Eduard Jose-Cunilleras

Equine piroplasmosis (EP), caused by the apicomplexan parasites Theileria equi and Babesia caballi , is a tick-borne disease of major clinical and economic importance in equids worldwide. This study reports, for the first time, the presence of EP and associated variables in the Menorca Purebred Horse (PRMe), an endangered breed of high genetic, cultural, and socio-economic value, based on a sample of participating horses from Menorca (Spain). A cross-sectional study was conducted on 213 horses, combining serological (cELISA), molecular (multiplex quantitative real-time PCR (qPCR) and nested PCR (nPCR)), and entomological analyses. An overall apparent seroprevalence of 56.34% was recorded, with a true seroprevalence of 59.31% (95% CI: 52.00-66.40) for Theileria equi and 3.26% (95% CI: 1.56–6.61%) for Babesia caballi . The overall molecular apparent positivity was 42.72% (95% CI: 36.27–49.44), confirming a substantial proportion of assessed horses with chronic subclinical infection at the time of sampling. Theileria equi was the predominant parasite in both serological and molecular analyses. Multivariate analysis identified different host-related and husbandry-related variables as potential risk factors. Entomological analysis revealed that Hyalomma was the dominant tick genus (96.43%) in the evaluated animals, detecting DNA of Theileria or Babesia species in 12/112 tick samples (10.71%), and more specifically DNA of EP-agents in 10/112 samples. Under the conditions evaluated, nested PCR detected more positive samples than qPCR, and column-based extraction yielded more qPCR-positive results than automated extraction. These findings establish a baseline for the epidemiological monitoring of EP in Menorca and provide evidence to inform vector-control and health-management strategies for the Menorca equine population.

Several studies investigated risk and prognostic parameters for horses with colic; however, the consensus is still debated. The present work aimed to investigate colic outcomes and to identify risk factors in horses referred for colic. In this multicenter retrospective study, 236 clinical records of equids referred for colic at three different equine centers were reviewed. The following data were collected: history, signalment, physical examination at the time of admission, hematological and biochemical analysis, diagnosis, SIRS status and 0–6 point-scale SIRS score, colic type, treatment attempted, and outcome. Descriptive statistics were performed, and distribution of continuous variables was reported as median and percentile. A multivariable logistic regression model was applied to assess parameters associated with colic outcomes in horses (p < 0.05). A total of 138/236 horses were included in the study. The univariate analysis identified as potentially associated with the outcome: sex (p = 0.046), colic type (p < 0.001), treatment type (p < 0.001), SIRS score (p = 0.049), age (p-value = 0.057), heart rate (p = 0.013), and respiratory rate (p = 0.017). The logistic regression model indicated that colic type (p < 0.001) and age (p = 0.004) were significantly associated with a negative outcome. Equine colic risks are multifactorial; prognosis declines with age and strangulating obstructive non-strangulating colic. Poor outcomes link to cardiovascular signs like elevated heart rate, SIRS status and score, and blood lactate. Heterogeneity from diverse sites limits generalizability, but standardized protocols, binarized data, and a multicenter approach enhance robustness and representativeness while reducing local biases.

The forest fly (Hippobosca equina) is an obligate haematophagous dipteran insect (order Diptera) that primarily infests horses and may contribute to the circulation of vector-borne pathogens. This study aimed to investigate the presence of Anaplasma phagocytophilum, Borrelia burgdorferi s.l., Babesia caballi, and Theileria equi, important vector-borne pathogens of equids, in forest flies collected from horses in endemic areas of Spain. A total of 170 forest flies were collected from 39 equids across four geographical regions in Spain (Segovia, Madrid, Toledo, and Menorca) and blood samples were collected from 27 of these horses. All flies were morphologically and molecularly identified as H. equina, and DNA extracted from flies and equine blood was screened using multiplex real-time and nested PCR, followed by sequencing and phylogenetic analysis. Neither flies nor horses tested positive for A. phagocytophilum, whereas one fly was positive for B. burgdorferi s.l. (0.6%). In contrast, T. equi and B. caballi DNA were detected in 11.2% and 1.2% of flies, respectively, and all positive flies were collected from horses positive for equine piroplasmosis (T. equi/B. caballi infection), with identical 18S rRNA sequences between hosts and flies. Nested PCR showed a higher detection rate than real-time PCR for the detection of these piroplasms in flies and blood samples. These findings provide the first molecular evidence of EP pathogens in H. equina and support further investigation into the epidemiological importance of forest flies in equine pathogen surveillance.

