Clinical Research Abstracts of the British Equine Veterinary Association Congress 2015.

REASONS FOR PERFORMING STUDY: Transcranial magnetic stimulation (TMS) is an electrophysiological technique used to elicit motor evoked potentials (MMEPs) to evaluate the functional integrity of the descending motor fibres in the spinal cord. Successful application of the technique was reported in horses with spinal cord compression. However, limited data are available on the correlation of TMS with histopathological changes. OBJECTIVES: To determine sensitivity of TMS for assessing the integrity of the spinal cord in horses with compressive lesions of the spinal cord. STUDY DESIGN: Case series. METHODS: The study was conducted on 6 horses with spinal ataxia grade III-V/V admitted to the Faculty of Veterinary Medicine, Ghent University. The horses underwent TMS, radiography of the cervical or thoracolumbar vertebral column, and myelography (4/6). All horses were subjected to euthanasia and the spinal cord examined histopathologically. RESULTS: In 5/6 horses MMEPs with abnormal onset latencies in both extensor carpi radialis muscles and cranial tibialis muscles were recorded, suggesting a cervical spinal cord lesion. Radiography revealed cervical vertebral malformation (4/5) with cervical vertebral canal stenosis (3/5) and tumour/osteomyelitis (1/5). In 1/6 horse MMEPs with normal onset latencies in extensor carpi radialis muscles and prolonged onset latencies in tibialis cranialis muscles were recorded, suggesting a thoracolumbar spinal cord lesion. Radiography revealed deformation of the 7th and 8th thoracic vertebrae. Myelography showed reduction in dural diameter and dorsal contrast column (4/4). Histopathological examination of the spinal cord confirmed compressive type lesions in all 6 horses with degenerative changes in the white matter of all funiculi, ballooning of myelin sheets, swollen axons, loss of axons and astroglial activation. CONCLUSIONS: In this case series abnormal function of descending motor pathways as registered by TMS showed 100% sensitivity with the histopathological characteristics of compressive lesions in the spinal cord, but the number of horses is limited and further research is warranted. Ethical animal research: Research ethics committee oversight not currently required by this conference: the study was performed on material collected during clinical procedures. Explicit owner informed consent for participation in this study was not stated. SOURCE OF FUNDING: Not applicable. Competing interests: None declared.

Acute and long-term cardiomyopathy and delayed neurotoxicity after accidental lasalocid poisoning in horses.

BACKGROUND: Horses are extremely susceptible to ionophore intoxication. Although numerous reports are available regarding monensin, little is known about lasalocid toxicity. OBJECTIVES: To describe accidental lasalocid poisoning on a farm in Belgium. ANIMALS: Eighty-one horses, of which 14 demonstrated clinical signs from day 0-21 after being fed a new concentrate batch. One horse died on day 20 and another on day 27. METHODS: The most severe cases (n = 7), admitted to the clinic on day 29-46, underwent cardiac examination and blood biochemical analysis, including determination of plasma cardiac troponin I (cTnI) at admission and during follow-up. On day 57-70, cardiac examination, cTnI determination or both were undertaken on 72 remaining horses. RESULTS: Short-term effects of lasalocid intoxication included inappetance, lethargy, sweating, and muscular weakness. All 7 horses admitted to the clinic demonstrated signs of myocardial degeneration such as increased cTnI, dysrhythmia and reduced myocardial contractility. Four horses developed ataxia on day 40-50. Five horses died or were euthanized on day 30-370, 2 horses recovered fully and returned to previous athletic use. None of the 72 remaining horses exhibited clinical signs between day 57-70, but 34 had dysrhythmia and 13 had increased cTnI concentrations. After a period of rest, all horses returned to their previous work. Lasalocid was detected in hepatic tissue of 2 necropsied horses. CONCLUSIONS AND CLINICAL IMPORTANCE: Lasalocid intoxication induced myocardial and neurological damage. Although uncommon, this should be included as differential diagnosis for unexplained inappetance, signs of depression, cardiomyopathy, and ataxia in horses.

