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vete la is a re The Veterinary Journal 248 2019 95 100 Contents lists available at ScienceDirect The Veterinary journal homepage www else Keywords Colic Diarrhea Fever Gastrointestinal Infectious 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 ndings 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 signi cantly lower white blood cell median 3 0 C2 10 9 L range 0 68 16 2 C2 10 9 L neutrophil median 1 26 C2 10 9 L range 0 15 14 4 C210 9 L and lymphocyte median 0 86 C2 10 9 L range 0 42 3 47 C2 10 9 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 in ammation and metabolic disturbances 2019 Elsevier Ltd All rights reserved Introduction Equine coronavirus ECoV is recognised as a cause of fever anorexia lethargy leukopenia and gastrointestinal disease in adult horses Pusterla et al 2018 Disease outbreaks have been reported in adult horses in boarding stables or competitive facilities 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 re ects increased awareness of the virus and increased availability and timeliness of testing through fecal real time quantitative polymerase chain reaction qPCR Pusterla et al 2013 Many cases of ECoV are self limiting with transient clinical signs however fatalities endotoxemia and hyperammonemia can occur Fielding et al 2015 Miniature horses may be more susceptible to severe disease and had a higher fatality rate in an outbreak Fielding et al 2015 In contrast foals 1 year of age examined by a veterinarian at the UC Davis Veterinary Medical Teaching Hospital or on the ambulatory service between 1 March 2012 and 31 March 2018 and with faeces positive for ECoV by qPCR were obtained Records of horses with negative fecal qPCR panels during the same time period were also obtained and negative controls were selected by matching to ECoV positive horses prioritising rst age then time and lastly sex Horses had fecal qPCRs performed due to presence of fever loose manure or leukopenia as dictated 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 05 001 1090 0233 2019 Elsevier Ltd All rights reserved Clinical presentation diagnostic ndings with equine coronavirus infection at a 33 cases 2012 2018 E H Berryhill K G Magdesia M Aleman N Puster Department of Medicine and Epidemiology School of Veterinary Medicine University of California A R T I C L E I N F O A B S T R A C T Equine coronavirus ECoV and outcome of adult horses rinary teaching hospital Davis One Shields Ave Davis CA 95616 USA cently described enteric virus with worldwide outbreaks however there are Journal vie t vjl 96 E H Berryhill et al The Veterinary Journal 248 2019 95 100 discomfort in addition to any of the former criteria Pathogens included in the fecal qPCR panel at the institution are ECoV Salmonella spp Clostridium dif cile toxin A and B Lawsonia intracellularis and Neorickettsia risticiii Fecal cultures for Salmonella spp were also performed at the institution on every hospitalized horse Respiratory qPCR panels are often performed as diagnostics for infectious disease screening including testing for equine In uenza A virus equine rhinitis A and B viruses 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 ndings hospitalisation and treatment and follow up Clumped platelet counts were excluded from analysis The last complete blood count CBC submitted for the visit in question was utilised for follow up data PCR analysis Nucleic acid extractions from faeces were performed using an automated nucleic acid extraction system CAS 1820 X tractor Gene according to the manufacturer s recommendation Faeces were tested for the molecular presence of ECoV as previously reported Pusterla et al 2013 Statistical analyses Data were summarised using descriptive statistics with the median and range reported for non parametric data Numerical data for ECoV qPCR positive only horses were compared to the qPCR negative control cohort using a Mann Whitney U test with P 0 05 considered signi cant Holm s Sequential Bonferroni Procedure was applied to control for type I error associated with the testing of multiple hypotheses with clinicopathological data Holm 1979 Differences in season prevalence were analysed with a Fisher s exact test with P 1 year of age were tested for ECoV by qPCR over the speci ed time period Thirty three adult horses met the study criteria including 12 