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Botulism
CHAPTER
43
Botulism
Pamela A. Wilkins
Botulism is a neuromuscular disorder of horses and other mammals caused by neurotoxins of Clostridium botulinum; similar disease has been described resulting from toxins produced by a few strains of Clostridium baratii and Clostridium butyricum. The first published reports of botulism in horses appeared in the early 1950s.1 Disease characterized by flaccid paralysis may occur in adult horses or in foals—this has been termed shaker foal syndrome.2-4 Botulinum toxin is considered to be one of the most potent toxins known.5 Although it has medicinal uses in the treatment of specific disorders,5,6 clinical botulism is a serious, frequently fatal disease. The exotoxin causes paresis and paralysis by interfering with acetylcholine release at the neuromuscular junction.6,7 Death is usually attributed to respiratory failure. Because of the pathogenicity of botulinum toxin, especially for humans, it has been placed on the list of select agents to facilitate control of dangerous biologic agents and toxins. Botulism can result from the ingestion of preformed botulinum toxin or the growth of C. botulinum in anaerobic tissues, with subsequent in vivo elaboration of toxin. Outbreaks of botulism resulting from consumption of contaminated feed can be devastating, with high morbidity and mortality.
Etiology Clostridium botulinum is a gram-positive, saprophytic, sporeforming, anaerobic rod-shaped bacterium. Eight neurotoxins isolated from C. botulinum (A, B, Ca, Cb, D, E, F, and G) are distinguished by neutralization of biologic activity with typespecific serologic reagents and are classified in seven serogroups designated A through G. Bacterial strains are usually identified on the basis of the type of toxin produced (Table 43-1). Although all types of botulinum toxin produce an identical clinical disease, determination of the toxin type is important if antitoxin is used for treatment. Clinical botulism in horses has been attributed to C. botulinum types A, B, C, and D. Clostridium botulinum spores are highly resistant to heat, light, and drying. Germination occurs under anaerobic conditions at temperatures of 15° C to 45° C (59° F-113° F). Toxin Table 43-1 Mammalian Species Affected by Each Type of Botulinum Toxin
364
Botulinum Toxin
Mammalian Species Affected
A B C D E F G
Horse, human, cattle, ferret, mink Horse, human, cattle Horse, cattle, sheep, dog, cat, mink, ferret Horse, cattle, sheep, dog Human, mink, ferret Human —
may be released from vegetative cells by cell lysis or by diffusion through the cell wall within several days of germination. After release, the single-chain toxins are inactive until cleaved by bacterial or tissue proteases to the active dichain neurotoxin. All serotypes of botulinum toxin are composed of a heavy chain with a molecular weight of approximately 100 kDa and a light chain of 50 kDa connected by a single disulfide bond.
Epidemiology Equine botulism is most frequently observed in Kentucky and the Mid-Atlantic region of the eastern United States, although the disease had been reported worldwide.3,8 In the United States, horses are most often affected with type B or C botulism, although type A botulism has been confirmed in adult horses and foals and is predominant in the Western United States.9 Type B C. botulinum spores can be isolated from the soil of most geographic regions of the United States, but they are most common in the soil of the northeastern and Appalachian regions. In contrast, C. botulinum type A spores are more common in the soil in the western United States. The frequency of occurrence of types A and B food-borne botulism in humans parallels the distribution of these types in the soil.10 A similar correlation between frequency of environmental isolation of a specific serotype of C. botulinum and the frequency of disease caused by that serotype is observed in horses in North America. Type B botulism is most often seen in the Mid-Atlantic states and Kentucky, type C occurs mainly in Florida, and type A has been observed predominantly in the western United States.
Pathogenesis Equine botulism may occur after ingestion of preformed botulinum toxin in contaminated feed (forage poisoning),11-15 ingestion of spores with elaboration of toxin within the gastrointestinal (GI) tract (toxicoinfectious botulism),16 or contamination of wounds with C. botulinum and subsequent in vivo toxin production (wound botulism).17 Ingestion of preformed botulinum toxin in decaying vegetable matter (grass, hay, grain, spoiled silage) or carcasses is the most common type of botulism observed in adult horses.3,8 An outbreak of botulism type C was associated with bird droppings and a horse burial site.18 Toxicoinfectious botulism is most common in foals 1 to 3 months of age, although it has been observed in foals as young as 7 days of age. Toxicoinfectious botulism may also be the cause of “grass sickness” in horses in Europe.19-21 Clostridial toxins are dichain structures with a molecular weight of approximately 150 kDa that are synthesized as single chains and posttranslationally cleaved into heavy (H) and light (L) chains. They are metalloproteinases that are structurally
Chapter 43 Botulism similar to tetanus toxin and that prevent the spontaneous or action potential–induced presynaptic release of acetylcholine at the neuromuscular junction. Botulinum neurotoxin (BoNT) consists of a light chain that functions as a zinc-dependent endopeptidase and a heavy chain with two functional domains of approximately the same size. The N-terminal section of the H chain is the translocation domain that forms ion channels spanning endosomal membranes to facilitate translocation and activation of light chains. The C-terminal section of the H chain is the ganglioside-binding domain to facilitate binding and internalization of the toxin at the cholinergic neuron.22 Botulism intoxication occurs by a multistep process, involving each of the functional domains of the toxin, and can be summarized as the outcome of three distinct stages: (1) binding to the target cell and internalization, (2) translocation, and (3) inhibition of neurotransmitter release. Binding to cholinergic nerve terminals is thought to require gangliosides and a protein receptor, possibly synaptotagmin for BoNT types A, B, and E.23,24 Once bound to the cell surface, BoNT is internalized into an acidic compartment by endocytosis that is temperature and energy dependent. After internalization, the toxin cannot be neutralized by antitoxin. Translocation is thought to involve a pH-dependent structural rearrangement of BoNT inside an acidic compartment within the cell, possibly synaptic vesicles or the endosomal compartment.25,26 The active light chain is translocated to the cytosol, where it interacts with and eventually cleaves SNARE proteins (soluble N-ethylmaleimide-sensitive factor attachment protein [SNAP] receptor). SNARE proteins are a group of proteins that are critical for membrane fusion and exocytosis of neurotransmitters from the cell and include synaptobrevin (vesicle-associated membrane protein family [VAMP]), syntaxin, and SNAP-25.22 The specific SNARE protein cleaved and the site of cleavage vary with the specific serotype of botulinum toxin. Botulinum neurotoxin types B, D, F, and G cleave members of the VAMP family of SNARE-complex proteins, whereas types A, C, and E cleave SNAP-25. Botulinum neurotoxin type C also has the capacity to cleave syntaxin. Botulinum neurotoxin prevents exocytosis of acetylcholine at the neuromuscular synapse by the cleavage of SNARE proteins involved in the fusion of synaptic vesicles with the plasma membrane. Cleavage of SNARE proteins creates a nonfunctional complex in which coupling between calcium ion (Ca++) influx and synaptic vesicle fusion is disrupted.27 The cleavage of the SNARE proteins allows docking of the synaptic vesicle but prevents exocytosis. Increasing intracellular Ca can partially overcome the effects of BoNT type A.22,28 The extended duration of activity of BoNT at the neuromuscular junction is the root of the clinical problem and the reason for its recent application as a therapeutic tool in human medicine. Specific BoNT serotypes vary in their duration of action. For example, BoNT type A induces long-term inhibition (months) of neurotransmission, whereas BoNT type E induces a comparatively short-term inhibition (weeks).29,30 The reasons for these differences have not been fully elucidated but may relate to the half-life of the light chain in the cytosol and to persistence of SNAP-25 fragments in the SNARE complex.
