Application of blood parameters for the early diagnosis of natural ascending placentitis in pregnant mares.

Placental infection is an important cause of late-term pregnancy loss and neonatal diseases in horses. Detection of changes in blood parameters especially during early placentitis could improve the diagnostic accuracy, treatment decision, and potential outcomes. The objectives of this 2-part study were to identify differences in circulating immunological, inflammatory, and hormonal parameters between mares with natural ascending placentitis and control mares; evaluate each and combination of parameters as predictors of placentitis; and determine how these parameters indicate severity of placentitis. Reproductive examination and blood sampling were prospectively performed on pregnant mares in a natural setting. Study 1 enrolled mares diagnosed with early stage of ascending placentitis based on ultrasonographic findings (n = 12), and gestationally age-matched mares with healthy pregnancies as controls (n = 12). Blood samples were classified as pre-onset (before) and early onset (at the time of ultrasonographic changes) of placentitis. There were no detected statistically significant differences between groups and timepoints in immunological and inflammatory parameters, including peripheral lymphocyte subpopulations, cytokine, and serum amyloid A concentrations. The placentitis group showed a reduced (P = 0.01) DHP/20α-DHP ratio when compared to the control group at the early onset timepoint. Plasma estradiol-17ß concentration <359 pg/mL predicted ascending placentitis with acceptable accuracy (area under the curve, AUC = 0.71). Combined albumin <3.7 g/dL, estradiol-17ß < 499 ng/mL, and DHP <33 ng/mL predicted 100 % of cases of ascending placentitis. In study 2, samples were classified according to the presence and severity of the abnormal ultrasonographic findings as mild (n = 11) and moderate-severe (n = 23), and gestationally age-matched with samples from control mares (n = 34). Mares with moderate-severe ascending placentitis had increased (P = 0.03) plasma 20α-DHP concentration and reduced (P = 0.03) DHP/20α-DHP ratio when compared to control mares. Our results suggest that the early events of ascending placentitis detected by ultrasonographic findings include hormonal alterations of feto-placental metabolism measurable in the mare's circulation, yet without obvious systemic immunological and inflammatory changes. Additional studies are warranted to further assess how hormonal markers and cutoff values could guide decisions for timely therapeutic intervention.

Seroprevalence of Toxoplasma gondii and Neospora spp. in horse population of Tehran, Iran.

Neospora spp. and Toxoplasma gondii are two closely related protozoan parasites that are widely distributed throughout the world. Horses can act as intermediate hosts for both parasites and can acquire disease. Blood samples were taken from 487 clinically healthy horses from 17 different mechanized stables in Tehran, the capital of Iran, during September and November of 2022. IFAT and ELISA were employed to detect antibodies directed against Neospora spp. and T. gondii. The anti-N. caninum antibodies were detected in 52 of the horses (10.67%) based on IFAT and in 86 of the 487 horses (17.65%) based on the ELISA test. Also, antibodies against T. gondii were detected in 41 horses (8.42%) based on IFAT and in 63 of 487 horses (12.94%) based on the ELISA test. Also, in 6 of the horses (1.23%) based on IFAT and in 13 of the 487 horses (2.67%) based on the ELISA test, double positivity suggested co-infection with both parasites. Gender, age groups, and the presence of dogs for neosporosis, and age groups and the presence of cats for toxoplasmosis, could be considered factors having an influence on the seroprevalences (P < 0.05). The results proved the importance of the urgent implementation of stringent regulatory measures to prevent and control the spread of these parasites.

Plasma concentration of serum amyloid A and lipopolysaccharide binding protein in horses with laminitis resulted from hindgut acidosis.

