key: cord-0003290-bdh9k0ja
authors: Smith, Fauna Leah; Watson, Johanna L.; Spier, Sharon J.; Kilcoyne, Isabelle; Mapes, Samantha; Sonder, Claudia; Pusterla, Nicola
title: Frequency of shedding of respiratory pathogens in horses recently imported to the United States
date: 2018-05-15
journal: J Vet Intern Med
DOI: 10.1111/jvim.15145
sha: d2652d3fd2fc1f01fabe7b5fff12aa99c377aae4
doc_id: 3290
cord_uid: bdh9k0ja

BACKGROUND: Imported horses that have undergone recent long distance transport might represent a serious risk for spreading infectious respiratory pathogens into populations of horses. OBJECTIVE: To investigate the frequency of shedding of respiratory pathogens in recently imported horses. ANIMALS: All imported horses with signed owner consent (n = 167) entering a USDA quarantine for contagious equine metritis from October 2014 to June 2016 were enrolled in the study. METHODS: Prospective observational study. Enrolled horses had a physical examination performed and nasal secretions collected at the time of entry and subsequently if any horse developed signs of respiratory disease during quarantine. Samples were assayed for equine influenza virus (EIV), equine herpesvirus type‐1, −2, −4, and −5 (EHV‐1, −2, −4, −5), equine rhinitis virus A (ERAV), and B (ERBV) and Streptococcus equi subspecies equi (S. equi) using quantitative PCR (qPCR). RESULTS: Equine herpesviruses were detected by qPCR in 52% of the study horses including EHV‐2 (28.7%), EHV‐5 (40.7%), EHV‐1 (1.2%), and EHV‐4 (3.0%). Clinical signs were not correlated with being qPCR‐positive for EHV‐4, EHV‐2, or EHV‐5. None of the samples were qPCR‐positive for EIV, ERAV, ERBV, and S. equi. The qPCR assay failed quality control for RNA viruses in 25% (46/167) of samples. CONCLUSIONS AND CLINICAL IMPORTANCE: Clinical signs of respiratory disease were poorly correlated with qPCR positive status for EHV‐2, −4, and −5. The importance of γ‐herpesviruses (EHV‐2 and 5) in respiratory disease is poorly understood. Equine herpesvirus type‐1 or 4 (EHV‐1 or EHV‐4) were detected in 4.2% of horses, which could have serious consequences if shedding animals entered a population of susceptible horses. Biosecurity measures are important when introducing recently imported horses into resident US populations of horses.

equine herpesvirus type-4 (EHV-4) continue to be prevalent viral respiratory pathogens throughout the United States and Europe. 1,2 These highly contagious and virulent respiratory viruses have major financial implications for the equine industry. 3, 4 Long distance horse transportation can result in substantial stress and alteration in immune function, including an acute phase response that results in impaired cell-mediated immunity. 5, 6 Imported horses also have increased exposure risk to infectious respiratory pathogens, because of comingling at sales/shipping barns, quarantine facilities, and during transports.

The goal of our study was to determine the detection frequency of selected infectious respiratory pathogens from horses recently imported to the United States. Specifically, we analyzed nasal swabs for shedding of EIV, equine herpesvirus type-1 (EHV-1), equine herpesvirus type-2 (EHV-2), equine herpesvirus type-4 (EHV-4), equine herpesvirus type-5 (EHV-5), Streptococcus equi subsp. equi (S. equi), equine rhinitis A virus (ERAV), and equine rhinitis B virus (ERBV).

All horses with owner consent (n 5 167) admitted to the contagious equine metritis (CEM) quarantine facility at the Center for Equine Health, University of California at Davis for CEM quarantine purposes were enrolled in our study from October 10, 2014 to June 30, 2016.

The USDA approved facility quarantines �200 recently imported horses on an annual basis. Examination and sample collection was performed �72 hours after having arrived in the United States. Horses generally spent 48 hours in USDA import quarantine upon arrival at Los Angeles International Airport, 12 hours in transit to the CEM quarantine facility, and 12-18 hours in CEM quarantine before sampling.

As part of the routine quarantine protocol, horses entering the quarantine facility had a physical examination performed and blood collected for a complete blood cell count. The physical examination and blood draw was performed by a veterinarian from the Equine Field Service of the UC Davis VMTH. Horses were defined as having clinical signs associated with respiratory disease if they had any one: nasal discharge, tachypnea (respiratory rate >24 breaths per minute), fever (>101.58F), cough, submandibular or retropharyngeal lymph node enlargement, or abnormal lung sounds. Data for hematocrit, white blood cell counts, and fibrinogen were collected from the complete blood counts for each horse. Additionally, 6 00 rayon-tipped nasal swabs were collected at the time of entrance from each horse with informed owner consent.

Horses that developed lethargy, anorexia, fever, excessive nasal discharge, lymphadenopathy, or cough during the quarantine period ( 6) primers (random hexamers, Invitrogen). The reaction was performed at 508C for 60 minutes. After inactivation at 958C for 5 minutes, the reaction volume was adjusted to 100 lL with nucleasefree water.

Nasal secretions were assayed for the presence of the EIV, EHV-1, EHV-2, EHV-4 EHV-5, S. equi, ERAV, and ERBV using previously reported quantitative TaqMan PCR assays. 1, 7 To determine the sample quality and efficiency of nucleic acid extraction all samples were analyzed for the presence of the housekeeping gene equine glyceraldehyde-3-phosphate dehydrogenase as previously described. 8 The laboratory

defines failure of quality control as inadequate genomic material or the presence of inhibitors of the qPCR reaction in samples.

