• Open Access

Repetitive Stimulation of the Common Peroneal Nerve as a Diagnostic Aid for Botulism in Foals


  • The study was performed at the University of California, Davis, Davis, CA.

Corresponding author: Monica Aleman, MVZ, PhD, Dipl ACVIM, William R. Pritchard Veterinary Medical Teaching Hospital, School of Veterinary Medicine, University of California, Davis, Davis, CA 95616; e-mail: mraleman@ucdavis.edu.


Background: Botulism is a potentially fatal paralytic disorder for which definitive diagnosis is difficult.

Objectives: To determine if repetitive stimulation of the common peroneal nerve will aid in the diagnosis of botulism in foals.

Animals: Four control and 3 affected foals.

Methods: Validation of the test in healthy foals for its comparison in foals with suspected botulism. Controls were anesthetized and affected foals were sedated to avoid risks of anesthesia. The common peroneal nerve was chosen for its superficial location and easy access. Stimulating electrodes were placed along the common peroneal nerve. For recording, the active and reference electrodes were positioned over the midpoint and distal end of the extensor digitorum longus muscle, respectively. Repeated supramaximal stimulation of the nerve was performed utilizing a range of frequencies (1–50 Hz). Data analysis consisted of measuring the amplitude and area under the curve for each M wave and converting these values into percentages of decrement or increment based on the comparison of subsequent potentials to the initial one (baseline) within each set.

Results: A decremental response was seen at all frequencies in control foals. Decremental responses also were observed in affected foals at low frequencies. An incremental response was seen in all affected foals at 50 Hz.

Conclusions and Clinical Importance: Decreased baseline M wave amplitudes with incremental responses at high rates are supportive of botulism. Repetitive nerve stimulation is a safe, simple, fast, and noninvasive technique that can aid in the diagnosis of suspected botulism in foals.


area under the curve


Clostridium botulinum neurotoxin


compound muscle action potential




repetitive nerve stimulation


streptococcal protein-G

Botulism is an often fatal, flaccid, paralytic disorder caused by the neurotoxins produced by Clostridium botulinum.1 Clinically relevant forms of botulism in horses include toxicoinfectious, food borne, and wound botulism.1–3 The toxicoinfectious form is more commonly seen in foals <6 months of age, and occurs when spores are ingested and germinate in the intestinal tract.4,5 Food borne is the most common form of botulism in adult horses, and occurs after the ingestion of feed contaminated with preformed toxin.1,4 Wound botulism occurs in wounds contaminated with C. botulinum spores, but it has been reported rarely in horses.3,4 Other forms such as inhalation and inadvertent or iatrogenic botulism have been described in humans.4 Eight antigenically distinct neurotoxins have been identified: A, B, C (C1, C2), D, E, F, and G.6 Botulism in horses has been reported to be caused by serotypes A–D.1,3,5,7,8

BoNT: Botulinum neurotoxins are produced and released under anaerobic conditions, enter the systemic circulation, migrate, and internalize into the presynaptic membrane of motor neuron nerve endings.9 The light chain is the neurotoxic fragment of BoNT that selectively cleaves specific proteins involved in vesicle fusion.10 The disruption of membrane fusion prevents the release of acetylcholine into the synaptic cleft, resulting in the interruption of neuromuscular transmission, which leads to functional denervation of the muscle.10 Clinical manifestations include progressive muscular weakness; dysphagia; dysphonia; ptosis; decreased pupillary light reflex; decreased tongue, tail, and anal tone; ileus; and respiratory failure.5,7 The diagnosis of botulism is predominantly clinical in both human and veterinary medicine because identification of C. botulinum or spores in feces or wounds, spores in canned food, or BoNT in serum, wound, feces, or contaminated food have a low yield.4 Furthermore, isolation of the pathogen in feces may be an incidental finding. The most reliable test in people is the mouse inoculation test in which mice exposed and unexposed to a specific type of botulinum antitoxin are injected with the patient's serum.4 Unexposed mice that die within 24–48 hours postinjection are considered positive.4 Other diagnostic modalities not commercially available include detection of specific anti-BoNT antibodies in serum samples by an ELISA, and identification of the BoNT gene in various samples including tissues, food, and feces by novel multiplex polymerase chain reaction techniques.8,11,12

