Motor Unit and Cauda Equina Diseases

Botulism

Robert J. MacKay

Botulism is a highly fatal disease characterized by muscle flaccidity and progressive generalized paralysis. Death results from euthanasia or respiratory failure.

C. botulinum spores are ubiquitous in the soil and marine sediments of most regions of the world. In general, soil-origin botulism in large domestic animals in the United States is caused by BoNT types A and B (and occasionally type C), whereas botulism caused by contamination of feed by animal carcasses is caused by BoNT types C and D. Botulism caused by C. botulinum type B is endemic among horses in Kentucky and in the mid-Atlantic states and accounts for most cases in the United States.⁵ Almost all type A equine cases and outbreaks in the United States have occurred in western states, with Oregon and Idaho overrepresented.⁶ Type C cases occur sporadically throughout the country, and type D botulism has rarely been reported in horses.⁵ BoNT is acquired either by ingestion of preformed toxin (forage poisoning), germination of ingested spores in the GI tract (toxicoinfectious botulism), or contamination of wounds with spores and subsequent vegetative growth and toxin production (wound botulism).⁶ Iatrogenic botulinum toxicity is an occasional cause of botulism in humans and is a potential side effect of the increasing use of BoNT for treatment of neurologic and muscular disorders in horses.⁷ Forage poisoning is the most common route of infection, although shaker foal syndrome, a form of toxicoinfec- tious botulism caused by C.

Horses are much more susceptible to the effects of BoNT than are cattle.⁵ In fact, horses and humans are among the mammals most sensitive to the toxin.¹ Botulism in horses typically affects one or a few members of a group. In contrast, the disease in cattle is usually in the form of outbreaks.⁵ These outbreaks in cattle have increasingly been associated with ingestion of poultry litter.^8-12

■ Pathogenesis The seven authentic BoNTs, BoNT mosaic toxins, and tetanus neurotoxin (tetanospasmin) are closely related 150-kDa proteins that differ in receptor and substrate specificities.¹ Unlike tetanospasmin, which is released alone, BoNTs are associated with a variable number of accessory proteins. It is believed that these proteins stabilize BoNTs during transit through the stomach and then may facilitate transport across intestinal epithelial cells. BoNTs are distributed hematogenously to bind to presynaptic cholinergic nerve terminals, most consequentially at the somatic neuromuscular end plate. Tissue proteases activate the toxin by cleavage into light and heavy chains connected by a disulfide bridge. After receptor-mediated internalization of BoNT in endosomes, the light chain, which is a zinc endopeptidase, is released into the cytosol, where it inactivates the SNARE (soluble N-ethylmaleimide-sensitive factor [NSF] attachment protein [SNAP] receptor) complex and thereby prevents docking and fusion of synaptic vesicles.¹ Vesicles containing acetylcholine are thus prevented from undergoing exocytosis and release of neurotransmitter at the neuromuscular junction. Flaccid paralysis of striated muscle is the main clinical result; however, paralysis of smooth muscle occurs, and parasympathetic effector functions fail. Once toxin is bound at the motor end plate, recovery is possible only by the sprouting of new axons and formation of new motor end plates, a process that requires weeks to complete.

Soil contamination of forage (including pasture) is the usual cause of botulism affecting one to several animals in the United States. In endemic areas of the United States, this is almost always caused by BoNT type B; cases associated with type A are also reported.^5,6 In small outbreaks or individual cases involving types C and D (or mosaic toxins), it is often not clear whether the source is carrion or soil contamination. Birds may play a role in carrying spores from a carcass to feeding areas.^5,13-15 Large outbreaks of foodborne botulism are of two types: germination of C. botulinum spores and bacterial growth in spoiled or moldy forage and point contamination of feed (forage or concentrate) by animal carcasses. Spoiled forage can be in the form of hay (especially large bales) or chaff or even grass clippings stored improperly or left out to rot, but usually it involves some type of improperly fermented forage.^5,16-18 Conditions of high pH, low oxygen, and high water content favor spore germination and toxin production.¹⁹

