Equine Coronavirus Research Articles

Proficiency test of SARS-CoV-2 Omicron variant detection in diagnostics samples by veterinary diagnostic laboratories

AbstractVeterinary diagnostic laboratories (VDLs) play a critical role in screening both human and animal samples for SARS-CoV-2. To evaluate the SARS-CoV-2 detection methods used by VDLs, a proficiency test was performed by the US Food and Drug Administration’s Veterinary Laboratory and Investigation and Response Network in collaboration with two other organizations. Thirty-two sets of 12 blind-coded samples were prepared by fortifying Molecular Transport Medium (MTM) or feline feces with SARS-CoV-2 Omicron variant or non-SARS-CoV-2 equine coronavirus RNA at various concentrations and shipped to 32 participants for blinded (unbiased) analysis. Results were analyzed according to the principles of International Organization for Standardization 16140-2:2016 using two approaches such as establishing the rate of detection (ROD) and the success rate by applying the analysis of binary outcome by logit approach. ROD provided the overall assessment of laboratories performance, whereas the novel logit approach provided an insight to more specific analysis based on the complexity of each sample. The ROD was 83% and 98% for MTM samples at 200 and 20000 genome copies per 100 µL, respectively. Fecal samples were classified as challenging exploratory, and results were not included in the assessment of performance but discussion purposes only. Fecal samples exhibited matrix interference impacting the performance. The ROD was 44% and 89% for fecal samples at 2000 and 20000 genome copies per 100 µL, respectively. The non-COVID coronavirus RNA, which was used to address the specificity, did not interfere with methods used. Establishing the success rate by evaluating the qualitative results (detected/not detected) applying a logit approach revealed that, out of thirty-two participants, twenty-eight had satisfactory results, one participant had unsatisfactory results, and three participants had questionable results for MTM samples. For fecal samples, three participants out of thirty-two did not meet the expectations at higher concentrations. Lower concentrations of fecal samples were excluded from this analysis. Again, the fecal samples were considered as challenge samples and the results were provided to assist participants in their continuous efforts to improve their performance and not to evaluate their performance.

Equine coronavirus infection and replication in equine intestinal enteroids

In this study, equine intestinal enteroids (EIEs) were generated from the duodenum, jejunum, and ileum and inoculated with equine coronavirus (ECoV) to investigate their suitability as in vitro models with which to study ECoV infection. Immunohistochemistry revealed that the EIEs were composed of various cell types expressed in vivo in the intestinal epithelium. Quantitative reverse-transcription PCR (qRT-PCR) and virus titration showed that ECoV had infected and replicated in the EIEs. These results were corroborated by electron microscopy. This study suggests that EIEs can be novel in vitro tools for studying the interaction between equine intestinal epithelium and ECoV.

A Review of Epithelial Ion Transporters and Their Roles in Equine Infectious Colitis.

Equine colitis is a devastating disease with a high mortality rate. Infectious pathogens associated with colitis in the adult horse include Clostridioides difficile, Clostridium perfringens, Salmonella spp., Neorickettsia risticii/findlaynesis, and equine coronavirus. Antimicrobial-associated colitis can be associated with the presence of infectious pathogens. Colitis can also be due to non-infectious causes, including non-steroidal anti-inflammatory drug administration, sand ingestion, and infiltrative bowel disease. Current treatments focus on symptomatic treatment (restoring fluid and electrolyte balance, preventing laminitis and sepsis). Intestinal epithelial ion channels are key regulators of electrolyte (especially sodium and chloride) and water movement into the lumen. Dysfunctional ion channels play a key role in the development of diarrhea. Infectious pathogens, including Salmonella spp. and C. difficile, have been shown to regulate ion channels in a variety of ways. In other species, there has been an increased interest in ion channel manipulation as an anti-diarrheal treatment. While targeting ion channels also represents a promising way to manage diarrhea associated with equine colitis, ion channels have not been well studied in the equine colon. This review provides an overview of what is known about colonic ion channels and their known or putative role in specific types of equine colitis due to various pathogens.

Positive selection, genetic recombination, and intra-host evolution in novel equine coronavirus genomes and other members of the Embecovirus subgenus.

