are making toxin, may be useful in the future for cases involving toxicoinfectious or wound botulism, where the organism may still be present. 7, 27, 31 For example, an assay has been developed that uses the nested polymerase chain reaction to identify the C 1 toxin gene in sediments from wetlands. 31 Diagnosis of botulism is still a difficult task. A thorough investigation to rule out the many possible causes of weakness and death in livestock is still indicated. The mouse protection bioassay is useful to the diagnostician primarily in cases where a possible source is identified in the feed or environment. New specific ELISA tests are helpful once the type of botulinum is known, but at this time, improved diagnostic methods in terms of sensitivity and specificity are needed, TREATMENT AND PREVENTION
Treatment of botulism largely relies on supportive care and injection of antiserum (polyvalent or monovalent depending on the type of toxin exposure). Injection of antisera, if done early in the course of the disease before recumbency occurs, may be of benefit, at least for type B botulism.29 Horses with mild disease that is slowly progressing may recover without antitoxin therapy. Antitoxin is not effective after the botulinum has entered the cells. 29 Obtaining antitoxin is not always easy, and the practitioner may want to contact a nearby veterinary school or other large program when seeking antitoxin. Supportive care includes use of antimicrobial therapy specific for the gram-positive bacillus. The antibiotics can be used to treat the botulism in cases where bacterial growth is suspected (e.g., wound botulism). Antibiotic therapy may also be indicated to treat complications of paralysis like aspiration pneumonia. Avoid the use of compounds that may impair the neuromuscular system. In addition, avoid the temptation to use neurostimulants on patients. Neurostimulants such as neostigmine deplete the acetylcholine and are likely to aggravate the situation. Whitlock and Buckley29 suggest that mineral oil may be used to combat ileus and constipation. In addition, histamine blockers may be used to prevent gastric ulcers. Supportive care is an important aspect of botulism treatment. Muz-
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zling helps to prevent aspiration of bedding in patients that are recumbent and still attempting to eat. Animals that are struggling can be carefully sedated w ith xylazine or diazepam. Ointments may be useful to help protect the eyes. Severely affected and recumbent animals may require assisted ventilation, bladder catheterization, and oral alimentation.29 As with many diseases, the best way to deal with botulism is prevention. Much of prevention involves good husbandry. It is suggested that horse owners be fastidious about how they feed . Examine forage closely as it is being fed for bits of carrion or other foreign matter (including poisonous plants). It is a good general practice not to feed poor-quality hay to horses. Keep surrounding premises clear of rotting carcasses, dead wildlife, and decayed vegetation. Also, provide strict control of mice, birds, and other potential carriers of disease or toxin. Properly care for penetrating wounds, and be aware of the potential for toxicoinfection in some foals. A toxoid vaccine is available for type B botulism. 29 Multiple doses are required for the vaccine to be effective. 14 Annual vaccination of horses in endemic areas is suggested. This booster is suggested in mares at about 4 to 6 weeks prepartum to provide foals with colostral protection.14 Currently, no multivalent vaccine is available. Case Report
An example of h ow botulism may run its course in a herd of horses was provided in a case from a guest ranch located in a mountainous area. Horses (n = 54) are generally brought in from a nearby state every year. In the middle of January, a single horse was found dead. A second horse (a mare) had mild colic with a paralytic ileus and an enlarged bladder. The mare seemed to be drowsy and died that evening. The following day, a 14-year-old mare was noticed having trouble holding her head up. Two more horses subsequently developed similar signs and became recumbent. A review of the history indicated that the horses had been fed a 1-ton bale of alfalfa hay that had been flooded from a nearby creek and was "rotten and partially fermented. " That bale had been fed 2 days before the index animal was found dead. The remaining fermented hay was removed from the horses 5 days after it was initially fed and 3 days after the first horse died. Over a period of approximately 3 weeks after the hay was fed, 15 of the 54 horses became ill, and 14 of the 15 affected horses died. Clinical signs were primarily of weakness. Horses were unable to hold up their heads. Incoordination, fasciculations in the triceps muscles, d ysphagia, drooling, and flaccid paralysis of the cervical muscles were all noticed. Horses retained control of their bladders and bowels. They also were aware of their surroundings and were not blind, although some seemed to be drowsy or lethargic. Two horses were treated using a polyvalent antiserum for C. botulin um type B and C toxins, without effect. Testing of the hay material using the mouse protection bioassay caused death in mice. Mice developed gastrointestinal signs in addition to weakness and death. Subsequent coadministration of extracted hay material with polyvalent antiserum to botulism toxins and the use of anti-A botulinum resulted in survival of some treated mice. Use of antitoxins to other types of botulism toxins was not effective. Filtration of hay extract to remove large molecules such as
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proteins was also protective of mice, as w as use of heat (70°C for 10 minutes, which would also destroy the toxin). It w as concluded that the hay contained type A botulinum toxin and perhaps another toxin that would have caused the gastrointestinal problem (perhaps C 2, an endotoxin, another bacterial toxin, or a mycotoxin). The clinical signs and history s upported the diagn osis of botulism as did the absence of gross and histologic lesion s in the central nervous system and musculoskeletal system.8 Additionally, infectious syndromes and poisonous diseases such as cholinesterase inhibition and metal poisoning were ruled out. The clinical progression of botulism and the challenge of this case are typical of most similar cases in horses. Visibly moldy or decayed alfalfa h ay has been identified as the cause of type B botulism in horses. 30 Decayed grass haylage and bagged fescue hay have caused type B botulism in cattle."'· 32 Pharyngeal paralysis (dysphagia) as rep orted in this case has been reported for horses with type B botulism. 30 This case is somewh at unique in the identification of type A botulism toxin in the source m aterial. As m en tion ed previously, typ es B, C, and D are the most common toxins associated with botulism in the horse. As a result, the need for diagnostic approaches that cover all types of botulism is underscored. Additionally, the lack of response of any animals to antiserum targeted to type B and C botulism is explained by the finding of type A botulinum in this case. The use of sp ecific toxoids or antitoxins is also complicated by the p ossibility that more than one typ e of botulinum may be present in any case of botulism .
SUMMARY
Botulism should be considered in cases where weakness, p aralysis, or intolerance to exercise might be seen in the horse. Dysphagia may also be present, although it is not a consistent finding. Potential sources include carrion in hay, moldy or otherw ise rotted vegetation or forage, birds carrying material from animal burial or other similar sites, and contaminated carcasses on-site. Horses, especially foals, may also suffer from toxicoinfectious botulism, a condition where the C. botulinum might colonize and produce toxin within the gastrointestinal tract. Wounds also may harbor the organism and otherw ise promote botulism . Diagnosis of botulism is often a clinical diagnosis backed up by elimination of other possible infectious, injurious, or toxic causes of weakness of the horse. Definitive diagnosis and type identification in the laboratory are difficult and usually require a suitable sample of the source material. Treatment often is unrewarding unless a case is identified early and the proper antitoxin is readily available. Prevention involves common sense approaches to feeding and care of the horse and, where possible, judicious use of vaccination in endemic areas. ACKNOWLEDGMENTS The author thanks Ors. Donal O'Toole and Merl Raisbeck (Wyoming State Veterinary Laboratory), Dr. Ken Griggs (practitioner in Wyoming), and Dr. Richard Walker (California Animal H ealth and Food Safety Laborato ry, Davis, CA) for input and collabora tion in p reparing the case report portion of this article.
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Address reprint requests to Francis D. Ga ley, DVM, PhD Wyoming State Veterinary Laboratory 1174 Snowy Range Road Laramie, WY 82070 e-mail: [email protected]