Traditionally, small-grain silage (i.e., rye, wheat, oats, and barley) has been a common source of botulism for cattle,²⁰ but corn silage has also been implicated in several outbreaks.¹⁵ The use of plastic film or bags to envelop (or encircle in the case of round bales) partially dried cut forage has become a popular way to produce and preserve silage and haylage for both cattle and horses. Unfortunately, plastic-wrapped forage has been the source of numerous outbreaks of botulism in both species.^5,20-22 These outbreaks have usually involved C. botulinum types B or A. In many cases, the plastic on the offending bale is found to be torn.⁵ Accidental incorporation of an animal corpse into feed can have devastating consequences. For example, a single cat carcass that contaminated feed caused the deaths of 420 cattle.^5,23 Carcass-origin botulism is usually caused by BoNT type C, type D, or, perhaps more often, either of the mosaic variants type DC or type CD.^16,24 Rodents can be caught in a grain auger and contaminate cereal grain or be killed by hay mowers and incorporated into the center of bales.

Poultry litter has become by far the most important source of carrion-based botulism. Processed broiler litter is widely used as a cheap source of nonprotein nitrogen in feed for beef cattle and as a fertilizer for pastures and hayfields. Accidental incorporation of chicken or rodent carcasses into these litter products has resulted in botulism outbreaks all over the world, affecting up to 5500 steers and 378 sheep and goats in separate outbreaks.^8-11,25,26 For these reasons, feeding of poultry litter to cattle is now banned in Australia and Brazil and guidelines are provided for resting of pasture after litter fertilization. Even free-ranging poultry were suspected as a source of botulism in outbreaks in dairy herds in Germany and The Nether­lands.^12,27-29 Type C and D botulism in cattle, including large outbreaks in Brazil, have been associated with osteophagia and sarcophagia (chewing on bones and soft tissues of decaying carcasses) by cattle with phosphorus deficiency.^19,30,31

C. botulinum can colonize the GI tract of normal infants or adult humans whose intestinal microflora has been disrupted by intercurrent disease or antimicrobial treatment. Shaker foal syndrome, an endemic form of toxicoinfectious botulism caused by C. botulinum type B, is of this type.³² The peak age at occurrence of shaker foal syndrome is 4 weeks; 70% of cases occurr between the ages of 2 and 6 weeks.⁵ The juvenile equine GI tract, possibly influenced by putative risk factors such as high milk intake, gastric ulceration, and heightened stress, is relatively hospitable to colonization and toxin production by the organism.^33,34 Toxin is detectable in the feces of approxi­mately 30% of shaker foals but only in the acute clinical phase of the condition.⁵ Normal intestinal flora of mature horses presumably inhibit colonization by C. botulinum. Evidence has been offered that chronic “visceral” botulism of dairy cattle in Germany is associated with GI dysbiosis and colonization of the GI tract by C.

■ Diagnosis Routine bloodwork is unhelpful, and abnor­malities found are nonspecific.

BoNT or spores must be present for diagnosis. The level of diagnostic support ranges from definitive to presumptive for detection of the following: (1) botulinum toxin in serum, GI contents, tissues, or wounds; (2) antibodies against botulinum toxin in the serum of convalescent animals; or (3) C. botulinum spores in the GI contents or suspect feed materials. Toxin is detected by the sensitive mouse bioassay and typed by mouse protection tests. In 139 horses with a clinical diagnosis of botulism, sensitivity of the mouse bioassay for detection of toxin in feces or GI contents was 32%, and specificity was 97%. In 43 cases in foals, sensitivity and specificity were 53% and 100%, respectively.³⁹ With mosaic toxins, mice may be protected by both type C and type D antisera, which probably explains why many outbreaks are recorded as involving both toxinotypes.⁴⁰ Detection of toxin in serum is possible only in animals with peracute onset and rapidly progressive clinical signs; the toxin is more likely to be detected in cattle than in horses.¹⁹ Spores are detected by inoculation of mice with culture filtrate after incubation of samples anaerobically for several days in enrichment media.⁶ ELISA and real-time PCR have been used on enriched samples to detect and distinguish botu­linum toxins or toxin genes, respectively.^3,41,42 Sensitive duplex (BoNT types A and B) and simplex (type C) quantitative PCR assays for toxin genes return results in 4 days, in comparison with 2 to 3 weeks for mouse bioassays, and do not require the sacrifice of laboratory animals.⁴³ Spores are found in the feces of approximately 34% of adult horses and 70% of foals affected with endemic type B botulism but are rarely detected in samples from normal foals or adult horses.⁵ The presence of toxin antibodies in survivors has been used diagnostically in outbreaks of type C and D botulism in cattle in Australia⁴⁴ but lacked sensitivity when examined in similar outbreaks in England and Wales.⁴⁵

Needle electromyography and decremental responses to repetitive nerve stimulation at low stimulus rates and incre­mental response at high stimulus rates can be used to support the diagnosis.^46-48 No obvious gross or histologic lesions are typically associated with botulism in most species.