Two atypical cases of granulosa cell tumours in Belgian Blue heifers.

This paper describes two atypical cases of granulosa cell tumours (GCTs) in Belgian Blue heifers. In the first case, an 18-month-old heifer died after ovariectomy, and a granulosa-theca cell tumour was diagnosed post-mortem. The second heifer (31 months of age) was near term and was successfully treated surgically for a malignant granulosa cell tumour. Unfortunately, no further reproductive data are available, as the heifer was sold for slaughter. This is the first report describing GCTs in Belgian Blue beef cattle.

Uterine fibrosarcoma in a Warmblood mare.

This paper describes a case of uterine fibrosarcoma in an 18-year-old Warmblood mare. The mare had exhibited bloody fluid accumulation inside the uterus and vaginal haemorrhagic discharge since the previous foaling. The mare was euthanized, and on pathological examination, in addition to the uterine neoplasia, multiple metastases were found in the lungs, liver and spleen. The histological and immunohistochemical examination determined that the tumour was a fibrosarcoma. To our knowledge, this is the first paper to describe a uterine fibrosarcoma in a mare.

Expression of P-glycoprotein in the intestinal epithelium of dogs with lymphoplasmacytic enteritis.

Inflammatory bowel disease (IBD) is an idiopathic chronic inflammatory disease of the stomach, the small intestine and/or the large intestine. Loss of integrity of the intestinal barrier may be an important factor in the pathogenesis of IBD. In dogs, lymphoplasmacytic enteritis (LPE) is one of the recognized forms of IBD. P-glycoprotein (P-gp) is a membrane-bound efflux pump constituting an important component of the intestinal barrier. Changes in P-gp expression at the level of the intestinal barrier may be important in the pathogenesis of canine LPE, as this may lead to variable protection against xenobiotics and bacterial products in the intestine. The aim of the present study was to evaluate the expression of epithelial P-gp in the intestine in dogs with LPE compared with disease-free animals. Formalin-fixed intestinal biopsy samples from 57 dogs with histopathological evidence of LPE were immunolabelled with anti-P-gp antibodies (C494 and C219). Endoscopic biopsy samples of the duodenum and colon from 16 healthy beagles were used as controls. None of the control dogs had P-gp expression in the apical membrane of duodenal enterocytes, but all had P-gp labelling at the colonic epithelial surface. Twenty out of 57 dogs with LPE had P-gp expression at the apical surface membrane of villus epithelial cells in the duodenum, jejunum and/or ileum. Six out of 16 colonic samples from dogs with LPE had decreased P-gp expression at the epithelial surface compared with controls. It is unclear whether these changes in P-gp expression in dogs with LPE are a cause or a consequence of the inflammation. The observed changes could affect bioavailability of therapeutic drugs used in LPE.

Expression of multidrug resistance-associated P-glycoprotein in feline tumours.

Permeability glycoprotein (P-gp) is a membrane-bound efflux pump that mediates the active transmembrane transport of a variety of substrates. Several studies in human and canine normal and neoplastic tissues indicate that P-gp is involved in resistance to chemotherapy and in the development of multidrug resistance (MDR). The purpose of this study was to evaluate P-gp expression with the monoclonal antibody C494 in common feline tumours from 88 patients not pretreated with chemotherapy. Tumours arising from tissues with intrinsic P-gp expression consistently showed positive labelling for P-gp in a cellular pattern identical to that described for the normal feline tissues. Both P-gp positive and negative tumour cells, however, were found in mammary gland tumours, lymphomas, mastocytomas and squamous cell carcinomas. Feline mammary gland tumours in particular showed strong membranous P-gp labelling, especially in areas with infiltrative growth and atypical cells, although this was not correlated with the grade of malignancy. These findings might have implications for future response to chemotherapy.

