【病毒外文文献】2019 Clinical presentation, diagnostic findings, and outcome of adult horses with equine coronavirus infection at a vete.pdf

Clinical presentation, diagnostic findings, and outcome of adult horseswith equine coronavirus infection at a veterinary teaching hospital:33 cases (2012–2018)E.H. Berryhill*, K.G. Magdesia, M. Aleman, N. PusterlaDepartment of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis, One Shields Ave., Davis, CA, 95616, USAA R T I C L E I N F OKeywords:ColicDiarrheaFeverGastrointestinalInfectiousA B S T R A C TEquine coronavirus (ECoV) is a recently described enteric virus with worldwide outbreaks; however, there arelittle data available on clinical presentation, diagnosis, and outcome. The study objective was to document casemanagement of ECoV in adult horses presented to a referral hospital and compare to a cohort of horses thattested negative for ECoV. A retrospective case series was performed based on positive real-time quantitativePCR results for ECoV on faeces from horses treated at the UC Davis Veterinary Medical Teaching Hospital from 1March 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 threehorses 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 hadsignificantly 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 werecommon. 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 horseswith fever, colic, anorexia, and leukopenia. The disease has a low mortality rate, but horses may requireintensive care to resolve severe leukopenia, systemic inflammation, and metabolic disturbances.© 2019 Elsevier Ltd. All rights reserved.IntroductionEquine coronavirus (ECoV) is recognised as a cause of fever,anorexia, lethargy, leukopenia, and gastrointestinal disease inadult horses (Pusterla et al., 2018). Disease outbreaks have beenreported in adult horses in boarding stables or competitivefacilities across the United States, Europe, and Japan (Oue et al.,2013; Pusterla et al., 2013; Miszczak et al., 2014; Pusterla et al.,2018). The recent increase in positive cases likely reflects increasedawareness of the virus, and increased availability and timeliness oftesting through fecal real-time quantitative polymerase chainreaction (qPCR) (Pusterla et al., 2013).Many cases of ECoV are self-limiting with transient clinicalsigns, however fatalities, endotoxemia and hyperammonemia canoccur (Fielding et al., 2015). Miniature horses may be moresusceptible to severe disease and had a higher fatality rate in anoutbreak (Fielding et al., 2015). In contrast, foals <1 year of age donot seem to be clinically affected as frequently as adult horses, andECoV is often present as a co-infection with other gastrointestinalpathogens in foals (Slovis et al., 2014).While data regarding ECoV epidemiology are available, there isa paucity of data on clinical presentation of cases, diagnosis, andoutcome from a clinical perspective. The purpose of the currentstudy was to document cases of ECoV diagnosed through fecalqPCR and presenting to a tertiary referral hospital.Materials and methodsStudy populationRecords of adult horses >1 year of age, examined by a veterinarian at the UCDavis Veterinary Medical Teaching Hospital or on the ambulatory service between 1March 2012 and 31 March 2018, and with faeces positive for ECoV by qPCR wereobtained. Records of horses with negative fecal qPCR panels during the same timeperiod were also obtained, and negative controls were selected by matching toECoV-positive horses, prioritising first age, then time, and lastly sex. Horses hadfecal qPCRs performed due to presence of fever, loose manure, or leukopenia asdictated by infectious disease protocol at the hospital, or due to signs of abdominal* Corresponding author.E-mail address: ehberryhill@vmth.ucdavis.edu (E.H. Berryhill).https://doi.org/10.1016/j.tvjl.2019.05.0011090-0233/© 2019 Elsevier Ltd. All rights reserved.The Veterinary Journal 248 (2019) 95–100Contents lists available at ScienceDirectThe Veterinary Journaljournal homepage: www.else vie /t vjldiscomfort in addition to any of the former criteria. Pathogens included