■ Clinical Signs The severity and rate of progression of clinical signs are dose dependent. Horses given low doses (e.g., 10³ mouse lethal dose units) or very high doses (e.g., 10⁸ units) of BoNT display the extremes of the clinical spectrum of botulism.⁴⁹ The low dose may cause only dysphagia with some progression over 5 to 7 days and then recovery with minimal treatment. In contrast, the high dose causes recumbency within 8 to 12 hours and death within 48 hours of the first detectable signs. After high experimental doses, signs may begin as soon as 12 hours after toxin challenge, whereas when low amounts of toxin have been ingested, cases may continue to develop for up to 14 days after removal of the suspect feed source.

Signs of dysphagia, lethargy, and generalized muscle weakness are the first indications of most naturally occurring cases.⁵⁰ In horses, an early sign that can be exploited diagnostically is slow, clumsy prehension and swallowing of concentrates. Normally, bright horses require less than 2 minutes to eat 250 mL of sweet feed from a shallow pan; horses with botulism might take at least twice as long. Efforts to swallow are made with the head extended and seem exaggerated and prolonged; mixtures of food and saliva are retained in the cheeks or between the teeth, or they drop out of the mouth. There are often thin trails of green-stained saliva from each nostril. Endoscopic examination of the nasopharynx usually reveals persistent displacement of the soft palate and aspiration of feed into the trachea. With progression, water runs freely from the nostrils during attempts to drink. For investigating suspected botulism in horses in areas endemic for equine grass sickness, it is important to remember that both these conditions interfere with grain ingestion, and both may result in “failure” of the grain test.⁵⁰ Drooling of saliva in affected cattle is continuous, although the volume is usually not great, perhaps because of inhibition of saliva production by BoNT. In foals, dysphagia is evident as spillage of milk from the mouth and regurgitation through the external nares during attempts to suckle. Some horses present with colic, and most have reduced auscultable borborygmi. Affected cows have decreased strength and fre­quency of rumen contractions and firm feces.

Although animals showing the first signs of botulism may appear obtunded, it is facial muscle weakness and lack of facial expression that creates this impression. Even mildly affected animals tend to lie down more frequently than usual. With stimulation, such an animal may stand and initially walk quite normally before moving in a progressively more stilted and hypometric manner. When the patient is made to stand in one place, tremors may be apparent in the triceps muscles. As the disease progresses, tremor spreads to other antigravity muscles and increases in intensity until the animal collapses into recumbency. Prominent, generalized tremors of this type in foals in the United States with endemic toxicoinfectious botulism gave rise to the description “shaker foal syndrome.”

Tongue weakness is an important and consistent early clinical sign and, at least for type B botulism, a reliable indicator of impending limb weakness in at-risk cattle and horses. Tongue tone is assessed for this purpose as the ability of the animal to withdraw its tongue back into the oral cavity after the tip has been pulled out several inches through the interdental space. Another sign of myasthenia around the head is jaw “looseness,” evinced by cattle as lack of resistance to forced lateral movements of the lower jaw. Mydriasis and sluggish pupillary light reflexes are variable early signs; the prominence and persistence of these signs probably corresponds well with the dose and type of botulinum toxin ingested.

Between episodes of recumbency, moderately affected animals walk with a slow, shuffling, toe-scuffing gait and hold the head progressively lower.²⁴ Recumbent animals mount increasingly more ineffectual attempts to stand and often adopt unusual positions while recumbent; severely affected cattle often lie in sternal recumbency with both pelvic limbs extended caudally. Other animals lie in lateral recumbency with intermit­tent weak voluntary limb movements. Heart and respiratory rates rise during these struggles and, in some cases, respiratory efforts may seem labored, with a prolonged expiratory lift. Animals often die within 3 days of becoming recumbent. In survivors that remain standing, signs of limb weakness, dys­phagia, and slow pupillary light reflexes completely reverse over several days to several weeks. Most persistently recumbent horses die, but in those that live and in the larger proportion of cattle that survive after becoming recumbent, efforts to stand typically resume after 1 to 4 weeks. Once such animals have successfully regained the ability to stand, they remain standing for increasingly longer periods until recovery is complete weeks to months later. Generalized but reversible muscle atrophy may be evident in long-standing cases.

