Equine Herpesvirus-1 Outbreak During a Show-Jumping Competition: A Clinical and Epidemiological Study.

A total of 752 horses were involved in the CES Valencia Spring Tour 2021. Due to an equine herpesvirus-1 (EHV-1) outbreak, the competition was cancelled and the site was locked down. The objective of this study was to describe epidemiological, clinical, diagnostic, and outcome data of the 160 horses remaining in Valencia. Clinical and quantitative polymerase chain reaction (qPCR) data were analysed for 60 horses in a retrospective case-control observational study. The risk of developing clinical manifestations was explored using a logistic regression approach. EHV-1 was detected by qPCR, genotyped as A2254 (ORF30) and isolated on cell culture. From the 60 horses, 50 (83.3%) showed fever, 30 horses (50%) showed no further signs and 20 (40%) showed neurological signs, with eight horses (16%) hospitalised, of which two died (3%). Stallions and geldings were six times more likely to develop EHV-1 infection compared to mares. Horses older than 9 years, or housed in the middle of the tent were more likely to develop EHV-1 myeloencephalopathy (EHM). These data show that for EHV-1 infection, the risk factor was male sex. For EHM the risk factors were age > 9-year old and location in the middle of the tent. These data highlight the crucial role of stable design, position, and ventilation in EHV-outbreaks. It also showed that PCR testing of the horses was important to manage the quarantine.

Equine Herpesvirus 1 Variant and New Marker for Epidemiologic Surveillance, Europe, 2021.

Equine herpesvirus 1 isolates from a 2021 outbreak of neurologic disease in Europe have a mutation, A713G, in open reading frame 11 not detected in 249 other sequences from equine herpesvirus 1 isolates. This single-nucleotide polymorphism could help identify horses infected with the virus strain linked to this outbreak.

Modelling the probability and impact of false-positive serology for Borrelia burgdorferi sensu lato: A case study.

BACKGROUND: Serological screening tests for Lyme borreliosis have poor specificity, with potential for misdiagnosis and unnecessary antimicrobial treatment. OBJECTIVES: To evaluate the impact of Lyme borreliosis seroprevalence and serologic test characteristics on the probability of obtaining a false-positive result and impact on antimicrobial use. STUDY DESIGN: Cross-sectional serological survey and modelling. METHODS: Sera from 303 horses in southern Belgium were analysed by enzyme-linked immunosorbent assay (ELISA). Apparent seroprevalence was derived from serological data and a Bayesian estimate of true seroprevalence was computed. These were a starting point to model the impact of test and population characteristics on the probability of obtaining false-positive results and consequently unnecessary treatments and complications. RESULTS: Apparent and true seroprevalence were 22% (95% CI 18%-27%) and 11% (credible interval with 95% probability 0.6%-21%) respectively. We estimate that two-thirds of positive samples are false positive in southern Belgium, with one in five of tested horses potentially misdiagnosed as infected. Around 5% of antimicrobial use in equine veterinary practice in Belgium may be attributable to treatment of a false-positive result. MAIN LIMITATIONS: There was uncertainty regarding the ELISA's sensitivity and specificity. CONCLUSIONS: This study highlights the importance of appreciating the poor diagnostic value of ELISA screening for Lyme borreliosis as demonstrated by this case study of seroprevalence in southern Belgium where we demonstrate that a nontrivial number of horses is estimated to receive unwarranted treatment due to poor appreciation of screening test characteristics by practitioners, contributing substantially to unnecessary use of antimicrobials.

Further Evidence for in Utero Transmission of Equine Hepacivirus to Foals.

(1) Background: Equine hepacivirus (EqHV), also referred to as non-primate hepacivirus (NPHV), infects horses-and dogs in some instances-and is closely related to hepatitis C virus (HCV) that has infected up to 3% of the world's human population, causing an epidemic of liver cirrhosis and cancer. EqHV also chronically infects the liver of horses, but does not appear to cause serious liver damages. Previous studies have been looking to identify route(s) of EqHV transmission to and between horses. (2) Methods: In this retrospective study, we sought to evaluate the prevalence of vertical transmission taking place in utero with measuring by quantitative RT-PCR the amounts of EqHV genome in samples from 394 dead foals or fetuses, paired with the allantochorion whenever available. (3) Results: Detection of EqHV in three foals most likely resulted from a vertical transmission from the mares to the fetuses, consistent with the in utero transmission hypothesis. In support of this observation, the presence of EqHV genome was found for the first time in two of the allantochorions. (4) Conclusions: As seemingly benign viruses could turn deadly (e.g., Zika flavivirus) and EqHV happens to have infected a significant proportion of the world's horse herds, EqHV infectious cycle should be further clarified.