in the fecalqPCR panel at the institution are ECoV, Salmonella spp., Clostridium difficile (toxin Aand B), Lawsonia intracellularis, and Neorickettsia risticiii. Fecal cultures forSalmonella spp. were also performed at the institution on every hospitalized horse.Respiratory qPCR panels are often performed as diagnostics for infectious diseasescreening, including testing for equine Influenza A virus, equine rhinitis A and Bviruses, equine herpesvirus-1 and -4, and Streptococcus equi ssp. equi.Data collected from records included signalment, presenting complaints,predisposing causes for infectious disease, physical examination and clinicopatho-logical findings, hospitalisation and treatment, and follow-up. Clumped plateletcounts were excluded from analysis. The last complete blood count (CBC) submittedfor the visit in question was utilised for follow-up data.PCR analysisNucleic acid extractions from faeces were performed using an automatednucleic acid extraction system (CAS-1820 X-tractor Gene) according to themanufacturer’s recommendation. Faeces were tested for the molecular presenceof ECoV as previously reported (Pusterla et al., 2013).Statistical analysesData were summarised using descriptive statistics, with the median and rangereported for non-parametric data. Numerical data for ECoV qPCR positive onlyhorses were compared to the qPCR negative control cohort using a Mann–WhitneyU test, with P < 0.05 considered significant. Holm’s Sequential Bonferroni Procedurewas applied to control for type I error associated with the testing of multiplehypotheses with clinicopathological data (Holm, 1979). Differences in seasonprevalence were analysed with a Fisher’s exact test, with P < 0.05 consideredsignificant.ResultsFecal samples from 498 equine patients >1 year of age weretested for ECoV by qPCR over the specified time period. Thirty-three adult horses met the study criteria, including 12 mares, 20geldings, and one stallion. Of these 33 horses, 30 were found topositive for ECoV only, and 3 were diagnosed with ECoV andadditional co-infections. Co-infections included one horse withSalmonella spp. infection (identified by fecal qPCR), one withActinobacillus equuli peritonitis (identified by abdominal fluidculture), and one with both rhinitis B virus (identified by nasalswab qPCR) and Salmonella spp. (Group E; identified by fecalculture) infections. Horses were a median of 11 (range 2–37) yearsof age and of mixed breeds (Supplementary material). Thirtyhorses were hospitalised, and 3 were seen as outpatients or by theambulatory service.Horses positive for ECoV were presented for a combination ofcomplaints elaborated in Table 1. Colic signs tended to be mild,with one horse presenting with net reflux following nasogastricintubation and two presenting with large colon impactions. Dataon housing were lacking in 13/33 horses. Nineteen of the 20 horses(95%) for which information was available were housed at a multi-horse facility with horse traffic, and one horse (5%) was distinctlynot from a high traffic environment. One horse was associated witha barn with five additional confirmed ECoV cases, two study horseswere from the same barn and hospitalised within the same week,and three separate study horses were associated with horses withfevers at their respective farms. Five of 22 horses (23%) for whichtravel history was available had traveled to a horse show within theprevious 3 weeks. Twenty-one of 33 (64%) cases occurred in coldermonths, with a significant difference between the number of casesseen between January and March and the other three seasons(Fig. 1; P < 0.02).Thirty-three horses negative for ECoV on fecal qPCR wereidentified as controls, including 15 mares, 17 geldings, and onestallion. Horses were a median of 10 (1–30) years of age and ofmixed breeds (Supplementary material). Presenting complaintsare found in Table 1. Two of 21 horses (10%) for which travel historywas available had recently been to a show. Housing informationwas available in 18 horses, of which 12/18 (67%) were housed at afacility with an open herd. Final