Negative Horses Research Articles

Quand le cheval paraît : représentations et fonctions diégétiques de la figure équine dans le roman contemporain français (1933-2016)

<p>Horses are the vectors of multiple representations related to their specific abilities and properties. These have endowed it, especially in the mythical narrative, with an image in turn positive (magic horses, divine, solar) or negative (horses of nightmare, infernal, psychopumps).</p><p>By examining a varied corpus of French short stories and novels published between 1933 and 2016 (Paul Morand, Jean Giono, Claude Simon, Richard Millet, Laurent Mauvignier), we will focus on the peculiarities of equine figures in contemporary french fiction, their functions in the story and their places in the construction of the narrative.</p><p>The appearance of the horse in the novel also allows us to question the notion of scene and to define the «equestrian scene», returning to the definitions and the means of analysis given by Gérard Genette (Figures III), or Stéphane Lojkine (La Scène de roman). This theoretical journey allows us to question the existence of a horse-type, in the Balzacian sense, an autonomous and characteristic figure that can be found in the various stories through the equestrian scenes. The analysis of these scenes, which presuppose an interaction between the horse and the other characters, will lead us to answer the question of how the presence of horses allows the dramatization of the story.</p>

Formation of ketoprofen methyl ester artifact in GC–MS analysis of basic drugs in horse urine using alkaline liquid-liquid extraction

This study investigates the unexpected formation of ketoprofen methyl ester (KME) during the routine alkaline liquid–liquid extraction (LLE) process for analyzing basic drugs in horse urine samples using GC–MS analysis. An unidentified peak in the GC–MS chromatogram was observed in certain horse urine samples, identified as KME through mass spectral comparison with in situ synthesized KME. Since the KME is not a metabolite of ketoprofen present in urine, it is proposed that its formation occurs during the LLE process due to the reaction between ketoprofen contained in the urine and methanol used as the solvent of the spiked internal standard. Surprisingly, no artifact was detected when negative quality control horse urine samples (absence of ketoprofen) even when spiked with standard ketoprofen and methanol. Further investigation indicated that the presence of lipase enzymes from bacteria in specific urine samples is the key factor in the formation of the KME artifact. This hypothesis was confirmed when negative quality control horse urines were spiked with ketoprofen, methanol, and a lipase enzyme and the KME artifact was detected. Additionally, the formation of a methyl ester artifact was also detected for flunixin, a carboxylic acid NSAID drug, when negative quality control horse urines were spiked with the drug, methanol, and lipase enzyme. These findings will be valuable for scientists analyzing drugs in urine.

A cross-sectional study on performance evaluation in Italian standardbred horses’ real-time PCR-positive for Theileria equi

BackgroundInflammatory myopathy and perivasculitis have been recently described in horses with chronic equine piroplasmosis (EP). These alterations may be linked to poor performances. The aims of this study were to evaluate the prevalence for EP in clinically healthy Italian Standardbred (IS) racehorses and to compare laboratory parameters and performance metrics between positive and negative horses. Real-time PCR was applied for the detection of T. equi and B. caballi positivity. Haematology parameters, blood chemistry results, subjective muscle mass scores, and performance metrics were compared between PCR-positive and -negative horses.ResultsThis cross-sectional study included 120 well-trained IS racehorses and was performed over a two-years period. The prevalence of T. equi was 36.3%, whereas all samples were negative for B. caballi. Red blood cells count, haemoglobin concentration, aspartate aminotransferase, alkaline phosphatase, and gamma-glutamyl transferase activities were significantly higher in PCR-positive horses, whereas blood urea nitrogen, globulin concentration and globulin-to-albumin ratio were significantly lower in PCR-positive horses compared to PCR-negative ones. Nonetheless, all values fell within the physiological range. The best racing time, which was selected as the most representative of the performance metrics at the principal component analysis, was not affected by PCR positivity, the muscle mass score or the training yard. The best racing time was significantly better in horses with a mild or no signs of muscular atrophy, within the PCR-positive group. The muscle mass score was associated with the training yard in PCR-negative horses.ConclusionsPrevalence of T. equi was high in IS racehorses in southern Italy. The absence of obvious changes in haematological and biochemical parameters, as well as performance metrics in positive horses, highlights the need for specific diagnostic tests to identify chronically infected horses.

