Amir Steinman

Pharmacokinetics of tramadol in horses after intravenous, intramuscular and oral administration

Tramadol is a centrally acting analgesic drug that has been used clinically for the last two decades to treat moderate to moderately severe pain in humans. The present study investigated tramadol administration in horses by intravenous, intramuscular, oral as immediate-release and oral as sustained-release dosage-form routes. Seven horses were used in a four-way crossover study design in which racemic tramadol was administered at 2 mg/kg by each route of administration. Altogether, 23 blood samples were collected between 0 and 2880 min. The concentration of tramadol and its M1 metabolite were determined in the obtained plasma samples by use of an LC/MS/MS method and were used for pharmacokinetic calculations. Tramadol clearance, apparent volume of distribution at steady-state, mean residence time (MRT) and half-life after intravenous administration were 26+/-3 mL/min/kg, 2.17+/-0.52 L/kg, 83+/-10 min, and 82+/-10 min, respectively. The MRT and half-life after intramuscular administration were 155+/-23 and 92+/-14 min. The mean absorption time was 72+/-22 min and the bioavailability 111+/-39%. Tramadol was poorly absorbed after oral administration and only 3% of the administered dose was found in systemic circulation. The fate of the tramadol M1 metabolite was also investigated. M1 appeared to be a minor metabolite in horses, which could hardly be detected in plasma samples. The poor bioavailability after oral administration and the short half-life of tramadol may restrict its usefulness in clinical applications.

West Nile virus infection in crocodiles.

To the Editor: Recently West Nile virus (WNV) infection has been reported in three alligators (Alligator sp.) from central Florida (1) and one captive crocodile monitor (Varanus salvadori) with neurologic signs from the District of Columbia and Maryland area (2). These first reports of the virus in American reptiles highlight the possible role of this group of vertebrates in the WNV life cycle. To our knowledge, WNV in a reptile was reported only once before in a serosurvey conducted in Israel from 1965 to 1966, in which 22 reptiles and 96 amphibians were tested for hemagglutination-inhibiting antibodies against several viruses, including WNV; one turtle (Clemmys caspica) was seropositive (3). Experimental infection of the lake frog (Rana ridibunda) with a Russian strain of WNV resulted in high levels of viremia (4). At present, the role of reptiles and amphibians in the life cycle and epidemiology of WNV is not known. We report, for the first time, WNV infection in crocodiles (Crocodylus niloticus). To assess the potential role of crocodiles in the life cycle of WNV in Israel, serum specimens were collected from 20 healthy crocodiles on a commercial farm in the Negev Desert, in southern Israel (31°14′N, 34°19′E). The crocodiles came from two separate breeding farms (32°03′N, 35°26′E and 30°18′N, 35°07′E) in the Syrian-African Rift Valley, which is on the main route of bird migration from Africa to Europe. Five males and 15 females, 1–2.5 years of age, were examined. Blood was withdrawn from the crocodiles’ ventral caudal vein, separated by centrifugation, and kept at –20°C until analyzed. Neutralizing antibody titers were determined against WN-goose-98 (5) and attempts to isolate the virus were performed by using Vero cell culture (6) and by using direct reverse transcription–polymerase chain reaction (RT-PCR) on the serum specimens. To eliminate the possibilities of nonspecific reaction, all serum samples were concurrently tested for the only other flavivirus known to be present in Israel; Israeli turkey meningo-encephalitis virus (ITV) (7). Because