Neuroelectrodiagnostic medicine refers to diagnoses based on electrical information obtained from neural conduction or needle electromyography (EMG) studies.13,14 Electrodiagnostic testing is considered an extension of the neurological examination.15 Specific neurophysiological studies such as repetitive nerve stimulation (RNS) have been particularly useful as a diagnostic aid for neuromuscular disease.13 The electrophysiological effects of BoNT have been reported in human botulism.4,16 In large animal neurology, the use of electrodiagnostics has been limited compared with other species. Currently, there are no reports of electrodiagnostic studies as a diagnostic aid in large animals with suspected botulism.17 Common neonatal diseases in foals may present with generalized weakness that could result in recumbency, dysphagia, and hypoventilation. These signs are commonly seen in foals with botulism, and differentiation of disorders could be challenging in the absence of other signs.5 In addition, other neuromuscular disorders may present with similar signs making the diagnosis of botulism difficult. Therefore, the aim of the study was to investigate whether repetitive stimulation of the common peroneal nerve would aid in the diagnosis of suspected botulism in foals.

Materials and Methods


Control Foals. Four healthy Quarter Horse foals were studied to validate the use of RNS of the common peroneal nerve as a diagnostic aid for neuromuscular disease. Two colts and 2 fillies ranging in age from 39 to 52 days old (mean, 47.5 days) were defined as healthy based on normal physical and neurological examinations. These foals were anesthetized for the study to avoid movement artifacts. A catheter was placed in the jugular vein and the mouth rinsed before anesthetic induction with propofol (3 mg/kg IV). Once unconscious, foals were immediately intubated orotracheally and connected to a small animal anesthetic circuit that was primed with 4% isoflurane. Foals were transferred to a padded table and maintained with 1.5–1.6% end-tidal isoflurane. A facial arterial catheter was placed for blood pressure and respiratory gas monitoring. Foals received intermittent positive pressure ventilation with a 20 cmH2O peak inspiratory pressure at a rate sufficient to maintain an α-stat PaCO2 between 40 and 50 mmHg. Lactated Ringer's solution was administered IV at 10 mL/kg/h throughout the study, and dobutamine hydrochloride was administered as needed to maintain mean arterial blood pressure >60 mmHg. Body temperature was measured with a nasoesophageal probe, and a warm air circulation device was used to maintain body temperature at 37 ± 1°C (98.6°F). The foals were positioned in lateral recumbency for the study. Left and right pelvic limbs were tested with and without manual limb restraint. The study was approved by the animal care and use committee.

Affected Foals. For this prospective study, foals <6 months of age with suspected botulism were examined over a 4-year period. The inclusion criteria consisted of the following: (1) Foals with suspected botulism based on clinical signs and exclusion of other causes (ie, systemic and metabolic disease, nutritional muscular dystrophy, neurological disorders). Signs included acute onset of generalized progressive weakness with one or more of the following: low carriage of the head and neck; muscle fasciculations; decreased muscle tone including tongue, eyelid, and tail; inability to stand; dysphagia; dysphonia; and hypoventilation. (2) Foals with complete minimum data base (CBC, serum biochemistry panel, arterial and venous blood gas analysis, urinalysis, thoracic and abdominal radiographs, and ultrasound examination). (3) Foals with RNS testing. Although needle EMG and muscle biopsy specimen were not part of the inclusion criteria, these procedures were performed to further evaluate neuromuscular alterations in affected foals. Selected foals were not anesthetized to conduct RNS because of safety concerns and were sedated according to the clinicians' discretion with all three of the following: diazepam (0.1–0.2 mg/kg IV), xylazine hydrochloride (0.1–0.2 mg/kg IV), and butorphanol tartrate (0.02–0.03 mg/kg IV). Drug administration was repeated as needed for electrodiagnostic testing. Owner consent was obtained. Specific description on case management of selected foals was not included.


Depending on availability, a Nicolet Viking IV evoked potential systema or a Nicolet Viking Questa was used to perform EMG and RNS. Needle insertion sites were cleaned with alcohol. EMG was performed in the triceps brachii, cleidobrachialis, extensor carporadialis, supraspinatus, infraspinatus, serratus, latissimus dorsi, lumbar paraspinalis, gluteus medius, biceps femoris, quadriceps femoris, cranial tibialis, intercostalis, and semimembranosus muscles in affected foals. A concentric needle electrode (26 G by 50 mm length, recording area of 0.07 mm2)b was used to perform the EMG, and a subdermal needle electrode (30 G by 20 mm)c served as the ground. At least 5 sites per muscle were examined. Abnormalities consisted of decreased, prolonged, or absent insertional activity, and spontaneous activity. The latter consisted of fibrillation potentials and positive sharp waves; scoring was based on that used in human EMG as described by Kimura.15