Clinical Findings Clinical signs in horses with botulism are related to inhibition of acetylcholine release at the neuromuscular junction and resultant generalized lower motor neuron and parasympathetic dysfunction.8,31 This includes dysphagia; flaccid paralysis; diminished pupillary reactivity; decreased eyelid, tongue, and tail
tone; and progressive flaccid tetraparesis and tetraplegia.3,8,32 The time to onset of clinical signs after exposure to toxin varies from 12 hours to several days. Sudden, unexplained death of one or more horses may be the initial signal of the onset of an outbreak. Decreased eyelid, tongue, and tail tone may be observed early in disease. Horses that walk may have a stilted, short-strided gait without ataxia. Muscle trembling and weakness may be apparent, particularly in foals. Pupillary dilation with sluggish pupillary light reflexes is common. There is normal cutaneous sensation with depressed spinal reflexes. Pharyngeal paralysis is frequently observed in adult horses with botulism and may be confirmed by endoscopic examination of the upper airway. Clinical signs may rapidly progress to recumbency. Tachycardia may occur, particularly in foals. Foals may appear or become constipated and dysuric. Signs of colic may be associated with diminished GI motility. Dyspnea and cyanosis may be present initially or terminally. Death is generally attributed to respiratory failure secondary to respiratory muscle paralysis.
Diagnosis Diagnosis of botulism is primarily made on the basis of history and clinical signs after exclusion of other diagnostic possibilities.3,8,32 Differential diagnoses include but are not limited to severe electrolyte imbalance (hyponatremia), tick paralysis, and postanesthetic myasthenic syndrome. Routine laboratory work, including complete blood count, serum biochemical profile, and urinalysis, is generally unremarkable unless secondary problems (e.g., infection, dehydration) have developed. Although characteristic electromyographic changes have been described in association with infant botulism and toxicoinfectious botulism in foals, electromyography (EMG) is not uniformly available, and most veterinarians are not trained to interpret this test properly.33 One recent study suggests that rapid nerve stimulation of the peroneal nerve in horses with botulism produces characteristic changes that include decreased baseline M-wave amplitudes with incremental responses at high rates.34 Definitive diagnosis is usually based on detection of toxin in serum, feces, GI contents, or feed. A variety of tests, including enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), passive hemagglutination, and polymerase chain reaction (PCR), have been described for identification of botulinum toxin; however, the diagnostic test of choice remains mouse inoculation. Serum or an extract of feed, feces, or GI contents is injected into the peritoneal cavity of mice, which are observed for classic clinical signs of botulism. The specific serotype of botulinum toxin present in a sample is determined by co-injection of mice with suspect samples and specific antisera. If appropriate antiserum is present, clinical signs will not occur. Although serum samples are occasionally positive for botulinum toxin by mouse inoculation assay, the quantity of circulating toxin is usually too small for detection.3 Isolation of C. botulinum or its toxin from feedstuffs, feces, or GI contents or from lesions or wounds in the patient is strong circumstantial evidence of infection.3,8
Pathologic Findings No pathologic findings are directly attributable to the effects of botulinum toxin. Pressure sores and regions of self-trauma may be seen in recumbent horses, and all patients, old or young, may have aspiration pneumonia secondary to dysphagia (Fig. 43-1). Gastric ulceration reported at postmortem examination in foals
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Section 3 Bacterial and Rickettsial Diseases
A
B
Figure 43-1 A, Mild pressure sores associated with recumbency in horse, caused by neurologic disease other than botulism. B, The same horse with severe pressure sores. (Courtesy Dr. Amy Bentz, Chadds Ford, PA.)
in the past may have been a reflection of the severity and progression of the disease rather than an inciting event. Gastric ulceration has been reported in association with severe illness in foals, but as intensive care techniques have improved, the incidence of gastric ulceration in nonsurviving patients at necropsy has decreased.35 In human infants, the large intestine is thought to be the site of colonization with C. botulinum and toxin-induced infection.36 “Grass sickness” (equine dysautonomia) appears to be a form of toxicoinfectious intestinal botulism in adult horses with fairly specific lesions, including degenerating neurons in peripheral autonomic ganglia, as well as in intramural intestinal ganglia, that stain positive for synaptophysin, a SNARE protein.21,37,38
Therapy The efficacy of early administration of antitoxin in improving survival and decreasing length of hospital stay has been clearly demonstrated for humans and horses and is the mainstay of therapy.3,8,32,39-44 Before the use of botulinum antitoxin in affected foals, the disease was almost uniformly fatal within 12 to 72 hours of the onset of clinical signs, except in very mildly affected horses.32 Currently, equine-origin polyvalent (anti-B and anti-C) botulinum antitoxin (Botulism Laboratory, New Bolton Center, Kennett Square, PA) and monovalent (anti-B) botulinum antitoxin (Veterinary Dynamics, Templeton, CA) are commercially available. Respiratory failure is almost uniformly the proximate cause of death in both adults and foals with botulism. Adults and foals with mild respiratory failure (normal pH and mild to moderate increase in arterial carbon dioxide tension [PaCO2]) can frequently be treated with intranasal oxygen insufflation, positioning in sternal recumbency, and repeated arterial blood gas (ABG) monitoring to detect worsening respiratory failure. Close ABG monitoring is required for the first 24 to 48 hours of treatment because administration of botulinum antitoxin does not remove toxin already bound to receptors within the terminal neuromuscular junction of the axon, and the equine patient may deteriorate further during this period. Arterial blood gas analysis should also be performed if the patient’s condition appears to change; these horses may suddenly alter their respiratory rate and pattern as respiratory failure worsens.