Many studies have shown a strong correlation between Hindgut Acidosis (HGA) and the occurrence of laminitis in horses; therefore, the early diagnosis of HGA is essential. In this study, we investigated changes in the plasma concentrations of lipopolysaccharide-binding protein (LBP) and serum amyloid A (SAA) as inflammatory markers in horses with laminitis. Sixteen healthy male Arabian horses that had cecal cannulation without visible laminitis or general symptoms were randomly divided into two groups. The horses were fed two different diets in a forage-to-concentrate ratio. Blood samples were collected on Days 1, 10, and 20. The primary objective of this study was to analyze plasma levels of LBP and SAA. Cecal specimens were obtained from each equine subject on three designated days: days 1, 10, and 20. The second objective was to assess the levels of pH and volatile fatty acids (VFA) in the samples. Throughout the study period, horses fed a high-concentrate diet exhibited a significantly elevated average lameness grade on days 10 and 20 compared to the initial stage (P < 0.001). On day 20, a significant increase in the concentration of SAA was observed in horses fed a high-concentrate diet, in contrast to the initial stage of the study. LBP levels in the plasma were significantly elevated on days 10 and 20 in horses fed a high-concentrate diet. Based on our findings, it is recommended that the evaluation of plasma LBP concentrations is more effective than SAA for the early identification of HGA in horses fed a high-grain diet.

Detection of Equine Parvovirus-Hepatitis Virus and Equine Hepacivirus in Archived Sera from Horses in France and Australia.

Reports of newly discovered equine hepatotropic flavi- and parvoviruses have emerged throughout the last decade in many countries, the discovery of which has stimulated a great deal of interest and clinical research. Although commonly detected in horses without signs of disease, equine parvovirus hepatitis (EqPV-H) and equine hepacivirus (EqHV) have been associated with liver disease, including following the administration of contaminated anti-toxin. Our aim was to determine whether EqPV-H and EqHV are present in Australian horses and whether EqPV-H was present in French horses and to examine sequence diversity between strains of both viruses amongst infected horses on either side of the globe. Sera from 188 Australian horses and 256 French horses from horses with and without clinical signs of disease were collected. Twelve out of 256 (4.7%) and 6 out of 188 (3.2%) French and Australian horses, respectively, were positive for the molecular detection of EqPV-H. Five out of 256 (1.9%) and 21 out of 188 (11.2%) French and Australian horses, respectively, were positive for the molecular detection of EqHV. Australian strains for both viruses were genomically clustered, in contrast to strains from French horses, which were more broadly distributed. The findings of this preliminary survey, with the molecular detection of EqHV and EqPV-H in Australia and the latter in France, adds to the growing body of awareness regarding these recently discovered hepatotropic viruses. It has provided valuable information not just in terms of geographic endemicity but will guide equine clinicians, carers, and authorities regarding infectious agents and potential impacts of allogenic tissue contamination. Although we have filled many gaps in the world map regarding equine hepatotropic viruses, further prospective studies in this emerging field may be useful in terms of elucidating risk factors and pathogenesis of these pathogens and management of cases in terms of prevention and diagnosis.

Limited association between stallion-like behavior and hormonal indicators of testicular remnants in geldings.

Persistent stallion-like behavior is a common sign of cryptorchidism in supposed geldings. The presence of testicular tissue can be evaluated by analyzing hormones such as testosterone and anti-Müllerian hormone (AMH). Here, we used hormonal analysis to investigate relationships between the likely presence of testicular tissue and stallion-like behavior in samples submitted from presumptive geldings (n = 1,202), retrospectively. Most geldings with stallion-like behaviors had serum concentrations of testosterone (851/1,056; 80.6 %) and AMH (682/877; 77.8 %) below the laboratory reference range for cryptorchids (< 60 pg/mL and ≤ 0.15 ng/mL for testosterone and AMH, respectively). A total of 13 samples (13/716; 1.8 %) showed AMH concentrations typical for geldings but testosterone above the cryptorchid range. Conversely, 31 samples (31/716; 4.3 %) had high AMH, suggesting cryptorchidism, but testosterone concentrations implied no testicular tissue. Among the cryptorchid stallions, the AMH and testosterone concentrations did not vary based on the season. However, age categories affected the concentration of both hormones among the presumptive true cryptorchid stallions. The results of this study demonstrate that undesirable behavior in geldings is rarely associated with the presence of testicular tissue, as assessed by these two hormonal biomarkers. This information highlights the complexity of behavior and demonstrates that persistent stallion-like behavior in geldings could be related to factors other than the presence of testicular tissue.

Serum concentrations of selenium, copper, and zinc in neonatal foals: Influence of failure of passive transfer and age-related changes.

