Effects of amitraz on nerve conduction and neuromuscular transmission in anaesthetised dogs

Ataxia is an occasional side effect of amitraz when used as a wash to treat dogs with demodectic mange. In the present study, successive doses of 0.5, 2, 5 and 10 mg kg-1 amitraz were given intravenously at intervals of nine minutes to thiopentone/methoxyflurane/oxygen anaesthetised dogs. The amplitude of the evoked muscle action potential to electrical stimulation of the right ulnar nerve and the muscle refractory period were unchanged by increasing doses of amitraz but there was a progressive and significant decrease in nerve conduction velocity. The minimum recorded nerve conduction velocity (50.7 +/- 1.5 m s-1) was still within an adequate range. From these results it appears that the ataxia following amitraz is unlikely to be attributable to peripheral mechanisms. The concurrent amitraz-induced rise in mean arterial pressure and bradycardia was consistent with previous findings in which alpha 2-adrenoceptors were shown to be the major mediators.     

Type C botulism in cattle being fed ensiled poultry litter

A botulinum toxin from ensiled poultry litter which caused a major outbreak of bovine botulism was characterised as type C1. The litter produced transient ataxia when fed to two experimental calves and the clinical signs were accompanied by a transient appearance of serum toxin. Type C1 toxin was demonstrated in muscle tissues which had been taken during the outbreak from an affected animal with high circulating serum toxin, and held frozen for seven months. Clostridium botulinum type C organisms were demonstrated in faeces from another affected animal and also in kidney tissue from a third animal. These observations have implications for the diagnosis and management of future outbreaks of botulism and for the potential health risk from the meat of affected animals.     

Failure of neuromuscular transmission after complete nerve section in the dog.

After the ulnar nerve was surgically transected, nerve conduction velocity in the distal segment and the evoked motor unit potential (EMUP) from the interosseous muscle were recorded until neuromuscular transmission failed. In five of the six dogs in the experiment, functional conduction ceased by 4.8 days, as determined by failure of both proximal and distal stimulation of the distal segment to evoke a muscle response. From the time of section until neuromuscular failure, the nerve conduction velocity remained unchanged. The amplitude of the EMUP from the interosseous muscle, however, decreased markedly during this time. Changes in other features of the EMUP are also presented. Fibrillation (denervation) potentials did not appear until the first day that muscle response could not be detected by stimulating the nerve. These data present a principle which would enable a determination of relative extent and progression of peripheral nerve damage.     

A new method for recording H-reflexes from the plantar muscles of dogs

H-reflexes were recorded consistently from the plantar muscles of pentobarbitone-anaesthetised dogs following supramaximal stimulation of the caudal cutaneous sural nerve (CCSN). As the amplitude, shape and latency of successive H-reflex potentials fluctuated from trial to trial, 16 consecutive sweeps were averaged to quantify the response. The averaged H-reflex had an amplitude of 1–6 ± 0–9 mV (mean ± SD] and a latency of 20 ± 2 ms. The CCSN-evoked H-reflex was recorded together with the CCSN-evoked compound muscle action potential (SurCMAP), which had a shorter latency (6 ± 1 ms) but comparable size (1–9 ± 1–3 mV). H-reflex afferents in the CCSN had overlapping but slightly higher electrical thresholds than plantar motoneurone axons. A ‘pure’ H-reflex could be obtained by injecting local anaesthetic below the site of nerve stimulation. Halothane/nitrous oxide anaesthesia substantially reduced the amplitude of H-reflex potentials in a reversible fashion.     

Motor nerve conduction parameters in the cat

The electrophysiological characteristics of motor conduction in normal cats have been determined using an alligator clip as a surface electrode to record the compound muscle action potential (CMAP) following stimulation of the tibial, ulnar and fibular nerves. Data on nerve conduction velocity, residual latency and the amplitude and area of the CMAP have been determined using a computerised electromyography unit. Motor nerve conduction was substantially faster in cats than dogs and the site of stimulation had less effect on the size and area of the CMAP. Although a small decline in the amplitude of successive CMAPs was observed following repetitive stimulation of the tibial and ulnar nerve at 20 Hz, the decrement was less marked than in the dog.     

Fatal Clostridium botulinum toxicosis in eleven Holstein cattle fed round bale barley haylage.