There are several examples of coronaviruses in the Betacoronavirus subgenus Embecovirus that have jumped from an animal to the human host. Studying how evolutionary factors shape coronaviruses in non-human hosts may provide insight into the coronavirus host-switching potential. Equids, such as horses and donkeys, are susceptible to equine coronaviruses (ECoVs). With increased testing prevalence, several ECoV genome sequences have become available for molecular evolutionary analyses, especially those from the United States of America (USA). To date, no analyses have been performed to characterize evolution within coding regions of the ECoV genome. Here, we obtain and describe four new ECoV genome sequences from infected equines from across the USA presenting clinical symptoms of ECoV, and infer ECoV-specific and Embecovirus-wide patterns of molecular evolution. Within two of the four data sets analyzed, we find evidence of intra-host evolution within the nucleocapsid (N) gene, suggestive of quasispecies development. We also identify 12 putative genetic recombination events within the ECoV genome, 11 of which fall in ORF1ab. Finally, we infer and compare sites subject to positive selection on the ancestral branch of each major Embecovirus member clade. Specifically, for the two currently identified human coronavirus (HCoV) embecoviruses that have spilled from animals to humans (HCoV-OC43 and HCoV-HKU1), we find that there are 42 and 2 such sites, respectively, perhaps reflective of the more complex ancestral evolutionary history of HCoV-OC43, which involves several different animal hosts.IMPORTANCEThe Betacoronavirus subgenus Embecovirus contains coronaviruses that not only pose a health threat to animals and humans, but also have jumped from animal to human host. Equids, such as horses and donkeys are susceptible to equine coronavirus (ECoV) infections. No studies have systematically examined evolutionary patterns within ECoV genomes. Our study addresses this gap and provides insight into intra-host ECoV evolution from infected horses. Further, we identify and report natural selection pattern differences between two embecoviruses that have jumped from animals to humans [human coronavirus OC43 and HKU1 (HCoV-OC43 and HCoV-HKU1, respectively)], and hypothesize that the differences observed may be due to the different animal host(s) that each virus circulated in prior to its jump into humans. Finally, we contribute four novel, high-quality ECoV genomes to the scientific community.

Equine coronavirus infection and replication in equine intestinal enteroids

Equine coronavirus infection and replication in equine intestinal enteroids

Seasonal trends of equine coronavirus in Sweden based on samples analysed at Swedish Veterinary Agency 2019–April 2024

Seasonal trends of equine coronavirus in Sweden based on samples analysed at Swedish Veterinary Agency 2019–April 2024

Investigation of the seroprevalence to equine coronavirus and SARS-CoV-2 in healthy adult horses recently imported to the United States

Adult horses are susceptible to equine coronavirus (ECoV) and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), although, only ECoV has been linked to clinical disease. Little information is available regarding the seroprevalence against ECoV and SARS-CoV-2 in adult healthy horses. The goal of the present study was to determine the seroprevalence against two coronaviruses known to infect horses using convenience samples collected from horses recently imported from Europe to the United States from 2019 to 2023. A total of 385 banked serum samples were tested against ECoV and SARS-CoV-2 using previously validated ELISA assays. Prevalence factors including date of arrival in the United States, signalment and country of origin were available for the majority of the horses. A total of 9/385 (2.3%) and 4/385 (1.0%) horses tested seropositive for ECoV and SARS-CoV-2, respectively. The ECoV seropositive horses were all mares, ages 4 to 26 years (median 9 years) and originated from Germany, the Netherlands, Ireland, Belgium and Italy. These mares were predominantly imported during the summer and fall months. All SARS-CoV-2 seropositive horses were mares ages 5 to 10 years (median 7.5 years) imported from the Netherlands and the United Kingdom. The majority of the SARS-CoV-2 seropositive horses were imported during the colder month of the year. The study results support the presence of ECoV in Europe and report on the first SARS-CoV-2 seropositive healthy adult horses outside the United States. Commingling for movements by air and close contact to humans may predispose transmission with ECoV and SARS-CoV-2, respectively.

