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.     

Outbreak of equine coronavirus infection among riding horses in Tokyo,Japan

In 2020, an outbreak of equine coronavirus (ECoV) infection occurred among 41 horses at a riding stable in Tokyo, Japan. This stable had 16 Thoroughbreds and 25 horses of other breeds, including Andalusians, ponies and miniature horses. Fifteen horses (37 %) showed mild clinical signs such as fever, lethargy, anorexia and diarrhoea, and they recovered within 3 days of onset. A virus neutralization test showed that all 41 horses were infected with ECoV, signifying that 26 horses (63 %) were subclinical. The results suggest that subclinical horses played an important role as spreaders. A genome sequence analysis revealed that the lengths from genes p4.7 to p12.7 or NS2 in ECoV differed from those of ECoVs detected previously, suggesting that this outbreak was caused by a virus different from those that caused previous outbreaks among draughthorses in Japan. Among 30 horses that tested positive by real-time RT-PCR, ECoV shedding periods of non-Thoroughbreds were significantly longer than those of Thoroughbreds. The difference in shedding periods may indicate that some breeds excrete ECoV longer than other breeds and can contribute to the spread of ECoV.     

Equine Coronavirus-Associated Colitis in Horses: A Retrospective Study

Equine coronavirus (ECoV) is a known cause of fever, anorexia, and lethargy in adult horses. Although there are multiple reports of ECoV outbreaks, less is known about the clinical presentation of individual horses during a nonoutbreak situation. The purpose of this study was to describe the clinical presentation of horses diagnosed with ECoV infection that were not associated with an outbreak. Medical records of all horses admitted to Washington State University, Veterinary Teaching Hospital, during an 8-year period were reviewed (2010–2018). The five horses included in this study were older than 1 year of age, were diagnosed with colitis, tested positive for ECoV using real-time polymerase chain reaction, and were negative to other enteric pathogens. Interestingly, 4 of 5 horses had moderate to severe diarrhea, 3 had abnormal large colon ultrasonography, 2 had transient ventricular tachycardia and 2 had clinicopathologic evidence of liver dysfunction. ECoV should be included as a differential diagnosis for individual horses presenting with anorexia, fever, lethargy, and colitis. Early identification of ECoV cases is key to implement appropriate biosecurity measures to prevent the potential spread of this disease.     

Portosystemic shunts: A diagnostic challenge

The case report in this issue by Woodford et al. (2017) describes the surgical management of an extrahepatic portosystemic shunt in a 5‐week old miniature foal. Only a handful of reports exist in the literature describing surgical procedures for repair of these congenital abnormalities in foals. One of the important aspects of the accompanying case report was the multidisciplinary approach taken in handling, imaging and surgical repair of the extrahepatic shunt. Small animal internal medicine specialists and surgeons diagnose portosystemic shunts much more frequently than equine veterinarians, regardless of whether they are general practitioners or specialists. It is therefore important to work as a team with small and large animal practitioners. The case described by Woodford et al. (2017) had a positive outcome.     

Lessons learned from a strangles outbreak on a large Standardbred farm

Streptococcus equi subspecies (ssp.) equi infection (strangles) remains one of the most frequently diagnosed and costly infectious diseases of horses. Large breeding herds, where a disease outbreak competes for personnel and financial resources needed for foaling management, present a special challenge for equine practitioners. A 15‐month outbreak involving 62 clinical cases of strangles occurred on a large Standardbred breeding farm (average population of 1400 horses). Sixteen asymptomatic horses were found to be PCR (polymerase chain reaction)‐positive for S. equi ssp. equi. During the outbreak, serological samples from 48 clinically normal horses were found to be seropositive for S. equi ssp. equi, confirming herd‐wide exposure. After several clinical cases of strangles had been diagnosed, an intranasal S. equi ssp. equi vaccine was administered to clinically normal horses (n = 558) considered to be at risk of exposure. Strangles complications included 7 fatalities (none in vaccinated horses) and 6 cases of purpura haemorrhagica (4 in vaccinated horses). Midway through the outbreak, injectable, sustained release ceftiofur crystalline free acid (CCFA), given as an initial dose followed by a second dose 4 days later, was used exclusively for systemic antimicrobial treatment of clinically affected and PCR‐positive horses. This antimicrobial regimen coincided with a reduction in disease incidence and eventual resolution of the outbreak. Two horses with persistent guttural pouch infection were endoscopically confirmed as carrier horses. The herd history demonstrated that a strangles outbreak will often result in asymptomatic carrier horses and that identification and treatment of these horses are necessary to eliminate long‐term sources of infection. Ceftiofur crystalline free acid was found to be a suitable antimicrobial due to its activity against S. equi ssp. equi and the efficiencies associated with twice parenteral dosing during a 10‐day treatment period. Occurrence of purpura in 4 vaccinated horses suggests that vaccination should be reserved for healthy seronegative horses and avoided during an active outbreak.     

