Evaluation of safety, humoral immune response and faecal shedding in horses inoculated with a modified-live bovine coronavirus vaccination.

Equine coronavirus (ECoV) is considered an emerging enteric virus with reported morbidity rates ranging from 10 to 83% and fatality rates ranging from 7 to 27% in adult horses; a vaccine for ECoV is currently not available. This study investigated the safety, humoral response and viral shedding in horses inoculated with a commercially available modified-live bovine coronavirus (BCoV) vaccine. Twelve healthy adult horses were vaccinated twice, 3 weeks apart, either orally, intranasally or intrarectally. Two healthy unvaccinated horses served as sentinel controls. Following each vaccine administration, horses were monitored daily for physical abnormalities whilst the onset and duration of BCoV shedding was determined by quantitative PCR (qPCR) in nasal secretions and faeces. Whole blood was collected every 3 weeks to determine BCoV-specific antibody response. With the exception of transient and self-limiting changes in faecal character observed in seven vaccinated and one control horse, no additional abnormal clinical findings were found in the study horses. Following the first and second vaccine administration, two and one horse, respectively, tested qPCR-positive for BCoV in nasal secretions 1-day post intranasal vaccination. No vaccinated horses tested qPCR-positive for BCoV in faeces following each vaccine administration. One of the two horses that shed BCoV seroconverted to BCoV after the first vaccine administration and an additional two vaccinated horses (oral and intrarectal) seroconverted to BCoV after the second vaccine administration. In conclusion, the results show that the modified-live BCoV is safe to administer to horses via various routes, causes minimal virus shedding and results in detectable antibodies to BCoV in 27% of the vaccinates.

Equine herpesvirus-1 genotype did not significantly affect clinical signs and disease outcome in 65 horses diagnosed with equine herpesvirus-1 myeloencephalopathy.

The objective of this study was to determine if the genotype of equine herpesvirus-1 (EHV-1) impacted clinical disease and outcome of horses with laboratory confirmed equine herpesvirus myeloencephalopathy (EHM). Medical records from 65 horses diagnosed with EHM from 2011 to 2019 were reviewed for signalment, presence and severity of clinical signs (lethargy, fever, ataxia, urinary incontinence) and outcome. Horses were further grouped based on the EHV-1 genotype into neuropathic (D752) or non-neuropathic (N752) EHV-1 infection. Between the two EHV-1 genotype groups, age and sex distributions were similar, although breed distribution was different (Quarter Horses and Saddlebreds were overrepresented and Warmbloods were underrepresented in the EHV-1 D752 group compared to the EHV-1 N752 group; P = 0.009). Lethargy, fever, ataxia and outcome were not significantly different between the two EHV-1 genotype groups (P > 0.05). However, urinary incontinence was significantly more frequently reported in horses infected with the D752 genotype of EHV-1 (P=0.04). Contrary to previous studies, the present study showed no difference in frequency of genotype (D752 or N752) among 65 horses with EHM and, with the exception of urinary incontinence, no difference in clinical disease or outcome related to the EHV-1 genotype.

Molecular detection of Streptococcus equi subspecies equi in face flies (Musca autumnalis) collected during a strangles outbreak on a Thoroughbred farm.

The objective of this study was to detect Streptococcus equi subspecies equi (S. equi) (Lactobacillales: Streptococcaceae) using quantitative polymerase chain reaction (qPCR) in flies collected from a farm with a documented outbreak of strangles. A total of 1856 face flies [Musca autumnalis (Diptera: Muscidae)] were collected using conventional fly traps. The flies were processed for nucleic acid purification and tested for the presence of S. equi by qPCR. A total of 10/1856 flies (0.54%) tested qPCR-positive for S. equi. The results may implicate the presence of face flies as a risk factor for the transmission of S. equi and highlight the need to institute proper husbandry measures, biosecurity protocols and fly control in order to reduce the potential for infection in at-risk horses.

Clinical presentation, diagnostic findings, and outcome of adult horses with equine coronavirus infection at a veterinary teaching hospital: 33 cases (2012-2018).

