Assessment of quantitative polymerase chain reaction for equine herpesvirus-5 in blood, nasal secretions and bronchoalveolar lavage fluid for the laboratory diagnosis of equine multinodular pulmonary fibrosis.

REASONS FOR PERFORMING STUDY: The ante mortem diagnosis of equine multinodular pulmonary fibrosis (EMPF) relies on histopathological results and polymerase chain reaction (PCR)-positive equine herpesvirus (EHV)-5 testing of lung tissue. Polymerase chain reaction detection of EHV-5 in bronchoalveolar lavage fluid (BALF) is commonly used to support a diagnosis of EMPF. However, the diagnostic power of EHV-5 testing on BALF and other biological samples such as blood and nasal secretions has yet to be shown to support a diagnosis of EMPF. OBJECTIVES: To determine the frequency of detection and the viral loads of EHV-5 by quantitative PCR (qPCR) in blood, nasal secretions and BALF from horses confirmed with EMPF, healthy horses and horses with non-EMPF pulmonary diseases. STUDY DESIGN: Prospective study. METHODS: The study population consisted of 70 adult horses divided into 4 groups based on a combination of clinical findings, cytology of BALF, imaging studies of the thoracic cavity and histopathology of pulmonary tissue: control group (n = 14), EMPF group (n = 11); inflammatory airway disease group (n = 32); and non-EMPF interstitial lung disease group (n = 13). For each horse, whole blood, nasal secretions and BALF were available for EHV-5 qPCR testing. Sensitivities, specificities and their respective 95% confidence intervals were calculated for viral loads from blood, nasal secretions and BALF. In addition, these measures were calculated for combined use of blood and nasal secretions. RESULTS: The detection of EHV-5 in BALF was strongly associated with EMPF (sensitivity 91%, specificity 98.3%). Detection of EHV-5 in blood was, independent of the viral loads, strongly associated with EMPF with a sensitivity of 91% and specificity of 83.1%. The detection of EHV-5 in nasal secretions displayed the highest sensitivity (72.7%) and specificity (83.1%) at a level of >245,890 glycoprotein B target genes/million cells to support a diagnosis of EMPF. Dually positive blood and nasal secretions at any viral loads in support of EMPF yielded a sensitivity and specificity of 90% and 89.8%, respectively. CONCLUSIONS: Although histopathological confirmation (lung biopsy) is considered the gold standard for EMPF diagnosis, results of qPCR testing of BALF or a combination of whole blood and nasal secretions should be regarded as clinically useful in support of this diagnosis. The latter testing may be relevant when dealing with horses in respiratory distress, for which invasive procedures such as BALF collection or lung biopsies may be detrimental to their health.

Equine coronavirus: An emerging enteric virus of adult horses.

Equine coronavirus (ECoV) is an emerging virus associated clinically and epidemiologically with fever, depression, anorexia and less frequently colic and diarrhoea in adult horses. Sporadic cases and outbreaks have been reported with increased frequency since 2010 from Japan, the USA and more recently from Europe. A faeco-oral transmission route is suspected and clinical or asymptomatic infected horses appear to be responsible for direct and indirect transmission of ECoV. A presumptive clinical diagnosis of ECoV infection may be suggested by clinical presentation, haematological abnormalities such as leucopenia due to lymphopenia and/or neutropenia. Confirmation of ECoV infection is provided by specific ECoV nucleic acid detection in faeces by quantitative PCR (qPCR) or demonstration of coronavirus antigen by immunohistochemistry or electron microscopy in intestinal biopsy material obtained ante or post mortem. The disease is generally self-limiting and horses typically recover with symptomatic supportive care. Complications associated with disruption of the gastrointestinal barrier have been reported in some infected horses and include endotoxaemia, septicaemia and hyperammonaemia-associated encephalopathy. Although specific immunoprophylactic measures have been shown to be effective in disease prevention for closely-related coronaviruses such as bovine coronavirus (BCoV), such strategies have yet not been investigated for horses and disease prevention is limited to basic biosecurity protocols. This article reviews current knowledge concerning the aetiology, epidemiology, clinical signs, diagnosis, pathology, treatment and prevention of ECoV infection in adult horses.