Emerging outbreaks associated with equine coronavirus in adult horses

The purpose of this study was to describe clinical, hematological and fecal PCR results from 161 horses involved in outbreaks associated with ECoV. The outbreaks happened at four separate boarding facilities between November 2011 and April 2012 in the States of CA, TX, WI and MA. Following the molecular detection of ECoV in the feces from the initial index cases, the remaining herdmates were closely observed for the development of clinical signs. Fecal samples were collected from sick and healthy horses for the PCR detection of ECoV. All four outbreaks involved primarily adult horses. Fifty-nine horses developed clinical signs with 12–16 sick horses per outbreak. The main clinical signs reported were anorexia, lethargy and fever. Four horses from 3 different outbreaks were euthanized or died due to rapid progression of clinical signs. The cause of death could not be determined with necropsy evaluation in 2 horses, while septicemia secondary to gastrointestinal translocation was suspected in 2 horses. Blood work was available from 10 horses with clinical disease and common hematological abnormalities were leucopenia due to neutropenia and/or lymphopenia. Feces were available for ECoV testing by real-time PCR from 44 and 96 sick and healthy horses, respectively. 38/44 (86%) horses with abnormal clinical signs tested PCR positive for ECoV, while 89/96 (93%) healthy horses tested PCR negative for ECoV. The overall agreement between clinical status and PCR detection of ECoV was 91%. The study results suggest that ECoV is associated with self-limiting clinical and hematological abnormalities in adult horses.     

Severe outbreak of bovine coronavirus infection in dairy cattle during the warmer season

A severe outbreak of enteric and respiratory disease associated with bovine coronavirus (BCoV) infection is described. The outbreak occurred in a dairy herd of southern Italy in the first decade of September 2006, when summer temperatures were still recorded, affecting calves, heifers and adult cows, with a marked decrease in milk production. By virus isolation and RT-PCR targeting the S gene, BCoV was identified as the etiological agent of the outbreak, whereas bacteriological, parasitological and toxicological investigations failed to detect other causes of disease. BCoV strains with 99-100% nucleotide identity in the S gene were isolated from nasal, ocular and rectal swabs, thus proving the absence of separate clusters of virus on the basis of tissue tropism. Sequence analysis of the haemagglutination-esterase and spike proteins of the strain detected in one rectal sample (339/06) showed a high genetic relatedness with recent BCoV isolates (98-99% amino acid identity), with several unique amino acid substitutions in the S protein. The BCoV outbreak described in this paper presents interesting aspects: (i) the occurrence of a severe form of disease in the warmer season; (ii) the simultaneous presence of respiratory and enteric disease; (iii) the involvement of young as well as adult cattle.     

Effect of body weight on the pharmacokinetics of flunixin meglumine in miniature horses and quarter horses

In most species, large variations in body size necessitate dose adjustments based on an allometric function of body weight. Despite the substantial disparity in body size between miniature horses and light‐breed horses, there are no studies investigating appropriate dosing of any veterinary drug in miniature horses. The purpose of this study was to determine whether miniature horses should receive a different dosage of flunixin meglumine than that used typically in light‐breed horses. A standard dose of flunixin meglumine was administered intravenously to eight horses of each breed, and three‐compartmental analysis was used to compare pharmacokinetic parameters between breed groups. The total body clearance of flunixin was 0.97 ± 0.30 mL/min/kg in miniature horses and 1.04 ± 0.27 mL/min/kg in quarter horses. There were no significant differences between miniature horses and quarter horses in total body clearance, the terminal elimination rate, area under the plasma concentration versus time curve, apparent volume of distribution at steady‐state or the volume of the central compartment for flunixin (P > 0.05). Therefore, flunixin meglumine may be administered to miniature horses at the same dosage as is used in light‐breed horses.     