clinical diagnoses included fever ofunknown origin (n = 6/33; 18%), colitis (n = 5/33; 15%), colic (n = 2/33; 6%), diarrhea (n = 2/33; 6%), colic and fever of unknown origin(n = 2/33; 6%); Anaplasmosis (n = 2/33; 6%); and large colonimpaction (n = 2/33; 6%).Initial examination and clinicopathological findingsInitial physical examination parameters and CBCs for ECoV-positive and negative horses are shown in Table 1. Seven of 30horses (23%) with ECoV only and 9 horses negative for ECoV hadloose manure/diarrhea. There were no significant differences in thenumber of horses with loose manure or lactate concentration>2 mmol/L between the ECoV-positive only and control groups.True thrombocytopenia was identified in four horses with ECoV, ofwhich one horse had a degenerative left shift with bands andmetamyelocytes, one had a regenerative left shift, and one had aleukocytosis of >15.0 cells ? 109/L. Horses positive for ECoV hadsignificantly lower total WBC, neutrophil, and lymphocyte countsthan negative controls (P = 0.0006, P = 0.004, P = 0.007, respective-ly). Serum amyloid A was evaluated in 3/30 horses positive forECoV only and found elevated in 3/3 (100%), with a medianconcentration of 1080 (191–1833) mg/mL. All 4/33 control horseswith serum amyloid A analysed had elevated concentrations, witha median of 1561 (77–2237) mg/mL.Results of serum biochemistry profiles are found in Table 2.There were no significant differences between horses positive forECoV only and negative controls. Abnormalities in horses withECoV only and the controls included, respectively, electrolytederangements (n = 27/28, 96%; n = 27/30, 90%), hyperbilirubinemia(n = 23/28, 82%; n = 15/30, 50%), hyperglycemia (n = 23/28, 82%;n = 14/30, 47%), hyperlipidemia (n = 13/28, 46%; n = 12/30, 40%),hypoproteinemia with hypoalbuminemia (n = 8/28, 29%; n = 9/30,30%), increased muscle enzymes (n = 8/28; 29%; n = 13/30, 43%),and decreased blood urea nitrogen (n = 4/28, 14%; n = 8/30, 27%).Azotemia was present in 3/28 (11%) of horses with ECoV and 2/30(7%) without ECoV. Hyperphosphatemia was associated withazotemia in two horses with ECoV. Blood ammonia concentrationsperformed in three horses with ECoV were within normal limits,with a median concentration of 22.1 (17.9–27.9) mmol/L (referencerange 3.57–42.1 mmol/L). The horse with the highest initialammonia concentration (27.9 mmol/L) had decreased concentra-tions (10.7 mg/dL) when measured 2 days later. Bile acids wereanalysed in one horse with ECoV and were elevated. This horse alsohad elevated liver enzymes and initial triglyceride concentrations>6.78 mmol/L, but ammonia concentrations were not performed.Additional diagnosticsAdditional diagnostics at intake in horses positive for ECoV onlyincluded abdominal ultrasound (n = 26/30), rectal examination(n = 16/30), abdominocentesis (n = 9/30), nasogastric intubation(n = 8/30), and abdominal radiographs (n = 5/30). Ultrasoundexamination yielded no significant findings in 19/26 horses(73%) with ECoV. Small intestinal abnormalities included hypo-motility (n = 5/26; 19%), increased wall thickness (n = 4/26; 15%),and hypermotility (n = 2/26; 8%); luminal distension was notapparent in any exam. Large intestinal abnormalities includedincreased wall thickness (n = 4/26; 15%), hypomotility (n = 2/26;8%), luminal distension with fluid (n = 2/26; 8%) or gas (n = 2/26;8%), and hypermotility (n = 1/26; 4%). Peritoneal effusion waspresent in 1 horse (1/26; 4%). Rectal examination indicated scantfaeces (n = 4/16; 25%), mild colon impaction (n = 3/16; 19%), mildcolonic gas distension (n = 2/16; 13%), taut bands (n = 2/16; 13%),soft manure (n = 1/16; 6%), an unrelated mass (n = 1/16; 6%), and nosignificant findings in 3 horses (3/16; 19%).96 E.H. Berryhill et al. / The Veterinary Journal 248 (2019) 95–100Abdominal ultrasound was performed in 23/33 control horses,and findings were similar to those described in the ECoV-positivegroup (Supplemental material). Four of 23 controls also receivedthoracic ultrasound exams, with pulmonary changes in 3/4. Therewere no significant differences