Background: An adequate supply of trace elements is very important for equine neonates, as deficiencies can lead to health problems and even death. Objective: This study investigated serum concentrations of selenium (Se), copper (Cu), and zinc (Zn) in neonatal foals up to the 8th day of life. The influences of disease, age, and failure of passive transfer (FPT) on these concentrations were analyzed. Animals and procedure: Serum concentrations of Se, Cu, and Zn were determined from blood samples of 93 foals by means of inductively coupled plasma mass spectrometry. The foals were divided into 2 groups based on health status: clinically sick (n = 51) and clinically healthy (n = 42). The latter group was further divided into foals with FPT (n = 20) and those without (n = 22). Results: Mean serum concentrations for Se, Cu, and Zn were 60 ± 40 µg/L, 0.25 ± 0.22 mg/L, and 605 ± 285 µg/L, respectively. A significant influence of age on serum Cu concentration was observed (P < 0.0001). No differences were observed between any of the serum concentrations in clinically sick and clinically healthy foals on the 1st day of life. The FPT status was not associated with reduced serum concentrations of Se, Cu, or Zn. Conclusion and clinical relevance: It is not necessary to supplement trace elements in all foals with FPT.

Concentrations sériques de sélénium, de cuivre et de zinc chez les poulains nouveau-nés : influence de l'échec du transfert passif et des changements liés à l'âge. Contexte: Un apport suffisant en oligo-éléments est très important pour les nouveau-nés équins, car des carences peuvent entraîner des problèmes de santé, voire la mort. Objectif: Cette étude a examiné les concentrations sériques de sélénium (Se), de cuivre (Cu) et de zinc (Zn) chez les poulains nouveau-nés jusqu'au 8ème jour de vie. Les influences de maladies, de l'âge et de l'échec du transfert passif (FPT) sur ces concentrations ont été analysées. Animaux et procédure: Les concentrations sériques de Se, Cu et Zn ont été déterminées à partir d'échantillons de sang de 93 poulains au moyen d'une spectrométrie de masse à plasma à couplage inductif. Les poulains ont été divisés en 2 groupes en fonction de leur état de santé: cliniquement malades (n = 51) et cliniquement sains (n = 42). Ce dernier groupe a été divisé en poulains avec FPT (n = 20) et ceux sans (n = 22). Résultats: Les concentrations sériques moyennes de Se, Cu et Zn étaient respectivement de 60 ± 40 µg/L, 0,25 ± 0,22 mg/L et 605 ± 285 µg/L. Une influence significative de l'âge sur la concentration sérique de Cu a été observée (P < 0,0001). Aucune différence n'a été observée entre les concentrations sériques chez les poulains cliniquement malades et cliniquement sains au premier jour de leur vie. Le statut FPT n'était pas associé à une réduction des concentrations sériques de Se, Cu ou Zn. Conclusion et pertinence clinique: Il n'est pas nécessaire de supplémenter tous les poulains en oligo-éléments avec FPT.(Traduit par Dr Serge Messier).

Effect of hospitalization on equine local intestinal immunoglobulin A (IgA) concentration measured in feces.

During hospitalization horses may develop gastrointestinal conditions triggered by a stress-associated weak local immune system. The prospective, clinical trial was conducted to find out whether fecal immunoglobulin A (IgA) concentrations could be determined in hospitalized horses and how they changed during hospitalization and in response to various stressors. Samples were obtained from 110 horses and a control group (n = 14). At arrival in the hospital, horses were categorized into pain grades (1-5), and elective versus strenuous surgery (> 2 hours, traumatic and emergency procedures). Feces were collected on day 1, day 2, day 3, and day 7 in all horses. Blood samples were obtained at the same intervals, but additionally after general anaesthesia in horses undergoing surgery (day 2). IgA concentration in feces was determined by ELISA and measured in optical density at 450nm. The control group showed constant IgA concentrations on all days (mean value 0.30 OD450 ±SD 0.11, 1.26 mg/g; n = 11). After general anaesthesia fecal IgA concentrations decreased considerably independent of duration and type of surgery (P < 0.001 for elective and P = 0.043 for traumatic surgeries). High plasma cortisol concentrations were weakly correlated with low fecal IgA on the day after surgery (P = 0.012, day 3, correlation coefficient r = 0.113). Equine fecal IgA concentrations showed a decline associated with transport, surgery, and hospitalization in general, indicating that stress has an impact on the local intestinal immune function and may predispose horses for developing gastrointestinal diseases such as enterocolitis.