Developing a safe antifungal treatment protocol to eliminate Batrachochytrium dendrobatidis from amphibians.

Batrachochytrium dendrobatidis is one of the most pathogenic microorganisms affecting amphibians in both captivity and in nature. The establishment of B. dendrobatidis free, stable, amphibian captive breeding colonies is one of the emergency measures that is being taken to save threatened amphibian species from extinction. For this purpose, in vitro antifungal susceptibility testing and the development of efficient and safe treatment protocols are required. In this study, we evaluated the use of amphotericin B and voriconazole to treat chytridiomycosis in amphibians. The concentration at which the growth of five tested B. dendrobatidis strains was inhibited was 0.8 µg/ml for amphotericin B and 0.0125 µg/ml for voriconazole. To completely eliminate a mixture of sporangia and zoospores of strain IA042 required 48 h of exposure to 8 µg/ml of amphotericin B or 10 days to 1.25 µg/ml of voriconazole. Zoospores were killed within 0.5 h by 0.8 µg/ml of amphotericin B, but even after 24 h exposure to 1.25 µg/ml of voriconazole they remained viable. Amphotericin B was acutely toxic for Alytes muletensis tadpoles at 8 µg/ml, whereas toxic side effects were not noticed during a seven-day exposure to voriconazole at concentrations as high as 12.5 µg/ml. The voriconazole concentrations remained stable in water during this exposure period. On the basis of this data, experimentally inoculated postmetamorphic Alytes cisternasii were sprayed once daily for 7 days with a 1.25 µg/ml solution of voriconazole in water which eliminated the B. dendrobatidis infection from all treated animals. Finally, treatment of a naturally infected colony of poison dart frogs (Dendrobatidae) using this protocol, combined with environmental disinfection, cleared the infection from the colony.

Haemorrhagic diathesis in neonatal calves: an emerging syndrome in Europe.

In 2008 and 2009 a large number of cases of haemorrhagic diathesis (HD) in neonatal calves were reported in different European countries. In Flanders, 84 cases of neonatal HD in 30 herds were reported in this period. The disease typically affects calves younger than 1 month old from different breed and gender. Prominent clinical signs are cutaneous bleeding, petechiae on all mucosae, melena and often high fever. Early in the disease, the mental state of the animals is uncompromised. The typical haematological finding is pancytopenia, with severe to complete thrombocytopenia being the cause of the increased susceptibility to bleeding. In seven of the affected herds blood samples of calves of the same age group as the clinical case were collected and on six of those farms at least one subclinical case could be identified. Necropsy findings were generalized petechiae, ecchymoses or haemorrhages and variable lymphadenopathy. Histopathology of haemorrhagic lesions revealed multifocal extravasation of red blood cells (haemorrhage) with preservation of tissue architecture and absence of other abnormalities. Total bone marrow aplasia and depletion of all lymphoid tissue was the most prominent finding on histology. Activated macrophages and haemophagocytosis were seen on bone marrow cytology from two live calves. Polymerase chain reaction for bovine viral diarrhoea virus, bluetongue and epizootic haemorrhagic disease virus was negative. Several attempts to isolate a viral agent were unsuccessful.

Inspiratory stridor secondary to palatolingual myokymia in a Maltese dog.

A nine-year-old male Maltese dog was presented with an eight-month history of inspiratory stridor leading to exertional dyspnoea and cyanosis. Myokymic contractions in the palatolingual muscles were noticed and confirmed by electromyography. Brain computer tomography-scan showed ventricular dilatation. Cerebrospinal fluid analysis revealed a slightly elevated protein level. Treatment with slow-release phenytoin was unsuccessful and symptoms gradually worsened over the next nine months. At post-mortem examination a small pituitary adenoma was found. Apart from a single canine report of facial myokymia, this is the only other description of spontaneous focal myokymia in animals. Palatolingual myokymia has only been reported in one human being. Although the co-occurrence with a pituitary adenoma might be incidental, a paraneoplastic pathogenetic mechanism is proposed. Its unique clinical presentation adds a new, albeit uncommon, syndrome to the differential diagnosis of upper airway complaints in dogs.