The common peroneal nerve was selected for the RNS test based on its superficial location and easy accessibility. The technique applied in this study was adapted from that described in dogs by Walker et al.18 Monopolar electrodes (28 G by 25 mm) were used for both stimulating and recording.d Stimulating electrodes were placed along the common peroneal nerve at the caudal border of the gastrocnemius muscle at the level ventral to the stifle (Fig 1). These electrodes were placed on either side of the nerve approximately 1 in. apart with the cathode caudal and deep with respect to the anode (Fig 1). For recording, the active electrode was positioned over the midpoint of the extensor digitorum longus muscle with the reference electrode at the distal end of this muscle. A subdermal needle electrodec was used as a ground and placed between the stimulating and recording electrodes. Repeated supramaximal stimulation of the nerve was performed utilizing a range of frequencies (1, 2, 3, 5, 7, 10, 15, 20, 30, and 50 Hz) in control foals. To minimize the time of sedation and possible discomfort in affected foals, the frequencies of stimulation were decreased (1, 3, 7, 15, 30, and 50 Hz). Stimulus duration was 0.2 ms with trains of 10 stimuli used for each stimulus rate. A minimum of 1 minute of recovery time elapsed between sets. Once the electrodes were properly placed, the total estimated time for the study was 11 and 6 minutes for each limb tested in control and affected foals, respectively. Compound muscle action potentials (CMAPs or M waves) were recorded. Data analysis consisted of measuring the amplitude and area under the curve (AUC) of each M wave and converting these values into percentages of decrement or increment based on the comparison of subsequent potentials to the initial 1 (baseline) within each set. The foals' limb temperature was maintained between 33 and 34°C (91.4–93.2°F) with a warm air blanket to avoid a decrease in limb temperature that may mask a decremental response.19

Figure 1.

 Electrode placement. (a) Lateral oblique view. Different colors of electrodes were used for this figure for easier recognition: Stimulating (top 2), recording (bottom 2 in red), and reference (small, middle 1). (b) Specimen depicting the common peroneal nerve (black arrow), and biceps femoris (A), gastrocnemius (B), extensor digitorum longus (C), and extensor digitorum lateralis (D) muscles.

Histochemical Evaluation of Skeletal Muscle

Muscle biopsies from triceps, quadriceps femoris, gluteus medius, and semimembranosus muscles were collected from affected foals after administering lidocaine hydrochloride (1.5 mL SC) as a local anesthetic. Immediately after collection, the specimens were flash frozen in isopentane precooled in liquid nitrogen and stored at −80°C until further processing. The following stains and reactions were performed: hematoxylin and eosin, modified Gomori trichrome, periodic acid Schiff, phosphorylase, esterase, Streptococcal protein G (SPG)-horseradish peroxidase, ATPase at pH 9.8, 4.6, and 4.3, nicotinamide adenine dinucleotide, succinic dehydrogenase, acid phosphatase, alkaline phosphatase, and oil red O. Additionally, to rule out a possible immune-mediated myasthenic disorder, serum was collected from affected foals, and dilutions were incubated with control external intercostalis muscle and stained for the detection of IgG antibodies (SPG assay). This muscle was selected because of its high concentration of neuromuscular junctions. If antibodies against acetylcholine receptors are present in the patient's serum, they would bind with the postsynaptic myofibers and be highlighted by the SPG reaction and the test would be considered positive. Positive (IgG present at acetylcholine receptors in a horse with suspected acquired myasthenic disorder) and negative (healthy horse) controls available from our tissue bank were processed simultaneously for quality control.

Statistical Analysis

In control foals, the results of RNS with manual restraint of the limb being tested were compared with those obtained with no limb restraint. The results of RNS were compared between right and left pelvic limbs in control foals. Comparison was performed by 2-way repeated measures analysis of variance. In order to avoid prolongation of sedation in affected foals, only 1 limb was studied.