in the number of horses with smallintestinal or large intestinal abnormalities between the ECoV onlyand negative control groups. Rectal examinations were performedin 19/33 control horses. Abnormalities included gas-distended(n = 3/19; 16%), fluid-filled (n = 2/19; 11%) or impacted large colon(n = 2/19; 11%); loose faeces (n = 2/19; 11%), loose (n = 1/19; 5%) ordry (n = 1/19; 5%) faeces and gas-distended colon, hard faeces(n = 1/19; 5%), and faeces with frank blood and rectal mucosaledema (n = 1/19; 5%). There were no significant findings on rectalexam in 6/19 (32%).Abdominocentesis findings for horses positive for only ECoVindicated a median lactate concentration of 1.5 (1–2.3) mmol/L,total protein of 22 (6–29) g/L, and total nucleated cell count of 1.52(0.42–4.6) cells ? 109/L. Horses negative for ECoV had a medianlactate concentration of 2 (0.2–13.9) mmol/L, total protein 15Table 1Presenting complaints, median (range) physical examination parameters and initial complete blood counts in cohorts of horses positive for equine coronavirus (ECoV)infection and a co-infection, ECoV only, and negative controls, with statistical comparison between horses positive for ECoV only and negative controls.RR ECoV ECoV + co-infection Control P-valuePresenting complaint (n = 30) (n = 3) (n = 33)Historic fever 25/30, 83% 3/3, 100% 18/33, 55%Anorexia 14/30, 47% 2/3, 67% –Colic 13/30, 43% 2/3, 67% 14/33, 42%Lethargy 8/30, 27% – 7/33, 21%Leukopenia 5/30, 17% – 1/33, 3%Diarrhea 1/30, 3% – 8/33, 24%Tachypnea 1/30, 3% – –Foot soreness 1/30, 3% – –Haemorrhagic rectal discharge – – 1/33, 3%Historic temperature (?C) (n = 25/30) (n = 2/3) (n = 18/33)40 (38.3–41.6) 39.8–40.3 39.3 (37.2–40.7)Physical examination (n = 29/30) (n = 3/3) (n = 32/33)Temperature (?C) 38.5 (37–39.9) 37.7 (37.5–38) 38 (36.9–40.6)Heart rate (beats/min) 48 (32–72) 54 (32–72) 48 (28–96)Resp. rate (breaths/min) 20 (12–60) 32 (24–40) 20 (10–88)Peripheral lactate (mmol/L) <2 (n = 21/30) (n = 3/3) (n = 19/33)1.3 (0.7–14.5) 1.4 (0.7–2.1) 1.6 (0.8–13.4)4 > RR 1 > RR 6 > RRCBC (n = 29/30) (n = 3/3) (n = 33/33)WBC (cells x 109/L) 5.0-11.6 3.0 (0.68–16.2) 5.0 (1.42–5.18) 5.68 (1.74–18.73) 0.000622 < RR, 3 >RR 1 < RR 22 < RR, 3 > RRMetamyelocytes (cells x 109/L) 0 0.17 (0.21–3.25) 0 02 > RRToxic bands (cells x 109/L) Rare 0.13 (0–7.96) 0.70 (0.31–0.83) 0 (0.10–2.77)19 > RR 3 > RR 13 > RRNeutrophils (cells x 109/L) 2.6-6.8 1.26 0.47 3.96 0.004(0.15–14.4) (0.23–1.90) (0.19–17.1)21 < RR, 2 > RR 3 < RR 12 < RR, 7 > RRLymphocytes (cells x 109/L) 1.6-5.8 0.86 (0.42–3.47) 1.6 (0.85–3.47) 1.4 (0.23–5.0) 0.007(n = 30/30) 1 < RR 21 < RR25 < RRPlatelet count (x109/L) 100–225 122 (58–164) 120 (117–137) 147 (86–248)4 < RR (n = 32/33)2 < RR, 1 > RRFibrinogen (mg/dL) 100–400 400 (200–600) 400 (300–500) 300 (100–900)1 > RR 1 > RR 5 > RRHCT (%) 30–46 37.0 (24.4–66.7) 40.3 34.6 (20.8–53.4)(n = 30/30) (36.6–45.2) 5 < RR, 3 > RR5 < RR 3 > RRTotal protein (g/dL) 58-87 62 (52–77) 62 (60–73) 63 (35–82)(n = 30/30) 7 < RR6 < RRLength of hospitalisation (days) (n = 27/30) (n = 3/3) (n = 24/33)5 (0.5–14) 7 3 (1–10)Survival to discharge 26/27 (96%) 3/3 (100%) 31/33 (94%)CBC, complete blood count; HCT, haematocrit; HR, heart rate; RR, reference range; RR, number of horses with values less than or greater than the reference range;Resp. rate, respiratory rate; WBC, white blood cell count.Fig. 1. Number of equine coronavirus positive cases over the months of the yearfrom 1 March 2012 through 31 March 2018. January through March hadsignificantly higher numbers of positive cases compared to other times of theyear (P < 0.05).E.H. Berryhill et al. / The Veterinary Journal 248 (2019) 95–100 97(5–38) g/L, and total nucleated cell count 1.15 (0.47–13.1) cells? 