Twenty-two lactating Holstein cattle in Tennessee had clinical signs of intoxication with preformed Clostridium botulinum toxin. These signs included weakness, paralysis of the tongue and chest muscles, abdominal breathing, and, in 11 of the 22 cows, death. Differential diagnoses included hypocalcemia, hypomagnesemia, carbohydrate overload, and several toxicoses including mycotoxin, lead, nitrate, organophosphate, atropine or atropine-like alkaloid, and botulism. A diagnosis of botulism by the ingestion of preformed C. botulinum type B toxin was made by eliminating these other diseases, by finding C. botulinum type B spores in 3 bales of round bale barley haylage fed to these cattle, and by isolating preformed type B toxin from 1 of the 3 bales. Confirmation of the toxin type was made by demonstrating mouse lethality by intraperitoneal injection of specimen extracts with neutralization by C. botulinum type B antitoxin. The haylage, harvested green and encased in black plastic bags to facilitate fermentation, was presumably contaminated by the botulinum toxin when fermentation failed to produce enough acid to lower the pH to 4.5, the pH below which C. botulinum growth is inhibited. Farmers and ranchers who use round hay balers to produce haylage should be alert to this potential problem.     

Partial tarsal arthrodesis and botulinum toxin A injection for correction of tarsal arthrogryposis in a cat

An 11-week-old, domestic shorthair cat was presented for evaluation of a congenital right tarsal deformity. The cat was non-weightbearing lame on the right hindlimb. There was severe tarsal hyperextension and concurrent spasticity/contracture/shortening of the gastrocnemius muscle-tendon. The cat was otherwise healthy. Radiographically there was luxation of the proximal intertarsal joint and talocrural hyperextension. Treatment with 10 mu/kg botulinum toxin A (Botox; Allergan) intramuscular injections directly into the gastrocnemius muscle and passive physiotherapy marginally improved the range of motion in the talocrural joint. Definitive surgical treatment with partial tarsal arthodesis produced an excellent clinical outcome. This case report demonstrates the successful treatment of a severe congenital tarsal abnormality and also documents the first clinical use of botulinum toxin in a feline patient.     

Usefulness of combined electrophysiological examinations for detection of neural dysfunction in cats with lumbar hematomyelia

We conducted combined electrophysiological examinations including F-wave, motor nerve conduction velocity (MNCV), spinal cord-evoked potential (SCEP), and needle electromyography (EMG) in two cats involved in traffic accidents that consequently developed hind limb paralysis caused by lumbar hematomyelia. F-wave could no longer be elicited within 3 days after the accident, and the MNCV and compound muscle action potential (CMAP) amplitude decreased in a time-dependent manner, with CMAP no longer being evoked after 7 or 8 days. EMG showed abnormalities such as fibrillation and positive sharp waves after 6 to 8 days. These results suggest that such combined electrophysiological examinations may provide objective, quantitative data for motor nerve dysfunction in cats with lumbar hematomyelia.     

Electrophysiologic Assessment of Acute Polyradiculoneuropathy in Dogs: Comparison with Guillain-Barré Syndrome in People

Electrophysiologic investigations of motor and sensory nerve as well as ventral nerve root function were performed on 12 dogs with suspected acute canine polyradiculoneuropathy (ACP) at different stages and with different severity of disease. The most reliable electrophysiologic indicators of ACP were electromyographic changes (occurring in 100% of affected dogs), significantly decreased compound muscle action potential amplitudes (in 75, 90, and 100% of affected dogs at all sites along the sciatic/tibial, radial, and ulnar nerves, respectively), increased minimum F-wave latencies (67%), increased F ratios (92%), and decreased F-wave amplitudes (67%). These findings suggest that ACP represents a peripheral motor axonopathy, with demyelination and axonal involvement also occurring in ventral nerve roots. Evidence of peripheral demyelination was present in some dogs although it was overshadowed by the prominent axonopathy. ACP more closely resembles the acute axonal or intermediate forms of Guillain-Barré syndrome in people.     

The occurrence of Clostridium botulinum type E in Finnish trout farms and the prevention of toxin formation in fresh-salted vacuum-packed trout fillets.

The occurrence of C. botulinum on two Finnish rainbow trout farms were studied. C. botulinum type E toxin was detected from samples of fish intestines in 10% and 4% of the samples and in 0% and 100% of dam bottom sediments, respectively. The toxin formation of inoculated C. botulinum type E in three different brands of commercial fresh-salted vacuum-packed trout fillets was also investigated. In the brand with a salt concentration of 2.7% (a w = 0.96) and no nitrate, the toxin was formed in two weeks at 10 and 20 degrees C. If the pH of the product was lowed to 4.90 and NaCl concentration increased to 5.67% (a w = 0.94%) no toxin was formed even without the use of nitrate. By adding 0.1% of sodium nitrate to the curing solution (0.046% nitrate in the product) the toxin formation was brought to an end. According to the results of the study C. botulinum type E presents a potential health risk for man as well as for fish also in Finland. Trout products must be manufactured and stored in such a way that the possibility of toxin formation is eliminated.     