Affected Foals

Three foals met the inclusion criteria for the study. Five foals with suspected botulism were admitted to our institution during the study period. However, only 3 foals had RNS testing. All 3 foals had acute progressive generalized weakness that resulted in recumbency and hypoventilation, which in 2 cases required mechanical ventilation. Aspiration pneumonia was the most common complication in these foals. Therapeutic management frequently was adjusted during hospitalization to fulfill the foals' requirements for hydration (IV maintenance polyionic fluids supplemented with dextrose and B vitamins), nutrition (enteral through a nasogastric tube), urination (urinary catheterization), defecation (enemas as needed), and to treat concurrent problems such as aspiration pneumonia. All foals received botulism antitoxin plasma (polyvalente for serotypes A–E [all 3 cases], bivalentf for serotypes B and C [cases 1 and 3]) within the first 2 days of admission.

Case 1. A 54-kg, 3-day-old Appaloosa filly presented with acute profound weakness, inability to stand or lie in sternal recumbency, and absence of tail tone that developed over 12 hours before admission. Arterial blood gas analysis performed during intranasal administration of 100% oxygen at 10 L/min indicated acidemia (pH 7.29; reference range, 7.35–7.40) because of respiratory acidosis (PaCO2 71.7 mmHg; reference range, 45–55 mmHg). The filly became progressively weaker, respirations became shallow, arterial blood pH decreased to 7.228, and PaCO2 increased to 101 mmHg. A nasotracheal tube was placed and mechanical ventilation was initiated and maintained for 6.5 days. The filly gradually improved and was discharged 14 days postadmission. Culture of an admission fecal sample did not yield C. botulinum. The filly appeared normal in a 4-month follow-up.

Case 2. A 97-kg, 33-day-old Thoroughbred filly was presented with acute, progressive, profound generalized weakness of 5 days' duration. Eyelid and tail tone were absent and swallowing appeared weak. The filly also had bilateral exposure ulcerative keratitis. Arterial blood gas analysis indicated respiratory acidemia (pH 7.2, PaCO2 80 mmHg) and hypoxemia (PaO2 70 mmHg; reference range, 80–98 mmHg on room air). The filly became progressively weaker, respirations became shallow, arterial blood pH decreased to 7.1, and PaCO2 increased to 100 mmHg. A nasotracheal tube was placed and mechanical ventilation was initiated and maintained for 17 days. Several attempts to wean the filly off mechanical ventilation were made but it was unable to ventilate unassisted, and the owners elected euthanasia. A mouse inoculation assay for botulism was negative. However, C. botulinum type A was isolated from a fecal sample collected on admission.

Case 3. A 59-kg, 10-day-old Gypsy Vanner filly had a history of 5 days of progressive weakness, inability to rise without assistance, severe exercise intolerance resulting in collapse, and requirement of the head to be supported. On presentation, the filly had dilated pupils, and decreased eyelid tone, corneal reflex, pupillary light reflex, and tail tone. The filly urinated without posturing while being supported. The muscle weakness progressed and the filly could no longer support its weight when assisted, and became dysphagic. Despite her ongoing generalized muscle weakness, mechanical ventilation was not required. The filly gradually improved and was discharged 12 days postadmission. Culture of an admission fecal sample did not yield C. botulinum. The filly's strength improved gradually over the next few months and a 3-year follow-up revealed no abnormalities.


Electrodiagnostics were performed on days 5, 15, and 2 of hospitalization (5, 20, and 7 days from onset of clinical signs) in cases 1, 2, and 3, respectively. Needle EMG was performed in cases 2 and 3. Findings consisted of fibrillation potentials and positive sharp waves in 6 (cleidobrachialis, extensor carporadialis, supraspinatus, infraspinatus, serratus, and intercostalis) of the 14 muscles tested in case 2. There were no abnormalities on EMG in case 3. CMAPs were decreased in affected foals (amplitude, 1.8 ± 0.92 mV [mean ± SD]; AUC, 7.56 ± 4.38 mV ms; versus amplitude, 13.04 ± 3.21 mV; AUC, 48.3 ± 13.22 mV ms in control foals).

Once the electrodes were properly placed in the foal under study, RNS testing was completed within the estimated time. RNS in control foals revealed a decremental response of <5% at low frequencies (1–10 Hz) and a larger decremental response at higher frequencies (20–50 Hz) for both amplitude and AUC compared with its own baseline at each frequency (Fig 2). Results from RNS were not statistically different when comparing limb restraint versus no restraint, and between right and left pelvic limb in control foals.

Figure 2.

 Decremental responses in control foals. The graphs represent the mean and SD for decremental responses compared with the baseline at each frequency stimuli for amplitude (A) and area under the curve (AUC) (B).