Most of the data is presented as descriptive results with population proportions, percentages, or both being reported. Odds ratios were calculated for differences between breed groups. Numerical data was analyzed for normality using the Shapiro-Wilk test. Hematologic variables were normally distributed and were compared using a one- 

One hundred sixty-seven individual horses were enrolled in the study.

The median age was 7 years with a range of 3 weeks-21 years. There were no yearlings; however, there were 3 foals under the age of 1 year (range 1-6 months) that were accompanying their dams. The population included 161 mares, 6 stallions and no geldings. Breed groups represented were European warmbloods (79.0%; 132/167), Friesians Table 1 .

The gB gene for EHV-1 non-neuropathogenic (A2254 genotype) was detected in nasal secretions from 1/167 horses on arrival (a 3-

week old filly) and in one additional animal (her dam) that developed clinical signs during the quarantine period. Both the foal and her mare had consistent clinical signs of tachypnea (respiratory rate 90-100 breaths per minute), mucoid nasal discharge, and fever (102.0-102.58F; Table 1 ). The viral load was high in the filly with 1.4 3 10 7 EHV-1 gB genes/million nasal cells. That filly was also positive in whole blood for the EHV-1 gB genes (9.7 3 10 2 gB genes/million WBCs). The filly's dam was positive in low numbers in nasal swab associated with clinical signs of respiratory disease (5.1 3 10 3 gB genes/million nasal cells) but qPCR-negative in blood. These horses were Andalusians from Spain. (1), and Ireland (1). There was no significant effect of country of origin on EHV-4 shedding.

All three foals enrolled in the study were qPCR-positive for herpesviruses on nasal swabs: EHV-1 (1), EHV-2 (2), and EHV-5 (2). All of the foals were shedding viral loads greater than 1. Of the nasal swab samples submitted for herpesvirus qPCR, 6.0% (10/167) for a-herpesviruses and 6.6% (11/167) for g-herpesviruses,

failed quality control, respectively.

Neither EIV nor ERVs were detected from any of the enrolled horses. 

Streptococcus equi was not recovered by qPCR from any nasal secretions. However, 12/167 horses failed quality control, including one horse with enlarged submandibular lymph nodes. One horse with fever and enlarged submandibular lymph nodes had multiple nasal swabs taken during the quarantine period and all came were negative for S. equi; however, it was reported to be bacterial culture positive from a lymph node aspirate performed after release from quarantine.

There were no significant differences in mean hematocrit (P 5 .11), WBC (P 5 .39), neutrophil count (P 5 .95), lymphocyte count (P 5 .092), monocyte count (P 5 .18), or fibrinogen (P 5 .80) between the four groups of horses as previously defined (Table 2 ).

Our study demonstrated that equine herpesviruses are frequently Our study found that g-herpesviruses were commonly shed by large numbers of horses and that a-herpesviruses were shed by only 4.2% of the horses. The role g-herpesviruses play in the development of respiratory disease has remained poorly understood. Equine herpesvirus-2 has been associated with outbreaks of upper respiratory tract disease in young horses 9 , but EHV-2 has also been isolated from clinically normal horses. 10 Equine herpesvirus-5 has been associated with the development of multinodular pulmonary fibrosis (EMPF), 11 herpetic ocular disease, 12 and lymphoma 13 in horses, however, these diseases are rare while of EHV-5 shedding in the equine population is common. 14 No horses were positive for S. equi, although 12 failed quality control. Of the horses that failed quality control, one had clinical signs consistent with strangles (enlarged lymph nodes and nasal discharge). In a clinical setting, it would have been prudent to collect a second sample for testing. Additionally, there was a single horse with consistent clinical signs, found to be negative on multiple nasal swabs, which was later found to be culture positive from a lymph node aspirate. It is currently recognized that nasal swabs are not as sensitive for recovery of S. equi as guttural pouch lavage or pharyngeal wash samples. 22, 23 It is recommended that in clinical cases with consistent signs the type of sample collected should be adjusted to maximize the chances of recovery.

In our study, complete blood count was not useful in the identification of horses shedding herpesviruses with or without the presence of clinical signs. It has previously been reported that horses with EHV-1

infections had an elevation in absolute monocyte count within the first 5 days of disease and increased neutrophil to lymphocytes ratios early in disease followed by a decrease later in the course of disease. 24 It may be that hematologic measurements over time may be more helpful in linking respiratory signs to a viral infection.

The study had several limitations. First the study population was only composed of mares and stallions 2 years of age and older, and a few nursing foals entering the CEM quarantine facility. Geldings imported from outside the United States and weaned fillies and colts <2 years or age were not available for testing as they directly enter the United States from the port of entry quarantine facility. The proposed study horses represent 20% of all horses imported through the USDA import facility located in Los Angeles. Based on the results of horses <2 years of age in our study this may represent a higher risk population that the general population of imported horses. Horses <2 years of age have been shown to be significantly more likely to have an increased rectal temperature than horses >2 years of age after arrival at an air import facilities in the United States. 16 Further investigation of respiratory pathogen shedding by young imported horses is warranted.

Additionally, because sampling took place at a secondary quarantine site the horses were sampled 72 hours after arrival in the country. It is possible that we missed horses that were shedding during the initial three days after air transport. We do not know if there is a difference in air versus overland travel for transmission of respiratory pathogens in horses. There is some evidence to suggest that horses are more stressed in air travel based on heart rate and heart rate variability. 25 In humans, air transportation appears important in influenza propagation and it is likely that ground transport systems and transport hubs play a role in influenza propagation. 26 Another limitation relates to the number of study horses. There is 

Authors declare no conflict of interest.

Authors declare no off-label use of antimicrobials.

Our study was approved by the IACUC Committee of the University of California, Davis.

Fauna Leah Smith http://orcid.org/0000-0001-5535-7726

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