Monitoring of inflammatory blood biomarkers in foals with Rhodococcus Equi pneumonia during antimicrobial treatment.

Rhodococcus equi (R. equi), a gram-positive facultative intracellular pathogen, is a common cause of pneumonia in foals and represents a major cause of disease and death. The aim of the present study was to investigate the time-depended changes in White Blood Cells (WBC), basophils (Baso), neutrophils (Neu), lymphocytes (Lymf), monocytes (Mon), eosinophils (Eos), platelet (PLT) counts, fibrinogen (Fbg) concentration, interferon (IFN-α, IFN-γ) and interleukins (IL-2 and IL-10) in foals with clinical R. equi pneumonia. The main treatment was with azithromycin-rifampicin for 14 days. Blood was sampled prior to, 7 and 14 days after starting therapy. Treatment was associated with significantly decreased counts of WBC, (25.6 ± 6.7 and 14.2 ± 2,7 × 103/ml), Neu (18.6 ±6.2 and 10.7 ± 3.1 × 103/ml), Mon (1.5 ± 0.5 and 0.9 ± 0.2 × 103/ml) and Fbg (539 ± 124 and 287 ± 26 g/dl) between day 0 and day 14. IL-2 and IL-10 concentrations were significantly increased (P = 0.028, P = 0.013, respectively) after treatment, whereas IFN-α and IFN-γ concentrations were not. The diagnostic potentials of INF-α, INF-γ, IL-2 and IL-10 per se seems not very high, however, the study suggests that the activity change of selected interleukins in the course of the disease may be associated with amelioration. We concluded that patterns of serum concentration changes of INF-α, INF-γ, IL-2 and IL-10 may help in the study of the innate immune response in foals during infection and treatment of R. equi pneumonia.

Evaluation of gastrointestinal tract lesions and serum malondialdehyde levels after repeated oral administration of phenylbutazone in horses.

Phenylbutazone (PBZ) is a widely used nonsteroidal anti-inflammatory drug for horses. However, because of its gastrointestinal side effects, its administration requires careful attention in veterinary practice. Malondialdehyde (MDA) is a serum biomarker associated with increased damage to the equine gastrointestinal system. This study investigated the hematological effects and alterations in the gastrointestinal tract and assessed serum MDA concentrations following repeated oral PBZ administration at clinical doses. Fourteen horses were randomly divided into control and treatment groups. All horses in the treatment group were administered 4.4 milligrams per kilogram of body weight of PBZ syrup orally twice a day for 7 days, whereas the control group received syrup as a placebo. The development of gastrointestinal side effects was investigated using gastroscopy, abdominal ultrasound, and fecal pH; serum MDA concentrations were assessed using a commercially available enzyme-linked immunosorbent assay kit. Data were compared between PBZ-treated and control horses before and after the treatment period. The treatment group exhibited decreased albumin and total protein concentrations. Moreover, this group exhibited a higher thickness of the right dorsal colon wall (p = 0.03) and had higher scores for squamous gastric ulcers (p = 0.01). Fecal pH was lower in the treatment group than in the control group after PBZ administration (p < 0.01). Although MDA concentrations were higher in the treatment group after PBZ administration, they did not differ significantly from those of the control group. This study highlighted the changes in hematological and gastrointestinal lesions resulting from PBZ administration in horses at clinical doses, even without clinical signs. However, MDA may not be an optimal biomarker for the early detection of gastrointestinal damage due to PBZ treatment in horses.

Nine-year seroepidemiological study of severe fever with thrombocytopenia syndrome virus infection in feral horses in Cape Toi, Japan.