Molecular and phylogenetic characterization of bovine coronavirus virus isolated from dairy cattle in Central Region,Thailand

Tropical Animal Health and Production - Bovine coronavirus (BCoV) is involved mainly in enteric infections in cattle. This study reports the first molecular detection of BCoV in a diarrhea outbreak...     

Upper respiratory signs associated with Anaplasma phagocytophilum infection in two horses

An adult Quarter Horse gelding (Case 1) was evaluated for tachypnoea and acute dysphagia. A 20-year-old Quarter Horse gelding (Case 2) was evaluated for respiratory stertor and severe, acute swelling of the head in the submandibular region. A physical examination, complete blood count, blood chemistry, upper airway endoscopy, and peripheral blood Anaplasma phagocytophilum polymerase chain reaction were completed for both horses. Both horses tested positive for A. phagocytophilum. The upper airway endoscopy for Case 1 revealed a feed contaminated pharynx, absent swallowing reflex, and left laryngeal hemiplagia. The upper airway endoscopy for Case 2 revealed severe diffuse pharyngeal swelling occluding the airway. Due to increased respiratory effort in Case 2, a tracheotomy was performed. In both horses, treatment consisted of intravenous oxytetracycline 6.6 mg/kg bwt i.v. q. 24 h for 2–3 days followed by minocycline 4 mg/kg bwt per os q. 12 h for 10–14 days. Both horses made full recoveries.     

Differentiating among horse (Equus caballus), donkey (Equus asinus) and their hybrids with combined analysis of nuclear and mitochondrial gene polymorphism

A novel and brief method of differentiating among horse (Equus caballus) and donkey (Equus asinus) and their hybrids (mule, E. asinus × E. caballus and hinny, E. caballus × E. asinus) with combined analysis of nuclear and mitochondrial gene polymorphism (CANMGP) was reported in the present report. A nuclear gene, protamine P1 gene of donkey was sequenced and compared with the known horse sequence from GenBank while a published equid mitochondrial gene, cytochrome b gene of donkey was compared with that of horse. In each of the two genes, a fixed nucleotide substitution within an exon that could be recognized by Dpn II restriction enzyme was found between the two species. Two pairs of primers were designed for amplifying the fragments within the two genes containing the informative nucleotide positions in 65 horses and 41 donkeys and 38 hybrids and conditions of polymerase chain reaction and restriction fragment length polymorphism (PCR‐RFLP) analysis were optimized. Horse, donkey and mule and hinny had their own specific cleavage patterns after the PCR‐RFLP analysis was performed, which made it very easy to identify them from each other. As multiplex PCR can be conducted with the two pairs of primers and only one restriction enzyme is involved in PCR‐RFLP analysis, the method described in the present study is a convenient way to identify horse and donkey and their hybrids. The idea involved in the method of CANMGP can be also used to differentiate other animal species or breeds and their hybrids.     

