Detection of equine herpesvirus-1 (EHV-1) in urine samples during outbreaks of equine herpesvirus myeloencephalopathy.

BACKGROUND: Real-time PCR is the diagnostic technique of choice for the diagnosis and control of equine herpesvirus-1 (EHV-1) in an outbreak setting. The presence of EHV-1 in nasal swabs (NS), whole blood, brain and spinal cord samples has been extensively described; however, there are no reports on the excretion of EHV-1 in urine, its DNA detection patterns, and the role of urine in viral spread during an outbreak. OBJECTIVES: To determine the presence of EHV-1 DNA in urine during natural infection and to compare the DNA detection patterns of EHV-1 in urine, buffy coat (BC) and NS. STUDY DESIGN: Descriptive study of natural infection. METHODS: Urine and whole blood/NS samples were collected at different time points during the hospitalisation of 21 horses involved in two EHV-1 myeloencephalopathy outbreaks in 2021 and 2023 in Spain. Quantitative real-time PCR was performed to compare the viral DNA load between BC-urine samples in 2021 and NS-urine samples in 2023. Sex, age, breed, presence of neurological signs, EHV-1 vaccination status and treatment data were recorded for all horses. RESULTS: A total of 18 hospitalised horses during the 2021 and 2023 outbreaks were positive for EHV-1, and viral DNA was detected in urine samples from a total of 11 horses in both outbreaks. Compared with BC samples, DNA presence was detected in urine samples for longer duration and with slightly higher concentration; however, compared with NS, detection of EHV-1 in urine was similar in duration with lower DNA concentrations. MAIN LIMITATIONS: Limited sample size, different sampling times and protocols (BC vs. NS) in two natural infection outbreak settings. CONCLUSIONS: EHV-1 was detected in the urine from naturally infected horses. Urine should be considered as complimentary to blood and NS in diagnosis of EHV-1 infection.

HISTORIAL: PCR en tiempo real es la técnica diagnostica de preferencia para el diagnóstico y control del herpes virus equino­1 (EHV­1) en una situación de brote. La presencia de EHV­1 en torulas nasales (TN), muestras de sangre entera, cerebro, y medula espinal ha sido descrita en forma extensa; sin embargo, no hay informes de excreción de EHV­1 en orina, la detección del patrón de ADN, y el rol de la orina en la propagación vírica durante un brote. OBJETIVOS: Determinar la presencia de ADN de EHV­1 en muestras de orina durante un brote infeccioso natural y comparar los patrones de detección de ADN de EHV­1 en orina, capa leucocitaria (CL) y TN. DISEÑO DEL ESTUDIO: Estudio prospectivo en una infección natural en caballos hospitalizados. MÉTODOS: Muestras de orina y sangre entera/TN fueron recolectadas a distintos tiempos durante la hospitalización de veintiún caballos involucrados en dos brotes de mielo encefalopatía por EHV­1 en 2021 y 2023 en España. PCR a tiempo real cuantitativo fue llevado a cabo para comparar la carga de ADN viral entre muestras de CL­orina en 2021 y muestras TN­orina en 2023. Sexo, edad, raza, presencia de síntomas neurológicos, estatus de vacunación y datos de tratamiento fueron anotados para todos los caballos. RESULTADOS: Un total de diez y ocho caballos hospitalizados durante los brotes de 2021 y 2023 resultaron positivos a EHV­1, y ADN viral fue detectado en muestras de orina en un total de 11 caballos de ambos brotes. En comparación a muestras de CL, la presencia de AND fue detectado por mas largo tiempo y con una concentración ligeramente mas alta; sin embargo, en comparación a TN, la detección de EHV­1 en orina fue similar en tiempo pero demostró menor concentración de ADN. LIMITACIONES PRINCIPALES: Tamaño de muestra limitado, tiempos de muestreo diferentes, y de protocolos (CL vs. TN) en dos situaciones de brotes naturales. CONCLUSIONES: Se detecto EHV­1 en orina de caballos infectados naturalmente. La recolección, no invasive, de orina debería considerarse como un complemento a las muestras de sangre y TN en el control de caballos infectados en situaciones de brote.

Haemato-biochemical characterization of equine piroplasmosis asymptomatic carriers and seropositive, real-time PCR negative horses.