Screening and evaluation of antiviral compounds against Equid alpha-herpesviruses using an impedance-based cellular assay.

Equid alpha-herpesviruses (EHV) are responsible for different diseases in equine population. EHV-1 causes respiratory diseases, abortions and nervous disorders, EHV-4 causes respiratory diseases and sporadic abortion, while EHV-3 is responsible of equine coital exanthema. In view of the lack of efficacy of vaccines against EHV-1 and EHV-4 and in the absence of vaccines against EHV-3, the use of antiviral treatment is of great interest. In this study, we documented the interest of the Real-Time Cell Analysis (RTCA) technology to monitor the cytopathic effects induced by these viruses on equine dermal cells, and established the efficacy of this method to evaluate the antiviral effect of aciclovir (ACV) and ganciclovir (GCV). In addition, the RTCA technology has also been found appropriate for the high-throughput screening of small molecules against EHV, allowing the identification of spironolactone as a novel antiviral against EHV.

Phenotypic and Molecular Characterization of Brucella microti-Like Bacteria From a Domestic Marsh Frog (Pelophylax ridibundus).

Several Brucella isolates have been described in wild-caught and "exotic" amphibians from various continents and identified as B. inopinata-like strains. On the basis of epidemiological investigations conducted in June 2017 in France in a farm producing domestic frogs (Pelophylax ridibundus) for human consumption of frog's legs, potentially pathogenic bacteria were isolated from adults showing lesions (joint and subcutaneous abscesses). The bacteria were initially misidentified as Ochrobactrum anthropi using a commercial identification system, prior to being identified as Brucella spp. by MALDI-TOF assay. Classical phenotypic identification confirmed the Brucella genus, but did not make it possible to conclude unequivocally on species determination. Conventional and innovative bacteriological and molecular methods concluded that the investigated strain was very close to B. microti species, and not B. inopinata-like strains, as expected. The methods included growth kinetic, antimicrobial susceptibility testing, RT-PCR, Bruce-Ladder, Suis-Ladder, RFLP-PCR, AMOS-ERY, MLVA-16, the ectoine system, 16S rRNA and recA sequence analyses, the LPS pattern, in silico MLST-21, comparative whole-genome analyses (including average nucleotide identity ANI and whole-genome SNP analysis) and HRM-PCR assays. Minor polyphasic discrepancies, especially phage lysis and A-dominant agglutination patterns, as well as, small molecular divergences suggest the investigated strain should be considered a B. microti-like strain, raising concerns about its environmental persistence and unknown animal pathogenic and zoonotic potential as for other B. microti strains described to date.

Validation of a new experimental model for assessing drug efficacy against infection with Trypanosoma equiperdum in horses.

Trypanosoma equiperdum, the causative agent of dourine, may affect the central nervous system, leading to neurological signs in infected horses. This location protects the parasite from most (if not all) existing chemotherapies. In this context, the OIE terrestrial code considers dourine as a non-treatable disease and imposes a stamping-out policy for affected animals before a country may achieve its dourine-free status. The use of practices as drastic as euthanasia remains controversial, but the lack of a suitable tool for studying a treatment's efficacy against dourine hampers the development of an alternative strategy for dourine infection management. The present study reports on the development of an experimental infection model for assessing drug efficacy against the nervous form of dourine. The model combines the infection of horses by Trypanosoma equiperdum and the search for trypanosomes in the cerebrospinal fluid (CSF) through an ultrasound-guided cervical sampling protocol. After a development phase involving four horses, we established an infection model that consists of inoculating 5 × 104T. equiperdum OVI parasites intravenously into adult Welsh mares (Equus caballus). To evaluate its efficacy, eight horses were infected according to this model. In all these animals, parasites were observed in the blood at 2 days post-inoculation (p.i.) and in CSF (12.5 ± 1.6 days p.i.) and seroconversion was detected (8.25 ± 0.5 days p.i.). All eight animals also developed fever (rectal temperature > 39 °C), low hematocrit (< 27%), and ventral edema (7.9 ± 2.0 days p.i.), together with other inconstant clinical signs such as edema of the vulva (six out of eight horses) or cutaneous plaques (three out of eight horses). This model provides a robust infection protocol that induces an acute trypanosome infection and that allows parasites to be detected in the CSF of infected horses within a period of time compatible with animal experimentation constraints. We conclude that this model constitutes a suitable tool for analyzing the efficacy of anti-Trypanosoma drugs and vaccines.

Multiple molecular detection of respiratory viruses and associated signs of airway inflammation in racehorses.