Increased nostril flare, decreased chest excursion, and restlessness may be physical indicators of worsening respiratory failure. Foals with botulism and respiratory failure can be mechanically ventilated successfully.44 Mechanical ventilation can ameliorate ABG abnormalities and allow time for the patient to recover cholinergic neuromuscular control. Volume-cycled ventilators tend to be better tolerated by foals. In synchronized intermittent mandatory ventilation (SIMV) mode, a minimum breath rate is set, along with a predetermined tidal volume and inspiratory flow rate, resulting in flow-controlled, volumecycled mandatory breaths.45 The sensitivity, related to the inspiratory effort the patient must generate to trigger a breath, should be set low in these horses. This is in recognition of their primary problem, botulism, and the muscular weakness associated with the disease. Antimicrobial administration, although not required for treatment unless wound botulism is suspected, is frequently employed in an effort to prevent or reduce some of the complications of the disease such as aspiration pneumonia caused by dysphagia. Antimicrobial choice in botulism is influenced by the disease process being treated. Antimicrobial drugs that might potentiate neuromuscular blockage (e.g., procaine penicillin, aminoglycosides, tetracyclines) should be avoided.46-48 Nutritional management must be considered in horses with botulism and can generally be achieved in foals by feeding milk or milk replacer via indwelling nasogastric or nasoesophageal tubes* as small, frequent meals (every 2 hours). In adult horses, periodic nasogastric intubation of slurry meals can be provided. In prolonged cases, it may be beneficial to consider commercially available liquid diets. Parenteral nutrition is generally not necessary. Intravenous fluid support may be required until patients are able to drink water safely. Nursing care is an important part of treatment, and equine patients should be protected as much as possible from development of decubital ulcers, corneal ulcers, and inadvertent aspiration. Frequent turning and slinging of adult horses are arduous tasks and require skill and persistence (Fig. 43-2). Ocular examination should be performed at least daily, and ocular lubricant ointments should be used to prevent exposure keratitis. Care should be taken to ensure the “down” eye is protected. Survival rate for botulinum neurointoxication in appropriately treated foals less than 6 months of age is greater than 90%. *Kangaroo, 12-French, 43-inch enteral feeding tube, Sherwood Medical, St. Louis, MO 63103.
Chapter 43 Botulism Box 43-1 Botulism Vaccination Protocol Adult Horses Broodmare: Initial three-dose series at 30-day intervals, with last dose 4 to 6 weeks before anticipated parturition date. Annually thereafter, 4 to 6 weeks prepartum. Other adult horses: Should consider vaccination, particularly if in endemic regions. Initial three-dose series, then annual booster.
Foals From vaccinated mares: Three-dose series of toxoid at 1-month intervals, starting at 2 to 3 months of age. From unvaccinated mares: Foal may benefit from (1) toxoid at 2, 4, and 8 weeks of age; (2) transfusion of plasma from vaccinated horse; or (3) antitoxin (efficacy needs further study).
Figure 43-2 “Slinging” a horse. The sling used here is the Anderson sling; the horse is not yet upright. Slings can be used to support horses in a standing position or can be used as an aid to change position of recumbency in “down” horses. This horse has neurologic disease, not botulism, but botulism cases would be similarly handled. (Courtesy Dr. Amy Bentz, Chadds Ford, PA.)
cholinergic neuromuscular control. Foals recovering from botulism are not protected after specific immunoglobulin G (IgG) from antitoxin is depleted, and they should be vaccinated. Adult horses with botulism that remain standing have a good prognosis for recovery; however, it may require several weeks to months before affected horses regain sufficient strength to return to work. Horses that become recumbent have a poorer prognosis even with antitoxin administration and excellent nursing care. This is related in part to their size and the secondary effects of prolonged recumbency. The degree of respiratory compromise can be severe, and long-term (days) mechanical ventilation of adult horses is a difficult undertaking.
Prevention
Figure 43-3 Weanling with botulism maintained in lateral recumbency on mattresses. Note intravenous line (coil) for fluid support and oxygen line providing intranasal oxygen insufflation. Two twin mattresses were required for this foal, and synthetic sheepskin was used to protect the body from pressure sores and skin maceration associated with prolonged recumbency. This weanling also received antitoxin and survived. (Courtesy Dr. Amy Bentz, Chadds Ford, PA.)
Approximately 50% of affected foals will require some form of ventilatory support, ranging from intranasal oxygen insufflation to mechanical ventilation, and all affected foals should have repeated ABG analysis performed during the first 48 hours of treatment (Fig. 43-3). Mechanical ventilation can ameliorate ABG abnormalities and allow time for the patient to recover
Appropriate vaccination (Clostridium botulinum type B toxoid, Neogen, Tampa, FL) is thought to be almost 100% protective in adult horses3,8 (Box 43-1). However, foals born to vaccinated dams can present with botulism, suggesting that reliance on passive transfer of immunity for protection of foals may be inadequate in endemic areas.32 Failure of transfer of specific immunity to botulinum toxin can originate from failure of passive transfer of immunity. However, foals less than 2 weeks of age with adequate blood concentration of IgG (>800 mg/dL) have also been diagnosed with botulism. In these cases, the dose of toxin may have overwhelmed the available antitoxin, or the dam may not have produced sufficient specific antibody in response to the vaccination to provide adequate protection to the foal through colostrum. Older foals from vaccinated dams may have lost specific passive immunity by the time of their exposure to the toxin and before their own vaccination. In humans, plasmapheresis of patients no longer responding to botulinum toxin administered for medicinal purposes can return them to responder status, most likely by decreasing available specific IgG.49
Public Health Considerations There is no zoonotic potential with equine botulism. The complete reference list is available online at www. expertconsult.com.