Performance of a microfluidic immunofluorescence assay kit for equine influenza virus antigen detection

Equine influenza virus (EIV) infection is one of the most important respiratory diseases in the equine industry around the world. Rapid diagnosis, facilitated by point-of-care testing, is essential to implement movement restrictions and control disease outbreaks. This study evaluated a microfluidic immunofluorescence assay kit, which detects influenza virus and SARS-CoV-2 antigens in human specimens with a 12 min turnaround time, for its potential use in detecting EIV. The microfluidic immunofluorescence assay kit succeeded in detecting 11 EIV strains. Using the real-time reverse transcription polymerase chain reaction as a reference assay, the microfluidic immunofluorescence assay kit showed a sensitivity of 60.7% when evaluating nasopharyngeal swab samples of three horses experimentally infected with EIV. Comparing with the other two rapid antigen detection kits based on immunochromatography and silver amplification immunochromatography, the microfluidic immunofluorescence assay kit exhibited higher sensitivity than the former assay (53.6%) and the same sensitivity as the latter (60.7%). The microfluidic immunofluorescence assay kit did not detect nine non-EIV viruses including one equine coronavirus strain and seven bacteria, suggesting a high specificity for EIV antigens. Similar to other rapid antigen detection kits, the microfluidic immunofluorescence assay kit could be an effective diagnostic tool to detect EIV in the field.

Presence of Equine and Bovine Coronaviruses, Endoparasites, and Bacteria in Fecal Samples of Horses with Colic.

Acute abdominal pain (colic) is one of the major equine health threats worldwide and often necessitates intensive veterinary medical care and surgical intervention. Equine coronavirus (ECoV) infections can cause colic in horses but are rarely considered as a differential diagnosis. To determine the frequency of otherwise undetected ECoV infections in horses with acute colic, fresh fecal samples of 105 horses with acute colic and 36 healthy control horses were screened for viruses belonging to the Betacoronavirus 1 species by RT-PCR as well as for gastrointestinal helminths and bacteria commonly associated with colic. Horses with colic excreted significantly fewer strongyle eggs than horses without colic. The prevalence of anaerobic, spore-forming, gram-positive bacteria (Clostridium perfringens and Clostridioides difficile) was significantly higher in the feces of horses with colic. Six horses with colic (5.7%) and one horse from the control group (2.8%) tested positive for Betacoronaviruses. Coronavirus-positive samples were sequenced to classify the virus by molecular phylogeny (N gene). Interestingly, in three out of six coronavirus-positive horses with colic, sequences closely related to bovine coronaviruses (BCoV) were found. The pathogenic potential of BCoV in horses remains unclear and warrants further investigation.

Postmortem examination of equids >1-year-old with enterotyphlocolitis in Ontario: a retrospective descriptive study.

Equine enterotyphlocolitis is an inflammatory process of the intestinal tract of horses that is associated with multiple etiologic agents and risk factors. Most clinical cases do not have an etiologic diagnosis. We describe here the pathogens detected and the histologic lesions found in horses with enterotyphlocolitis in Ontario that were submitted for postmortem examination, 2007-2019. We reviewed the medical records of 208 horses that fulfilled inclusion criteria. Cultures were positive in 67 of 208 (32%) equids for Clostridium perfringens, in 16 of 208 (8%) for Clostridioides difficile, and in 14 of 208 (7%) for Salmonella spp.; 6 of 208 (3%) were positive for Neorickettsia risticii by PCR assay. One horse was positive in a Rhodococcus equi PCR assay. All horses tested by PCR assay for equine coronavirus and Lawsonia intracellularis were negative. The histologic lesions were characterized as follows: 6 of 208 (3%) enteritis, 5 of 208 (2%) typhlitis, 104 of 208 (50%) colitis, 37 of 208 (18%) enterocolitis, 45 of 208 (22%) typhlocolitis, and 11 of 208 (5%) enterotyphlocolitis. We strongly recommend standardized testing of diarrheic horses during and/or after postmortem examination, as well as standardized reporting of histologic lesions in enterotyphlocolitis cases.

Risk factors associated with an outbreak of equine coronavirus at a large farm in North Carolina.

Equine coronavirus (ECoV) leads to outbreaks with variable morbidity and mortality. Few previous reports of risk factors for infection are available in the literature. To describe unique clinical findings and risk factors for infection and development of clinical disease. 135 horses on a farm affected by ECoV outbreak. Retrospective cohort study. Data obtained included age, breed, gender, activity level, housing, and feed at the onset of the outbreak. Factors were evaluated for assessment of risk of infection using simple logistic regression or Fisher's exact test. Significance was set at p ≤ 0.05. Forty-three of 54 (79.6%) horses tested on the farm were positive on fecal PCR for ECoV, and 17 horses (12.6%) developed clinical signs consistent with ECoV. Out of 17 horses in which the presence or absence of signs of colic was noted, 6 of 17 (35.3%) showed signs of colic. Three of these horses had small colon impactions, 2 of which required surgical intervention. Significant risk factors for having positive PCR results included being primarily stalled (OR 167.1, 95% CI 26.4-1719), housing next to a positive horse (OR 7.5, 95% CI 3.1-19.0), being in work (OR 26.9, 95% CI 4.6-281.9), being fed rationed hay vs. ad libitum (OR 1,558, 95% CI 130.8-15,593), and being fed alfalfa hay (OR 1,558, 95% CI 130.8-15,593). This report describes risk factors for ECoV infection many of which were associated with intensive management of show horses. Clinicians should be aware that clinical signs vary and can include severe colic.