BACKGROUND: The potential involvement of viruses in inflammatory airway disease (IAD) was previously investigated through either serology or PCR from nasopharyngeal swabs (NS). The aims of this study were to determine the prevalence and incidence of viral genome detection by qPCR in the equine airways, and their association with respiratory clinical signs. METHODS: Both NS and tracheal washes (TW) were collected monthly on 52 Standardbred racehorses at training, over 27 consecutive months (581 samples). Equid herpesviruses (EHV)-1, -4, -2 and -5, equine rhinitis virus-A and -B (ERBV), equine adenovirus-1 and -2, equine coronavirus and equine influenza virus were systematically investigated in both NS and TW. Nasal discharge, coughing, tracheal mucus score and TW neutrophil proportions were simultaneously recorded. RESULTS: Genome for 7/10 viruses were detected at least once throughout the study; up to 4 different viruses being also concomitantly detected. Monthly incidence in TW was respectively 27.9% (EHV-5), 24.8% (EHV-2), 7.1% (ERBV), 3.8% (EHV-4), 1.9% (EAdV1) and 0.2% (EHV-1; ERAV). Neither agreement nor correlation between NS and TW was found for respectively genome detection and viral loads. Detection of viral genome in NS was not associated with any clinical sign. Coughing was significantly associated with TW detection of EHV-2 DNA (OR 3.1; P = 0.01) and ERBV RNA (OR 5.3; P < 0.001). Detection of EHV-2 DNA in TW was also significantly associated with excess tracheal mucus (OR 2.1; P = 0.02). CONCLUSIONS: Detection and quantification of EHV-2 and ERBV by qPCR in TW, but not in NS, should be considered when investigating horses with IAD.

Influence of bronchoalveolar lavage volume on cytological profiles and subsequent diagnosis of inflammatory airway disease in horses.

The aim of the study was to determine whether instillation of either 250 mL or 500 mL of saline for bronchoalveolar lavage (BAL) would influence cytological confirmation of inflammatory airway disease (IAD). Thirty client-owned Standardbred racehorses were sampled via endoscopy with 250 mL of saline in one lung and 500 mL in the contralateral lung. The procedure was repeated 72 h later, reversing the volume per lung. The proportions of BAL fluid (BALF) recovered were significantly higher and neutrophil percentages significantly lower with the larger volume. A poor agreement was found between methodologies in terms of final diagnosis, when based on proportions of neutrophils (>10% from at least one lung). Within the recommended range (250­500 mL), the instilled volume significantly influenced cytological profiles. Establishing specific BALF reference values is warranted.

Bronchoalveolar lavage fluid in Standardbred racehorses: influence of unilateral/bilateral profiles and cut-off values on lower airway disease diagnosis.

The aim of this study was to determine whether the lung side being sampled would significantly influence bronchoalveolar lavage (BAL) cytological profiles and subsequent diagnosis in Standardbred racehorses. One hundred and thirty-eight French Trotters in active training and racing were included in a prospective observational study. BAL was performed using videoendoscopy in both right and left lungs during summer meetings in 2011 (64 horses) and 2012 (74 horses). Cytological data performed 24h later from right and left lungs were compared and specifically used to classify horses as affected with exercise-induced pulmonary haemorrhage (EIPH), inflammatory airway disease (IAD), or were 'controls'. For IAD, cytological definition was based on two different cut off values. Neutrophil percentages, haemosiderophage percentages and the haemosiderophage/macrophage (H/M) ratios were significantly higher in the right compared to the left lung. Measures of intra-class correlation coefficients revealed a fair agreement between left and right lungs for percentages of mast cells, eosinophils, and for the H/M ratio, and a moderate agreement for neutrophil percentages. Fair to moderate agreements were observed between left and right lungs for the diagnosis of IAD and/or EIPH based on kappa coefficients. When sampling one lung only, the risk of incorrectly classifying a horse as a 'control' increased with the use of the restraint cut-off values for IAD. As BAL from one lung is not representative of the other lung in the same horse, both lungs should be sampled for a better assessment of lung cellularity and for a precise diagnosis of lower airway diseases.

Stimulation of airway neutrophils following dexamethasone administration and equid herpesvirus-2 challenge in horses.

The aim of this study was to investigate neutrophil stimulation following experimentally-induced airway inflammation in healthy horses. Six horses received dexamethasone and four were then inoculated with equid herpesvirus-2 (EHV-2). Significant neutrophilia was detected in tracheal wash and bronchoalveolar lavage fluid for up to 6 days. Concentrations of neutrophil elastase (NE) and myeloperoxidase (MPO) were significantly increased compared to baseline for up to 14 days in tracheal washes and both markers were significantly correlated with neutrophil counts. Serum levels of surfactant protein D were not significantly modified throughout the study. These results suggest that dexamethasone administration with or without EHV-2 inoculation is associated with a sustainable activation and degranulation of neutrophils in the trachea along with moderate modifications detectable in the lower airways.