Severe fever with thrombocytopenia syndrome (SFTS) is a fatal zoonosis caused by ticks in East Asia. As SFTS virus (SFTSV) is maintained between wildlife and ticks, seroepidemiological studies in wildlife are important to understand the behavior of SFTSV in the environment. Miyazaki Prefecture, Japan, is an SFTS-endemic area, and approximately 100 feral horses, called Misaki horses (Equus caballus), inhabit Cape Toi in Miyazaki Prefecture. While these animals are managed in a wild-like manner, their ages are ascertainable due to individual identification. In the present study, we conducted a seroepidemiological survey of SFTSV in Misaki horses between 2015 and 2023. This study aimed to understand SFTSV infection in horses and its transmission to wildlife. A total of 707 samples from 180 feral horses were used to determine the seroprevalence of SFTSV using enzyme-linked immunosorbent assay (ELISA). Neutralization testing was performed on 118 samples. In addition, SFTS viral RNA was detected in ticks from Cape Toi and feral horses. The overall seroprevalence between 2015 and 2023 was 78.5% (555/707). The lowest seroprevalence was 55% (44/80) in 2016 and the highest was 92% (76/83) in 2018. Seroprevalence was significantly affected by age, with 11% (8/71) in those less than one year of age and 96.7% (435/450) in those four years of age and older (p < 0.0001). The concordance between ELISA and neutralization test results was 88.9% (105/118). SFTS viral RNA was not detected in ticks (n = 516) or feral horses. This study demonstrated that horses can be infected with SFTSV and that age is a significant factor in seroprevalence in wildlife. This study provides insights into SFTSV infection not only in horses but also in wildlife in SFTS-endemic areas.

Precision and accuracy of a point of care glucometer for detection of hypoglycaemia in horses.

Point-of-care (POC) glucometry is commonly used in horses; however, measurement error with this method when analysing hypoglycaemic samples (<4 mmol/L) is unknown. The objective of this study was to determine the precision and accuracy of glucometry in hypoglycaemic horses in comparison to a laboratory method of glucose measurement (LAB). Repeatability coefficients were 0.47 mmol/L for POC and 0.09 mmol/L for LAB, and coefficients of variation were 10 % and 2.11 %, for the POC and LAB methods, respectively. Systemic bias with the POC method was present, with a mean bias of -0.26 mmol/L (95 % limits of agreement: -0.88 - 0.37) in comparison to LAB, and <70% of measurements were within 20 % of paired LAB results. Prior to use of glucometers, assessment of the diagnostic performance of the equipment is necessary, including determination of acceptable criteria and reference ranges for hypoglycaemic samples.

Selecting potential biomarkers of plasma proteins in mares with endometritis.

BACKGROUND: Endometritis is a common condition in mares that causes significant economic loss. Lacking obvious clinical signs, the clinical diagnosis of endometritis in mares relies on case-by-case clinical examinations, which can be particularly inefficient in large-scale farms. Therefore, the identification of potential biomarkers can serve as a non-invasive and efficient screening technique for endometritis in mares. OBJECTIVES: To compare the blood proteome between fertile mares and mares with endometritis to identify biomarkers potentially associated with the development of endometritis and validate their predictive potential. STUDY DESIGN: Observational and experimental study. METHODS: Differentially expressed proteins were identified via Data Independent Acquisition (DIA) proteomic profiling in a screening cohort composed of eight healthy mares and eight mares with endometritis. Subsequently, enzyme-linked immunosorbent assay was employed that included a validation cohort of 40 healthy mares and 40 mares with endometritis to verify the accuracy and sensitivity of the identified proteins, thereby establishing a diagnostic threshold. RESULTS: In the screening cohort, 12 proteins were significantly differentially expressed between endometritis mares and healthy controls (p < 0.05, outside the 1/1.2 to 1.2-fold). In the validation experiment, all six screened proteins were assessed with area under the curve (AUC) >0.8. MAIN LIMITATIONS: The samples displayed certain levels of individual heterogeneity, and the number of samples analysed was limited. Additionally, the identified biomarkers were primarily associated with generalised inflammation, which potentially limited their specificity for endometritis. CONCLUSION: Levels of plasma proteins are sensitive indicators of equine endometritis and potential tools for endometritis screening. In plasma, fetuin B, von Willebrand factor, vitamin K-dependent protein C, insulin-like growth factor binding protein 3, interleukin 1 receptor accessory protein, and type II cell cytoskeleton showed great predictive ability, with fetuin B being the best predictor (AUC = 0.93, 95% CI: 0.89-0.98), which performs better when combined with all six detected proteins (AUC = 1, 95% CI: 0.99-1.00).

Equine brucellosis in Iran: serological, bacteriological and molecular analysis.