A pilot phase II study of the efficacy and biosafety of doxorubicin chemotherapy in tumor-bearing equidae.

BACKGROUND: The efficacy and biosafety of a previously established tolerable dosage of doxorubicin have not been established in horses. OBJECTIVES: To provide preliminary evidence of the efficacy of doxorubicin in tumor-bearing horses, explore drug pharmacokinetics profile, and estimate period of risk of exposure to drug residues. ANIMALS: Twelve horses with 37 tumors. PROCEDURES: Treatment protocol included 6 treatments at 3-week intervals. Eight horses were uniformly treated at a dosage of 70 mg/m(2) and 4 horses received 4 of 6 treatment cycles at 70 mg/m(2) . Clinical signs, tumor responses, and toxicoses were evaluated. Drug residue concentrations were quantitated in 3 horses receiving of 65, 70, and 75 mg/m(2) by high-performance liquid chromatography with ultraviolet detection (plasma, feces) and liquid chromatography and tandem mass spectrometry (urine). RESULTS: Thirty tumors, including lymphomas, carcinomas, sarcoids, and melanoma, were evaluated for efficacy. The overall response rate was 47% (95% CI, 28-65%). Doxorubicin was not found to be effective against melanomas. Lymphomas and carcinomas were most responsive. Pooled serum Cmax and half-life of doxorubicin were 121.3 ng/mL and 12.9 hours, respectively. There were no detectable residues in fecal samples up to 3 weeks after treatment and in plasma and urine after 2 and 3 days, respectively. CONCLUSION AND CLINICAL RELEVANCE: This study provides preliminary evidence that single-agent doxorubicin at a dosage of 70 mg/m(2) has a broad spectrum of activity. The risk of exposure to drug residues in plasma and feces was low. Direct contact with urine-contaminated wastes should be avoided for 2 days after treatment.

Phase I dose escalation of doxorubicin chemotherapy in tumor-bearing equidae.

BACKGROUND: There is no information on the use of doxorubicin in horses with tumors. OBJECTIVE: To determine dose-limiting toxicosis (DLT) and maximum tolerated dose (MTD) of doxorubicin in tumor-bearing horses. ANIMALS: Seventeen horses with 34 localized or multicentric advanced tumors. METHODS: Two-stage dose-ranging design involving intrapatient and interpatient dose escalation. Treatment protocol included 6 treatment cycles given at 3-week intervals with dosages ranging from 40 to 85 mg/m(2). Clinical signs, hematologic, and nonhematologic changes were evaluated. RESULTS: Total doses ranged from 1,127 to 2,900 mg in 12 horses that completed the assigned treatment protocols. The MTD was 75 mg/m(2). Hypersensitivity reactions and neutropenia were dose limiting. Hypersensitivity was dose-dependent but schedule invariant. Neutropenia was dose- and cycle-dependent but dose-escalation schedule invariant. Cardiotoxicity was not observed. CONCLUSION AND CLINICAL RELEVANCE: The recommended dosage of doxorubicin to treat horses is 70 mg/m(2) given at 3-week intervals as single agent. Adjunctive treatment with antihistamines and nonsteroidal anti-inflammatory drugs is recommended to control hypersensitivity.

Characterization of the interferon gamma response to Lawsonia intracellularis using an equine proliferative enteropathy challenge (EPE) model.