Laryngeal rhinosporidiosis in a Belgian warmblood horse.

In Belgium and even in northern Europe Rhinosporidium seeberi has not been reported in autochtonous people or animals. In this paper, the authors report the first observation of laryngeal masses, caused by Rhinosporidium seeberi, in a Belgian Warmblood horse. Moreover, laryngeal rhinosporidiosis is extremely rare since this localisation is only described in four human cases.

Bluetongue virus serotype 8-associated congenital hydranencephaly in calves.

Hydranencephaly, the almost complete absence of the cerebral parenchyma, induced by infection with modified live bluetongue virus (BTV) crossing the placenta has previously been reported in sheep and rarely in cattle in the USA and in South Africa. The current study describes 29 cases of hydranencephaly in bovine foetuses and 'dummy' calves up to 3 months of age in Belgium associated with natural BTV serotype 8 infection very early in gestation. Histological examination of the remaining cerebral parenchyma showed moderate to severe atrophy of the neural tissue. The lesions observed support the hypothesis of BTV-induced destruction of precursor cells. However, in several calves a slight infiltration of the walls of venules and arterioles with T lymphocytes (vasculitis) was observed as well, which seems to be responsible for at least some of the lesions. Bluetongue viral RNA was detected in 15 animals using a BTV-specific real-time RT-PCR with a much higher success rate in brain tissues compared with blood and spleen samples. Virus isolation in embryonated eggs was unsuccessful. In conclusion, hydranencephaly in calves can be associated with natural wild-type BTV-8 intra-uterine infection.

Concurrent atypical myopathy and equine dysautonomia in two horses.

This report concerns 2 horses that suffered typical clinical signs of atypical myopathy (AM) and equine grass sickness (EGS) concurrently. Clinical details and pathological lesions of the cases are described. EGS and AM are relatively rare diseases and the concurrency of the diseases in the same animals is therefore considered unlikely to be a coincidence. However, it is not suggested that the evidence shows a common aetiology but rather the existence of common predisposing causes.

True hermaphroditism in six female littermates after administration of synthetic androgens to a pregnant bitch.

A pregnant bitch was treated with a synthetic testosterone mixture on approximately day 40. The female offspring (six pups) showed an increased anogenital distance, vaginal enlargement and a variable amount of vaginal discharge. The urinary orifice was found dorsally in the vestibulum, stooled on a protruding phallus-like structure. All six pups underwent a laparotomy and subsequent spaying and a modified ventral episioplasty technique to lift up the labia to a more vertical position in order to prevent urine accumulation. Histopathological examination of the genital tracts demonstrated the presence of bilateral ovotestis and remnants of the Wolffian duct system in all cases. The finding of true hermaphroditism of the offspring after exogenous androgen administration during gestation of the bitch has not yet been reported elsewhere.

Comparative evaluation of eight commercial enzyme linked immunosorbent assays and 14 simple assays for detection of antibodies to HIV.

To evaluate the performance of 22 assays for the detection of antibodies to HIV. Twenty-two assays for the combined detection of antibodies to HIV-1 and HIV-2, were evaluated on the same panel of serum specimens of diverse origin. Eight of the assays were ELISAs and the remaining 14 were simple, assays read visually. The specimen panel consisted of anti-HIV positive and negative samples from Africa (n=192), Europe (n=206), Asia (n=99) and Latin America (n=98). In addition to estimations of sensitivity and specificity, the assays were assessed, using a novel scoring system, for their ease of performance and for their suitability for use in small laboratories and clinics. The sensitivities of the assays in terms of seroconversion were assessed using series of specimens collected from nine individuals undergoing seroconversion. Eight ELISAs and eight of 14 simple assays had sensitivities and specificities of >99 and 95%, respectively. The results of these evaluations will be of assistance to those responsible for the selection of appropriate anti-HIV assays according to laboratory circumstances, the purpose of the testing and the population being tested.