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Chapter 43 Botulism References 1. Willems R: Botulism in horses. Rev Belg Pathol Med Exp 21:257, 1951. 2. Rooney JR: Shaker foal syndrome. Mod Vet Pract 48:44, 1967. 3. Whitlock RH, Buckley C: Botulism. Vet Clin North Am Equine Pract 13:107, 1997. 4. Fenger CK: Diseases of foals. In Reed SM, Bayley WM, editors: Equine internal medicine, Philadelphia, 1999, Saunders, p 956. 5. Rossetto O, Seveso M, Caccin P, et al: Tetanus and botulinum neurotoxins: turning bad guys into good by research. Toxicon 39:27, 2001. 6. Pellizzari R, Rossetto O, Schiavo G, et al: Tetanus and botulinum neurotoxins: mechanism of action and therapeutic uses. Philos Trans R Soc Lond B Biol Sci 354:259, 1999. 7. Humeau Y, Doussau F, Grant NJ, et al: How botulinum and tetanus neurotoxins block neurotransmitter release. Biochimie 82:427, 2000. 8. Galey FD: Botulism in the horse. Vet Clin North Am Equine Pract 17:579, 2001. 9. Johnson AL, McAdams SC, Whitlock RH: Type A botulism in horses in the United States: a review of the past ten years (1998-2008). J Vet Diagn Invest 22(2):165–173, 2010. 10. Smith LDS: Clostridium botulinum: characteristics and occurrence. Rev Infect Dis 1:637, 1979. 11. Ricketts SW, Greet TR, Glyn PJ, et al: Thirteen cases of botulism in horses fed big bale silage. Equine Vet J 16:515, 1984. 12. Divers TJ, Bartholomew RC, Messick JB, et al: Clostridium botulinum type B toxicosis in a herd of cattle and a group of mules. J Am Vet Med Assoc 188:382, 1986. 13. Franzen P, Gustafsson A, Gunnarsson A: Botulism in horses associated with feeding big bale silage. Svensk Veterinartidning 44:555, 1992. 14. Wichtel JJ, Whitlock RH: Botulism associated with feeding alfalfa hay to horses. J Am Vet Med Assoc 199:471, 1991. 15. Kinde H, Bettey RL, Ardans A, et al: Clostridium botulinum type-C intoxication associated with consumption of processed alfalfa hay cubes in horses. J Am Vet Med Assoc 199:742, 1991. 16. Swerczek TW: Experimentally induced toxicoinfectious botulism in horses and foals. Am J Vet Res 41:348, 1980. 17. Bernard W, Divers TJ, Whitlock RH, et al: Botulism as a sequel to open castration in a horse. J Am Vet Med Assoc 191:73, 1987. 18. Schoenbaum MA, Hall SM, Glock RD, et al: An outbreak of type C botulism in 12 horses and a mule. J Am Vet Med Assoc 217:365, 2000. 19. McGorum BC, Kyles KW, Prince D, et al: Clinicopathological features consistent with both botulism and grass sickness in a foal. Vet Rec 152:334, 2003. 20. McCarthy HE, French NP, Edwards GB, et al: Equine grass sickness is associated with low antibody levels to Clostridium botulinum: a matched case-control study. Equine Vet J 36:123, 2004. 21. Hilbe M, Guscetti F, Wunderlin S, et al: Synaptophysin: an immunohistochemical marker for animal dysautonomias. J Comp Pathol 132:223, 2005. 22. Turton K, Chaddock JA, Acharya K, et al: Botulinum and tetanus neurotoxins: structure, function and therapeutic utility. Trends Biochem Sci 27:552, 2002. 23. Montecucco C: How do tetanus and botulinum toxins bind to neuronal membranes? Trends Biochem Sci 11:314, 1986. 24. Li L, Singh BR: Isolation of synaptotagmin as a receptor for types A and E neurotoxin and analysis of their comparative binding using a new microtiter plate assay. J Nat Toxins 7:215, 1998.
25. Montecucco C, Papini E, Schiavo G: Bacterial protein toxins penetrate cells via a four-step mechanism. FEBS Lett 346:92, 1994. 26. Blaustein RO, Germann WJ, Finkelstein A, et al: The N-terminal half of the heavy chain of botulinum type A neurotoxin forms channels in planar phospholipid bilayers. FEBS Lett 226:115, 1987. 27. Humeau Y, Doussau F, Grant NJ, et al: How botulinum and tetanus neurotoxins block neurotransmitter release. Biochimie 82:427, 2000. 28. Meunier FA, Lisk G, Sesardic D, et al: Dynamics of motor nerve terminal remodeling unveiled using SNARE-cleaving botulinum toxins: the extent and duration are dictated by the sites of SNAP-25 truncation. Mol Cell Neurosci 22:454, 2003. 29. Eleopra R, Tugnoli V, Rossetto O, et al: Different time courses of recovery after poisoning with botulinum neurotoxin serotypes A and E in humans. Neurosci Lett 256:135, 1998. 30. Foran PG, Mohammed N, Lisk GO, et al: Evaluation of the therapeutic usefulness of botulinum neurotoxin B, C1, E, and F compared with the long lasting type A: basis for distinct durations of inhibition of exocytosis in central neurons. J Biol Chem 278:1363, 2003. 31. Merz B, Bigalke H, Stoll G, et al: Botulism type B presenting as pure autonomic dysfunction. Clin Auton Res 13:337, 2003. 32. Wilkins PA, Palmer JE: Botulism in foals less than 6 months of age: 30 cases (1989-2002). J Vet Intern Med 17:702, 2003. 33. Chaudhry V, Crawford TO: Stimulation single-fiber EMG in infant botulism. Muscle Nerve 22:1698, 1999. 34. Aleman M, Williams DC, Jorge NE, et al: Repetitive stimulation of the common peroneal nerve as a diagnostic aid for botulism in foals. J Vet Intern Med 25(2):365–372, 2011. 35. Barr BS, Wilkins PA, Del Piero F, et al: Is prophylaxis for gastric ulcers necessary in critically ill equine neonates? A retrospective study of necropsy cases, 1995-1999. J Vet Intern Med 14:328, 2000. 36. Mills DC, Arnon SS: The large intestine as the site of Clostridium botulinum colonization in human infant botulism. J Infect Dis 156:997, 1987. 37. Newton JR, Wylie CE, Proudman CJ, et al: Equine grass sickness: are we any nearer to answers on cause and prevention after a century of research? Equine Vet J 42(6):477– 481, 2010. 38. Waggett BE, McGorum BC, Shaw DJ, et al: Evaluation of synaptophysin as an immunohistochemical marker for equine grass sickness. J Comp Pathol 142(4):284–290, 2010. 39. Valla WE: Diagnosis and treatment of Clostridium botulinum infection in foals: a review of fifty-three cases. In Proceedings of the 9th Annual Veterinary Medical Forum, New Orleans, 1991, p 379. 40. Bohnel H, Behrens S, Loch P, et al: Is there a link between infant botulism and sudden infant death? Bacteriological results obtained in central Germany. Eur J Pediatr 160:623, 2001. 41. Shapiro RL, Hatheway C, Swerdlow DL: Botulism in the United States: a clinical and epidemiologic review. Ann Intern Med 129:221, 1998. 42. Sandrock CE, Murin S: Clinical predictors of respiratory failure and long-term outcome in black tar heroin– associated wound botulism. Chest 120:562, 2001. 43. Tacket CO, Shandera WX, Mann JM, et al: Equine antitoxin use and other factors that predict outcome in type A foodborne botulism. Am J Med 76:794, 1984.