109/L when measured in 12/33, 11/33, and 14/33 horses,respectively (Supplementary material). There were no significantdifferences in lactate, protein, or nucleated cell count betweenhorses positive for ECoV and controls.Nasogastric intubation performed in those positive for ECoVonly yielded net reflux in 1/7 horses (14%), with 7 L recovered. Anasogastric tube was passed in 17/33 (52%) of the control horses,with 11 L of net reflux in one horse. Abdominal radiographsshowed mild to moderate sand accumulation in 2/5 horses (40%)with ECoV only, fluid lines in 2/5 (40%), and no significantfindings in 1/5 (20%). Radiographs were performed in 12/33control horses and indicated scant to moderate sand accumula-tion in 5/12 (42%).Among the ECoV-positive horses, 30/33 horses were tested forat least one additional pathogen (including gastrointestinal,respiratory, or blood-borne agents), with 3/30 (10%) diagnosedwith known co-infections (salmonellosis, Actinobacillus sp. perito-nitis, and salmonellosis with concurrent Rhinitis B virus).Diagnostics to assess for the direct presence of other infectiousagents in horses positive for ECoV and controls are found in Table 3.A complete list of diagnostic tests performed ca be found in theAppendix: Supplementary Material.Therapy and hospitalisationMedical therapy instituted was available in 28/30 (93%) horsespositive for ECoV only and 33/33 negative horses (see Appendix:Supplementary Material). Follow-up complete blood counts (CBC)were performed in 21/30 (70%) horses positive for ECoV only and13/33 (39%) negative horses and are shown in Table 4. The last CBCwas performed a median of 4 (2–14) and 3 (2–9) days after theintake CBC for horses positive and negative for ECoV, respectively.Follow-up fecal qPCRs were performed in 15/33 (45%) of all horsespositive for ECoV. Seven of the 15 (47%) horses retested remainedECoV qPCR positive when tested a median of 3 (2–8) days after theinitial qPCR. Eight of the 15 (53%) horses retested had negativeECoV qPCR results when tested a median of 7 (5–16) days after theinitial qPCR.Length of hospitalisation and survival rate for horses with andwithout ECoV are shown in Table 1. One horse with ECoV died 12 hafter admission and had a 6-day history of fever, diarrhea, andleukopenia, a peripheral lactate of 14.5 mmol/L, haematocrit of66.7%, degenerative left shift with metamyelocytes, and evidenceof multi-organ dysfunction syndrome and disseminated intravas-cular coagulation. Necropsy revealed necrohaemorrhagic colitisand enteritis with disseminated vascular thrombi, congestion andpetechiation of the heart and other organs, and renal infarcts.Immunohistochemistry identified ECoV within the contents of thecolon. Thirty-two of 33 control horses were managed medicallywhile one required colic surgery. Two control horses did notsurvive, with diagnoses at necropsy of (1) colitis and mega-esophagus and (2) toxic shock syndrome secondary to S. aureusinfection. There was no significant difference in survival todischarge between horses with ECoV and the control population.Horses positive for ECoV and co-infectionsDiagnostic findings for horses positive for ECoV and a co-infection were similar to horses positive for ECoV only andincluded small intestinal hypomotility and colonic fluid onultrasound (n = 1/3), scant (n = 2/3) or malodorous (n = 1/3) faeceson rectal examination, and sand on radiographs (n = 1/3). The horseTable 2Serum biochemistry profiles in 28 horses with equine coronavirus (ECoV) only, three horses with ECoV and a co-infection, and 30 negative control horses, with median (range)presented. Statistically significant differences were not demonstrated between the group with ECoV only and negative controls (P > 0.05).RR ECoV (n = 28/30) ECoV + co-infection (n = 3/3) Control (n = 30/33)iMagnesium (mmol/L) 0.47–0.70 0.35 (0.29–0.56) 0.33 (0.25–0.41) 0.4 (0.24–0.61)Sodium (mmol/L) 125–137 132 (117–136) 129 (129–132) 134 (122–139)Potassium (mmol/L) 3.0–5.6 3.0 (1.6–4.4) 3.4 (3.0–3.4) 3.2 (2.7–4.8)Chloride (mmol/L) 91–104 96 (77–101) 94 (89–101) 97 (77–103)Phosphorus (mmol/L) 0.68–1.52 0.68 (0.23–3.94) 0.68 (0.48–1.39) 0.74 (0.48–4.68)Calcium (mmol/L) 2.85–3.53 2.7 (2.4–2.95) 2.7 (2.68–2.85) 2.7 (1.83–3.28)Anion Gap (mmol/L) 9–17 14 (11–46) 16 (13–18) 14 (11–31)Bicarbonate (mmol/L) 23–32 25 (11–28) 25 (15–28) 25 (19–30)Blood urea nitrogen (mmol/L) 4.28–9.64 5.36 (2.86–11.78) 12 (9–14) 5.36 (3.21–25.7)Creatinine (mmol/L) 