Lawsonia intracellularis is the etiological agent of infectious intestinal hyperplasia for which several clinical diseases have been described including proliferative enteropathy (PE), intestinal adenomatosis, and ileitis. While initially recognized as the causative agent of PE in pigs, L. intracellularis is now viewed as an emerging cause of intestinal hyperplasia in a wide range of mammalian species, including horses. Equine proliferative enteropathy (EPE) has been reported worldwide though definitive diagnosis is difficult and the epidemiology of the disease remains poorly understood. Weanlings, in particular, appear to be most at risk for infection, though the reasons for their particular susceptibility is unknown. Using an infectious challenge model for EPE, we demonstrate that EPE, like porcine proliferative enteropathy, can exhibit three clinical forms: classical, subclinical and acute. Out of six pony weanlings, one developed signs of classic EPE, one developed acute EPE, and two developed subclinical EPE. Attempts to induce pharmacological stress through the use of dexamethasone failed to have any effect on outcome. Peripheral blood cells collected from those weanlings that developed clinical EPE exhibited decreased expression of interferon-gamma (IFN-γ) following in vitro stimulation with L. intracellularis. By contrast, those weanlings that did not develop clinical disease generated a robust IFN-γ response. These results indicate IFN-γ likely plays a significant role in protection from disease caused by L. intracellularis in the equid.

Gastric neoplasia in horses.

BACKGROUND: Gastric neoplasia of horses is incompletely described. OBJECTIVE: Provide history, clinical signs, and clinicopathological and pathological findings associated with gastric neoplasia in horses. ANIMALS: Twenty-four horses with gastric neoplasia. METHODS: Retrospective study. History, clinical signs, and clinicopathological and pathologic findings in horses diagnosed histologically with gastric neoplasia were reviewed. RESULTS: Horses ranged in age from 9 to 25 years (median 18 years at presentation). There was no apparent breed or sex predisposition. The most common presenting complaints were inappetance (17/24), weight loss (14/24), lethargy (7/24), hypersalivation (7/24), colic (5/24), and fever (5/24). The most consistent clinical signs were tachypnea (10/19), decreased borborygmi (8/19), and low body weight (7/17). Useful diagnostic tests included rectal examination, routine blood analysis, gastroscopy, abdominocentesis, and transabdominal ultrasound examination. Anemia was the most common hematologic abnormality encountered (7/19), and hypercalcemia of malignancy was seen in 4/16 horses. Squamous cell carcinoma was the most common tumor identified (19/24), and was most often (14/19) found as a single ulcerated, necrotic mass in the nonglandular portion of the stomach. Other gastric neoplasms encountered were leiomyoma (n=2), mesothelioma (n=1), adenocarcinoma (n=1), and lymphoma (n=1). Metastatic neoplasia was found in 18/23 horses. The median time from onset of clinical signs to death was 4 weeks, and all horses died or were euthanized because of gastric neoplasia. CONCLUSIONS: Squamous cell carcinoma is the most common primary gastric neoplasia in horses. The survival time after diagnosis of gastric neoplasia in horses is short.

Intestinal neoplasia in horses.

BACKGROUND: Intestinal neoplasia of horses is inadequately described. HYPOTHESIS: Intestinal neoplasia of horses has characteristic clinicopathologic features. ANIMALS: Thirty-four horses with intestinal neoplasia. METHODS: Retrospective study. RESULTS: Anamnesis, clinical signs, clinicopathologic and pathologic findings in 34 adult horses diagnosed histologically with intestinal neoplasia were reviewed. The horses ranged in age from 2 to 30 years (mean 16.6 years at presentation). The Arabian breed was most represented and there was no sex predisposition. The most common presenting complaints were weight loss, colic, anorexia, and fever. The most consistent clinical signs were poor body condition, tachycardia, tachypnea, fever, and diarrhea. Useful diagnostic tools included rectal examination, routine blood analyses, abdominocentesis, ultrasonographic examination, rectal biopsy, and exploratory laparotomy. Alimentary lymphoma was the most common intestinal neoplasia identified, followed by adenocarcinoma and smooth muscle tumors. The small intestine was the most common segment of intestine affected for all neoplasms. Intestinal neoplasia was diagnosed antemortem in 13 of 34 (38%) horses. The median time from onset of clinical signs to death or euthanasia was 1.9 months. The discharge rate was 15%. Although the longest survival was observed in horses with jejunal adenocarcinoma, all horses were eventually euthanized because of intestinal neoplasia. CONCLUSIONS: Arabian horses were 4.5 times more likely to have intestinal neoplasia diagnosed than were other breeds.