Equine piroplasmosis (EP) is caused by Theileria equi and Babesia caballi, transmitted by tick vectors. Horses can suffer an acute, subacute, and chronic forms of the disease, with clinical signs such as poor performance, fever, pale mucosal membranes, and jaundice. The diagnosis of EP subclinical cases is complex due to the sensitivity of real-time PCR and the limited parasite load in some carriers, making it challenging to differentiate them from seropositive, PCR negative (S+PCR-) individuals. This study aimed to describe haematological and biochemical changes in asymptomatic EP carriers, EP S+PCR- horses and control horses (EP seronegative and PCR negative). It also investigated potential haemato-biochemical markers to aid in distinguishing true EP carriers alongside molecular and serological tests. A comprehensive haematology and biochemistry profile was conducted on 410 sera and EDTA blood samples, comprising 130 EP positives by real-time PCR and competitive ELISA (cELISA) (carriers), 130 EP negatives by real-time PCR but positive to cELISA (S+PCR-) and 150 EP negative horses to real-time PCR and c-ELISA (controls). Our study confirmed that a haematological and biochemistry profile could help to differentiate between EP carriers/S+PCR- from healthy horses. Carriers and S+PCR- horses showed significant increases in the white blood cell count (WBC), high total proteins (TP) and total globulins (GLOB) concentration, and liver function markers compared to controls. Additionally, the evaluation of uric acid (UA) suggested oxidative stress in carrier horses. However, no useful haemato-biochemical diagnostic markers were identified to aid the challenging differentiation of EP carriers and S+PCR- horses, highlighting the need for improvement in molecular/serological diagnosis for these horses.

First report and molecular characterization of cases of natural Taylorella asinigenitalis infection in three donkey breeds in Spain.

Taylorella asinigenitalis is a non-pathogenic bacteria isolated from the genital tract of donkeys but also a cause of metritis and vaginal discharge in mares. It is closely related to Taylorella equigenitalis, the cause of Contagious Equine Metritis (CEM) in horses, and has been present in different countries in Europe since 1995. Up to date, there are no studies on the prevalence of T. asinigenitalis in the equine or asinine populations in Spain; this is the first report of the presence of T. asinigenitalis in donkeys (Equus asinus) from different breeds in three regions of Spain. A total of 106 healthy animals of three different Spanish donkey breeds: Andaluza (26), Majorera (12) and Zamorano-Leonés (68) were sampled between June and July 2017 and a real-time PCR was used to detect T. asinigenitalis in all samples. A total of 39/221 (17,65 %) samples from 22/106 (20,75 %) animals yielded a positive result and were further characterized by MLST; an allelic profile and Sequence Type (ST) could be assigned to 11 of the 39 positive samples, resulting in four novel STs and no clonal complexes within the PubMLST database. There were statistically significant differences in the percentage of positive animals by breed and sex, and also in the variability of STs between farms. Breeding management would have an influence on the percentage of positives in a farm; artificial insemination and separating jacks from jennies should be implemented. Further studies to detect and characterize T. asinigenitalis in donkeys and horses from Spain would be required to obtain a broader epidemiological picture in this country.

Taylorella asinigenitalis: raising awareness of its importance and presence in equine and asinine populations.

Taylorella equigenitalis has long been recognised as a causative agent of contagious equine metritis, but practitioners may be less familiar with Taylorella asinigenitalis, which has been identified more recently. Here, Abel Dorrego, Consuelo Serres and Fatima Cruz-Lopez of the Universidad Complutense de Madrid describe T asinigenitalis and report the findings of a survey they carried out in donkeys in Spain.

Influence of multiple factors on hematologic reference intervals in horses residing in livery yards in Spain.

The hemogram is a routine analysis for equine veterinary practitioners in the assessment of patient clinical status. Reference intervals (RIs) of hematologic constituents vary according to different horse populations and are often described for a particular breed or horse type. The aims of this study were to determine RIs for hematologic constituents in a mixed-breed horse population residing in livery yards in central Spain and evaluate the associations between estimated RIs and multiple phenotypic and management characteristics. A total of 122 healthy horses from different breeds in central Spain were included in the study. RIs were calculated following the American Society for Veterinary Clinical Pathology guidelines. Significant associations between red blood cell (RBC) counts, packed cell volumes (PCVs), hemoglobin (HGB) concentrations, white blood cell (WBC) counts, and phenotypic and management features were evaluated using a novel multiple linear regression model analysis. Reference intervals were 5.8-10.0 × 1012 /L for RBCs, 97-164 g/L for HGB, 0.27-0.46 L/L for PCVs, 37.1-53.6 fL for MCVs, 3.8-10.8 × 109 /L for WBCs, and 76.1-377.9 × 109 /L for platelet counts. The season, discipline, and housing when and where the horses were sampled were factors significantly associated with WBC counts and/or red cell values (HGB, RBC, and PCV). Hematologic RIs for these horses were comparable to the RIs of warm-blooded horses and influenced by husbandry. These location-specific RIs should allow veterinary practitioners to make better-informed decisions for their patients residing in livery yards.