mares 20 geldings and one stallion Of these 33 horses 30 were found to positive for ECoV only and 3 were diagnosed with ECoV and additional co infections Co infections included one horse with Salmonella spp infection identi ed by fecal qPCR one with Actinobacillus equuli peritonitis identi ed by abdominal uid culture and one with both rhinitis B virus identi ed by nasal swab qPCR and Salmonella spp Group E identi ed by fecal culture infections Horses were a median of 11 range 2 37 years of age and of mixed breeds Supplementary material Thirty horses were hospitalised and 3 were seen as outpatients or by the ambulatory service Horses positive for ECoV were presented for a combination of complaints elaborated in Table 1 Colic signs tended to be mild with one horse presenting with net re ux following nasogastric intubation and two presenting with large colon impactions Data on 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 traf c and one horse 5 was distinctly not from a high traf c environment One horse was associated with a barn with ve additional con rmed ECoV cases two study horses were from the same barn and hospitalised within the same week and three separate study horses were associated with horses with fevers at their respective farms Five of 22 horses 23 for which travel history was available had traveled to a horse show within the previous 3 weeks Twenty one of 33 64 cases occurred in colder months with a signi cant difference between the number of cases seen between January and March and the other three seasons Fig 1 P 2 mmol L between the ECoV positive only and control groups True thrombocytopenia was identi ed in four horses with ECoV of which one horse had a degenerative left shift with bands and metamyelocytes one had a regenerative left shift and one had a leukocytosis of 15 0 cells C2 10 9 L Horses positive for ECoV had signi cantly lower total WBC neutrophil and lymphocyte counts than negative controls P 0 0006 P 0 004 P 0 007 respective ly Serum amyloid A was evaluated in 3 30 horses positive for ECoV only and found elevated in 3 3 100 with a median concentration of 1080 191 1833 mg mL All 4 33 control horses with serum amyloid A analysed had elevated concentrations with a median of 1561 77 2237 mg mL Results of serum biochemistry pro les are found in Table 2 There were no signi cant differences between horses positive for ECoV only and negative controls Abnormalities in horses with ECoV only and the controls included respectively electrolyte derangements 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 with azotemia in two horses with ECoV Blood ammonia concentrations performed in three horses with ECoV were within normal limits with a median concentration of 22 1 17 9 27 9 mmol L reference range 3 57 42 1 mmol L The horse with the highest initial ammonia concentration 27 9 mmol L had decreased concentra tions 10 7 mg dL when measured 2 days later Bile acids were analysed in one horse with ECoV and were elevated This horse also had elevated liver enzymes and initial triglyceride concentrations 6 78 mmol L but ammonia concentrations were not performed Additional diagnostics Additional diagnostics at intake in horses positive for ECoV only included 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 Ultrasound examination yielded no signi cant ndings 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 not apparent in any exam Large intestinal abnormalities included increased wall thickness n 4 26 15 hypomotility n 2 26 8 luminal distension with uid n 2 26 8 or gas n 2 26 8 and hypermotility n 1 26 4 Peritoneal effusion was present in 1 horse 1 26 4 Rectal examination indicated scant faeces n 4 16 25 mild colon impaction n 3 16 19 mild colonic 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 no signi cant ndings in 3 horses 3 16 19 Table 1 Presenting complaints median range physical examination parameters and initial complete infection and a co infection ECoV only and negative controls with statistical comparison RR ECoV Presenting complaint n 30 Historic fever 25 30 83 Anorexia 14 30 47 Colic 13 30 43 Lethargy 8 30 27 Leukopenia 5 30 17 Diarrhea 1 30 3 Tachypnea 1 30 3 Foot soreness 1 30 3 Haemorrhagic rectal discharge Historic temperature C14 C n 25 30 40 38 3 41 6 Physical examination n 29 30 Temperature C14 C 38 5 37 39 9 Heart rate beats min 48 32 72 Resp rate breaths min 