Cytokine gene signatures in neural tissue of horses with equine protozoal myeloencephalitis or equine herpes type 1 myeloencephalopathy.

This study was designed to determine the relative levels of gene transcription of selected pathogens and cytokines in the brain and spinal cord of 12 horses with equine protozoal myeloencephalitis (EPM), 11 with equine herpesvirus type 1 (EHV-1) myeloencephalopathy, and 12 healthy control horses by applying a real time pcr to the formalin-fixed and paraffin-embedded tissues. Total rna was extracted from each tissue, transcribed to complementary dna (cDNA) and assayed for Sarcocystis neurona, Neospora hughesi, EHV-1, equine GAPDH (housekeeping gene), tumour necrosis factor (TNF)-alpha, interferon (IFN)-gamma, interleukin (IL)-1beta, IL-2, IL-4, IL-6, IL-8, IL-10 AND IL-12 p40. S neurona cdna was detected in the neural tissue from all 12 horses with EPM, and two of them also had amplifiable cDNA of N hughesi. The relative levels of transcription of protozoal cdna ranged from 1 to 461 times baseline (mean 123). All the horses with ehv-1 myeloencephalopathy had positive viral signals by PCR with relative levels of transcription ranging from 1 to 1618 times baseline (mean 275). All the control horses tested negative for S neurona, N hughesi and EHV-1 cdna. The cytokine profiles of each disease indicated a balance between pro- and anti-inflammatory markers. In the horses with epm the pro-inflammatory Th1 cytokines (IL-8, TNF-alpha and IFN-gamma) were commonly expressed but the anti-inflammatory Th2 cytokines (IL-4, IL-6 AND IL-10) were absent or rare. In the horses with ehv-1 the proinflammatory cytokine IL-8 was commonly expressed, but IL-10 and IFN-gamma were not, and TNF-alpha was rare. Tissue from the control horses expressed only the gene GAPDH.

Multicentric T-cell lymphosarcoma in an alpaca.

A two-year-old female alpaca with multicentric lymphosarcoma presented because of progressive weakness and recumbency. The diagnosis of lymphosarcoma was based on the detection of immature and atypical lymphocytes in a lumbosacral CSF sample. Post mortem examination confirmed multicentric lymphosarcoma involving multiple organs. Immunophenotyping using cross reactive T- and B-cell antibodies characterized the tumour as a T-cell lymphosarcoma.

Acute hemoperitoneum in horses: a review of 19 cases (1992-2003).

The medical records of 19 horses with acute hemoperitoneum were reviewed. The causes for the hemoperitoneum were idiopathic (8 horses), splenic hematoma with capsular tear (7), bleeding from the reproductive tract (3), multicentric hemangiosarcoma (1), and systemic amyloidosis (1). The affected horses were between 4 and 32 years of age (median 11.5 years). The most consistent findings on initial examination were depression, tachycardia, tachypnea, pale mucous membranes, prolonged capillary refill time, colic, and abdominal discomfort. Less common clinical signs included abdominal distention, profuse sweating, ataxia, and broad ligament mass palpated on rectal examination. Clinicopathologic abnormalities commonly detected were anemia, neutrophilia, lymphopenia, thrombocytopenia, hypoproteinemia, hypocalcemia, azotemia, increased creatinine kinase, and sorbitol dehydrogenase activity. Hemoperitoneum was diagnosed on the basis of abdominocentesis, transabdominal ultrasonography, and postmortem examination. Sixteen horses were treated, and 3 horses were euthanized at owners' request because of severe clinical signs. The treatment consisted of the administration of intravenous fluids, plasma or blood transfusion, nonsteroidal drugs, antimicrobial drugs, and antifibrinolytic and procoagulant agents. Rapid clinical deterioration was observed in 2 horses, necessitating euthanasia. The remaining 14 horses survived the abdominal bleeding (survival rate 74%) and were discharged 3-15 days (median 7.0 days) after presentation. Postmortem examination of the 6 nonsurvivors showed massive abdominal hemorrhage from splenic hematoma with capsular tear (2 horses), multicentric hemangiosarcoma with liver rupture (1), systemic amyloidosis with splenic hematoma and capsular tear (1), and bilateral ruptured ovarian hematomas (1). In one horse, no origin of the bleeding could be determined during postmortem examination.