79.56–176.8 114.92 (79.56–327.08) 1 (1–1.3) 114.92 (61.88–318.24)Glucose (mmol/L) 2.78–5.94 7.27 (5.38–10.38) 6.16 (5.99–8.32) 6.22 (2.83–9.82)Total protein (g/L) 58–77 61 (46–74) 57 (55–73) 60 (42–76)Albumin (g/L) 27–42 30 (19–36) 31 (25–36) 30 (22–37)Globulin (g/L) 16–50 29 (22–38) 30 (26–37) 31 (21–52)AST (IU/L) 168–494 270 (186–1881) 299 (293–372) 259 (146–6980)Creatine kinase (IU/L) 119–287 239 (86–813) 484 (125–596) 211 (79–30,451)ALP (IU/L) 86–285 157 (91–655) 459 (98–550) 130 (22–316)GGT (IU/L) 8–22 12 (8–85) 18 (10–22) 12 (7–83)SDH (IU/L) 0–8 3 (0–173) 0 (0–6) 3 (0–47)Triglycerides (mmol/L) 0.02–0.46 0.49 (0.24–8.12) 2.19 (2.01–4.61) 0.59 (0.17–3.3)Total bilirubin (mmol/L) 8.55–39.33 44.46 (18.81–188.1) 46.17 (42.75–141.93) 37.62 (10.26–141.93)Direct bilirubin (mmol/L) 3.42–10.26 3.42 (1.71–15.39) 3.42 (1.71–3.42) 3.42 (1.71–8.55)Indirect bilirubin (mmol/L) 5.13–29.07 42.75 (15.39–172.71) 42.75 (39.33–140.22) 34.2 (6.84–138.51)ALP, Alkaline phosphatase; AST, Aspartate aminotransferase; GGT, Gamma-glutamyltransferase; RR, reference range; SDH, Sorbitol dehydrogenase.Table 3Additional diagnostic testing to assess for the direct presence of other infectiousagents performed in horses positive for equine coronavirus (ECoV) compared tonegative controls.ECoV n = 33 Control n = 33Salmonella culture 30 (91%) (1 positive) 25 (76%) (all negative)Respiratory qPCR panel 11 (33%) (1 positive) 8 (24%) (all negative)A. phagocytophilum PCRand/or buffy coat smear6 (18%) (all negative) 6 (18%) (2 positive)C. difficile ELISA 0 3 (9%) (all negative)Abdominal fluid culture 1 (3%) (positive) 2 (6%) (all negative)Blood culture 1 (3%) (negative) 1 (3%) (negative)C. pseudotuberculosis PCR 0 1 (abdominal fluid)(3%) (negative)98 E.H. Berryhill et al. / The Veterinary Journal 248 (2019) 95–100co-infected with Actinobacillus spp. peritonitis had 5 L of net refluxupon initial nasogastric intubation and had markedly abnormalabdominocentesis results (lactate 17 mmol/L, total protein 61 g/L,total nucleated cell count 165 cells ? 109/L). Follow-up CBCs weresimilar to the rest of the ECoV-positive cohort, except for the horsewith Actinobacillus peritonitis with persistent thrombocytopenia,hyperproteinemia, and hyperfibrinogenemia.DiscussionThis study showcases common presentations and the range ofdisease severity in horses positive for ECoV. It is the first study todocument advanced diagnostic findings and outcomes for a cohortof hospitalised horses positive for ECoV and provides a comparisonto a matched group of horses negative for ECoV. A recentseroprevalence study showed an increased proportion of ECoVinfection in draft horses (17.6%), which was not apparent in thecurrent study and likely reflects a difference in populations(Kooijman et al., 2017). Similar to other studies, ECoV infectionrates were increased in the cooler months of the year, with peakprevalences in January through March likely related to husbandrychanges and possible decreased viability of the virus in hot, drytemperatures (Kooijman et al., 2017; Pusterla et al., 2018).The clinical presentations and clinicopathological findings inthe ECoV-positive group were similar to those observed in horsesafter experimental inoculation with ECoV (Nemoto et al., 2014).Additionally, the diarrhea qPCR panel and salmonella fecal cultureperformed are considered sensitive screening tools for otherinfectious gastrointestinal pathogens. Thus, with the exception ofthe co-morbidities found, disease presentations observed in ourequine patients are considered likely associated with ECoVinfection. However, horses can shed ECoV subclinically andintermittently, and causation between clinical signs and fecalqPCR positivity for ECoV cannot be claimed. Furthermore,diagnostics performed were based on clinical findings and caseprogression and were often incomplete in ruling out additionalpathogens (e.g. 11/33 positive for ECoV had respiratory qPCRpanels performed). There is the potential for co-infections to havebeen underrepresented and their contribution to clinical signsunaccounted for. The likelihood of