Globetrotting strangles: the unbridled national and international transmission of Streptococcus equi between horses.

The equine disease strangles, which is characterized by the formation of abscesses in the lymph nodes of the head and neck, is one of the most frequently diagnosed infectious diseases of horses around the world. The causal agent, Streptococcus equi subspecies equi, establishes a persistent infection in approximately 10 % of animals that recover from the acute disease. Such 'carrier' animals appear healthy and are rarely identified during routine veterinary examinations pre-purchase or transit, but can transmit S. equi to naïve animals initiating new episodes of disease. Here, we report the analysis and visualization of phylogenomic and epidemiological data for 670 isolates of S. equi recovered from 19 different countries using a new core-genome multilocus sequence typing (cgMLST) web bioresource. Genetic relationships among all 670 S. equi isolates were determined at high resolution, revealing national and international transmission events that drive this endemic disease in horse populations throughout the world. Our data argue for the recognition of the international importance of strangles by the Office International des Épizooties to highlight the health, welfare and economic cost of this disease. The Pathogenwatch cgMLST web bioresource described herein is available for tailored genomic analysis of populations of S. equi and its close relative S. equi subspecies zooepidemicus that are recovered from horses and other animals, including humans, throughout the world. This article contains data hosted by Microreact.

Sero-molecular survey and risk factors of equine piroplasmosis in horses in Spain.

BACKGROUND: Theileria equi and Babesia caballi cause equine piroplasmosis (EP), one of the most important tick-borne diseases of horses due to its high negative impact to the equine industry. Although infections with these parasites have been reported for decades in Spain, epidemiological studies have only been carried out in certain regions. OBJECTIVES: To determine the (sero)prevalence of these parasites in asymptomatic horses nationwide in Spain and to identify potential individual and environmental factors associated with seropositivity to EP. STUDY DESIGN: Sample size was calculated according to the horses registered in Spain in 2013 and by autonomous community using a random stratified sampling. A questionnaire was used to collect data on factors associated with EP seropositivity. METHODS: Serological (cELISA and complement fixation test) and molecular (real-time PCR) analyses were carried out in 740 horses. Risk factors were identified computing two independent logistic regression models with the collated data. RESULTS: Antibodies against EP were detected in 42.9% (95% CI 39.4-46.5) of horses, whereas 30.3% (95% CI 27.0-33.6) were EP positive by PCR. A substantial strength of agreement (k = 0.721) was estimated between serological tests. Exposure to T. equi was significantly higher than to B. caballi and the highest (sero)prevalence was detected in the northern communities. Increasing horse age, presence of ticks and contact with cows were factors related to EP seropositivity in the horses, whereas tetanus vaccination and fairs attendance were associated with lower seropositivity. CONCLUSIONS: Almost half of the horses residing in Spain had antibodies against EP or circulating parasitaemia. Appropriate prevention measures and implementation of a EP surveillance programme should be considered in order to reduce and control the infection.

Phylogenetic analysis and geographical distribution of Theileria equi and Babesia caballi sequences from horses residing in Spain.

The intraerythrocytic protozoans Theileria equi and Babesia caballi are the causative agents of equine piroplasmosis (EP), one of the most important equine tick-borne diseases due to its significant impact on global international horse trade. Although EP is known to be endemic in Spain, previous phylogenetic studies have only been conducted for limited geographical regions. Therefore, the objective of this study was to evaluate the genetic diversity and distribution of these parasite species nationwide. This was performed by amplification of the 18S small subunit (SSU) rRNA gene from 100 EP positive equine blood samples using a nested PCR protocol, and sequencing the obtained amplicons. Seventy-seven T. equi and six B. caballi isolates were successfully sequenced and phylogenetic analysis revealed that the T. equi isolates grouped into the previously described clades A (n = 21/77), D (n = 1/77) and E (n = 55/77), while B. caballi isolates were placed into clades A (n = 5/6) and B (n = 1/6). Isolates from T. equi clade D and B. caballi clade B have not previously been reported in Spain. A greater intra-clade diversity (97.3-98.3 % identity) was observed between T. equi clade E isolates compared to those within clade A (99.7-100 % identity). Additionally, a multivariable logistic regression model was used to analyse associations between the clade of T. equi infection and available epidemiological data. Horses residing in Spanish northern regions were statistically more likely to be infected with T. equi clade E (p = 0.01). We conclude that while extensive sequence variation of equine piroplasms exists in Spanish infected horses, a requirement for increased equine movement controls between Spain and EP-endemic countries should be considered.