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Section 3 Bacterial and Rickettsial Diseases 44. Wilkins PA, Palmer JE: Mechanical ventilation in foals with botulism: 9 cases (1989-2002). J Vet Intern Med 17:708, 2003. 45. Palmer JE: Ventilatory support of the newborn foal. Vet Clin North Am Equine Pract 10:167, 1994. 46. Singh YN, Marshall IG, Harvey AL: Pre- and postjunctional blocking effects of aminoglycoside, polymyxin, tetracycline and lincosamide antibiotics. Br J Anaesth 54: 1295, 1982.
47. Snavely SR, Hodges GR: The neurotoxicity of antibacterial agents. Ann Intern Med 101:92, 1984. 48. L’Hommedieu C, Stough R, Brown L, et al: Potentiation of neuromuscular weakness in infant botulism by aminoglycosides. J Pediatr 95:1065, 1979. 49. Naumann M, Toyka KV, Mansouri Taleghani B, et al: Depletion of neutralising antibodies resensitises a secondary nonresponder to botulinum A neurotoxin. J Neurol Neurosurg Psychiatry 65:924, 1998.
43
Botulism
Pamela A. Wilkins
Botulism is a neuromuscular disorder of horses and other mammals caused by neurotoxins of Clostridium botulinum; similar disease has been described resulting from toxins produced by a few strains of Clostridium baratii and Clostridium butyricum. The first published reports of botulism in horses appeared in the early 1950s.1 Disease characterized by flaccid paralysis may occur in adult horses or in foals—this has been termed shaker foal syndrome.2-4 Botulinum toxin is considered to be one of the most potent toxins known.5 Although it has medicinal uses in the treatment of specific disorders,5,6 clinical botulism is a serious, frequently fatal disease. The exotoxin causes paresis and paralysis by interfering with acetylcholine release at the neuromuscular junction.6,7 Death is usually attributed to respiratory failure. Because of the pathogenicity of botulinum toxin, especially for humans, it has been placed on the list of select agents to facilitate control of dangerous biologic agents and toxins. Botulism can result from the ingestion of preformed botulinum toxin or the growth of C. botulinum in anaerobic tissues, with subsequent in vivo elaboration of toxin. Outbreaks of botulism resulting from consumption of contaminated feed can be devastating, with high morbidity and mortality.
Etiology Clostridium botulinum is a gram-positive, saprophytic, sporeforming, anaerobic rod-shaped bacterium. Eight neurotoxins isolated from C. botulinum (A, B, Ca, Cb, D, E, F, and G) are distinguished by neutralization of biologic activity with typespecific serologic reagents and are classified in seven serogroups designated A through G. Bacterial strains are usually identified on the basis of the type of toxin produced (Table 43-1). Although all types of botulinum toxin produce an identical clinical disease, determination of the toxin type is important if antitoxin is used for treatment. Clinical botulism in horses has been attributed to C. botulinum types A, B, C, and D. Clostridium botulinum spores are highly resistant to heat, light, and drying. Germination occurs under anaerobic conditions at temperatures of 15° C to 45° C (59° F-113° F). Toxin Table 43-1 Mammalian Species Affected by Each Type of Botulinum Toxin
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Botulinum Toxin
Mammalian Species Affected
A B C D E F G
Horse, human, cattle, ferret, mink Horse, human, cattle Horse, cattle, sheep, dog, cat, mink, ferret Horse, cattle, sheep, dog Human, mink, ferret Human —
may be released from vegetative cells by cell lysis or by diffusion through the cell wall within several days of germination. After release, the single-chain toxins are inactive until cleaved by bacterial or tissue proteases to the active dichain neurotoxin. All serotypes of botulinum toxin are composed of a heavy chain with a molecular weight of approximately 100 kDa and a light chain of 50 kDa connected by a single disulfide bond.
Epidemiology Equine botulism is most frequently observed in Kentucky and the Mid-Atlantic region of the eastern United States, although the disease had been reported worldwide.3,8 In the United States, horses are most often affected with type B or C botulism, although type A botulism has been confirmed in adult horses and foals and is predominant in the Western United States.9 Type B C. botulinum spores can be isolated from the soil of most geographic regions of the United States, but they are most common in the soil of the northeastern and Appalachian regions. In contrast, C. botulinum type A spores are more common in the soil in the western United States. The frequency of occurrence of types A and B food-borne botulism in humans parallels the distribution of these types in the soil.10 A similar correlation between frequency of environmental isolation of a specific serotype of C. botulinum and the frequency of disease caused by that serotype is observed in horses in North America. Type B botulism is most often seen in the Mid-Atlantic states and Kentucky, type C occurs mainly in Florida, and type A has been observed predominantly in the western United States.