Bioavailability and pharmacokinetics of metronidazole in fed and fasted horses

Britzi, M., Gross, M., Lavy, E., Soback, S., Steinman, A. Bioavailability and pharmacokinetics of metronidazole in fed and fasted horses. J. vet. Pharmacol. Therap. 33 , 511–514. Metronidazole (1‐[2‐hydroxyethyl]‐2‐methyl‐5‐nitroimidazole) is a bactericidal antimicrobial agent used for treatment of infectious diseases caused by anaerobic bacteria and protozoa. Pharmacokinetics of metronidazole following its administration to horses was previously described ( Sweeney et al., 1986 ; Baggot et al., 1988 ; Specht et al., 1992 ; Steinman et al., 2000 ). The bioavailability (F) was 85% (ranging from 57% to 105%) and the time to reach maximum serum concentration (t_max) was 1–2 h after oral dose at 25 mg/kg body weight ( Sweeney et al., 1986 ). Baggot et al. (1988) found that F was 74.5% (ranging from 58.4% to 91.5%) and t_max was 1.5 h after oral dose at 20 mg/kg body weight. Specht et al. (1992) reported that F was 97% (ranging from 79% to 111%) and t_max was 40 min after oral dose at 15 mg/kg body weight. In an earlier study by our group F was 74% and t_max was 65 min after oral dose at 20 mg/kg body weight ( Steinman et al., 2000 ). These individual variations in F might be partially explained by the effect of feed, among other factors, mainly on metronidazole absorption. Interactions between food and drugs may reduce or increase the drug effect. The majority of clinically relevant food–drug interactions are caused by food‐induced changes on the bioavailability of the drug ( Schmidt & Dalhoff, 2002 ). In dogs, absorption of metronidazole is enhanced when given with food, but delayed in humans ( Plumb, 1995 ). Although, metronidazole is used commonly to treat various clinical conditions in horses with relatively little adverse effects ( Sweeney et al., 1991 ), narrow margin of safety was suggested because histological evidence of peripheral neurotoxicity and hepatotoxicity were noted in horses treated with doses as low as 30 mg/kg body weight every 12 h orally for 30 days ( White et al., 1996 ). For drugs with a narrow therapeutic index, even small changes in dose–response effects can have significant consequences ( Schmidt & Dalhoff, 2002 ).     

Dairy calf rearing unit and infectious diseases: diarrhea outbreak by bovine coronavirus as a model for the dispersion of pathogenic microorganisms

Dairy calf rearing unit is a management system that is only recently being implemented by some milk producer’s cooperatives in southern Brazil. However, aspects related to the health profile of the heifer calves that arrive in the rearing unit as well as about biosecurity practices and microbiological challenges have not yet been evaluated in this rearing system in a tropical country. Diarrhea is the main and most frequent consequence of enteric infections in newborn calves. This study, through some etiological and epidemiological characteristics of an outbreak of neonatal diarrhea, has the aim to alert to the possibility of pathogenic microorganism spread in a dairy heifer calf rearing unit. The diarrhea outbreak presented some non-regular characteristics observed in bovine coronavirus (BCoV) enteric infections in dairy calves. The spread of infection was extremely rapid (1 week); the attack rate (>?50%) was much higher than that observed in calves subjected to conventional rearing; and the age range (5 to 90 days) of the affected heifer calves was much broader than that often observed in the BCoV diarrhea worldwide. These unusual epidemiological characteristics observed in this BCoV diarrhea outbreak raise awareness of the health threat present in calf rearing units as well as of the easy and rapid viral spread in a population of young animals from different dairy herds and, therefore, with very distinct immunological status.

     

Equine coronavirus induces apoptosis in cultured cells

Equine coronavirus (ECoV) was first isolated from a diarrheic foal and was found genetically similar to group II coronaviruses. However, its pathological characteristics were not adequately investigated. In our preliminary in vitro investigation, ECoV-induced cell death was observed in bovine kidney-derived MDBK cells. Based on this finding, we investigated whether the ECoV-induced CPE was apoptosis. Following ECoV infection, MDBK cells showed morphological changes such as cell rounding and detachment from the culture surface. Moreover, syncytium formation was observed as the other type of cytopathic effect in ECoV infection. Morphologic and biochemical features of apoptosis, such as nuclear fragmentation and DNA ladder formation, were also detected in ECoV-infected cells. Moreover, as is commonly observed in coronavirus infection in other animals, the activities of effecter caspases – caspase-3/7 – and initiator caspases – caspase-8 and caspase-9 – that are representative factors in the death receptor-mediated apoptotic pathway and mitochondrial apoptotic pathway, respectively, were increased in ECoV-infected MDBK cells. Therefore, it was suggested that ECoV can induce apoptosis in MDBK cells via a caspase-dependent pathway. Apoptotic death of infected cells is detrimental because it causes cell and tissue destruction and inflammatory responses. Although the pathological characteristics of ECoV are largely unknown, apoptosis may be the pathological basis of lesions of the digestive system in ECoV infection.     

Disease Associated with Equine Coronavirus Infection and High Case Fatality Rate

Background

Equine coronavirus (ECoV) is associated with clinical disease in adult horses. Outbreaks are associated with a low case fatality rate and a small number of animals with signs of encephalopathic disease are described.

