A serosurvey of selected cystogenic coccidia in Spanish equids: first detection of anti-Besnoitia spp. specific antibodies in Europe.

BACKGROUND: Equine besnoitiosis, caused by Besnoitia bennetti, and equine protozoal myeloencephalitis (EPM), caused by Sarcocystis neurona and Neospora hughesi are relevant equine diseases in the Americas that have been scarcely studied in Europe. Thus, a serosurvey of these cystogenic coccidia was carried out in Southern Spain. A cross-sectional study was performed and serum samples from horses (n = 553), donkeys (n = 85) and mules (n = 83) were included. An in-house enzyme-linked immunosorbent assay (ELISA) was employed to identify a Besnoitia spp. infection and positive results were confirmed by an a posteriori western blot. For Neospora spp. and Sarcocystis spp., infections were detected using in-house ELISAs based on the parasite surface antigens N. hughesi rNhSAG1 and S. neurona rSnSAG2/3/4. Risk factors associated with these protozoan infections were also investigated. RESULTS: Antibodies against Besnoitia spp., Neospora spp. and Sarcocystis spp. infections were detected in 51 (7.1%), 46 (6.4%) and 20 (2.8%) of 721 equids, respectively. The principal risk factors associated with a higher seroprevalence of Besnoitia spp. were the host species (mule or donkey), the absence of shelter and the absence of a rodent control programme. The presence of rodents was the only risk factor for Neospora spp. infection. CONCLUSIONS: This study was the first extensive serosurvey of Besnoitia spp. infection in European equids accomplished by two complementary tests and gives evidence of the presence of specific antibodies in these populations. However, the origin of the infection is still unclear. Further parasite detection and molecular genotyping are needed to identify the causative Besnoitia and Neospora species. Finally, cross-reactions with antibodies directed against other species of Sarcocystis might explain the positive reactions against the S. neurona antigens.

Ultrasonographic visualization of colonic mesenteric vasculature as an indicator of large colon right dorsal displacement or 180° volvulus (or both) in horses.

Visualization of colonic mesenteric vasculature during transabdominal ultrasonographic examination of horses with colic can be a predictor of right dorsal displacement of the large colon or 180° large colon volvulus, or both. Medical records of 82 horses having had surgical treatment of colic and having received a transabdominal ultrasonographic examination on admission were reviewed. Colonic mesenteric vessels were sonographically identified coursing laterally on the right side of the abdomen in 24 of the 82 cases. Horses with colonic vessels identified on ultrasound were 32.5 times more likely to be diagnosed at surgery with either large colon right dorsal displacement or 180° large colon volvulus than those in which vessels were not seen (P < 0.001). Visualization of colonic mesenteric vessels on ultrasound provided a sensitivity of 67.7%, specificity of 97.9%, positive predictive value of 95.8%, and negative predictive value of 81% for large colon right dorsal displacement or 180° large colon volvulus, or both.

Randomized placebo-controlled study of the effects of Yunnan Baiyao on hemostasis in horses.

OBJECTIVE To determine effects of oral administration of Yunnan Baiyao on platelet activation, coagulation, and fibrinolysis in healthy horses. ANIMALS 12 healthy adult horses. PROCEDURES In a randomized blinded crossover study that included a 4-week washout period between treatments, horses were orally administered a paste containing Yunnan Baiyao (15 mg/kg) or placebo at 12-hour intervals for 3 days. Blood samples were collected before start of treatment (time 0) and at 24 and 72 hours for a CBC, measurement of fibrinogen concentration, coagulation screening tests, and a panel of assays to assess platelet activation (including ADP- and collagen-induced aggregation and closure times, flow-cytometric variables of platelet-leukocyte aggregates, platelet membrane P-selectin and phosphatidylserine expression, and microparticle release), von Willebrand factor (vWF) concentration, and cofactor activity. In addition, thrombelastography was used to evaluate fibrin formation in tissue factor-activated whole blood and plasma and to assess tissue plasminogen activator-induced plasma fibrinolysis. For each treatment, values obtained before and 72 hours after start of administration were compared by use of Wilcoxon signed rank tests. RESULTS Yunnan Baiyao treatment had no significant effect on any hemostatic variable, compared with results for the placebo treatment. CONCLUSIONS AND CLINICAL RELEVANCE Administration of Yunnan Baiyao at a dosage typically used in clinical practice had no effect on in vitro measures of platelet or vWF function and no enhancement of fibrin-clot formation or stability. Any hemostatic actions of Yunnan Baiyao may require higher dosages or result from cell-surface interactions at sites of vascular and tissue injury not examined in this study.

Equine Mesenchymal Stromal Cells from Different Sources Efficiently Differentiate into Hepatocyte-Like Cells.

Adult equine hepatocytes have proven challenging to culture long term in vitro as they rapidly lose their morphology and functionality, thus limiting studies on liver function and response to disease. In this study, we describe for the first time the differentiation of equine mesenchymal stromal cells (MSC) from a variety of sources into functional hepatocyte-like cells (HLC). First, we differentiated equine umbilical cord blood (UCB)-derived MSC into HLC and found that these cells exhibited a distinct polygonal morphology, stored glycogen as visualized by periodic acid Schiff's reagent staining, and were positive for albumin and other hepatocyte-specific genes. Second, we demonstrated that UCB-HLC could be revived following cryopreservation and retained their phenotype for at least 10 days. Third, we differentiated three sets of MSC from bone marrow (BM), adipose tissue (AT), and peripheral blood (PB), matched within the same horse. We achieved a 100% differentiation success rate with BM, 0% with AT, and 66% with PB. An additional set of nine PB-MSC samples resulted in an overall success rate of 42% (n = 12), and age or gender did not seem to have an effect on the success of hepatic differentiation from that source. In a final set of experiments, we evaluated the use of these HLC as tools in different fields of biomedical research like virology, to study viral growth, and toxicology, to study chemicals with hepatic toxicity. Equine HLC were found susceptible for infection with the equine herpesviruses type 1 (EHV-1), -2, and -5, and exhibited a more sensitive dose-dependent response to arsenic toxicity than the commonly used human hepatocellular cell line HepG2. Taken together, these data indicate that equine MSC can be efficiently differentiated into HLC and these equine HLC could be a useful tool for in vitro studies.