Endocytosis of Clostridium botulinum type B neurotoxin into rat brain synaptosomes

Clostridium botulinum type B neurotoxin cleaves VAMP (vesicle-associated membrane protein)/synaptobrevin into two fragments, which results in inhibition of neurotransmitter release. The induced fragment did not react to the antibody raised against the synthetic peptide of the amino-terminal 20 residues of VAMP-2, suggesting that the toxin treatment has caused antigenical alteration in the amino-terminal region of VAMP-2. In rat brain synaptosomes, type B neurotoxin was reduced presumably with sulfhydryls in the membrane and detected in the synaptic vesicle fraction which involved the degradation of VAMP-2 and the inhibition of neurotransmitter release. The light chain in a free form was present in the cytosol fraction. These findings suggest a possibility that type B neurotoxin endocytoses into synaptic vesicles by the recycling pathway and the light chain is penetrable through synaptic vesicle membrane. However, the amount of type B neurotoxin entrapped into synaptic vesicles appears to be extremely small, which may be attributed to a lower sensitivity of the toxin to brain synaptosomes than to peripheral nerve endings.     

Proteases of Clostridium botulinum. VI. The role of trypsin, Clostridium botulinum proteases and protease inhibitors in the formation and activation of toxin in growing cultures of Clostridium botulinum

In this study the influence of bovine serum protease inhibitors, trypsin and proteases produced by different types of Clostridium botulinum has been investigated. Trypsin and botulinum proteases had the capability of increasing the toxicity in growing cultures in Clostridium botulinum types A, B and E. Trypsin increased the toxin level to a greater extent than proteases from Clostridium botulinum types A, B, C and F. Protease inhibitors did not influence the toxin formation to any extent compared with the controls. The combined effects of proteases and protease inhibitors on the development of toxin in Clostridium botulinum type B were also investigated by adding proteases and protease inhibitors to the same culture at different time intervals. Protease inhibitors did not reduce the toxicity of the cultures as compared to the controls. Altogether a complex relationship seems to exist between protoxin, toxin, proteases and inhibitors in the culture, and the order and time sequence of addition seem to be of importance. The results obtained in this investigation indicate that proteases of Clostridium botulinum play a part in the formation and/or activation of toxin in growing cultures of proteolytic strains such as Clostridium botulinum types A and B. As to the activation of protoxin and progenitor toxin produced by non-proteolytic Clostridium botulinum types B and E, botulinum proteases showed a marked capability of increasing the toxicity in these cultures. Trypsinization may be valuable for the detection of Clostridium botulinum types A and B in foods, as well as for type E, where it is commonly used.     

A new method for recording and analysing evoked motor potentials from dogs

Normal values for motor conduction in the tibial, ulnar and fibular nerves of dogs have been determined using a new method for recording and analysing evoked motor potentials. The use of an alligator clip as a roving surface electrode for recording, and a personal computer to analyse the evoked potentials has facilitated faster and more reproducible motor conduction studies. Compound muscle action potential (CMAP) and motor nerve conduction velocity data are in good agreement with previous studies. Normal values for CMAP area, residual latency and proximal to distal ratios for CMAP area and amplitude are presented for the first time.     

Myasthenia gravis in two cats

A case of the autoimmune form of myasthenia gravis and a case of what is probably a congenital form of myasthenia gravis were diagnosed in 2 unrelated cats. Neuromuscular weakness exacerbated by exercise was a prominent feature in both cats. Weakness was eliminated temporarily by administration of anticholinesterase drugs. Serum autoantibodies to acetylcholine receptors of skeletal muscle were present in the 1st cat and were not detected in the 2nd cat. A characteristic decrement in the amplitude of the compound muscle action potential during repetitive stimulation of the motor nerve was elicited in the 2nd cat. There was marked electromyographic improvement in response to anticholinesterase drugs. Electromyography was not performed in the 1st cat.     

Determination of the median toxic dose of type C botulinum toxin in lactating dairy cows.