Equine Coronavirus: An Infectious Disease Affecting Florida Horses

This publication provides an overview of equine coronavirus and discusses prevalence, transmission, clinical disease, diagnostic sampling, testing, and handling, treatment, prognosis, prevention and biosecurity, and potential for transmission to other species. Written by Sally DeNotta and Linda Mittel, and published by the UF/IFAS Veterinary Medicine—Large Animal Clinical Sciences Department, January 2023.

Prevalence of serum and salivary virus-neutralizing antibodies against equine coronavirus in four riding stables in Japan.

To assess the prevalence of equine coronavirus infection in riding horses, virus-neutralizing tests were performed on serum and saliva samples collected at four facilities in Japan. Seropositivity rates ranged from 79.2% to 94.6%, suggesting widespread circulation of the virus in these populations. Antibody prevalence in saliva samples from two facilities that had experienced outbreaks in the previous year (67.6% and 71.4%) was significantly higher than at the other facilities without reported outbreaks (41.7% and 45.2%, P<0.05). The presence of salivary antibodies in a high proportion of horses is therefore suggestive of recent exposure to the virus.

Characterization of an outbreak of equine coronavirus infection in adult horses in Switzerland.

Outbreaks of equine coronavirus (ECoV) infections have been described in different parts of the world including Europe. The aim of this report was to describe clinical signs, diagnostic work-up and outcome of the first documented outbreak of ECoV in Switzerland in order to raise the awareness for the disease and its various clinical presentations. The outbreak occurred on a farm with 26 horses. Of these, seven horses developed clinical disease ranging from mild signs such as fever and anorexia to severe signs of acute colitis. One horse died due to severe endotoxemia and circulatory shock secondary to severe acute necrotizing enteritis and colitis. Out of the 26 horses, five horses tested positive for ECoV, including two ponies without any clinical signs of infection. The low number of positive cases should nevertheless be interpreted with caution as testing was only performed on one occasion, over a month after the onset of clinical signs in the first suspected case. This report highlights the importance of diagnostic testing and early implementation of biosecurity measures on a farm with an ECoV outbreak. It should furthermore raise the awareness for unspecific and mild clinical signs such as fever and anorexia in affected animals that are potentially able to spread the disease.

Serosurveillance of equine coronavirus infection among Thoroughbreds in Japan.

Equine coronavirus (ECoV) causes fever, lethargy, anorexia and gastrointestinal signs in horses. There has been limited information about the prevalence and seasonality of ECoV among Thoroughbreds in Japan. To understand the epidemiology and to evaluate the potential risk of ECoV infection to the horse industry in Japan. Longitudinal. The virus-neutralisation (VN) test was performed using sera collected three times a year at 4months intervals from 161 yearlings and at 6-7months intervals from 181 active racehorses in Japan in 2017-2018, 2018-2019 and 2019-2020. VN titre ≥1:8 was defined as seropositive, and ≥4-fold increase in titres between paired sera was regarded as indicative of infection. The VN test showed that 44.1% (71/161) of yearlings were seropositive in August, when they first entered the yearling farm. The infection rate was significantly higher between August and December (60.9%, 98/161) than between December and the following April (5.6%, 9/161; p=0.002). Among the racehorses, it was significantly higher between November and the following May (15.5%, 28/181) than between the preceding April/May and November (0%; p=0.02). The morbidity rates during the estimated periods of viral exposure were 39.2% in the yearlings and 4% in the racehorses. No horses showed any severe clinical signs. Clinical records did not cover the period during horses' absence from the training centre. ECoV was substantially prevalent in Thoroughbred yearlings and racehorses in Japan, and there was a difference in epizootic pattern between these populations in terms of predominant periods of infection. ECoV infection was considered to be responsible for some of the pyretic cases in the yearlings. However, no diseased horses were severely affected in either population, suggesting that the potential risk of ECoV infection to the horse industry in Japan is low.