Background: Equine Infectious Anemia (EIA) is a notifiable disease caused by Equine Infectious Anemia Virus (EIAV), with major sanitary and economic importance for equids worldwide. There is limited and outdated information on the circulation of EIAV in many European countries. In the present study, we aimed to assess virus exposure in different equid species in Western Europe. Methods: Between 2011 and 2023, a cross-sectional study was conducted to investigate the seroprevalence of EIAV in 1676 equids (1444 horses, 106 donkeys and 126 mules/hinnies) from four European regions: Andalusia (Southern Spain; n = 808), Catalonia (Northeastern Spain; n = 437), Southeastern United Kingdom (UK; n = 209), and Ireland (n = 222). Results: None of the analyzed equines tested positive for antibodies against EIAV (0%; 95 %CI: 0.0–0.18), indicating limited virus exposure of the equid populations from these European regions. Conclusions: This finding is consistent with the low EIAV circulation previously reported in Western Europe and the few official EIA outbreaks recorded in the region during the last decade. To the best of the author’s knowledge, this serosurvey represents the first investigation assessing EIAV exposure in equids from Ireland and the UK. Our results not only provide novel epidemiological evidence for the four European regions assessed but also reinforce the effectiveness of surveillance strategies and control measures implemented by European countries, in accordance with national and international regulations, to successfully limit EIAV circulation.

This study aims to generate foundational metabolomic data of aqueous humor (AH) in healthy horses and donkeys, and to investigate potential changes or trends in the metabolomic profile associated with age, sex or ocular pathology in horses. The AH metabolomic fingerprint from 5 donkeys and 35 equine eyes (17 controls, 8 with cataracts, 6 with retinal disease and 4 with anterior chamber disease (ACD)) were analyzed using nuclear magnetic resonance (NMR) spectroscopy. A linear mixed-effects model, with individual horse as a random effect and group as a fixed effect, with multiple testing correction using the Benjamini–Hochberg false discovery rate (FDR) method was used to compare groups. The metabolomic profile of the donkeys and horse’s AH is very similar to that of other mammals. Threonine was higher in young horses (p = 0.04), and creatinine was elevated in males (p = 0.04). Compared with control groups, dimethyl sulfone was higher in the retina (p < 0.00) and cataract (p = 0.05) groups. Arginine (p = 0.05) and valine (p = 0.03) were lower in the retina group compared to controls. This study successfully characterized the AH metabolomic profile in healthy horses and donkeys and identified several metabolites that could be associated with ocular pathology, warranting further investigation to determine their potential as biomarkers of ocular disease.

Background: Equid alphaherpesvirus 1 (EHV‐1) remains a major concern for the equine industry, with recent outbreaks at international equestrian events highlighting the need for improved surveillance during competitions. Objectives: To investigate EHV‐1 and ‐4 presence in shared airspaces and environmental surfaces in direct contact with horses at international equestrian events; and to evaluate air sampling as a surveillance alternative to individual horse testing. Study Design: Cross‐sectional field study. Methods: Air (37) and surface samples (205) were collected from temporary stabling facilities across six events in Spain and 2 in the United States at different seasons over 10 months. Viral DNA (genomic copies) was measured and quantified by quantitative and digital PCR. During all the Spanish events, we sampled the air twice, early evening and night. Results: EHV‐1 was frequently detected in the air in 20/28 samples in Spain, in all 3 samples from Florida and in 3/6 samples from Kentucky. Surface samples were positive 15.6% in Spain, 26.3% in Florida and 0.0% in Kentucky. EHV‐4 was found in air samples at rates of 12/28 in Spain, 5/6 in Kentucky and 0/3 in Florida, with surface detection of 1.6% in Spain and 7% in Kentucky. No significant differences were observed between daytime (high activity) and nighttime (low activity) airborne viral loads, suggesting that daytime horse movement and management practices had minimal impact on detection levels in our study. A positive correlation (Cohen's K = 0.401) and moderate agreement between surface and air positivity for EHV‐1 emphasised the potential of air sampling as a non‐invasive, cohort‐based surveillance tool. Main Limitations: Virus isolation was not used; only molecular detection was employed, which limits conclusions about transmission risk. Conclusions: These findings underscore the utility of air sampling for early EHV‐1 detection at mass gatherings; although further validation is needed to connect genomic detection to active shedding and viral infectivity.