Antibody reactions of horses against various domains of the EHV-1 receptor-binding protein gD1.

Equid alphaherpesviruses 1 (EHV-1) and 4 (EHV-4) are closely related and both endemic in horses worldwide. Both viruses replicate in the upper respiratory tract, but EHV-1 may additionally lead to abortion and equine herpesvirus myeloencephalopathy (EHM). We focused on antibody responses in horses against the receptor-binding glycoprotein D of EHV-1 (gD1), which shares a 77% amino acid identity with its counterpart in EHV-4 (gD4). Both antigens give rise to cross-reacting antibodies, including neutralizing antibodies. However, immunity against EHV-4 is not considered protective against EHM. While a diagnostic ELISA to discriminate between EHV-1 and EHV-4 infections is available based on type-specific fragments of glycoprotein G (gG1 and gG4, respectively), the type-specific antibody reaction against gD1 has not yet been sufficiently addressed. Starting from the N-terminus of gD1, we developed luciferase immunoprecipitation system (LIPS) assays, using gD1-fragments of increasing size as antigens, i.e. gD1_83 (comprising the first 83 amino acids), gD1_160, gD1_180, and gD1_402 (the full-length molecule). These assays were then used to analyse panels of horse sera from Switzerland (n = 60) and Iceland (n = 50), the latter of which is considered EHV-1 free. We detected only one true negative horse serum from Iceland, whereas all other sera in both panels were seropositive for both gG4 (ELISA) and gD1 (LIPS against gD1_402). In contrast, seropositivity against gG1 was rather rare (35% Swiss sera; 14% Icelandic sera). Therefore, a high percentage of antibodies against gD1 could be attributed to cross-reaction and due to EHV-4 infections. In contrast, the gD1_83 fragment was able to identify sera with type-specific antibodies against gD1. Interestingly, those sera stemmed almost exclusively from vaccinated horses. Although it is uncertain that the N-terminal epitopes of gD1 addressed in this communication are linked to better protection, we suggest that in future vaccine developments, type-common antigens should be avoided, while a broad range of type-specific antigens should be favored.

Diagnostic approaches, aetiological agents and their associations with short-term survival and laminitis in horses with acute diarrhoea admitted to referral institutions.

An international description of the diagnostic approaches used in different institutions to diagnose acute equine diarrhoea and the pathogens detected is lacking. To describe the diagnostic approach, aetiological agents, outcome, and development of laminitis for diarrhoeic horses worldwide. Multicentre retrospective case series. Information from horses with acute diarrhoea presenting to participating institutions between 2016 and 2020, including diagnostic approaches, pathogens detected and their associations with outcomes, were compared between institutions or geographic regions. One thousand four hundred and thirty-eight horses from 26 participating institutions from 4 continents were included. Overall, aetiological testing was limited (44% for Salmonella spp., 42% for Neorickettsia risticii [only North America], 40% for Clostridiodes difficile, and 29% for ECoV); however, 13% (81/633) of horses tested positive for Salmonella, 13% (35/262) for N. risticii, 9% (37/422) for ECoV, and 5% (27/578) for C. difficile. C. difficile positive cases had greater odds of non-survival than horses negative for C. difficile (OR: 2.69, 95%CI: 1.23-5.91). In addition, horses that were positive for N. risticii had greater odds of developing laminitis than negative horses (OR: 2.76, 95%CI: 1.12-6.81; p = 0.029). Due to the study's retrospective nature, there are missing data. This study highlighted limited diagnostic investigations in cases of acute equine diarrhoea. Detection rates of pathogens are similar to previous reports. Non-survival and development of laminitis are related to certain detected pathogens.

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.