Equine coronavirus (ECoV) is a recently described enteric virus with worldwide outbreaks; however, there are little data available on clinical presentation, diagnosis, and outcome. The study objective was to document case management of ECoV in adult horses presented to a referral hospital and compare to a cohort of horses that tested negative for ECoV. A retrospective case series was performed based on positive real-time quantitative PCR results for ECoV on faeces from horses treated at the UC Davis Veterinary Medical Teaching Hospital from 1 March 2012 to 31 March 2018. Horses negative for ECoV were matched to the ECoV-positive group as controls. Data collected included signalment, history, exam findings, diagnostics, treatment, and follow-up. Thirty-three horses (median age, 11 years; range, 2-37 years) tested ECoV-positive, including three horses with co-infections. Presenting complaints for ECoV-infected horses included historic fevers (n = 25/30; 83%), anorexia (n = 14/30; 47%), and colic (n = 13/30; 43%). ECoV-positive horses had significantly lower white blood cell (median, 3.0 × 109/L; range, 0.68-16.2 × 109/L), neutrophil (median, 1.26 × 109/L; range, 0.15-14.4 × 109/L), and lymphocyte (median, 0.86 × 109/L; range, 0.42-3.47 × 109/L) counts than ECoV-negative horses. Electrolyte and metabolic derangements and scant faeces were common. Twenty-seven horses were hospitalised for a median of 5 days (range, 0.5-14 days), with 26/27 (96%) horses surviving to discharge. ECoV infection should be a differential diagnosis for adult horses with fever, colic, anorexia, and leukopenia. The disease has a low mortality rate, but horses may require intensive care to resolve severe leukopenia, systemic inflammation, and metabolic disturbances.

Enteric coronavirus infection in adult horses.

A new enteric virus of adult horses, equine coronavirus (ECoV), has recently been recognized. It is associated with fever, lethargy, anorexia, and less frequently, colic and diarrhea. This enteric virus is transmitted via the feco-oral route and horses become infected by ingesting fecally contaminated feed and water. Various outbreaks have been reported since 2010 from Japan, Europe and the USA. While the clinical signs are fairly non-specific, lymphopenia and neutropenia are often seen. Specific diagnosis is made by the detection of ECoV in feces by either quantitative real-time PCR, electron microscopy or antigen-capture ELISA. Supportive treatment is usually required, as most infections are self-limiting. However, rare complications, such as endotoxemia, septicemia and hyperammonemia-associated encephalopathy, have been reported, and have been related to the loss of barrier function at the intestinal mucosa. This review article will focus on the latest information pertaining to the virus, epidemiology, clinical signs, diagnosis, pathology, treatment and prevention of ECoV infection in adult horses.

Use of quantitative real-time PCR to determine viability of Streptococcus equi subspecies equi in respiratory secretions from horses with strangles.

BACKGROUND: In recent years, molecular approaches have been able to characterise the viability of equine upper respiratory tract pathogens using absolute molecular quantitation as well as detection of transcripts for virulence genes. OBJECTIVES: The objective of this study was to investigate molecular surrogates for S. equi subspecies equi (S. equi) viability in biological samples from horses with strangles. STUDY DESIGN: Retrospective cross-sectional study. METHODS: S. equi culture-positive and culture-negative upper airway secretions were assessed by qPCR at the genomic (gDNA) and complimentary DNA (cDNA) level for various target genes (SeM, SEQ2190, eqbE and szpSe). Absolute quantitation was performed using standard curves, and the results were expressed as number of S. equi target genes per µl of gDNA or cDNA. Additionally, the presence or absence of S. equi gene expression for the various target genes was assessed and compared with the culture results. RESULTS: While all 21 culture-positive samples tested S. equiqPCR positive, up to 43.7 and 18.9% of 64 culture-negative samples tested qPCR positive at the gDNA and cDNA level, respectively. Significant differences in absolute quantitation for S. equi at the gDNA level were found between culture-positive and culture-negative samples. When absolute quantitation of S. equi target genes at the gDNA level was assessed with the presence or absence of transcripts, there was a significantly higher S. equi target gene number in samples with expression of transcripts compared with samples with no expression of transcripts. MAIN LIMITATIONS: The lack of standardisation of samples collected in the field and the delay from sample collection to samples processing may have negatively affected the cultivability of S. equi and mRNA quality. CONCLUSIONS: Molecular viability for S. equi can be investigated by determining absolute quantitation and/or by detecting mRNA for specific target genes. However, veterinarians have to be cautioned that any qPCR-positive result for S. equi needs to be taken seriously and trigger biosecurity protocols aimed at reducing spread.