Molecular detection of Theileria equi and Babesia caballi in the bone marrow of asymptomatic horses.

Equine piroplasmosis is a tick-borne disease, the aetiological agents of which are either Theileria equi or Babesia caballi parasites. Piroplasmosis is commonly encountered in acute or sub-acute clinical forms although clinically recovered horses may remain asymptomatic but infected for several years. The clinical detection of such apparently healthy carrier horses (that serve as a host for subsequent infecting ticks), remains a worldwide challenge for controlling the spread of the disease. The aim of the present paper is to report on the detection of both T. equi and B. caballi by PCR in the bone marrow of naturally infected asymptomatic horses. Among 35 bone marrow samples evaluated for orthopaedic clinical research purposes, three samples from clinically healthy horses were found to be positive for T. equi, one of which was also positive for B. caballi. Even if the precise localisation of these parasites as well as the underlying mechanisms for persistence still remains unknown, one should not exclude bone marrow as a potential reservoir site for T. equi and B. caballi in infected asymptomatic horses. We suggest that, this possible localisation site (the bone marrow) should be considered as a therapeutic target when treating parasitic infection in apparently healthy horses.

Sub-clinical diseases affecting performance in Standardbred trotters: diagnostic methods and predictive parameters.

The objectives of this study were to determine the prevalence of sub-clinical diseases in poorly-performing Standardbred horses, compare their physiological response to exercise with control horses, and identify predictive parameters of poor-performance. Fifty horses underwent thorough clinical and ancillary examinations, including haematological and biochemical evaluation, Doppler echocardiography, standardised exercise tests (SETs) on both treadmill and racetrack, treadmill video-endoscopy and collection of respiratory fluids. Most of the poorly-performing horses exhibited many concomitant diseases. The most frequently diagnosed problems involved the lower and upper respiratory tract and the musculoskeletal system. Poor-performers had lower speeds at a blood lactate (LA) concentration of 4mmol/L (V(LA4)) and a heart rate (HR) of 200bpm (V(200)) on treadmill and racetrack, as well as lower values for haematological parameters, plasma angiotensin-converting enzyme and antioxidants, compared to control horses. Problems of the respiratory system were the most frequently diagnosed sub-clinical diseases affecting performance. SETs, together with some blood markers, may be useful as a non-specific diagnostic tool for early detection of diseases that may affect performance.

Anaplasma phagocytophilum infection in cattle in France.

Anaplasma phagocytophilum is the agent of pasture fever or tick-borne fever, a disease of ruminants and humans in the United States and in Europe. Although several hundred cases have been suspected to occur in cattle in France, none has yet been microbiologically confirmed. We report the first identification of A. phagocytophilum 16S RNA gene sequence in a case of TBF in France. This indicates that the diagnosis of tick-borne fever should be also evoked in cattle exposed to Ixodes ticks in France.

Investigation of Neospora sp. antibodies in aborted mares from Normandy, France.

Neospora caninum, an apicomplexan protozoan parasite, is recognized as a major cause of abortion in cattle while limited information is presently available on association between equine Neospora infections and abortions. The aim of the present study was to document prevalence of antibodies against Neospora sp. in aborted mares as a clue to the role of N. caninum in mare reproductive failure in Normandy, France. Using an agglutination test, the number of animals with elevated (>80) anti-Neospora sp. antibody titer was higher in a group of 54 aborted mares than in randomly chosen groups of 45 mares and 76 horses sampled for equine arteritis virus and Fasciola hepatica antibodies, respectively (P<0.001). N. caninum DNA was found in 3/91 fetal brains, 2/77 fetal hearts, and 1/1 placenta, and present in both brains and hearts of two fetuses. In 13 cases for which both mare serum and fetus were available, no fetal N. caninum amplification product was present while a large variation of maternal antibody titers was found. Data prompt at additional surveys of association between equine reproductive failure and Neospora sp. infection.

Borna disease: current knowledge and virus detection in France.

For over two centuries, Borna disease (BD) has been described as a sporadically occurring infectious meningoencephalomyelitis affecting horses and sheep in Central Europe. Over the last decade, the BD epidemiology has been discussed. Firstly, its geographical distribution seems larger than what was previously thought. Secondly, the disease can affect a large number of warm-blooded animal species, including humans. The aetiological agent is the Boma disease virus (BDV), an enveloped, nonsegmented negative-stranded RNA virus classified in the new virus family Bornaviridae (Mononegavirales order). It can induce severe clinical signs of encephalitis with striking behavioural disturbances and may cause death. BDV genome has recently been detected in France in the blood and brain of several animal species (horses, bovines, foxes).