Seroprevalence Study of Glanders in South Part of Iraq

The study was conducted to investigate the Seroprevalence of Glanders in south part or Iraq. Eight hundred (800) local and different horse breeds aged from (1.8–25) years old and from both sexes was examine during the period started from a15.oct. 2020 to 20 April 2021. The 184 serum samples were drained from suspected horses to examined by using double antigen multispecies Eliza of Burkholderia mallei. Results show that only 4 (2.17%) horses give a positive result with the Elisa test in Maysan governorate. Whereas negative horses for glanders included 180 (97.82%) in the south of Iraq, and that Basrah and Thi-Qar governorates were negatively against glanders. Such favorable result help for equestrian regions and horse breeding. It has been concluded that, although a very low infection rate was indicated in maysan governorate, However, Glanders could be re-emerging compared with other south governorates (Basrah and Thi-Qar) which being free. Moreover, Basrah and Thi-Qar governorates can be used as free areas

Possibilities of horse-breeding sustainable development

Up until the middle of the 20th Century equine breeding was considered an industry that directly reflected the national wealth and status of any given country. Since then, due to the upheaval of mechanisation the number of horses gradually declined, primarily in Europe. The recorded declining numbers concerning draft horse breeds are particularly alarming, in comparison to breeds used for sport and recreation which have increased notably during the last thirty years or so. Thus it can also be remarked how the numbers and quality of horses in many European countries like Slovenia and Croatia had improved noticeably. However, in Bosnia and Herzegovina, horse breeding practices continue to deteriorate: the numbers of these magnificent creatures plummet progressively. Former state stud farms are being shut down and the once abundant population of autochthonous Bosnian mountain horses have reached a breaking point. The Arab, Lipizzan and cold blooded horse breeds have met a similar fate. Despite that, horses do hold an important position in the folk traditions of Bosnia and Herzegovina, especially with the people of the Republic of Srpska. The people cherish numerous traditions in relation to these animals and genuinely care for horses, which is how and why the horse breeding industry lives on - nurtured by the enthusiasm of individuals. Equine breeding is not economically unsustainable, on the contrary, its economical significance is in fact great in developed countries. Sustainable development possibilities can be sought in equestrian sports certainly, but also in tourism, recreational practices, hippotherapy, landscape maintenance (pastures), the production of mare milk, etc. Observing the economic potential of the equine industry which includes trade and consumerism, employment opportunities, food production, the related pharmaceutical and equestrian equipment industry, as well as services, it becomes clear that it’s in the absolute interest of the state to stop and reverse the negative horse breeding trend. Moreover, it is essential to keep in mind that the preservation of the autochthonous horse breeds and stud farms, developed throughout the decades, is beneficial not only for the obvious economic reasons, but for the sake of heritage and culture, as well as the conservation of folkcustoms and traditional values in general. The autochthonous horse breeds could be used in all of these areas. This effort, the conservation of autochthonous genetic resources, requires state support. With the support of the state, by promoting professional equine breeding practices primarily in relation to selection and veterinary care, the decline of horse populations may be overturned which would consequently rehabilitate the horse breeding industry, and additionally contribute to the conservation of agrobiodiversity and autochthonous animal resources, environmental protection, and reverse the depopulation of rural municipalities through means of undeniable economic potential.

Role of Leptospira spp. testing and ocular examination in horses with equine recurrent uveitis: A retrospective study of 63 horses

SummaryBackgroundThere is little information on the correlation between equine recurrent uveitis (ERU) and leptospiral infection status as it relates to ocular examination findings and testing recommendations.ObjectiveTo evaluate the role of leptospiral testing in horses with ERU by correlating results to signalment, clinical findings and visual outcome.Study designRetrospective case series.MethodsRecords of horses presenting for ERU at NC State University Veterinary Health Complex (NCSU‐VHC) between 2014 and 2019 were reviewed. Signalment, initial visual status, ocular examination findings, treatments and visual outcome were collected. Serum and aqueous humour (AH) leptospiral titres were assessed for six different leptospirosis serovars and polymerase chain reaction (PCR) testing. Goldmann‐Whitmer (C) values were calculated.ResultsRecords of 63 horses and 78 eyes with ERU were evaluated. Horses with a positive aqueous humour PCR were significantly younger in age (mean 9.25 ± 1.14) than PCR negative horses (mean 13.3 ± 0.95) (p = 0.01). A positive aqueous humour titre of any serotype was significantly correlated to blindness at presentation (p = 0.04). A positive serum titre was significantly correlated to presence of ocular posterior segment disease (p = 0.01). Positivity to L. bratislava in the serum correlated to posterior segment disease (p = 0.04) and AH positivity correlated to blindness at presentation (p = 0.002).Main limitationsLimitations of this study include that it is a retrospective and information gathered relies on data included within medical records.ConclusionsPositive leptospiral testing results were associated with younger horses and ocular posterior segment clinical disease. Horses with aqueous humour titres to any leptospiral serotype, especially L. bratislava serovar, were correlated to poor prognosis for vision.