Pathogenesis Equine botulism may occur after ingestion of preformed botulinum toxin in contaminated feed (forage poisoning),11-15 ingestion of spores with elaboration of toxin within the gastrointestinal (GI) tract (toxicoinfectious botulism),16 or contamination of wounds with C. botulinum and subsequent in vivo toxin production (wound botulism).17 Ingestion of preformed botulinum toxin in decaying vegetable matter (grass, hay, grain, spoiled silage) or carcasses is the most common type of botulism observed in adult horses.3,8 An outbreak of botulism type C was associated with bird droppings and a horse burial site.18 Toxicoinfectious botulism is most common in foals 1 to 3 months of age, although it has been observed in foals as young as 7 days of age. Toxicoinfectious botulism may also be the cause of “grass sickness” in horses in Europe.19-21 Clostridial toxins are dichain structures with a molecular weight of approximately 150 kDa that are synthesized as single chains and posttranslationally cleaved into heavy (H) and light (L) chains. They are metalloproteinases that are structurally
Chapter 43 Botulism similar to tetanus toxin and that prevent the spontaneous or action potential–induced presynaptic release of acetylcholine at the neuromuscular junction. Botulinum neurotoxin (BoNT) consists of a light chain that functions as a zinc-dependent endopeptidase and a heavy chain with two functional domains of approximately the same size. The N-terminal section of the H chain is the translocation domain that forms ion channels spanning endosomal membranes to facilitate translocation and activation of light chains. The C-terminal section of the H chain is the ganglioside-binding domain to facilitate binding and internalization of the toxin at the cholinergic neuron.22 Botulism intoxication occurs by a multistep process, involving each of the functional domains of the toxin, and can be summarized as the outcome of three distinct stages: (1) binding to the target cell and internalization, (2) translocation, and (3) inhibition of neurotransmitter release. Binding to cholinergic nerve terminals is thought to require gangliosides and a protein receptor, possibly synaptotagmin for BoNT types A, B, and E.23,24 Once bound to the cell surface, BoNT is internalized into an acidic compartment by endocytosis that is temperature and energy dependent. After internalization, the toxin cannot be neutralized by antitoxin. Translocation is thought to involve a pH-dependent structural rearrangement of BoNT inside an acidic compartment within the cell, possibly synaptic vesicles or the endosomal compartment.25,26 The active light chain is translocated to the cytosol, where it interacts with and eventually cleaves SNARE proteins (soluble N-ethylmaleimide-sensitive factor attachment protein [SNAP] receptor). SNARE proteins are a group of proteins that are critical for membrane fusion and exocytosis of neurotransmitters from the cell and include synaptobrevin (vesicle-associated membrane protein family [VAMP]), syntaxin, and SNAP-25.22 The specific SNARE protein cleaved and the site of cleavage vary with the specific serotype of botulinum toxin. Botulinum neurotoxin types B, D, F, and G cleave members of the VAMP family of SNARE-complex proteins, whereas types A, C, and E cleave SNAP-25. Botulinum neurotoxin type C also has the capacity to cleave syntaxin. Botulinum neurotoxin prevents exocytosis of acetylcholine at the neuromuscular synapse by the cleavage of SNARE proteins involved in the fusion of synaptic vesicles with the plasma membrane. Cleavage of SNARE proteins creates a nonfunctional complex in which coupling between calcium ion (Ca++) influx and synaptic vesicle fusion is disrupted.27 The cleavage of the SNARE proteins allows docking of the synaptic vesicle but prevents exocytosis. Increasing intracellular Ca can partially overcome the effects of BoNT type A.22,28 The extended duration of activity of BoNT at the neuromuscular junction is the root of the clinical problem and the reason for its recent application as a therapeutic tool in human medicine. Specific BoNT serotypes vary in their duration of action. For example, BoNT type A induces long-term inhibition (months) of neurotransmission, whereas BoNT type E induces a comparatively short-term inhibition (weeks).29,30 The reasons for these differences have not been fully elucidated but may relate to the half-life of the light chain in the cytosol and to persistence of SNAP-25 fragments in the SNARE complex.
Clinical Findings Clinical signs in horses with botulism are related to inhibition of acetylcholine release at the neuromuscular junction and resultant generalized lower motor neuron and parasympathetic dysfunction.8,31 This includes dysphagia; flaccid paralysis; diminished pupillary reactivity; decreased eyelid, tongue, and tail
tone; and progressive flaccid tetraparesis and tetraplegia.3,8,32 The time to onset of clinical signs after exposure to toxin varies from 12 hours to several days. Sudden, unexplained death of one or more horses may be the initial signal of the onset of an outbreak. Decreased eyelid, tongue, and tail tone may be observed early in disease. Horses that walk may have a stilted, short-strided gait without ataxia. Muscle trembling and weakness may be apparent, particularly in foals. Pupillary dilation with sluggish pupillary light reflexes is common. There is normal cutaneous sensation with depressed spinal reflexes. Pharyngeal paralysis is frequently observed in adult horses with botulism and may be confirmed by endoscopic examination of the upper airway. Clinical signs may rapidly progress to recumbency. Tachycardia may occur, particularly in foals. Foals may appear or become constipated and dysuric. Signs of colic may be associated with diminished GI motility. Dyspnea and cyanosis may be present initially or terminally. Death is generally attributed to respiratory failure secondary to respiratory muscle paralysis.
Diagnosis Diagnosis of botulism is primarily made on the basis of history and clinical signs after exclusion of other diagnostic possibilities.3,8,32 Differential diagnoses include but are not limited to severe electrolyte imbalance (hyponatremia), tick paralysis, and postanesthetic myasthenic syndrome. Routine laboratory work, including complete blood count, serum biochemical profile, and urinalysis, is generally unremarkable unless secondary problems (e.g., infection, dehydration) have developed. Although characteristic electromyographic changes have been described in association with infant botulism and toxicoinfectious botulism in foals, electromyography (EMG) is not uniformly available, and most veterinarians are not trained to interpret this test properly.33 One recent study suggests that rapid nerve stimulation of the peroneal nerve in horses with botulism produces characteristic changes that include decreased baseline M-wave amplitudes with incremental responses at high rates.34 Definitive diagnosis is usually based on detection of toxin in serum, feces, GI contents, or feed. A variety of tests, including enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), passive hemagglutination, and polymerase chain reaction (PCR), have been described for identification of botulinum toxin; however, the diagnostic test of choice remains mouse inoculation. Serum or an extract of feed, feces, or GI contents is injected into the peritoneal cavity of mice, which are observed for classic clinical signs of botulism. The specific serotype of botulinum toxin present in a sample is determined by co-injection of mice with suspect samples and specific antisera. If appropriate antiserum is present, clinical signs will not occur. Although serum samples are occasionally positive for botulinum toxin by mouse inoculation assay, the quantity of circulating toxin is usually too small for detection.3 Isolation of C. botulinum or its toxin from feedstuffs, feces, or GI contents or from lesions or wounds in the patient is strong circumstantial evidence of infection.3,8
Pathologic Findings No pathologic findings are directly attributable to the effects of botulinum toxin. Pressure sores and regions of self-trauma may be seen in recumbent horses, and all patients, old or young, may have aspiration pneumonia secondary to dysphagia (Fig. 43-1). Gastric ulceration reported at postmortem examination in foals
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A
B
Figure 43-1 A, Mild pressure sores associated with recumbency in horse, caused by neurologic disease other than botulism. B, The same horse with severe pressure sores. (Courtesy Dr. Amy Bentz, Chadds Ford, PA.)