Analytical validation of a new point-of-care assay for serum amyloid A in horses.

BACKGROUND: Serum amyloid A (SAA) is a major acute phase protein in horses. A new point-of-care (POC) test for SAA (Stablelab) is available, but studies evaluating its analytical accuracy are lacking. OBJECTIVES: To evaluate the analytical performance of the SAA POC test by 1) determining linearity and precision, 2) comparing results in whole blood with those in serum or plasma, and 3) comparing POC results with those obtained using a previously validated turbidimetric immunoassay (TIA). STUDY DESIGN: Assay validation. METHODS: Analytical validation of the POC test was done in accordance with American Society of Veterinary Clinical Pathology guidelines using residual equine serum/plasma and whole blood samples from the Clinical Pathology Laboratory at the University of California-Davis. A TIA was used as the reference method. We also evaluated the effect of haematocrit (HCT). RESULTS: The POC test was linear for SAA concentrations of up to at least 1000 µg/mL (r = 0.991). Intra-assay CVs were 13, 18 and 15% at high (782 µg/mL), intermediate (116 µg/mL) and low (64 µg/mL) concentrations. Inter-assay (inter-batch) CVs were 45, 14 and 15% at high (1372 µg/mL), intermediate (140 µg/mL) and low (56 µg/mL) concentrations. SAA results in whole blood were significantly lower than those in serum/plasma (P = 0.0002), but were positively correlated (r = 0.908) and not affected by HCT (P = 0.261); proportional negative bias was observed in samples with SAA>500 µg/mL. The difference between methods exceeded the 95% confidence interval of the combined imprecision of both methods (15%). MAIN LIMITATIONS: Analytical validation could not be performed in whole blood, the sample most likely to be used stall side. CONCLUSION: The POC test has acceptable accuracy and precision in equine serum/plasma with SAA concentrations of up to at least 1000 µg/mL. Low inter-batch precision at high concentrations may affect serial measurements, and the use of the same test batch and sample type (serum/plasma or whole blood) is recommended. Comparison of results between the POC test and the TIA is not recommended.

Toxoplasma gondii seroprevalence and association with equine protozoal myeloencephalitis: A case-control study of Californian horses.

While toxoplasmosis is not commonly considered a clinical disease of equines, previous seroprevalence studies have reported differing background rates of Toxoplasma gondii infection in horses globally. The objective of this study was to evaluate possible associations between T. gondii seroprevalence and clinical signs of equine protozoal myeloencephalitis (EPM) in horses. Using a case-control study design, 720 Californian horses with neurologic signs compatible with EPM were compared to healthy, non-neurologic horses for the presence of T. gondii antibodies (using indirect fluorescent antibody tests [IFAT]). Toxoplasma gondii seroprevalence among cases and controls was determined at standard serum cut-offs: 40, 80, 160, 320, and 640. At a T. gondii titre cut-off of 320, horses with clinical signs compatible with EPM had 3.55 times the odds of a seropositive test compared to those without clinical signs (P<0.01) when adjusted for covariates. When restricted to the autumn season and at the same titre cut-off, an EPM suspect horse had 6.4 times the odds of testing seropositive to T. gondii, compared to non-neurologic horses. The association between high T. gondii titres and clinical signs compatible with EPM is potentially reflective of toxoplasmosis in equines. Serologic testing of cerebrospinal fluid and isolation of T. gondii in EPM suspect cases should be considered. Future studies investigating the relationship between T. gondii and EPM are warranted.

Seroprevalence and risk factors for infection with equine coronavirus in healthy horses in the USA.

Equine coronavirus (ECoV) is considered an enteric pathogen of foals and has only recently been associated with infections in adult horses. Seroprevalence data is needed to better understand the epidemiology of ECoV in adult horses, evaluate diagnostic modalities and develop preventive measures. The objective of this study was to investigate the seroprevalence and selective risk factors for ECoV in 5247 healthy adult horses in the USA, using a recently established and validated IgG enzyme-linked immunosorbent assay. Prevalence factors analysed in this study included geographic region, age, breed, sex and use. A total of 504/5247 horses (9.6%) horses tested seropositive. Geographic region (Mid-West; P = 0.008), breed (Draft horses; P = 0.003) and specific uses of horses (ranch/farm, P = 0.034; breeding use, P = 0.016) were all statistically significant risk factors for seropositivity.