Importance of equine piroplasmosis antibody presence in Spanish horses prior to export.

Serological analysis of equine piroplasmosis (EP), caused by Theileria equi and Babesia caballi, is included in the export testing requirements for most of the countries worldwide, thus involving a high economic impact on equine industry of EP-endemic countries, such as Spain. A total of 3368 serum samples from healthy horses collected prior to export between 2015 and 2018 in Spain were tested for antibodies against T. equi and B. caballi by using a competitive inhibition enzyme-linked immunosorbent assay (cELISA). The overall seroprevalence results in Spain revealed that almost a quarter of the horses analysed (24.1 %; 95% CI 22.6-25.5) could not be exported to countries free from EP. The implementation of prevention measures such as the use of acaricides and daily checks for ticks in horses, as well as regular serological screening of horses in Spain would aid to increase the number of horses exported to other countries.

Serological, molecular and hematological diagnosis in horses with clinical suspicion of equine piroplasmosis: Pooling strengths.

Equine piroplasmosis (EP) is a tick-borne protozoan disease caused by Theileria equi and/or Babesia caballi. Clinical signs (fever, pale mucosal membranes, jaundice), anemia and hyperbilirubinemia have been associated with the disease. EP is widespread, has a significant economic impact on the equine industry and remains endemic in Spain. This study was carried out with samples belonging to 140 horses residing in Spain and showing common clinical signs of EP. A blood smear microscopic examination and a comparison between the different results obtained by competitive Enzyme-Linked Immunosorbent Assay (cELISA), real-time Polymerase Chain Reaction (PCR) and hematological and biochemical (direct and total bilirubin) screening were conducted. EP positivity rates by cELISA and PCR were 50.7% and 42.9%, respectively, whereas only 9% of the horses were positive in the microscopic analysis. A significantly higher number of B. caballi-positive horses were detected by cELISA than PCR, and Kappa value was higher for T. equi (k = 0.575) than for B. caballi (k = 0.401). For the first time, an association between a high ELISA inhibition percentage (IP) and a positive PCR result for B. caballi was determined. Although most authors have described T. equi as more pathogenic than B. caballi, we found that horses parasitized by B. caballi showed a more severe hemolytic anemia, whereas T. equi infections were mostly associated with leukocytosis. The hemogram and clinical chemistry could guide the veterinary surgeon towards the diagnosis of T. equi or B. caballi since horses showed a significant leukocytosis or anemia and hyperbilirubinemia, respectively; however PCR would be the test of choice in order to confirm the diagnosis. Information about the importance of a correct diagnosis of EP using a combination of techniques is essential in order to allow the early detection of cases and prevent the spread of the disease, as well as to avoid the common practice of treating horses without a laboratory diagnosis.

Equine viral arteritis in breeding and sport horses in central Spain.

Equine viral arteritis (EVA) may have a high economic impact on breeding stud farms due to the occurrence of EVA-associated abortion outbreaks and the ability of the virus to persist in carrier stallions. While the consequences of EVA in premises with sport horses are usually less severe, the first confirmed outbreak of EVA in Spain occurred in a riding club in Barcelona, but no data on the seroprevalence of EVA in sport horses have been reported in Spain. Given the importance of both Spanish Purebred (SP) breeding horses and sport horses for Spain's equine industry, the aim of this study was to determine and compare the seroprevalence of EVA in these two horse populations in central Spain. Serum samples from 155 SP breeding horses residing in 16 stud farms and 105 sport horses of different breeds housed in 12 riding clubs, collected between September 2011 and November 2013, were tested using a commercial EVA antibody ELISA test with a 100% sensitivity, and confirmed by seroneutralisation (SN) test. EVA seroprevalence in SP breeding horses was higher 21.1% (95% CI 15.3-26.8%) than that in sport horses (6.7%, 95% CI 1.89-11.45%). However, the primary use (breeding vs. sport) was not significantly associated with seropositivity to Equine Arteritis Virus (EAV), suggesting that different management factors do not affect EVA circulation in these two horse populations.