in the past may have been a reflection of the severity and progression of the disease rather than an inciting event. Gastric ulceration has been reported in association with severe illness in foals, but as intensive care techniques have improved, the incidence of gastric ulceration in nonsurviving patients at necropsy has decreased.35 In human infants, the large intestine is thought to be the site of colonization with C. botulinum and toxin-induced infection.36 “Grass sickness” (equine dysautonomia) appears to be a form of toxicoinfectious intestinal botulism in adult horses with fairly specific lesions, including degenerating neurons in peripheral autonomic ganglia, as well as in intramural intestinal ganglia, that stain positive for synaptophysin, a SNARE protein.21,37,38
Therapy The efficacy of early administration of antitoxin in improving survival and decreasing length of hospital stay has been clearly demonstrated for humans and horses and is the mainstay of therapy.3,8,32,39-44 Before the use of botulinum antitoxin in affected foals, the disease was almost uniformly fatal within 12 to 72 hours of the onset of clinical signs, except in very mildly affected horses.32 Currently, equine-origin polyvalent (anti-B and anti-C) botulinum antitoxin (Botulism Laboratory, New Bolton Center, Kennett Square, PA) and monovalent (anti-B) botulinum antitoxin (Veterinary Dynamics, Templeton, CA) are commercially available. Respiratory failure is almost uniformly the proximate cause of death in both adults and foals with botulism. Adults and foals with mild respiratory failure (normal pH and mild to moderate increase in arterial carbon dioxide tension [PaCO2]) can frequently be treated with intranasal oxygen insufflation, positioning in sternal recumbency, and repeated arterial blood gas (ABG) monitoring to detect worsening respiratory failure. Close ABG monitoring is required for the first 24 to 48 hours of treatment because administration of botulinum antitoxin does not remove toxin already bound to receptors within the terminal neuromuscular junction of the axon, and the equine patient may deteriorate further during this period. Arterial blood gas analysis should also be performed if the patient’s condition appears to change; these horses may suddenly alter their respiratory rate and pattern as respiratory failure worsens.
Increased nostril flare, decreased chest excursion, and restlessness may be physical indicators of worsening respiratory failure. Foals with botulism and respiratory failure can be mechanically ventilated successfully.44 Mechanical ventilation can ameliorate ABG abnormalities and allow time for the patient to recover cholinergic neuromuscular control. Volume-cycled ventilators tend to be better tolerated by foals. In synchronized intermittent mandatory ventilation (SIMV) mode, a minimum breath rate is set, along with a predetermined tidal volume and inspiratory flow rate, resulting in flow-controlled, volumecycled mandatory breaths.45 The sensitivity, related to the inspiratory effort the patient must generate to trigger a breath, should be set low in these horses. This is in recognition of their primary problem, botulism, and the muscular weakness associated with the disease. Antimicrobial administration, although not required for treatment unless wound botulism is suspected, is frequently employed in an effort to prevent or reduce some of the complications of the disease such as aspiration pneumonia caused by dysphagia. Antimicrobial choice in botulism is influenced by the disease process being treated. Antimicrobial drugs that might potentiate neuromuscular blockage (e.g., procaine penicillin, aminoglycosides, tetracyclines) should be avoided.46-48 Nutritional management must be considered in horses with botulism and can generally be achieved in foals by feeding milk or milk replacer via indwelling nasogastric or nasoesophageal tubes* as small, frequent meals (every 2 hours). In adult horses, periodic nasogastric intubation of slurry meals can be provided. In prolonged cases, it may be beneficial to consider commercially available liquid diets. Parenteral nutrition is generally not necessary. Intravenous fluid support may be required until patients are able to drink water safely. Nursing care is an important part of treatment, and equine patients should be protected as much as possible from development of decubital ulcers, corneal ulcers, and inadvertent aspiration. Frequent turning and slinging of adult horses are arduous tasks and require skill and persistence (Fig. 43-2). Ocular examination should be performed at least daily, and ocular lubricant ointments should be used to prevent exposure keratitis. Care should be taken to ensure the “down” eye is protected. Survival rate for botulinum neurointoxication in appropriately treated foals less than 6 months of age is greater than 90%. *Kangaroo, 12-French, 43-inch enteral feeding tube, Sherwood Medical, St. Louis, MO 63103.
Chapter 43 Botulism Box 43-1 Botulism Vaccination Protocol Adult Horses Broodmare: Initial three-dose series at 30-day intervals, with last dose 4 to 6 weeks before anticipated parturition date. Annually thereafter, 4 to 6 weeks prepartum. Other adult horses: Should consider vaccination, particularly if in endemic regions. Initial three-dose series, then annual booster.
Foals From vaccinated mares: Three-dose series of toxoid at 1-month intervals, starting at 2 to 3 months of age. From unvaccinated mares: Foal may benefit from (1) toxoid at 2, 4, and 8 weeks of age; (2) transfusion of plasma from vaccinated horse; or (3) antitoxin (efficacy needs further study).
Figure 43-2 “Slinging” a horse. The sling used here is the Anderson sling; the horse is not yet upright. Slings can be used to support horses in a standing position or can be used as an aid to change position of recumbency in “down” horses. This horse has neurologic disease, not botulism, but botulism cases would be similarly handled. (Courtesy Dr. Amy Bentz, Chadds Ford, PA.)
cholinergic neuromuscular control. Foals recovering from botulism are not protected after specific immunoglobulin G (IgG) from antitoxin is depleted, and they should be vaccinated. Adult horses with botulism that remain standing have a good prognosis for recovery; however, it may require several weeks to months before affected horses regain sufficient strength to return to work. Horses that become recumbent have a poorer prognosis even with antitoxin administration and excellent nursing care. This is related in part to their size and the secondary effects of prolonged recumbency. The degree of respiratory compromise can be severe, and long-term (days) mechanical ventilation of adult horses is a difficult undertaking.