Equine brucellosis significantly impacts the health and functionality of horses, leading to complications such as bursitis infection, septic tenosynovitis, septic arthritis, and non-specific lameness resulting from joint infections. In the present study, we used the Rose Bengal plate agglutination test (RBPT), serum agglutination test (SAT), and the 2-mercaptoethanol (2-ME) assays to find equine brucellosis. From June 2018 to September 2022, 876 blood samples were randomly taken from apparently healthy racing horses in certain parts of Iran, such as Kerman, Isfahan, Tehran, Qom, and Kurdistan. DNA extraction was carried out directly on all 63 serum samples identified as seropositive through RBPT. An additional 30 seronegative serum samples were also randomly chosen for study. Bacterial culture was also done on milk, blood, and vaginal swabs taken from seropositive horses.The bacteria that were found in the samples were then put through Bruce-ladder PCR. Our results indicated that 63 (7.1%), 21 (2.3%), and 2 (0.2%) of horses were seropositive using RBPT, SAT, and 2-ME, respectively. Also, none of the 30 DNA-extracted serum samples from seronegative horses tested positive for Brucella DNA, while 44.5% (28/63) of the DNA samples from seropositive horses yielded positive results for Brucella DNA. Out of the seropositive samples, 26 had DNA from Brucella abortus and 2 had DNA from Brucella melitensis. Also, B. melitensis biovar 1 was found in two milk samples from mares in the provinces of Kerman and Isfahan. It was identified using classical biotyping, and molecular assays. It was seen that some of healthy racing horses in some parts of Iran had antibodies against Brucella. The bacteriology and PCR methodologies provide a more comprehensive and reliable means of identifying Brucella spp. infections in horse, especially when the RBPT test came back positive. This underscores the imperative for employing molecular, bacterial, and serological methods in the diagnosis and monitoring of this zoonotic infection. Additionally, this finding suggests that Brucella is being transmitted to equine hosts as a result of its presence in ruminants. The mechanism of transmission may involve interactions between infected ruminants and susceptible equines. This discovery is significant as it underscores the potential cross-species transmission of Brucella and highlights the importance of understanding and managing the spread of the pathogen in both ruminant and equine populations.

Efficacy of the oral supplement, Equine Omega Complete, for the prevention of gastric ulcers and alpha-tocopherol supplementation in horses.

BACKGROUND: Omega-3 fatty acid and alpha-tocopherol supplementation reduces gastric ulcer formation in humans and rodents; however, efficacy of prevention in horses is unknown. Equine Omega Complete (EOC) is an oral supplement containing omega-3 fatty acids and alpha-tocopherol. HYPOTHESIS/OBJECTIVE: Determine if EOC supplementation prevents gastric ulcers and increases serum alpha-tocopherol concentrations in healthy horses. ANIMALS: Nine thoroughbred geldings; 5-13 years old. METHODS: Prospective randomized block design, repeated in crossover model. Horses were administered EOC, omeprazole, or water PO for 28 days. Horses underwent an established gastric ulcer induction protocol from days 21-28 via intermittent feed deprivation. Gastroscopies were performed on days 0, 21, and 28. Serum alpha-tocopherol concentrations were measured on days 0 and 28. The effects of treatment and time on ulcer grades were assessed with ordinal logistic regression, with significance at P-value <.05. RESULTS: Ulcer grades increased during ulcer induction in control and EOC but not omeprazole groups (P = .02). Grades increased in EOC-treated horses after ulcer induction from a median of 1 [95% confidence interval 0-2.5] (day 0) to 2.5 [1.5-3.5] (day 28) and were similar to the control group (P = .54). Serum alpha-tocopherol increased in EOC-treated horses from day 0 to day 28 (mean 2.2 ± 0.43 µg/mL to 2.96 ± 0.89 µg/mL; P < .001) with high individual variation; this increase was not different from omeprazole or control groups. CONCLUSION AND CLINICAL IMPORTANCE: Supplementation with EOC for 28 days did not prevent gastric ulcer formation nor increase alpha-tocopherol concentrations relative to the control group.

Development of a Test Card Based on Colloidal Gold Immunochromatographic Strips for Rapid Detection of Antibodies against Theileria equi and Babesia caballi.