20 12 60 Peripheral lactate mmol L RR CBC n 29 30 WBC cells x 10 9 L 5 0 11 6 3 0 0 68 16 2 22 RR Metamyelocytes cells x 10 9 L 0 0 17 0 21 3 25 2 RR Toxic bands cells x 10 9 L Rare 0 13 0 7 96 19 RR Neutrophils cells x 10 9 L 2 6 6 8 1 26 0 15 14 4 21 RR Lymphocytes cells x 10 9 L 1 6 5 8 0 86 0 42 3 47 n 30 30 25 RR Platelet count x10 9 L 100 225 122 58 164 4 RR HCT 30 46 37 0 24 4 66 7 n 30 30 5 RR Total protein g dL 58 87 62 52 77 n 30 30 6 RR Length of hospitalisation days n 27 30 5 0 5 14 Survival to discharge 26 27 96 CBC complete blood count HCT haematocrit HR heart rate RR reference range RR or Resp rate respiratory rate WBC white blood cell count Fig 1 Number of equine coronavirus positive cases over the months of the year from 1 March 2012 through 31 March 2018 January through March had signi cantly higher numbers of positive cases compared to other times of the year P RR 6 RR Abdominal ultrasound was performed in 23 33 control horses and ndings were similar to those described in the ECoV positive group Supplemental material Four of 23 controls also received thoracic ultrasound exams with pulmonary changes in 3 4 There were no signi cant differences in the number of horses with small intestinal or large intestinal abnormalities between the ECoV only and negative control groups Rectal examinations were performed in 19 33 control horses Abnormalities included gas distended n 3 19 16 uid lled n 2 19 11 or impacted large colon n 2 19 11 loose faeces n 2 19 11 loose n 1 19 5 or dry n 1 19 5 faeces and gas distended colon hard faeces n 1 19 5 and faeces with frank blood and rectal mucosal edema n 1 19 5 There were no signi cant ndings on rectal exam in 6 19 32 Abdominocentesis ndings for horses positive for only ECoV indicated 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 C2 10 9 L Horses negative for ECoV had a median lactate concentration of 2 0 2 13 9 mmol L total protein 15 n 3 3 n 33 33 5 0 1 42 5 18 5 68 1 74 18 73 0 0006 1 RR 22 RR 0 0 0 70 0 31 0 83 0 0 10 2 77 3 RR 13 RR 0 47 3 96 0 004 0 23 1 90 0 19 17 1 3 RR 12 RR 1 6 0 85 3 47 1 4 0 23 5 0 0 007 1 RR 21 RR 120 117 137 147 86 248 n 32 33 2 RR 400 300 500 300 100 900 1 RR 5 RR 40 3 34 6 20 8 53 4 36 6 45 2 5 RR 62 60 73 63 35 82 7 RR number of horses with values less than or greater than the reference range Table 2 Serum biochemistry pro les in 28 horses with equine coronavirus ECoV only three horses presented Statistically signi cant differences were not demonstrated between the group RR ECoV n 28 30 iMagnesium mmol L 0 47 0 70 0 35 0 29 0 56 Sodium mmol L 125 137 132 117 136 Potassium mmol L 3 0 5 6 3 0 1 6 4 4 Chloride mmol L 91 104 96 77 101 Phosphorus mmol L 0 68 1 52 0 68 0 23 3 94 Calcium mmol L 2 85 3 53 2 7 2 4 2 95 Anion Gap mmol L 9 17 14 11 46 Bicarbonate mmol L 23 32 25 11 28 Blood urea nitrogen mmol L 4 28 9 64 5 36 2 86 11 78 Creatinine mmol L 79 56 176 8 114 92 79 56 32 Glucose mmol L 2 78 5 94 7 27 5 38 10 38 Total protein g L 58 77 61 46 74 Albumin g L 27 42 30 19 36 Globulin g L 16 50 29 22 38 88 72 7 98 E H Berryhill et al The Veterinary Journal 248 2019 95 100 5 38 g L and total nucleated cell count 1 15 0 47 13 1 cells C2 10 9 L when measured in 12 33 11 33 and 14 33 horses respectively Supplementary material There were no signi cant differences in lactate protein or nucleated cell count between horses positive for ECoV and controls Nasogastric intubation performed in those positive for ECoV only yielded net re ux in 1 7 horses 14 with 7 L recovered A nasogastric tube was passed in 17 33 52 of the control horses with 11 L of net re ux in one horse Abdominal radiographs showed mild to moderate sand accumulation in 2 5 horses 40 with ECoV only uid lines in 2 5 40 and no signi cant ndings in 1 5 20 Radiographs were performed in 12 33 control horses and indicated scant to moderate sand accumula AST IU L 168 494 270 186 1881 Creatine kinase IU L 119 287 239 86 813 ALP IU L 86 285 157 91 655 GGT IU L 8 22 12 8 85 SDH IU L 0 8 3 0 173 Triglycerides mmol L 0 02 0 46 0 49 0 24 8 12 Total bilirubin mmol L 8 55 39 33 44 46 18 81 1 Direct bilirubin mmol L 3 42 10 26 3 42 1 71 15 39 Indirect bilirubin mmol L 5 13 29 07 42 75 15 39 1 ALP Alkaline phosphatase AST Aspartate aminotransferase GGT Gamma glutamyltransfera tion in 5 12 42 Among the ECoV positive horses 30 33 horses were tested for at least one additional pathogen including