Immunoglobulin A monoclonal gammopathy in two horses with multiple myeloma.

The clinical findings in two horses with secretory multiple myeloma and secondary immunoglobulin A (IgA) monoclonal gammopathy were non-specific and included weight loss, pale mucous membranes, limb oedema and bacterial respiratory tract infection. Consistent laboratory abnormalities included hyperproteinaemia, hyperglobulinaemia, hypoalbuminaemia and hypercalcaemia. The diagnosis was based on the presence of IgA monoclonal gammopathy in serum and urine and bone marrow plasmacytosis (> 10 per cent). One horse was euthanased; it had neoplastic plasma cell infiltrates in its kidneys, spleen, liver, bone marrow, myocardium and adrenal glands. The other horse was treated for a bacterial pneumonia and was still alive six months after it was first examined.

Idiopathic granulomatous pneumonia in seven horses.

The history, clinical signs and pathological findings in seven adult horses with histologically confirmed idiopathic granulomatous disease, primarily of the lungs, are reviewed. They ranged in age from eight to 21 years, five were geldings and two were females, they belonged to five breeds and there were no seasonal or geographical associations. The primary clinical signs were chronic weight loss, exercise intolerance and respiratory distress which did not respond to conventional treatment. The most consistent physical findings were depression, anorexia, tachycardia, tachypnoea and adventitious lung sounds. Thoracic radiographs revealed a diffuse, structured, nodular, interstitial pulmonary pattern in each horse. Haematological measurements suggested a chronic inflammatory process and the cytology of transtracheal washes was consistent with a mild suppurative inflammation. Idiopathic granulomatous pneumonia was confirmed histologically in each of the horses, either postmortem or by a lung biopsy. The horses responded poorly to medical treatment and only one of three treated horses is still alive.

Transmission of Ehrlichia risticii, the agent of Potomac horse fever, using naturally infected aquatic insects and helminth vectors: preliminary report.

Ehrlichia risticii, the agent of Potomac horse fever (PHF), has been recently detected in trematode stages found in snail secretions and in aquatic insects. Based on these findings, horses could conceivably be exposed to E. risticii by skin penetration with infected cercariae, by ingestion of infected cercariae in water or via metacercariae in a second intermediate host, such as an aquatic insect. In order to test this hypothesis, horses were challenged with infectious snail secretions and aquatic insects collected from a PHF endemic region in northern California. Two horses stood with their front feet in water harbouring E. risticii-infected cercariae, 2 horses drank water harbouring E. risticii-infected cercariae, and 6 horses were fed pools of different aquatic insects harbouring E. risticii-infected metacercariae. In this preliminary study, only the one horse infected orally with mature caddisflies (Dicosmoecus gilvipes) developed the clinical and haematological disease syndrome of PHF. The agent was isolated from the blood of the infected horse in a continuous cell line and identified as E. risticii by characterisation of the 16S rRNA gene. Therefore, E. risticii is maintained in nature in a complex aquatic ecosystem and transmission to horses can occur through accidental ingestion of insects such as caddisflies containing infected metacercariae. At present, the small number of horses used in this study does not exclude other insects and free trematode stages as potential sources of infection.