Evaluation of an agglutination HIV-1 + 2 antibody assay.

Studies have shown that an HIV (HIV-PA) agglutination assay (Serodia) for the detection of antibody to human immunodeficiency virus (HIV) can be as sensitive and as specific as enzyme-linked immunosorbent assay (ELISA). However, since this HIV assay was designed to detect antibody to the HIV-1 virus, a substantial number of HIV-2 positive sera are missed by this assay. Since the HIV-2 has now been found throughout the world this test is becoming less suitable. The new HIV-1 + 2 assay version (HIV-1 + 2 PA) was evaluated in 300 sera, which contained 50 HIV-1, 40 HIV-2 and 10 HIV-1/HIV-2 antibody positive samples, and a sensitivity and specificity of 100% and 99%, respectively, was obtained. Whereas all HIV-2 positive sera were detected by the new HIV-1 + 2 version, 26% (13/50) were missed by the old version of the agglutination test. It is concluded that the HIV-1 + 2 PA assay is a promising instrument free assay which can be used for screening purposes in areas where both HIV-1 and HIV-2 are present.

Selective increase of activation antigens HLA-DR and CD38 on CD4+ CD45RO+ T lymphocytes during HIV-1 infection.

Infection with HIV results in a progressive depletion of CD4+ T cells and leads to significant in vivo lymphocyte phenotype changes. In this regard, the expression of HLA-DR and CD38 on CD8+ T cells has been shown to increase dramatically with disease progression. We investigated the expression of both activation markers on CD4+ T cells in HIV-1-infected subjects at different clinical stages of infection and compared the in vivo activation of CD4+ T cells with parameters of viral activity and CD8+ T cell activation. Fresh peripheral venous blood was obtained from 54 HIV-infected subjects and from 28 uninfected healthy controls. Three-colour immunophenotyping of the CD4+ T cell subset showed that the proportion of CD4+ T cells expressing HLA-DR (10% in HIV-negative controls) or CD38 (62% in HIV-negative controls) was higher in asymptomatic (P < 0.05 for CD38) and symptomatic (P < 0.001 for HLA-DR and CD38) HIV-infected subjects than in controls, whereas the proportion of CD4+ T cells expressing CD45RO (54% in controls) remained relatively unchanged. Simultaneous expression of HLA-DR and CD38 on CD4+ T cells increased from 2.3% in controls to 11% (P < 0.001) in asymptomatic and 22% (P < 0.001) in symptomatic HIV-infected subjects. This relative increase of CD38 and HLA-DR expression occurred mainly on CD4+ T cells co-expressing CD45RO. Changes in expression of HLA-DR and CD38 on CD4+ T cells correlated with similar changes on CD8+ T lymphocytes, with the presence of HIV antigen in the circulation, and with the disease stage of HIV infection.

Diagnosis of human immunodeficiency virus (HIV) infection: multicenter evaluation of a newly developed anti-HIV 1 and 2 enzyme immunoassay.

A new anti-human immunodeficiency virus type 1 and 2 (anti-HIV 1 and 2) test is described. It uses recombinant p24 and peptides covering gp32, gp41, and gp120 to identify HIV-1 and HIV-2 infections. This test has been shown to be specific (99.5%) and sensitive (99.8%). In this respect, the assay was equal or superior to anti-HIV 1 and 2 tests run as references. The test was able to discriminate sera from patients with HIV infections from those from uninfected individuals with excellence; it also exerted high intra- and interassay precisions. The "modular" concept of the test allows the use of single components (gp32 or gp41) to separate between HIV-2 and HIV-1 infections, respectively.