ITV does not produce cytopathic effects (CPE) in Vero cells, virus neutralization was conducted on BHK cells for both WNV and ITV by using WN-goose-98 and ITV (vaccine strain). In this case, the virus stocks (10-4.2 50% tissue culture infective dose) were diluted 1:400, and virus neutralization titers were checked 3 days later. Viral RNA was extracted from serum samples with the QIAamp RNA blood kit (QIAGEN, Valencia, CA) , according to the manufacturer’s protocol and resuspended in 30 μl of RNase-free water. The primer pair WN240-Kun848 (respective genome positions 5′: 848 and 1,645) was used to synthesize an 800-bp product in the E gene region (8,9). The resulting DNA fragment was visualized on 1.5% agarose gel stained with ethidium bromide. The seroprevalence rate in the first set of virus neutralization assays in Vero cells was 14/20 (70%, with titers ranging from 1:20 to 1:320 [3x1:20, 3x1:40, 3x1:80, 2x1:160, 3x1:320]). No differences were discernible in either the seroprevalence rate or in the average titers of crocodiles from two different breeding farms. In BHK cells, a similar seroprevalence rate was observed, with titers ranging from 1:40 to 1:1,280 (3x1:40, 2x1:80, 1x1:160, 4x1:320, 3x1:640, 1x1:1280). All serum samples, except one, were <1:10 against ITV virus, which had a titer of 1:640 against WNV and 1:10 against ITV. Viremia was not detected in any of the 20 samples in Vero cell culture or by RT-PCR. These results demonstrate a high rate of infection with WNV in crocodiles in Israel. The crocodiles may have been exposed to the virus during the summer at their present location, since no difference in prevalence was seen between the two groups (which differed only in the farm of origin) and since the younger crocodiles had been hatched in the spring of 2002. Furthermore, a cross-reaction with the other prevalent flavivirus in Israel, ITV, was ruled out. Preliminary results from an equine seroprevalence study (involving 800 horses over a 3-year period) of virus neutralization antibodies to WNV collected during fall 2002, indicate that most horses sampled in Israel’s Arava Valley (a desert in the Syrian-African Rift near the Jordanian and Egyptian borders) and the Gulf of Aqaba/Eilat (30°59′N, 35°18′E to 29°34′N, 34°57′E) (85%, 79/90) were positive (A. Steinman and S. Tal, unpub. data,). WNV was also isolated from mosquitoes in the same region (10). The seroprevalence of WNV antibodies among horses and local birds from the Negev Desert is not known nor is the time when the horses acquired WNV infection. However, the isolation of WNV from mosquitoes (10) and the presence of antibodies to WNV in young crocodiles demonstrate arboviral activity in this region in the summer of 2002, although clinical cases were few. That virus was not isolated from crocodiles in late November (past outbreaks of WNV in Israel mainly occurred between August and October) (6,11). WNV has been endemic in Israel since the early 1950s (12). More recently, in the summer of 2000, an extensive outbreak occurred, affecting hundreds of people (11), dozens of horses (6), and several flocks of geese (5). However, no deaths of crocodiles were reported. This contrasts with the report from Florida (1), where WNV was isolated from dead alligators, and where hundreds of cases of sudden death had been reported in previous years; these deaths are now suspected to result, at least in part, from WNV disease. The role of various reptile species in the epidemiology of other arboviruses such as western equine encephalitis, eastern equine ecephalitis, and Venezuelan equine encephalitis is well documented (13–15). At present, the role of reptiles and amphibians in the life cycle and epidemiology of WNV is not known, and further research is necessary.