Equine piroplasmosis (EP) is a serious problem in the horse industry, and controlling EP is critical for international horse trading. EP is caused by two apicomplexan protozoan parasites, Theileria equi and Babesia caballi. Rapid and accurate methods that are suitable for detecting these parasites in the field are crucial to control the infection and spread of EP. In this study, we developed a card to detect antibodies against T. equi and B. caballi based on two colloidal gold immunochromatographic strips according to the principle of the double-antigen sandwich. The proteins equi merozoite antigen 1 (EMA1) and rhoptry protein BC48 are commonly used as diagnostic antigens against T. equi and B. caballi, respectively. On the strip, the purified EMA1 or BC48 protein labeled with colloidal gold was used as the detector, and nitrocellulose membranes were coated with EMA1 or BC48 and the corresponding MAb as the test and control lines, respectively. The protocol takes 10 to 15 min and requires no specialized equipment or chemical reagents, and one test can detect two EP pathogens in one card. Specificity tests confirmed there was no cross-reactivity with sera positive for common equine pathogens. Using a commercial competitive enzyme-linked immunosorbent assay (cELISA) kit for comparison, 476 clinical samples were tested with the card. The coincidence rates were 96.43% and 97.90% for T. equi and B. caballi, respectively. The field trial feedback was uniformly positive, suggesting that this diagnostic tool may be useful for controlling the spread of T. equi and B. caballi. IMPORTANCE Equine piroplasmosis (EP), caused by Theileria equi and Babesia caballi, is an important tick-borne disease of equines that is prevalent in most parts of the world. EP is considered a reportable disease by the World Organization for Animal Health (OIE). The accurate diagnosis and differentiation of T. equi and B. caballi are very important for the prevention, control, and treatment of EP. Therefore, we developed a double-antigen sandwich colloidal gold immunochromatography assay (GICG) to detect T. equi and B. caballi. Two GICG strips were assembled side by side on one card for the detection of T. equi and B. caballi, and the two EP pathogens could be detected in one test. This method was simple, rapid, and specific for the detection of EP; therefore, compared to the previous methods, this method is more suitable for pathogen diagnosis in the field.

Investigating the pathogenesis of high-serum gamma-glutamyl transferase activity in Thoroughbred racehorses: A series of case-control studies.

BACKGROUND: High-serum γ-Glutamyl Transferase (GGT) activity has been associated with and thought to be a marker of maladaptation to training and possibly poor performance in racehorses, but the cause is unknown. OBJECTIVES: To investigate possible metabolic and infectious causes for the high GGT syndrome. STUDY DESIGN: Pilot case-control study and nested case-control study. METHODS: The case-control study in 2017 included 16 horses (8 cases and 8 controls with median [range] serum GGT 82 [74-148] and 22 [19-28] IU/L, respectively) from the same stable. In 2018, similar testing was performed in a nested case-control study that identified 27 case (serum GGT 50 ≥ IU/L)-control pairs from three stables for further testing. Serum liver chemistries, selenium measurements, viral PCR and metabolomics were performed. RESULTS: No differences were found in frequency of detection of viral RNA/DNA or copy numbers for equine hepacivirus (EqHV) and parvovirus-hepatitis (EqPV-H) between cases and controls. Mild increases in hepatocellular injury and cholestatic markers in case vs control horses suggested a degree of liver disease in a subset of cases. Metabolomic and individual bile acid testing showed differences in cases compared with controls, including increased abundance of pyroglutamic acid and taurine-conjugated bile acids, and reduced abundance of Vitamin B6. Selenium concentrations, although within or above the reference intervals, were also lower in case horses in both studies. MAIN LIMITATIONS: Observational study design did not allow us to make causal inferences. CONCLUSIONS: We conclude that high GGT syndrome is likely a complex metabolic disorder and that viral hepatitis was not identified as a cause for this syndrome in this cohort of racehorses. Our results support a contribution of oxidative stress and cholestasis in its pathophysiology.

Diagnostic and prognostic potential of eight whole blood microRNAs for equine sarcoid disease.