gastrointestinal respiratory or blood borne agents with 3 30 10 diagnosed with known co infections salmonellosis Actinobacillus sp perito nitis and salmonellosis with concurrent Rhinitis B virus Diagnostics to assess for the direct presence of other infectious agents in horses positive for ECoV and controls are found in Table 3 A complete list of diagnostic tests performed ca be found in the Appendix Supplementary Material Table 3 Additional diagnostic testing to assess for the direct presence of other infectious agents performed in horses positive for equine coronavirus ECoV compared to negative controls ECoV n 33 Control n 33 Salmonella culture 30 91 1 positive 25 76 all negative Respiratory qPCR panel 11 33 1 positive 8 24 all negative A phagocytophilum PCR and or buffy coat smear 6 18 all negative 6 18 2 positive C dif cile ELISA 0 3 9 all negative Abdominal uid culture 1 3 positive 2 6 all negative Blood culture 1 3 negative 1 3 negative C pseudotuberculosis PCR 0 1 abdominal uid 3 negative Therapy and hospitalisation Medical therapy instituted was available in 28 30 93 horses positive 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 and 13 33 39 negative horses and are shown in Table 4 The last CBC was performed a median of 4 2 14 and 3 2 9 days after the intake CBC for horses positive and negative for ECoV respectively Follow up fecal qPCRs were performed in 15 33 45 of all horses positive for ECoV Seven of the 15 47 horses retested remained ECoV qPCR positive when tested a median of 3 2 8 days after the initial qPCR Eight of the 15 53 horses retested had negative with ECoV and a co infection and 30 negative control horses with median range with ECoV only and negative controls P 0 05 ECoV co infection n 3 3 Control n 30 33 0 33 0 25 0 41 0 4 0 24 0 61 129 129 132 134 122 139 3 4 3 0 3 4 3 2 2 7 4 8 94 89 101 97 77 103 0 68 0 48 1 39 0 74 0 48 4 68 2 7 2 68 2 85 2 7 1 83 3 28 16 13 18 14 11 31 25 15 28 25 19 30 12 9 14 5 36 3 21 25 7 7 08 1 1 1 3 114 92 61 88 318 24 6 16 5 99 8 32 6 22 2 83 9 82 57 55 73 60 42 76 31 25 36 30 22 37 30 26 37 31 21 52 299 293 372 259 146 6980 484 125 596 211 79 30 451 459 98 550 130 22 316 18 10 22 12 7 83 0 0 6 3 0 47 2 19 2 01 4 61 0 59 0 17 3 3 1 46 17 42 75 141 93 37 62 10 26 141 93 3 42 1 71 3 42 3 42 1 71 8 55 1 42 75 39 33 140 22 34 2 6 84 138 51 se RR reference range SDH Sorbitol dehydrogenase ECoV qPCR results when tested a median of 7 5 16 days after the initial qPCR Length of hospitalisation and survival rate for horses with and without ECoV are shown in Table 1 One horse with ECoV died 12 h after admission and had a 6 day history of fever diarrhea and leukopenia a peripheral lactate of 14 5 mmol L haematocrit of 66 7 degenerative left shift with metamyelocytes and evidence of multi organ dysfunction syndrome and disseminated intravas cular coagulation Necropsy revealed necrohaemorrhagic colitis and enteritis with disseminated vascular thrombi congestion and petechiation of the heart and other organs and renal infarcts Immunohistochemistry identi ed ECoV within the contents of the colon Thirty two of 33 control horses were managed medically while one required colic surgery Two control horses did not survive with diagnoses at necropsy of 1 colitis and mega esophagus and 2 toxic shock syndrome secondary to S aureus infection There was no signi cant difference in survival to discharge between horses with ECoV and the control population Horses positive for ECoV and co infections Diagnostic ndings for horses positive for ECoV and a co infection were similar to horses positive for ECoV only and included small intestinal hypomotility and colonic uid on ultrasound n 1 3 scant n 2 3 or malodorous n 1 3 faeces on rectal examination and sand on radiographs n 1 3 The horse e for rang follow 21 30 2 9 8 5 0 6 1 20 50 26 8 99 30 E H Berryhill et al The Veterinary Journal 248 2019 95 100 99 co infected with Actinobacillus spp peritonitis had 5 L of net re ux upon initial nasogastric intubation and had markedly abnormal abdominocentesis results lactate 17 mmol L total protein 61 g L total nucleated cell count 165 cells C2 10 9 L Follow up CBCs were similar to the rest of the ECoV positive cohort except for the horse with Actinobacillus peritonitis with persistent thrombocytopenia hyperproteinemia and hyper brinogenemia Discussion This study showcases common presentations