The differential diagnosis for horses include any disease associated with muscular weakness and dysphagia, including infectious encephalomyelitis (EPM; EEE, WEE, and VEE; EHM; WNE); generalized myopathy (nutritional, seasonal pasture, polysaccharide storage myopathy); equine motor neuron disease (EMND); grass sickness; hyperkalemic periodic paralysis; and heavy metal toxicity. Diagnoses in cattle that should be ruled out include hypocalcemia, hypokalemia, sulfur toxicity, listeriosis, epidural lymphosarcoma, and organophosphate toxicity.

■ Treatment Treatment involves nursing care and the use of specific anti-BoNT. Early treatment with specific antitoxin is a critical determinant of survival in horses. Of 91 untreated horses of various ages, 89 died of presumptive type B botulism,⁴⁹ whereas 27 of 28 foals³² and more than 70% of adults⁴⁹ treated early with antitoxin survived. Among 92 adult horses with botulism that were transported to a referral hospital for treatment, the survival rate among those given antitoxin was 59%, in comparison with 10% among horses not given antitoxin.⁵¹ The trivalent antitoxin commercially available in the United States (C. botulinum A, B, C [Antigen Select, Lake Immunogenetics, Ontario, N.Y.) covers cases typically observed in this country. Only one dose of antitoxin is necessary because the half-life of botulinum antitoxin (equine origin) is approxi­mately 12 days in normal horses.⁵ Unfortunately, antitoxin has minimal efficacy if used after the onset of recumbency in adult horses. The doses of antitoxin are 200 mL (≈20,000 IU) for a foal and 500 mL (≈50,000 IU) for an adult horse.⁴⁹ Clinical signs may progress for 12 to 24 hours after antitoxin administra­tion. Horses with mild, slowly progressive disease may survive without antitoxin, as long as they are confined to stalls to restrict activity as much as possible.

Recumbent animals need to be kept on deep, clean bedding and, if in lateral recumbency, need to be rolled to the opposite side several times daily. Bony prominences need to be padded with pillows or circular inflatable tubes in an effort to prevent decubital ulcers. Standing horses with prominent neck weakness may suffer life-threatening edema of the head and upper respiratory tract as a result of persistently hanging their heads near to the ground. This was noted to be a feature of the presentation during an outbreak of type C botulism in horses.²² These horses need to have their heads supported above the level of the heart for several hours daily to allow mobilization of tissue fluid. Adults that are dysphagic can be fed and watered via an indwelling stomach tube. Thoroughly soaked and suspended complete pelleted feed (e.g., Equine Senior, Purina Mills, St. Louis) can be administered indefinitely by this route. It is important to establish the patient's tolerance for bolus feedings and adjust volume and frequency of feedings accord­ingly. A reasonable starting point is 50% maintenance caloric requirement in 50 mL/kg water divided over four to six feedings. Caloric intake is progressively increased, and feeding frequency decreased as the animal's GI motility allows. Foals are initially fed at least 100 mL/kg of milk daily via this route, in 6 to 12 feedings, increasing to 200 mL/kg if tolerated. All recumbent animals must be in sternal position during feeding and remain sternal for at least 30 minutes after feeding. Patients without auscultable or other evidence of GI motility may be hydrated intravenously and may also be given intravenous parenteral nutrition. Recumbent adults that are being fed by nasogastric tube are at increased risk for large intestinal impactions and should be given 1 to 2 L mineral oil daily with feedings (or the equivalent twice weekly) in an effort to lubricate digesta. Recumbent male horses, including foals, are prone to retention of urine and bladder distention and must be catheterized with meticulously clean technique at least twice daily to prevent pressure necrosis and failure of the bladder wall. Animals that are able to support their own weight when assisted to a standing position should be lifted several times daily by an abdominal sling and allowed to stand with sling support for brief periods (or simply helped to their feet in the case of small ruminants). Flotation tanks may be used intermittently for this purpose in recovering cattle, especially those that are limited by recumbency-associated peripheral neuropathies.