MicroRNAs have been proposed as biomarkers for equine sarcoids, the most prevalent equine skin tumors globally. This study served to validate the diagnostic and prognostic potential of whole blood microRNAs identified in a previous study for long-term equine sarcoid diagnosis and outcome prediction. Based on findings of a clinical examination at the age of 3 years and a follow-up following a further 5-12 years, 32 Franches-Montagnes and 45 Swiss Warmblood horses were assigned to four groups: horses with regression (n = 19), progression (n = 9), new occurrences of sarcoid lesions (n = 19) and tumor-free control horses (n = 30). The expression levels for eight microRNAs (eca-miR-127, eca-miR-432, eca-miR-24, eca-miR-125a-5p, eca-miR-134, eca-miR-379, eca-miR-381, eca-miR-382) were analyzed through reverse transcription quantitative polymerase chain reaction in whole blood samples collected on initial examination. Associations of sex, breed, diagnosis, and prognosis with microRNA expression levels were examined using multivariable analysis of variance. Sex and breed influenced the expression level of five and two microRNAs, respectively. Eca-miR-127 allowed discrimination between sarcoid-affected and tumor-free horses. No variation in microRNA expression was found when comparing horses with sarcoid regression and progression. Expression levels of eca-miR-125a-5p and eca-miR-432 varied in male horses that developed sarcoids throughout the study period in comparison to male control horses. While none of the investigated miRNAs was validated for predicting the prognosis of sarcoid regression / progression within young horses with this condition, two miRNAs demonstrated potential to predict if young male (though not female) tumor-free horse can develop sarcoids within the following years. Sex- and breed- biased miRNAs exist within the equine species and have an impact on biomarker discovery.

Widespread Circulation of Flaviviruses in Horses and Birds in Northeastern Spain (Catalonia) between 2010 and 2019.

The surveillance for West Nile virus (WNV) in Catalonia (northeastern Spain) has consistently detected flaviviruses not identified as WNV. With the aim of characterizing the flaviviruses circulating in Catalonia, serum samples from birds and horses collected between 2010 and 2019 and positive by panflavivirus competition ELISA (cELISA) were analyzed by microneutralization test (MNT) against different flaviviruses. A third of the samples tested were inconclusive by MNT, highlighting the limitations of current diagnostic techniques. Our results evidenced the widespread circulation of flaviviruses, in particular WNV, but also Usutu virus (USUV), and suggest that chicken and horses could serve as sentinels for both viruses. In several regions, WNV and USUV overlapped, but no significant geographical aggregation was observed. Bagaza virus (BAGV) was not detected in birds, while positivity to tick-borne encephalitis virus (TBEV) was sporadically detected in horses although no endemic foci were observed. So far, no human infections by WNV, USUV, or TBEV have been reported in Catalonia. However, these zoonotic flaviviruses need to be kept under surveillance, ideally within a One Health framework.

Development of a sensitive competitive enzyme-linked immunosorbent assay for serodiagnosis of Burkholderia mallei, a Tier 1 select agent.

Glanders is a highly contagious and potentially serious disease caused by Burkholderia mallei, a Tier 1 select agent. In this study, we raised a monoclonal antibody (mAb) against the lipopolysaccharide (LPS) of B. mallei and developed a competitive enzyme-linked immunosorbent assay (cELISA) for B. mallei infection. Using the titrated optimal conditions of B. mallei-LPS (2 ng) for microtiter plate coating, sample serum dilution at 1:20 and 3.5 ng/µL anti-LPS mAb B5, the cutoff value of the cELISA was determined using serum samples from 136 glanders-free seronegative horses in Hong Kong. All calculated percentage inhibition (PI) values from these seronegative samples were below 39.6% inhibition (1.5 standard deviations above mean PI) and was used as the cutoff value. The diagnostic sensitivity of the developed LPS-based cELISA was first evaluated using sera from donkeys and mice inoculated with B. mallei. An increasing trend of PI values above the defined cELISA cutoff observed in the donkey and mouse sera suggested positive detection of anti-LPS antibodies. The sensitivity and specificity of the LPS-based cELISA was further evaluated using 31 serologically positive horse sera from glanders outbreaks in Bahrain and Kuwait, of which 30 were tested positive by the cELISA; and 21 seronegative horse sera and 20 seronegative donkey sera from Dubai, of which all were tested negative by the cELISA. A cELISA with high sensitivity (97.2%) and specificity (100%) for the detection of B. mallei antibodies in different animals was developed.