Pharmacokinetic parameters for single- and multi-dose regimens for subcutaneous administration of a high-dose ceftiofur crystalline-free acid to neonatal foals.

The objective of this study was to determine the pharmacokinetics of single- and multi-dose ceftiofur crystalline-free acid (CCFA) administered subcutaneously at a dose of 13.2 mg/kg to 12 neonatal foals 1-3 days of age. Six foals received a single subcutaneous dose, while 6 additional foals received 4 doses of CCFA at 48-h intervals. Blood samples were collected at pre-determined times following drug administration, and plasma concentrations of ceftiofur free acid equivalents (CFAE) were measured using high-performance liquid chromatography. Following single-dose administration of CCFA, the mean ± standard deviation maximum observed plasma concentration was 3.1 ± 0.6 µg/mL and observed time to maximal plasma concentration was 14.0 ± 4.9 h. Following multi-dose administration of CCFA, the mean ±standard deviation times above CFAE concentrations of ≥0.5 µg/mL and ≥2.0 µg/mL were 192.95 ± 15.86 h and 78.80 ± 15.31 h, respectively. The mean ± standard deviation area under the concentration vs time curve (AUC0→∝ ) was 246.2 ± 30.7 h × µg/mL and 172.7 ± 27.14 h × µg/mL following single- and multi-dose CCFA administrations, respectively. Subcutaneous administration of CCFA at 13.2 mg/kg in neonatal foals was clinically well- tolerated and resulted in plasma concentrations sufficient for the treatment of most bacterial pathogens associated with neonatal foal septicemia. Multi-dose administration of four doses at dosing interval of 48 h between treatments maintains appropriate therapeutic concentrations in neonatal foals.

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.

Field use of N-butylscopolammonium bromide to facilitate thorough ophthalmic examination in horses.

N-butylscopolammonium bromide (NBB), an anticholinergic muscarinic antagonist, was assessed as a mydriatic agent for field examination of equine eyes. Six adult horses were randomly assigned to four treatments with 2 weeks washout between treatments: (1) topical saline/IV saline (negative control); (2) topical tropicamide/IV saline (positive control); (3) topical NBB/IV saline; or (4) topical saline/IV NBB. Horizontal and vertical pupil diameters, temperature, pulse, respiration, pupillary light reflexes (PLRs) and mydriasis sufficient to perform complete fundic examination were recorded. Tropicamide induced mydriasis in all horses. Topical NBB induced mydriasis in one horse, and IV NBB enabled thorough fundic examination in two horses, delayed PLRs without allowing thorough examination in two horses and had no effect in two horses.

Equine Protozoal Myeloencephalitis: An Updated Consensus Statement with a Focus on Parasite Biology, Diagnosis, Treatment, and Prevention.

Equine protozoal myeloencephalitis (EPM) remains an important neurologic disease of horses. There are no pathognomonic clinical signs for the disease. Affected horses can have focal or multifocal central nervous system (CNS) disease. EPM can be difficult to diagnose antemortem. It is caused by either of 2 parasites, Sarcocystis neurona and Neospora hughesi, with much less known about N. hughesi. Although risk factors such as transport stress and breed and age correlations have been identified, biologic factors such as genetic predispositions of individual animals, and parasite-specific factors such as strain differences in virulence, remain largely undetermined. This consensus statement update presents current published knowledge of the parasite biology, host immune response, disease pathogenesis, epidemiology, and risk factors. Importantly, the statement provides recommendations for EPM diagnosis, treatment, and prevention.

Prevalence factors associated with equine herpesvirus type 1 infection in equids with upper respiratory tract infection and/or acute onset of neurological signs from 2008 to 2014.