Distribution of equine coronavirus RNA in the intestinal and respiratory tracts of experimentally infected horses

Equine coronavirus (ECoV) causes pyrexia, anorexia, lethargy, and sometimes diarrhoea. Infected horses excrete the virus in their faeces, and ECoV is also detected in nasal samples from febrile horses. However, details about ECoV infection sites in the intestinal and respiratory tracts are lacking. To identify the ECoV infection sites in the intestinal and respiratory tracts, we performed an experimental infection study and analysed intestinal and respiratory samples collected from four infected horses at 3, 5, 7, and 14 days post-inoculation (dpi) by real-time reverse transcription polymerase chain reaction (real-time RT-PCR) and in situ hybridization (ISH). Two horses became febrile, but the other two did not. None of the horses had diarrhoea or respiratory signs, and severe cases were not observed in this study. None of the horses showed obvious abnormalities in their intestinal or respiratory tracts. Real-time RT-PCR and ISH showed that ECoV RNA was present throughout the intestinal tract, and ECoV-positive cells were mainly detected on the surface of the intestine. In one horse showing viremia at 3 dpi, ECoV RNA was detected in the lung by real-time RT-PCR, but not by ISH. This suggests that the lung cells themselves were not infected with ECoV and that real-time RT-PCR detected viremia in the lung. The other three horses were positive for ECoV RNA in nasal swabs but were negative in the trachea and lung by real-time RT-PCR and ISH. This study suggests that ECoV broadly infects the intestinal tract and is less likely to infect the respiratory tract.

Outbreak of equine coronavirus disease in adult horses, Switzerland 2021

Coronaviruses are causing severe respiratory and enteric diseases in humans and animals. Here, we report an outbreak of equine coronavirus disease in adult horses, detected by a voluntary syndromic surveillance scheme for equine diseases in Switzerland. This scheme allowed a rapid concerted action to diagnose and contain the disease.

Effect of season and geographic location in the United States on detection of potential enteric pathogens or toxin genes in horses ≥6-mo-old.

We investigated the effects of season and geographic location on detection of nucleic acids of potential enteric pathogens (PEPs) or their toxins (PEP-Ts) in feces of horses ≥6-mo-old in the United States. Results of 3,343 equine diarrhea PCR panels submitted to Idexx Laboratories for horses >6-mo-old were reviewed. Submission months were grouped into 4 seasons, and states were grouped into 4 geographic regions. Logistic regression was performed to assess effects of season and region on detection rates of PEPs and PEP-Ts. Agresti-Coull CIs were determined. Detection rate of Salmonella enterica was higher in the South in summer compared to all other regions, and was also higher in the South in fall compared to the Midwest and Northeast. The Neorickettsia risticii detection rate was lower during summer in the West and higher in fall in the Midwest. Detection of Cryptosporidium spp. was lower during spring, summer, and winter in the West. Differences were not identified for detection rates of Clostridioides difficile, Clostridium perfringens, Lawsonia intracellularis, Rhodococcus equi, equine rotavirus, and equine coronavirus. Overall, our data support seasonal and regional differences in detection rates of S. enterica, N. risticii, and Cryptosporidium spp. in horses ≥6-mo-old in the United States.

Bacterial and viral enterocolitis in horses: a review.

Enteritis, colitis, and enterocolitis are considered some of the most common causes of disease and death in horses. Determining the etiology of these conditions is challenging, among other reasons because different causes produce similar clinical signs and lesions, and also because some agents of colitis can be present in the intestine of normal animals. We review here the main bacterial and viral causes of enterocolitis of horses, including Salmonella spp., Clostridium perfringens type A NetF-positive, C. perfringens type C, Clostridioides difficile, Clostridium piliforme, Paeniclostridium sordellii, other clostridia, Rhodococcus equi, Neorickettsia risticii, Lawsonia intracellularis, equine rotavirus, and equine coronavirus. Diarrhea and colic are the hallmark clinical signs of colitis and enterocolitis, and the majority of these conditions are characterized by necrotizing changes in the mucosa of the small intestine, colon, cecum, or in a combination of these organs. The presumptive diagnosis is based on clinical, gross, and microscopic findings, and confirmed by detection of some of the agents and/or their toxins in the intestinal content or feces.

Distribution of equine coronavirus RNA in the intestinal tracts of experimentally infected horses

Distribution of equine coronavirus RNA in the intestinal tracts of experimentally infected horses