Clinical signs of West Nile virus encephalomyelitis in horses during the outbreak in Israel in 2000

Between August and October 2000, 76 horses were reported by veterinary practitioners as having signs of a neurological disorder, varying from an involvement of the spinal cord alone to the entire central nervous system; 15 of the horses died or were euthanased as a result of their grave prognosis or secondary complications. At the same time, an outbreak of West Nile virus infection affected people and birds, principally domestic geese. West Nile virus was isolated from four of the horses with encephalomyelitis and five other horses seroconverted, indicating that the virus was the probable cause of the outbreak in horses. Three of the cases from which the virus was isolated are described briefly and one case is described in detail. This horse behaved abnormally and had general proprioceptive deficits in all four limbs. Its neurological condition deteriorated after two days and severe inspiratory dyspnoea due to a failure to abduct the arytenoids necessitated a tracheostomy. It died on the fourth day and histological lesions were observed in the brain stem and grey matter of the spinal cord.

Quantitative analysis of levels of serum immunoglobulin G against botulinum neurotoxin type D and association with protection in natural outbreaks of cattle botulism.

ABSTRACT The recent outbreaks of cattle botulism in vaccinated Israeli dairy cattle prompted us to determine vaccine efficacy and reasons for vaccine failure. Analysis of clinical signs, feeding practice, vaccination history, and epidemic curves enabled us to define a study population in two outbreaks, where high doses of Clostridium botulinum neurotoxin type D (BoNT/D) were evenly consumed by the affected animal groups. Attack rates among unvaccinated 6- to 24-month-old heifers were 96% (55/57) and 85% (53/62). The attack rates in vaccinated parity 1, 2, and ≥3 cows were 40.4% (21/52), 14.3% (4/28), and 5.6% (3/54), respectively. Vaccine efficacies for these cow groups were 52.5%, 83.2%, and 93.4%, respectively. In younger, unvaccinated 2- to 6-month-old calves, presumably protected by maternal antibodies, the attack rate was 24% (17/71). These differences correlated with significant differences in levels of specific anti-BoNT/D antibody in serum by an enzyme-linked immunosorbent assay (ELISA). The ELISA performance for predicting protection was analyzed by receiver operating characteristic analysis and was found to be highly significant, with an area under the curve of 0.941 (standard error, 0.034; 95% confidence interval, 0.875 to 1.008; P < 0.000). No animals with serum ELISA unit levels above 0.33 were affected in these exposed groups. At this cutoff level, the specificity of the ELISA was 100%, sensitivity was 67%, and accuracy was 92%. We concluded that botulinum toxoids can confer adequate protection against natural exposure to lethal doses of BoNT/D; however, the vaccination protocols should be optimized. Our in-house ELISA system will enable us to optimize vaccination protocols in the animal population.

Efficacy of antiepileptic tetramethylcyclopropyl analogues of valproic acid amides in a rat model of neuropathic pain

Antiepileptic drugs (AEDs) are widely utilized in the management of neuropathic pain. The AED valproic acid (VPA) holds out particular promise as it engages a variety of different anticonvulsant mechanisms simultaneously. However, the clinical use of VPA is limited by two rare but potentially life-threatening side effects: teratogenicity and hepatotoxicity. We have synthesized several tetramethylcyclopropyl analogues of VPA amides that are non-teratogenic, and are likely to be non-hepatotoxic, and that exhibit good antiepileptic efficacy. In the present study we have assessed the antiallodynic activity of these compounds in comparison to VPA and gabapentin (GBP) using the rat spinal nerve ligation (SNL) model of neuropathic pain. TMCA (2,2,3,3-tetramethylcyclopropanecarboxylic acid, 100-250 mg/kg), TMCD (2,2,3,3-tetramethylcyclopropanecarboxamide, 40-150 mg/kg), MTMCD (N-methyl-TMCD, 20-100 mg/kg), and TMCU (2,2,3,3-tetramethylcyclopropanecarbonylurea, 40-240 mg/kg) all showed dose-related reversal of tactile allodynia, with ED(50) values of 181, 85, 41, and 171 mg/kg i.p., respectively. All were more potent than VPA (ED(50)=269 mg/kg). An antiallodynic effect was obtained for TMCD, MTMCD and TMCU at plasma concentrations as low as 23, 6 and 22 mg/L, respectively. MTMCD was found to be non-toxic, non-sedative and equipotent to gabapentin, currently the leading AED in neuropathic pain treatment. Tetramethylcyclopropyl analogues of VPA amides have potential to become a new series of drugs for neuropathic pain treatment.

Epidemiological study of Culicoides hypersensitivity in horses in Israel.

The variations with breed, gender, age and coat colour in the prevalence of Culicoides hypersensitivity were studied in 408 horses on 18 farms in Israel. Data were gathered by means of questionnaires and the diagnoses were confirmed by direct physical examinations. The prevalence of Culicoides hypersensitivity was 28 per cent. The disease was rare on farms more than 800 m above sea level but was more prevalent at lower altitudes. The most important factors affecting the prevalence of Culicoides hypersensitivity were the farm, breed and age, but gender and colour were not significantly correlated with its prevalence.

Demographic and environmental risk factors for infection by Theileria equi in 590 horses in Israel.

The prevalence of Theileria equi infection as well as the environmental and demographic risk factors for infection was studied in 590 healthy horses from 46 farms in Israel. The prevalence of T. equi DNA was assessed using a polymerase chain reaction for a segment of the Theileria 18S rRNA gene. The overall prevalence was 26.4% (156/590). There was a significant geographical variation in the prevalence of T. equi infection, ranging from 9.3% (25/270) in the central lowlands to 81.7% (49/60) in the Golan Heights. The prevalence of T. equi infection was found to be significantly associated with management types with more horses with access to pasture being positive. Breed was identified as a risk factor for T. equi infection in a univariate analysis with relatively high infection rates in the Quarter horse and local breeds (41.1% and 36.3% respectively), while ponies and Arabian horses had a relatively low prevalence (10% and 9.1%, respectively). However, since a correlation between geographic location and breed was found, it is difficult to draw definite conclusions regarding this risk factor. Age and gender were not found as risk factors for T. equi infection in this study. The environmental variables that were significantly associated with positivity were relative humidity and minimum land surface temperature at day which both showed negative correlation with T. equi prevalence. In conclusion, Israel was found to be enzootic for T. equi infection, as indicated by the high sub-clinical infection rate, which differed between geographical areas.