At minimum, laboratory monitoring in recumbent horses with botulism should include daily measurements of hematocrit and total solids for hydration status, twice-weekly urine dipstick and specific gravity measurements to detect urinary tract infections and monitor hydration, and twice-weekly chemistry panels to assess organ function and electrolyte homeostasis. Total CO₂ values should be accorded particular attention. Values below 18 mEq/L indicate metabolic acidosis, whereas values above 35 mEq/L indicate significant metabolic alkalosis. The latter finding suggests compensation for chronic respiratory acidosis caused by respiratory paralysis or aspiration pneumonia and may prompt management changes such as improved sternal body position, frequent monitoring of respiratory function via arterial blood gas testing, or even initiation of mechanical ventilation. Horses with partial pressure of arterial oxygen (PaO₂) persistently below 65 mm Hg should be given humidified oxygen by nasal insufflation; those with partial pressure of arterial carbon dioxide (PaCO₂) persistently above 60 mm Hg need to be mechanically ventilated. This is generally practicable only in foals, although successful ventilation, including weaning from the ventilator, of a 425-kg horse over a period of 14 days has been reported.⁵² Of 30 foals with type B toxicoinfectious botulism admitted to a referral hospital and given antitoxin, 16 required nasal insufflation with oxygen, and 8 of those 16 foals also required mechanical ventilation.^32,53

Antibiotics are generally used only to treat known infec­tions (e.g., aspiration pneumonia or cystitis) or to prevent or treat bacterial sepsis in heavily instrumented foals. In cases in which wound botulism is suspected, the responsible wound must be opened, drained, cleaned, and debrided, in the same approach as is used for wounds associated with tetanus. Antibiotics such as metronidazole and penicillin G that are clostridiocidal are generally not recommended for treatment of wound or infant botulism in humans, out of concern that lysed organisms will release another pulse of BoNT into the circula- tion.⁵⁴ Pretreatment of large animal patients with antitoxin is presumed to remove this theoretical risk. Aminoglycosides should also be avoided if possible because they may potenti­ate the action of BoNT and were a significant risk factor for a subsequent requirement for ventilation in cases of infant botulism.⁵⁴ Because gastric ulcers were an important feature of the original postmortem descriptions of shaker foals,^33,34 such foals are usually on a regimen of acid suppression (e.g., omeprazole, 4 mg/kg PO once daily, or sucralfate, 40 mg/kg PO every 6 hours), although the importance of this approach is unknown.

The rate of recovery from botulism depends on the toxin type and dose, the species and body weight of the patient, and the dose and timing of antitoxin. Dysphagic horses that are able to stand and are given antitoxin will gradually regain the ability to swallow over 1 to 14 days, with most cases resolving within 7 days.^5,49 Mydriasis usually resolves within several days, but the pupillary light reflexes may remain sluggish for weeks. Return to full limb strength often takes more than 1 month.^5,49 Few adult horses that become persistently recumbent recover unless they are provided meticulous nursing care, and most such horses should be euthanized. Decubital ulcers and second­ary respiratory problems are the major complications. Recum­bent foals that were treated with antitoxin and intensive care at a referral hospital were able to stand in an average of 7 days if they did not need ventilation and in 10 days if they did.^32,53 There is a belief, based on small numbers of cases, that horses with type A botulism have a worse prognosis than those with type B; in fact, only one horse with type A botulism, a 10-day- old foal, has been reported to survive.⁴⁸ If the controversial category of chronic visceral botulism is excluded,³⁶ death rates among cattle and other livestock with botulism are close to 100%.^30,31 This relatively high death rate among livestock needs to be interpreted in view of the general implausibility of provid­ing specific BoNT antitoxin and intensive nursing care to affected cattle.

■ Prevention BoNT type B toxoid (BotVax B, Neogen Corporation, Lexington, Ky.) is a safe and highly effective vaccine. The primary series is three inoculations, each 4 weeks apart, and then annual revaccination of broodmares 4 to 6 weeks before foaling to provide passive immunity to their foals. Although multivalent and BoNT type C toxoid vaccines for horses and cattle are available in many parts of the world, no vaccine of this type is licensed for horses or livestock in the United States.