The objective of the present case-control study was to determine prevalence factors associated with the detection of equine herpesvirus type 1 (EHV-1) by quantitative PCR (qPCR) in horses presented to veterinarians with clinical signs related to an upper respiratory tract infection and/or acute onset of neurological disease from March 2008 to December 2014. Nasal secretions and whole blood from 4228 equids with acute onset of fever, respiratory signs and/or neurological deficits were tested by qPCR for EHV-1. Categorical analyses were performed to determine the association between observations and EHV-1. A total of 117/4228 (2.7 per cent) equids tested qPCR-positive for EHV-1, with most of the isolates belonging to the non-neuropathogenic genotype (N752). EHV-1 PCR-positive equids were over-represented in racing horses. Depression, anorexia, nasal discharge and coughing were significantly less frequently reported in the EHV-1 qPCR-positive equids compared with the EHV-1 qPCR-negative cases. Neurological deficits were more frequently reported in the EHV-1 qPCR-positive cases. This study provides contemporary information on the frequency of EHV-1 detection by qPCR in blood and nasal secretions from horses with fever, respiratory signs and neurological deficits.

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.

The Use of Recombinant Tissue Plasminogen Activator (rTPA) in The Treatment of Fibrinous Pleuropneumonia in Horses: 25 Cases (2007-2012).

BACKGROUND: Information about treatment protocols, adverse effects and outcomes with intrapleural recombinant tissue plasminogen activator (rTPA) use in horses with fibrinous pleuropneumonia is limited. HYPOTHESIS/OBJECTIVES: Describe factors that contribute to clinical response and survival of horses treated with rTPA intrapleurally. ANIMALS: Horses with bacterial pneumonia and fibrinous pleural effusion diagnosed by ultrasonography, that were treated with rTPA intrapleurally. METHODS: Retrospective multicenter case series from 2007-2012. Signalment, history, clinical and laboratory evaluation, treatment, and outcome obtained from medical records. Regression analysis used to identify associations between treatments and outcomes. RESULTS: Thirty three hemithoraces were treated in 25 horses, with 55 separate treatments. Recombinant tissue plasminogen activator (375-20,000 µg/hemithorax) was administered 1-4 times. Sonographically visible reduction in fibrin mat thickness, loculations, fluid depth, or some combination of these was seen in 32/49 (65%) treatments. Response to at least 1 treatment was seen in 17/20 (85%) horses with sonographic follow-up evaluation after every treatment. Earlier onset of rTPA treatment associated with increased survival odds. No association was found between cumulative rTPA dose or number of rTPA doses and survival, development of complications, duration of hospitalization or total charges. Clinical evidence of hypocoagulability or bleeding was not observed. Eighteen horses (72%) survived to discharge. CONCLUSIONS AND CLINICAL IMPORTANCE: Treatment with rTPA appeared safe and resulted in variable changes in fibrin quantity and organization within the pleural space. Recombinant tissue plasminogen activator could be a useful adjunct to standard treatment of fibrinous pleuropneumonia, but optimal case selection and dosing regimen remain to be elucidated.

Necrotizing Enteritis and Hyperammonemic Encephalopathy Associated With Equine Coronavirus Infection in Equids.

Equine coronavirus (ECoV) is a Betacoronavirus recently associated clinically and epidemiologically with emerging outbreaks of pyrogenic, enteric, and/or neurologic disease in horses in the United States, Japan, and Europe. We describe the pathologic, immunohistochemical, ultrastructural, and molecular findings in 2 horses and 1 donkey that succumbed to natural infection with ECoV. One horse and the donkey (case Nos. 1, 3) had severe diffuse necrotizing enteritis with marked villous attenuation, epithelial cell necrosis at the tips of the villi, neutrophilic and fibrinous extravasation into the small intestinal lumen (pseudomembrane formation), as well as crypt necrosis, microthrombosis, and hemorrhage. The other horse (case No. 2) had hyperammonemic encephalopathy with Alzheimer type II astrocytosis throughout the cerebral cortex. ECoV was detected by quantitative polymerase chain reaction in small intestinal tissue, contents, and/or feces, and coronavirus antigen was detected by immunohistochemistry in the small intestine in all cases. Coronavirus-like particles characterized by spherical, moderately electron lucent, enveloped virions with distinct peplomer-like structures projecting from the surface were detected by negatively stained transmission electron microscopy in small intestine in case No. 1, and transmission electron microscopy of fixed small intestinal tissue from the same case revealed similar 85- to 100-nm intracytoplasmic particles located in vacuoles and free in the cytoplasm of unidentified (presumably epithelial) cells. Sequence comparison showed 97.9% to 99.0% sequence identity with the ECoV-NC99 and Tokachi09 strains. All together, these results indicate that ECoV is associated with necrotizing enteritis and hyperammonemic encephalopathy in equids.