Clonal transmission of a rare methicillin-resistant Staphylococcus aureus genotype between horses and staff at a veterinary teaching hospital.

Methicillin-resistant Staphylococcus aureus (MRSA) infection or colonization has become a serious emerging condition in equine hospitals. Following the detection of MRSA in asymptomatic hospitalized horses and in two horses with post-operative wound infections, an investigation was conducted. Twelve of 84 horses (14.3%) and 16 of 139 personnel (11.5%) were MRSA carriers. The profile of the dominant MRSA strain common to horses and staff was multi-drug-resistant, spa-type t535, SCCmec type V, pvl-negative. MLST of a representative isolate yielded sequence type (ST) 5. The risk of MRSA carriage among veterinary personnel was greater in equine veterinarians and full-time technicians in comparison to part-time technicians and to other personnel not working with horses. Strict infection control measures were implemented, horses infected or colonized with MRSA were isolated and decolonization of personnel was attempted. Six months after the intervention, the large animal department personnel and hospitalized horses were all MRSA-negative and the decolonization was considered successful. This outbreak, caused by a rare MRSA strain and involving both hospitalized horses and personnel, further demonstrates the ability of MRSA to spread between animals and humans and emphasizes the importance of infection control measures to decrease the risk for MRSA colonization and infection of both horses and personnel.

Toxicological, bacteriological and serological diagnosis of botulism in a dog

neostigmine test failed to produce any subsequent evident clinical improvement. The presence of BoNT/C was identified in the dog’s serum by the mouse inoculation bioassay (Elad and others 2004), and toxigenic C botulinum serotype C bacteria were found in the faeces of the dog; both samples were collected on the day of admission. The dog was hospitalised and received supportive treatment that included the administration of intravenous fluids; a urinary catheter was placed due to the lack of voluntary voiding, and the dog was turned from side to side every four hours. Tachypnoea developed on the second day of hospitalisation, and blood gas analysis revealed mild hypoxia (oxygen partial pressure of 81·7 mmHg with oxygen saturation of 95 per cent) and normal carbon dioxide partial pressure. The dog was given oxygen via a nasal catheter. On the third day of hospitalisation, a serous discharge was observed from the dog’s eyes. Fluorescein staining revealed small and superficial corneal ulcers, which had developed due to reduced blinking. Topical antibiotics (5 per cent chloramphenicol) and a local antiseptic (benzalkonium chloride) were administered. The dog was discharged from the hospital for further care at home six days after admission. At this time, the dog was in sternal recumbency, and its withdrawal reflexes were present but weak. Ten days after discharge, the dog was re-examined, and voluntary movements were present in all four legs. Neurological examination revealed the dog to be quadriparetic, with a normal withdrawal reflex in all four legs. The sciatic reflex was present but was weak in both hindlegs. Twenty days after the onset of the clinical signs, the dog was able to walk and apparently fully recovered, and showed no neurological deficits. The level of specific anti-BoNT/C antibodies in the dog’s serum was determined using an ELISA as described previously in human beings with minor modifications (Montgomery and others 2000). The wells of an immuno-ELISA plate (F96 Maxisorb; Nunc) were filled with 100 μl/well of 1 μg/ml

Surgical management of sand colic impactions in horses: a retrospective study of 41 cases

OBJECTIVE A retrospective review of the medical records of 41 horses requiring abdominal surgery for sand colic. RESULTS The diagnosis of sand colic was made when sand was found to be the cause of impaction of the gastrointestinal tract during surgical exploration. The most common clinical signs at presentation were abdominal pain, abdominal distension and diarrhoea. A statistically significant association was found between the respiratory rate on arrival and short-term survival. Sand impaction at multiple locations was detected in one-third of the horses. Concurrent pathology was detected in half of the horses. Four horses were euthanased during surgery; of those that recovered from surgery, 35/37 (95%) were discharged from hospital. Short- and long-term complications were similar to those previously reported. Long-term (1 year) survival of the horses discharged was 100%. CONCLUSION The good prognosis for horses undergoing surgery for the treatment of sand impaction supports early surgical intervention in cases where large amounts of sand are suspected.