Serological diagnosis of Besnoitia bennetti infection in donkeys (Equus asinus).

Besnoitiosis is an emerging infectious disease of donkeys (Equus asinus) in the United States for which there are currently no serologic methods of diagnosis. A study was performed to evaluate physical examination findings and 3 serologic assays for the detection of Besnoitia bennetti infection in donkeys. A prospective study of 416 donkeys from 6 privately owned herds across 5 U.S. states (New York, Pennsylvania, Vermont, Oregon, and Washington) was performed. Donkeys were examined for clinical lesions suggestive of besnoitiosis and evaluated for antibodies against B. bennetti using a fluorescent antibody test (FAT) and 2 immunoblot assays specific for bradyzoite and tachyzoite antigens, respectively. Donkeys were confirmed to be infected with B. bennetti by histology (cases; n = 32) and were compared to those with no clinical signs of besnoitiosis (controls; n = 384). Identifying clinical lesions in 2 or more locations correctly identified infected donkeys 83% of the time. Donkeys with besnoitiosis had significantly higher FAT titers (P < 0.001) and numbers of bradyzoite (P < 0.001) and tachyzoite (P < 0.001) immunoblot bands than control donkeys. The sensitivity and specificity of the serologic assays for detecting besnoitiosis was 88% and 96% for FAT, 81% and 91% for bradyzoite immunoblot, and 91% and 92% for tachyzoite immunoblot, respectively. Fluorescent antibody and immunoblot assays are effective at identifying donkeys with besnoitiosis and provide a more efficient and less invasive diagnostic alternative to histology.

Serum antibodies from a subset of horses positive for Babesia caballi by competitive enzyme-linked immunosorbent assay demonstrate a protein recognition pattern that is not consistent with infection.

Tick-borne pathogens that cause persistent infection are of major concern to the livestock industry because of transmission risk from persistently infected animals and the potential economic losses they pose. The recent reemergence of Theileria equi in the United States prompted a widespread national survey resulting in identification of limited distribution of equine piroplasmosis (EP) in the U.S. horse population. This program identified Babesia caballi-seropositive horses using rhoptry-associated protein 1 (RAP-1)-competitive enzyme-linked immunosorbent assay (cELISA), despite B. caballi being considered nonendemic on the U.S. mainland. The purpose of the present study was to evaluate the suitability of RAP-1-cELISA as a single serological test to determine the infection status of B. caballi in U.S. horses. Immunoblotting indicated that sera from U.S. horses reacted with B. caballi lysate and purified B. caballi RAP-1 protein. Antibody reactivity to B. caballi lysate was exclusively directed against a single ∼50-kDa band corresponding to a native B. caballi RAP-1 protein. In contrast, sera from experimentally and naturally infected horses from regions where B. caballi is endemic bound multiple proteins ranging from 30 to 50 kDa. Dilutions of sera from U.S. horses positive by cELISA revealed low levels of antibodies, while sera from horses experimentally infected with B. caballi and from areas where B. caballi is endemic had comparatively high antibody levels. Finally, blood transfer from seropositive U.S. horses into naive horses demonstrated no evidence of B. caballi transmission, confirming that antibody reactivity in cELISA-positive U.S. horses was not consistent with infection. Therefore, we conclude that a combination of cELISA and immunoblotting is required for the accurate serodiagnosis of B. caballi.

Investigation of an outbreak of besnoitiosis in donkeys in northeastern Pennsylvania.

OBJECTIVE: To describe the clinical, endoscopic, and serologic features of an outbreak of besnoitiosis in 2 donkey operations in northeastern Pennsylvania and to report the outcome of attempted treatment of 1 naturally infected individual. DESIGN: Observational study. ANIMALS: 29 donkeys (Equus asinus) in northeastern Pennsylvania. PROCEDURES: Donkeys were examined for lesions suggestive of besnoitiosis in an outbreak investigation. Information was collected regarding the history and signalment of animals on each premises. Rhinolaryngoscopy was performed to identify nasopharyngeal and laryngeal lesions. Serum samples were collected for immunofluorescent antibody testing and immunoblotting for Besnoitia spp. Skin biopsy samples were obtained from 8 animals with lesions suggestive of besnoitiosis for histologic examination. Quantitative real-time PCR assay for Besnoitia spp was performed on tissue samples from 5 animals. RESULTS: Besnoitiosis was confirmed in 6 of the 8 suspected cases. The most common lesion site was the nares, followed by the skin and sclera. Donkeys with clinical signs of disease had higher serum antibody titers and tested positive for a greater number of immunoblot bands than did donkeys without clinical signs of disease. All animals evaluated by PCR assay tested positive. Putative risk factors for disease included age and sex. Ponazuril was not effective at treating besnoitiosis in a naturally infected donkey. CONCLUSIONS AND CLINICAL RELEVANCE: Knowledge of clinical and serologic features of besnoitiosis in donkeys will assist clinicians in the diagnosis and prevention of this disease in donkey populations. Besnoitiosis may be an emerging disease of donkeys in the United States.