false negative results in horsestested for multiple pathogens through qPCR is consideredrelatively low unless the pathogen is shed intermittently, as qPCRis known to have high sensitivity and specificity for identifying andquantifying genetic material if present. If anything, the likelihoodof identifying pathogens that might be present but not toxigenicmay be a risk if qPCR is the sole test utilised (e.g. C. difficile toxingene positive on qPCR but not elaborating toxin).Horses positive for ECoV had similar presentations, diagnostics,and management to many of the horses negative for ECoV. Thisresult is not surprising since submitting fecal samples for qPCR onhorses that present with fever, colic, or loose manure is one of thefirst steps performed at the hospital as part of the diagnostic planand for infectious disease control. Horses positive for ECoV hadsignificantly lower WBC, neutrophil, and lymphocyte counts thanthe negative controls, suggesting a more florid acute inflammatoryresponse in those with ECoV. The majority of the biochemistryabnormalities in both the ECoV-positive and ECoV-negative groupscan be explained by inappetance leading to decreased electrolyteintake and possible electrolyte secretion into the gastrointestinallumen. Metabolic derangements (e.g. hyperlipidemia) were alsocommon and attributed to a negative energy balance, as well assystemic inflammation (Barsnick et al., 2014). Ammonia concen-trations were not evaluated in most of the ECoV cases and wouldhave been interesting to document, as hyperammonemia is areported complication of ECoV associated with higher mortality(Fielding et al., 2015). The three horses that were evaluated forammonia had concentrations within laboratory references. How-ever, a horse positive for ECoV only with the highest ammoniaconcentration at 27.9 mmol/L showed a decrease to 10.7 mmol/Lwhen measured 2 days later, possibly indicating a relativehyperammonemia. The fatality rate for all horses with ECoV waslow at 3%, which is less than the reported 7% in one outbreak and27% in another (Pusterla et al., 2013; Fielding et al., 2015). Thus,prognosis can be considered good with appropriate supportivecare even in more severe cases of ECoV, although owners should bewarned about the possibility of complications and death,comparable to that seen in horses presenting for fever, lethargyor colic for other reasons.This case series is the first known documentation of co-infections in adult horses positive for ECoV. Co-infections betweenECoV and other gastrointestinal pathogens have been reported indiarrheic foals but were not reported in previous studiesdocumenting outbreaks of ECoV (Slovis et al., 2014; Fieldinget al., 2015; Pusterla et al., 2018). The reasons for co-infection withECoV remain unknown. The authors speculate that ECoV allows forreduced colonisation resistance of the gastrointestinal microbiomeor reduced immune function, permitting colonisation withpathogenic organisms like salmonellae, or allowing Actinobacillusequuli to translocate across the gut wall. As mentioned earlier,diagnostic testing to evaluate for the presence of co-infectingpathogens was not complete in many cases in this study, andfurther research is required prior to drawing conclusions about theimpact ECoV and co-infections have on treatment and prognosis inadult horses.Approximately 50% of horses positive for ECoV in the currentstudy were still shedding the organism when retested ?8 daysafter admission. This is similar to studies where horses inoculatedwith infected faeces shed for >9 days, independent of clinical signs,or ranged in shedding from 5 to 9 days (Nemoto et al., 2014;Schaefer et al., 2018). At the current hospital, clinicians tend toTable 4Median (range) follow-up and admission complete blood cell counts in horses positive for equine coronavirus (ECoV) only by quantitative polymerase chain reaction and 13control horses. Follow-ups occurred a median of 4 days (range, 2–14 days) and 3 days (range, 2–9 days) after hospital intake for ECoV-positive and control horses, respectively.RR n >RR n