Objectives

The aim of this study is to describe the epidemiological and clinical features of two outbreaks of ECoV infection that were associated with an high case fatality rate.

Animals

14 miniature horses and 1 miniature donkey testing fecal positive for ECoV from two related disease outbreaks.

Methods

Retrospective study describing the epidemiological findings, clinicopathological findings, and fecal viral load from affected horses.

Results

In EcoV positive horses, 27% (4/15) of the animals died or were euthanized. Severe hyperammonemia (677 μmol/L, reference range ≤60 μmol/L) was identified in one animal with signs of encephalopathic disease that subsequently died. Fecal viral load (ECoV genome equivalents per gram of feces) was significantly higher in the nonsurvivors compared to animals that survived (P = .02).

Conclusions and Clinical Importance

Equine coronavirus had a higher case fatality rate in this group of miniature horses than previously reported in other outbreaks of varying breeds. Hyperammonemia could contribute to signs of encephalopathic disease, and the fecal viral load might be of prognostic value in affected horses.     

Tuberculosis caused by Mycobacterium bovis infection in a donkey

Mycobacterial infections are rare in equines. Mycobacterium bovis (M. bovis) is an important zoonotic bacterial pathogen causing disease in a wide range of animal species and sporadically causes severe disseminated disease in horses. This report describes the clinical, gross post‐mortem examination and histopathological findings in a case of disseminated M. bovis infection in a donkey which to the authors’ knowledge has not been previously documented in the scientific literature.     

Frequency of BCoV detection by a semi-nested PCR assay in faeces of calves from Brazilian cattle herds

Bovine coronavirus (BCoV) is one of the main causes of neonatal calf diarrhoea. Several diagnostic assays have been employed to detect the presence of the virus in stool samples from calves. Despite this, the frequency of BCoV infection among Brazilian and even South American cattle herds has yet to be well characterised. This study describes the occurrence of BCoV infection among calves from dairy and beef herds in four Brazilian states. A total of 282 stool samples from 1 to 60-day-old calves were evaluated for the presence of BCoV by a semi-nested (SN) PCR assay. The animals were from herds (n = 23) located in three geographical regions in Brazil (south, southeast, and center-west). The specific BCoV amplicon was detected in 15.6% (44/282) of the faecal specimens examined, of which 95.4% (42/44) were from diarrhoeic and 4.6% (2/44) from asymptomatic calves. The specificity of the SN-PCR amplicons was evaluated by restriction fragment length polymorphism (RFLP) analysis. The results show that the BCoV is widespread, mainly among calves from 16 to 30-days-old (p = 0.0023), and verify the association between BCoV infection and clinical signs of diarrhoea (p = 0.005). These findings emphasise the importance of this virus in enteric infections of Brazilian cattle herds.     

Congenital stationary night blindness is associated with the leopard complex in the Miniature Horse

Objective To determine if congenital stationary night blindness (CSNB) exists in the miniature horse in association with leopard complex spotting patterns (LP), and to investigate if CSNB in the miniature horse is associated with three single nucleotide polymorphisms (SNPs) in the region of TRPM1 that are highly associated with CSNB and LP in Appaloosas. Animals studied Three groups of miniature horses were studied based on coat patterns suggestive of LP/LP (n = 3), LP/lp (n = 4), and lp/lp genotype (n = 4). Procedures Horses were categorized based on phenotype as well as pedigree analysis as LP/LP, LP/lp, and lp/lp. Neurophthalmic examination, slit‐lamp biomicroscopy, indirect ophthalmoscopy, and scotopic flash electroretinography were performed on all horses. Hair samples were processed for DNA analysis. Three SNPs identified and associated with LP and CSNB in the Appaloosa were investigated for association with LP and CSNB in these Miniature horses. Results All horses in the LP/LP group were affected by CSNB, while none in the LP/lp or lp/lp groups were affected. All three SNPs were completely associated with LP genotype (χ² = 22, P << 0.0005) and CSNB status (χ² = 11, P < 0.0005). Conclusions The Miniature Horse breed is affected by CSNB and it appears to be associated with LP as in the Appaloosa breed. The SNPs tested could be used as a DNA test for CSNB until the causative mutation is determined.