Background: Equid alphaherpesvirus 1 (EHV-1) remains a major concern for the equine industry, with recent outbreaks at international equestrian events highlighting the need for improved surveillance during competitions. Objectives: To investigate EHV-1 and -4 presence in shared airspaces and environmental surfaces in direct contact with horses at international equestrian events, and to evaluate air sampling as a surveillance alternative to individual horse testing. Study design: A cross-sectional field study. Methods: Air (37) and surface samples (205) were collected from temporary stabling facilities across six events in Spain and 2 in the U.S. at different seasons over 10 months. Viral DNA (genomic copies) was measured and quantitated by quantitative and digital PCR. During all Spain events, we sampled air twice, early evening vs nighttime. Results: EHV-1 was frequently detected in the air in 20/28 samples in Spain, in all 3 samples from Florida, and in 3/6 samples from Kentucky, U.S. Surface samples were positive 15.6% in Spain, 26.3% in Florida, and 0.0% in Kentucky. EHV-4 was found in air samples at rates of 12/28 in Spain, 5/6 in Kentucky, and 0/3 in Florida, with surface detection of 1.6% in Spain and 7% in Kentucky. No significant differences were observed between daytime (high activity) and nighttime (low activity) airborne viral loads, suggesting that daytime horse movement and management practices had minimal impact on detection levels in our study. A positive correlation (Cohen’s κ= 0.401) and moderate agreement between surface and air positivity for EHV-1 emphasized the potential of air sampling as a non-invasive, cohort-based surveillance tool. Main limitations: The study did not isolate infectious viruses, only molecular detection was employed, which limits conclusions about transmission risk. Conclusions: These findings underscore the utility of air sampling for early EHV-1 detection at mass gatherings, although further validation is needed to connect genomic detection to active shedding and viral infectivity.

The growing awareness of the diet–health connection drives interest in natural dog diets, which replace synthetic additives like antioxidants with natural ingredients. In Trial 1 of this study, preference for diets containing powdered Fucus algae (1.5%), powdered clove (0.45%), or whole blueberries (3%) was evaluated using the two-bowl method. Dogs exhibited a clear preference for the blueberry diet. In Trial 2, the impact of blueberries on apparent digestibility was assessed. Twelve Beagles were fed either a control diet (CON) or the same diet with 3% blueberries (BLU). No differences were observed, except for cellulose digestibility. Trial 3 evaluated the effect of blueberries on oxidative status during submaximal exercise. The same dogs were fed either CON or BLU diets for 4 weeks, with eight dogs completing treadmill exercises at the start and end of this period. Blood samples were collected before and after exercise. Trial 3 was replicated in summer and winter, reversing diet groups. Exercise increased creatine kinase (CK), urea, and malondialdehyde and decreased glutathione peroxidase, with stronger effects in summer, suggesting heat stress. Although the BLU diet did not mitigate exercise-induced changes, lower resting CK levels after 4 weeks in summer suggest protection against heat stress, warranting further study.

Background: Equine herpesvirus myeloencephalopathy (EHM) is a persistent threat to horses, with unclear risk factors and disease severity. Objectives: To evaluate risk factors, effective reproduction rate (Rt), and long-term athletic outcomes of an EHM outbreak. Methods: Retrospective study of the 2021 EHM outbreak in Valencia, Spain, examining associations between risk factors (sex, age, breed, country of origin, and vaccination status) and case fatality rate, EHM development, and odds of returning to competition using odds ratios [95% CI] and Rt via the Robert Kochs Institute method. Results: Among 191 horses, 38 (20%) were clinically normal, 13 (7%) were subclinical, and 140 (73%) presented clinical signs (89 EHM, 64%). One hundred sixty horses were isolated at the show, while 47 were treated in hospitals. The mean age was 9.8 ± 3.0 years; 85 (45%) were mares, 79 (41%) geldings, and 27 (14%) stallions. The EHM case fatality rate was 11/89 (12%). Vaccination was associated with EHM development (4.54[2.23–9.27]; OR[95% CI]; p < 0.001) and case fatality rate (3.9[1.1–14.4]; OR[95% CI]; p < 0.043). EHV-1-infected horses without EHM were more likely to return to competition (54/61; 89%) than those recovering from EHM (65/89; 73%; p = 0.024). It was initially 4.2 and decreased to < 1 within 2 weeks of the outbreak. Conclusions: During the 2021 EHV-1 outbreak in Valencia, vaccination status appears to be associated with EHM development. Horses recovering from EHM had slightly lower chances of returning to competition than those shedding EHV-1 without EHM. The high Rt value underscores the contagious nature of EHV-1.

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.