Prevention
Figure 43-3 Weanling with botulism maintained in lateral recumbency on mattresses. Note intravenous line (coil) for fluid support and oxygen line providing intranasal oxygen insufflation. Two twin mattresses were required for this foal, and synthetic sheepskin was used to protect the body from pressure sores and skin maceration associated with prolonged recumbency. This weanling also received antitoxin and survived. (Courtesy Dr. Amy Bentz, Chadds Ford, PA.)
Approximately 50% of affected foals will require some form of ventilatory support, ranging from intranasal oxygen insufflation to mechanical ventilation, and all affected foals should have repeated ABG analysis performed during the first 48 hours of treatment (Fig. 43-3). Mechanical ventilation can ameliorate ABG abnormalities and allow time for the patient to recover
Appropriate vaccination (Clostridium botulinum type B toxoid, Neogen, Tampa, FL) is thought to be almost 100% protective in adult horses3,8 (Box 43-1). However, foals born to vaccinated dams can present with botulism, suggesting that reliance on passive transfer of immunity for protection of foals may be inadequate in endemic areas.32 Failure of transfer of specific immunity to botulinum toxin can originate from failure of passive transfer of immunity. However, foals less than 2 weeks of age with adequate blood concentration of IgG (>800 mg/dL) have also been diagnosed with botulism. In these cases, the dose of toxin may have overwhelmed the available antitoxin, or the dam may not have produced sufficient specific antibody in response to the vaccination to provide adequate protection to the foal through colostrum. Older foals from vaccinated dams may have lost specific passive immunity by the time of their exposure to the toxin and before their own vaccination. In humans, plasmapheresis of patients no longer responding to botulinum toxin administered for medicinal purposes can return them to responder status, most likely by decreasing available specific IgG.49
Public Health Considerations There is no zoonotic potential with equine botulism. The complete reference list is available online at www. expertconsult.com.
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Chapter 43 Botulism References 1. Willems R: Botulism in horses. Rev Belg Pathol Med Exp 21:257, 1951. 2. Rooney JR: Shaker foal syndrome. Mod Vet Pract 48:44, 1967. 3. Whitlock RH, Buckley C: Botulism. Vet Clin North Am Equine Pract 13:107, 1997. 4. Fenger CK: Diseases of foals. In Reed SM, Bayley WM, editors: Equine internal medicine, Philadelphia, 1999, Saunders, p 956. 5. Rossetto O, Seveso M, Caccin P, et al: Tetanus and botulinum neurotoxins: turning bad guys into good by research. Toxicon 39:27, 2001. 6. Pellizzari R, Rossetto O, Schiavo G, et al: Tetanus and botulinum neurotoxins: mechanism of action and therapeutic uses. Philos Trans R Soc Lond B Biol Sci 354:259, 1999. 7. Humeau Y, Doussau F, Grant NJ, et al: How botulinum and tetanus neurotoxins block neurotransmitter release. Biochimie 82:427, 2000. 8. Galey FD: Botulism in the horse. Vet Clin North Am Equine Pract 17:579, 2001. 9. Johnson AL, McAdams SC, Whitlock RH: Type A botulism in horses in the United States: a review of the past ten years (1998-2008). J Vet Diagn Invest 22(2):165–173, 2010. 10. Smith LDS: Clostridium botulinum: characteristics and occurrence. Rev Infect Dis 1:637, 1979. 11. Ricketts SW, Greet TR, Glyn PJ, et al: Thirteen cases of botulism in horses fed big bale silage. Equine Vet J 16:515, 1984. 12. Divers TJ, Bartholomew RC, Messick JB, et al: Clostridium botulinum type B toxicosis in a herd of cattle and a group of mules. J Am Vet Med Assoc 188:382, 1986. 13. Franzen P, Gustafsson A, Gunnarsson A: Botulism in horses associated with feeding big bale silage. Svensk Veterinartidning 44:555, 1992. 14. Wichtel JJ, Whitlock RH: Botulism associated with feeding alfalfa hay to horses. J Am Vet Med Assoc 199:471, 1991. 15. Kinde H, Bettey RL, Ardans A, et al: Clostridium botulinum type-C intoxication associated with consumption of processed alfalfa hay cubes in horses. J Am Vet Med Assoc 199:742, 1991. 16. Swerczek TW: Experimentally induced toxicoinfectious botulism in horses and foals. Am J Vet Res 41:348, 1980. 17. Bernard W, Divers TJ, Whitlock RH, et al: Botulism as a sequel to open castration in a horse. J Am Vet Med Assoc 191:73, 1987. 18. Schoenbaum MA, Hall SM, Glock RD, et al: An outbreak of type C botulism in 12 horses and a mule. J Am Vet Med Assoc 217:365, 2000. 19. McGorum BC, Kyles KW, Prince D, et al: Clinicopathological features consistent with both botulism and grass sickness in a foal. Vet Rec 152:334, 2003. 20. McCarthy HE, French NP, Edwards GB, et al: Equine grass sickness is associated with low antibody levels to Clostridium botulinum: a matched case-control study. Equine Vet J 36:123, 2004. 21. Hilbe M, Guscetti F, Wunderlin S, et al: Synaptophysin: an immunohistochemical marker for animal dysautonomias. J Comp Pathol 132:223, 2005. 22. Turton K, Chaddock JA, Acharya K, et al: Botulinum and tetanus neurotoxins: structure, function and therapeutic utility. Trends Biochem Sci 27:552, 2002. 23. Montecucco C: How do tetanus and botulinum toxins bind to neuronal membranes? Trends Biochem Sci 11:314, 1986. 24. Li L, Singh BR: Isolation of synaptotagmin as a receptor for types A and E neurotoxin and analysis of their comparative binding using a new microtiter plate assay. J Nat Toxins 7:215, 1998.
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47. Snavely SR, Hodges GR: The neurotoxicity of antibacterial agents. Ann Intern Med 101:92, 1984. 48. L’Hommedieu C, Stough R, Brown L, et al: Potentiation of neuromuscular weakness in infant botulism by aminoglycosides. J Pediatr 95:1065, 1979. 49. Naumann M, Toyka KV, Mansouri Taleghani B, et al: Depletion of neutralising antibodies resensitises a secondary nonresponder to botulinum A neurotoxin. J Neurol Neurosurg Psychiatry 65:924, 1998.