Limb restraint was performed in affected foals during RNS testing. There was a decremental response in affected foals at low frequencies (case 1 shown, Fig 3A). However, this decrement was larger than that observed in control foals. An incremental response was first observed in case 1 at 3 Hz with higher incremental responses in subsequent frequency stimuli. Decremental responses were recorded in cases 2 and 3 but as the frequency stimuli increased, there was less of a decrement. At 50 Hz, an increment in amplitude and AUC were observed in affected foals but not in control foals (case 1 shown, Fig 3C). The recordings from 1 control foal and case 1 are shown in Figure 4. A summary of the of the CMAPs' amplitudes and AUC at 1 and 50 Hz in both groups (control and affected foals) is shown in Table 1 (only trains 1, 3, 5, and 10 for each rate are shown).

Figure 3.

 Comparison of decremental/incremental responses in control foals and case 1. Control foals are shown as the mean with SD compared with case 1 at frequencies 1 Hz (A) and 50 Hz (B).

Figure 4.

 Recordings in 1 control foal and case 1. Control foal at 1 Hz (A), case 1 at 1 Hz (B), control foal at 50 Hz (C), and case 1 at 50 Hz (D). Notice the increment in case 1 at 50 Hz.

Table 1.   Compound muscle action potentials.
Rate (Hz)CMAPAmp (mV)AUC (mV ms)
  1. CMAP, compound muscle action potential; AUC, area under the curve.

  2. The table shows the amplitude (Amp) in millivolts (mV) and AUC in millivolts multiplied by millisecond (mV ms) of trains 1, 3, 5, and 10 of stimuli at 1 and 50 Hz in control and affected foals.

  3. The values represent mean ± SD.

1113.04 ± 3.211.80 ± 0.9248.30 ± 13.227.56 ± 4.38
312.89 ± 3.211.67 ± 0.9248.09 ± 12.897.07 ± 4.20
512.87 ± 3.201.60 ± 0.9848.11 ± 12.876.60 ± 4.44
1011.77 ± 3.631.50 ± 0.9343.11 ± 9.326.21 ± 4.23
50111.85 ± 3.452.32 ± 1.9643.66 ± 8.297.15 ± 5.30
311.58 ± 3.632.68 ± 2.2633.69 ± 7.407.14 ± 3.72
510.38 ± 3.762.68 ± 2.3128.34 ± 8.637.50 ± 4.79
109.30 ± 3.643.22 ± 1.9824.91 ± 5.4811.01 ± 2.04

Histochemical Evaluation of Skeletal Muscle

Muscle biopsies were collected from the triceps brachii (longissimus and lateralis) in case 2; and gluteus medius, semimembranosus, and quadriceps femoris in case 3. All muscle specimens in both fillies had marked myofiber size variation with neurogenic myofiber atrophy of both fiber types (1 and 2). Hypertrophic fibers were intermixed with atrophic fibers in case 3. Intramuscular nerve branches appeared normal. Neuromuscular junctions were highlighted with esterase, and no abnormalities were seen. The serum samples from these foals (cases 2 and 3) were negative for antibodies against acetylcholine receptors.


This study showed the value of RNS testing for the investigation of neuromuscular dysfunction in foals. RNS was validated in control foals and proved to be safe, simple, fast, and noninvasive. In this study, RNS of the common peroneal nerve was used to aid in the diagnosis of suspected botulism in foals. Concomitantly, other causes of acute progressive muscle weakness that resulted in recumbency and hypoventilation in affected foals were ruled out and further excluded by the results of RNS testing. The study also showed that RNS can be performed under sedation to avoid potential risks associated with anesthesia in neurologically compromised foals. Once the electrodes were positioned and supramaximal stimulus identified, the study required <10 minute to be completed. Furthermore, an advantage of RNS testing was that it provided results while performing the study. Additionally, this study documented electromyographic and muscle histochemical findings in 2 foals at different stages (acute versus subacute) and severity of disease. Motor nerve conduction velocity studies and nerve biopsy specimen analysis would have further assisted in ruling out other disorders that may result in progressive generalized weakness but were not performed because of safety concerns in compromised foals.