Because of the difficulty in identifying botulinum toxin in cattle, it is hypothesized that cattle are sensitive to levels of toxin below the detection limits of current diagnostic techniques (the mouse protection bioassay and the immunostick enzyme-linked immunosorbent assay [ELISA] for type C botulinum toxin). Using an up-down method for toxicologic testing, the median toxic dose (MTD50) for cattle was determined. Four lactating Holstein cows were dosed at 0.125 or 0.25 ng/kg with Clostridium botulinum type C toxin and failed to develop clinical signs of botulism during the 7-day observation period. Three cows given 0.50 ng/kg of toxin developed clinical signs of botulism. From these results, the MTD50 was calculated at 0.388 ng/kg (3.88 mouse lethal doses/kg) using the trim-logit method. These results suggest that cattle are 12.88 times more sensitive to type C botulinum toxin than a mouse on a per kilogram weight basis. The mouse protection bioassay and the immunostick ELISA for type C botulinum toxin failed to identify the presence of the toxin in the serum, blood, and milk samples taken from all 7 animals.     

Susceptibility of mink to Clostridium botulinum type E toxin

Experiments aiming at elucidation of the toxicity of Clostridium botulinum type E for mink are described. The observations indicate that amounts in the order of 2 x10^s intraperitoneal MLD (mice) or approximately 200 MLD per g of type E toxin will kill a mink after oral administration. The symptoms observed in the animals were atypical as there was an unusually short period between administration of the toxin and the onset of symptoms and deaths of the animals. Similar results were obtained when Clostridium botulinum type E toxin was fed to Swiss mice. When mice were protected by subcutaneous injections of type E antitoxin prior to feeding the animals survived without showing any symptoms.Subcutaneous injection of type E toxin in amounts of the order of 2 x10^s intraperitoneal MLD (mice) killed mink, and typical symptoms of botulism were observed. This quantity corresponds to ap-proximately 2 intraperitoneal MLD (mice) per g.Comparison is made with previous observations obtained in similar experiments made with Clostridium botulinum type C toxin. It is shown that mink arc substantially less susceptible to type E than to type C toxin when the toxins are given by mouth. On this basis previous results in reports on outbreaks of botulism in mink caused by Clostridium botulinum type E may be regarded as questionable.     

The use of botulinum toxin A for treatment of possible essential blepharospasm in a dog

A 3-year-old Great Dane with bilateral possible essential blepharospasm resulting in ocular complications is described. Conservative treatment was not successful and the disease was treated with local injections of botulinum toxin A into the orbicularis oculi muscle. Blepharospasm disappeared completely 5 to 6 days after injection and did not reappear until 3 to 4 months later, at which time the injection was repeated. After several treatments over a period of more than 3 years no side effects have occured. Botulinum toxin A appeared to be effective in the treatment of essential blepharospasm in this dog.     

Type C botulism in young dogs

A diffuse lower motor neurone paralysis developed in a 6-month-old male Australian cattle dog pup 4 days after it had eaten the carcase of a rotting duck in Centennial Park, Sydney. Two other dogs which ate smaller portions of the same carcase were less severely affected. Clostridium botulinum type C was isolated from and C. botulinum type C toxin was detected in faeces from the severely affected dog. The serum contained 25 LD50 of toxin/ml. The high C. botulinum count and toxin level in the faeces declined progressively during the ensuing weeks, but 114 days after ingesting the carcase C. botulinum type C was still present in faeces and a low toxin titre persisted. Soil, mud and water samples in the area of the duck ponds in the park contained C. botulinum type C spores. Spores and high toxin titres were also found in the intestine of the carcases of 2 birds in the area.     

Spinal Nerve Root Origin of the Median, Ulnar and Musculocutaneous Nerves and their Muscle Nerve Branches to the Canine Forelimb

The contribution individual ventral spinal nerve roots made to the canine median nerve, ulnar nerve, musculocutaneous nerve, and their muscle nerve branches was determined electrophysiologically. Each spinal nerve root was sequentially stimulated. Utilizing quantitative signal averaging techniques, the evoked potential was measured at each tested peripheral nerve. Evoked potential to the median nerve originated from the seventh cervical spinal root (C7) through the second thoracic spinal root (T2) with most input from C8 and T1. The ulnar nerve received evoked potential from C7-T2. Although T1 provided the major input to both the median and ulnar nerves, the relative contribution of T1 was greater in the ulnar nerve. The musculocutaneous nerve received input from ventral spinal roots C6-T1 with C6 and C7 providing most of the evoked potential. The ventral spinal roots which supplied the bulk of the evoked potential to a particular muscle nerve were consistent between individual dogs. Variation of evoked potential input was greatest from spinal roots which supplied less than 10% of the total potential.