Electromyographic studies in people with botulism usually reveal no abnormal spontaneous activity.13 Abnormal spontaneous activity, however, has been described in severely affected individuals.13,16 In humans, BoNT type A usually is associated with more severe disease, longer recovery period, and higher mortality rate than other types.4,13 Similarly, botulism caused by BoNT type A appears to be more severe and have high mortality in horses.3 In this study, the most severely affected foal (case 2) was euthanized and presumed to have BoNT type A. This foal had abnormal spontaneous activity consisting of fibrillation potentials and positive sharp waves. Fibrillation potentials and positive sharp waves are indicators of myofiber membrane instability and can be seen in peripheral neuropathies with axonal loss.14 Impaired neurotransmission at neuromuscular junctions caused by BoNT also results in functional axonal loss. Abnormal EMG findings may not be detectable early in the course of the disease.14 If axonal loss occurs, fibrillation potentials and positive sharp waves appear within 2–3 weeks.14 In this study, an EMG was performed at earlier stages of disease in case 3 (7 days from onset) as compared with case 2 (20 days from onset). Severity and stage of disease likely contributed to the observed EMG findings in these foals.

RNS in control foals showed amplitude decrements at rates of 10–50 Hz. These results are similar to those reported in normal muscle in humans.20 RNS of the common peroneal nerve showed a decrement in CMAP amplitude at 1, 3, 7, 15, and 30 Hz in cases 2 and 3. However, the decrement in amplitude at 30 Hz was smaller than those observed at lower frequencies. Stimulation of the nerve at 50 Hz resulted in an incremental response from baseline in cases 2 and 3. In case 1, incremental responses were already observed at lower rates (3 Hz). Furthermore, the incremental response was greater at higher frequencies (30 and 50 Hz). Similar incremental responses at higher stimulus frequencies have been reported in humans with botulism.16 Depending on severity of disease and age of the individual, several patterns of the CMAP response to repetitive stimulation may be observed.13,21,22 At low frequency rates of RNS, approximately 56% of infants demonstrate a decremental response, 24% have no change, and 20% have an incremental change from baseline.13 Incremental responses are observed in 73 and 92% of infants at 20 and 50 Hz, respectively.13 Similarly in adults, stimulation at higher frequencies induces incremental responses.13 Therefore, if increments are not observed at 20 Hz in people with suspected botulism, the RNS test is performed at higher frequencies.2,13 All affected foals in this study had an incremental response in both amplitude and AUC at 50 Hz, different from control foals which had a decremental response at higher rates including 50 Hz.

The initial evoked CMAP in resting muscles usually is decreased in patients with botulism.13,16 This finding also was observed in our affected foals. However, decreased CMAP can be observed with other neuromuscular disorders.13 Decrement and increment of the CMAP are quantified in amplitude, AUC, and commonly reported as a percentage decrement or increment when compared with the patient's first potential (baseline).13 Decrements or increments may vary with the severity of disease.13,22 Decremental responses may not always be observed in patients with less severe disease, however, increments may be found in the majority of the patients.13,22 An incremental response to RNS is known as facilitation. Facilitation is a reproducible increase in the amplitude and AUC of successive CMAPs during RNS and represents the activation of previously inactive muscle fibers as the stimulus increases.13 Facilitation has only been demonstrated in a few presynaptic disorders: botulism, Lambert-Eaton myasthenic syndrome, and magnesium-induced neuromuscular transmission disorders.13,23 Facilitation was observed in affected foals. Plasma ionized magnesium concentrations in these foals were within reference range at the time of electrodiagnostic testing. Lambert-Eaton myasthenic syndrome is a rare chronic progressive disorder that does not fit the clinical course and outcome of these foals. The important feature of facilitation unique to botulism is its persistence for a few minutes because of calcium accumulation at the presynaptic membrane.15,24 A normal phenomenon called pseudofacilitation can be easily confused with facilitation by the inexperienced examiner.15 Pseudofacilitation can be observed in healthy individuals and consists of CMAPs with increased amplitude resulting from better synchronization of different muscle fibers without recruitment of more muscle fibers as seen by an unchanged AUC.15 Pseudofacilitation was observed in this study as shown in Figure 2 in which decrements in amplitude and AUC were different (larger percentage decrement in AUC than in amplitude at 50 Hz).

The effect of age on RNS testing was not evaluated and needs further investigation. At the time of RNS evaluation, the affected foals were 8 (case 1), 48 (case 2), and 12 (case 3) days old. Cases 1 and 3 were younger than the controls (mean, 47.5 days old). Despite these differences, the youngest foal (case 1) had larger CMAPs than did cases 2 and 3. Case 2 had a decreased CMAP baseline (amplitude, 2.01 mV; AUC, 9.18 mV ms) compared with age-matched control foals (mean amplitude, 13.04; AUC, 48.3 mV ms), which was similar to results in cases 1 and 3. Furthermore, decrements or increments in CMAP are compared with its initial potentials at various frequency rates.13,15,16 Another limitation of the present study was RNS testing was performed by different chemical restraint methods (sedation versus anesthesia). However, the determination of motor points within the muscle and interpretation of results during RNS evaluation are difficult or obscured by movement in healthy foals despite sedation. Sedation in affected foals was sufficient to prevent movement artifacts, perhaps because of disease. Additionally, anesthetizing foals with progressive generalized neuromuscular dysfunction involving respiratory muscles was a major concern for both clinicians and owners. Studies in humans have shown that inhalation anesthesia has no effect on CMAP amplitude.25 Other electrophysiologic features in people with botulism include normal motor and sensory nerve conduction velocities.16 These procedures were not performed because of client compliance, safety concerns, and prioritizing the most indicated test based on the suspicion of botulism.

Histopathological alterations in muscle specimens from people with botulism are similar to those seen after severing the nerve supply or with other forms of axonal loss.13 These alterations include angular atrophy of all fiber types.13 The establishment of functional neuromuscular junctions through reinnervation takes about 2 weeks and histologically is observed as the absence of a mosaic myofiber pattern.26,27 Evidence of reinnervation in these foals was not observed. Severity and stage of disease could have played a role in the observed histological findings.21 Neighboring surviving motor units may not have been available to reinnervate affected muscle fibers because of the severity of disease in case 2 or more time may have been needed for reinnervation to occur. Muscle specimens were collected 7 days after the onset of clinical signs in case 3, likely not long enough to detect reinnervation. In addition, follow-up electrodiagnostics and muscle biopsies to document reinnervation were not performed.

Differential diagnosis for botulism in any species should include acute inflammatory or immune-mediated polyneuropathies, demyelinating polyneuropathies, tick paralysis, myasthenic disorders, and magnesium toxicity.13,20 Drugs such as aminoglycosides could cause or contribute to neuromuscular blockade.13 Fluids containing magnesium should be avoided in patients with botulism because magnesium may interfere with neuromuscular transmission and worsen clinical signs.13 Muscle weakness is one of the most common clinical signs of botulism and frequently manifests as muscle fasciculations. Therefore, disorders that may present with this sign, such as hyperkalemic periodic paralysis, West Nile virus, equine motor neuron disease (chronic), lead toxicity, and electrolyte derangements, must be ruled out. Other common manifestations of weakness in horses include exercise intolerance, toe drag, low carriage of the head, collapse, and recumbency. Dysphagia and decreased muscle tone (facial, eyelid, tongue, anal, tail) also should prompt the clinician to consider botulism as a possible diagnosis. Muscle weakness, low carriage of the head, dysphagia, and gastrointestinal dysfunction also can be observed in subacute and chronic forms of equine grass sickness.28 An association of the presence of BoNT type C in the ileum of affected horses and disease has been reported.28 Common neonatal diseases in foals may present with weakness, dysphagia, and recumbency and must be ruled out. Therefore, accurate diagnosis is essential for appropriate patient management.

In conclusion, RNS testing proved to be safe, simple, fast, and noninvasive, with acquisition of results as the examiner performs the study. Although RNS did not provide a definitive diagnosis of botulism, it documented a neuromuscular presynaptic disorder of which botulism was the most likely cause. Results supportive of botulism included decreased baseline CMAP (amplitude and AUC) with an incremental response in both amplitude and AUC at high rates of stimulation (50 Hz). This test should be considered as a valuable diagnostic aid in the evaluation of neuromuscular disorders in foals.


a Nicolet Biomedical Inc, Madison, WI

b Disposable concentric needle electrode, VIASYS Healthcare, Madison, WI

c Subdermal Grass electrode, Astro-Med Inc, West Warwick, RI

d Disposable EasyGrip monopolar electrode, Nicolet Biomedical Inc

e Botulism polyvalent antitoxin (A–E), Botulism Laboratory, New Bolton Center, Kennett Square, PA

fClostridium botulinum antitoxin types B and C of equine origin, Veterinary Dynamics Inc, Templeton, CA


Funding provided by the Comparative Gastroenterology Laboratory and gift from anonymous private donor.