Evaluation of Plasmatic Procalcitonin in Healthy, and in Systemic Inflammatory Response Syndrome (SIRS) Negative or Positive Colic Horses.

Simple SummaryProcalcitonin (PCT) increased in the case of systemic inflammatory response syndrome (SIRS), especially due to bacterial infection. The correlation between SIRS score and plasma PCT levels in horses have not been evaluated, and no studies investigated plasma PCT concentration over time. In the present study, PCT and SIRS score were evaluated in colic horses at admission to the hospital and at 24, 48, 72 and 96 h. Statistically differences were detected between healthy vs. all colic horses and between healthy vs. SIRS positive or SIRS negative horses. No correlation was observed between SIRS score and PCT. This suggests a role of plasmatic PCT as good biomarker for colic.Colic horses show systemic inflammatory response syndrome (SIRS) clinical signs. Procalcitonin (PCT) showed increased circulating levels in sick horses. This study compares plasma PCT concentrations in healthy vs. SIRS negative/positive colic horses over time, and evaluates PCT and SIRS score potential correlation, to verify the usefulness of PCT for the evaluation of SIRS severity. Ninety-one horses were included; 43/91 were healthy, on basis of physical examination, blood work and SIRS score (score = 0), while 48/91 were sick colic horses, classified as SIRS-negative (score < 2) and positive (score ≥ 2). Moreover, a 0–6 point-scale SIRS score was calculated (assessing mucous membrane color and blood lactate concentration). PCT was evaluated at admission, and at 24, 48, 72 and 96 h, using a commercial kit for equine species. We verified by the ANOVA test PCT differences between healthy vs. colic horses, healthy vs. SIRS-negative or SIRS-positive colic horses, at all sampling times, and the correlation between the SIRS score at admission with the SIRS score. Statistically significant differences were detected between healthy vs. all colic horses and between healthy vs. SIRS-positive or negative horses at all sampling times. No correlation was observed between the SIRS score at admission and PCT values. PCT was statistically higher in colic horses compared to the healthy ones, suggesting a role as a biomarker for colic.

Epidemiology and Genomic Analysis of Equine Encephalosis Virus Detected in Horses with Clinical Signs in South Africa, 2010-2017.

Equine encephalosis virus (EEV) is a neglected virus endemic to South Africa and is considered to generally result in mild disease in equines. Specimens were analyzed from live horses that presented with undefined neurological, febrile, or respiratory signs, or sudden and unexpected death. Between 2010 and 2017, 111 of 1523 (7.3%) horse samples tested positive for EEV using a nested real-time reverse transcriptase polymerase chain reaction (rRT-PCR). Clinical signs were reported in 106 (7.2%) EEV positive and 1360 negative horses and included pyrexia (77/106, 72.6%), icterus (20/106, 18.9%) and dyspnea (12/106, 11.3%). Neurological signs were inversely associated with EEV infection (OR < 1, p < 0.05) relative to EEV negative cases despite a high percentage of animals presenting with neurological abnormalities (51/106, 48.1%). Seventeen of the EEV positive horses also had coinfections with either West Nile (5/106, 4.7%), Middelburg (4/106, 3.8%) or African Horse sickness virus (8/106, 7.6%). To investigate a possible genetic link between EEV strains causing the observed clinical signs in horses, the full genomes of six isolates were compared to the reference strains. Based on the outer capsid protein (VP2), serotype 1 and 4 were identified as the predominant serotypes with widespread reassortment between the seven different serotypes.

Reactivity of Horse Sera to Antigens Derived From Sarcocystis falcatula-Like and Sarcocystis neurona.

Sarcocystis neurona and Sarcocystis falcatula are protozoan parasites endemic to the Americas. The former is the major cause of equine protozoal myeloencephalitis, and the latter is associated with pulmonary sarcocystosis in birds. The opossum Didelphis virginiana is the definitive host of these parasites in North America. Four Didelphis species are found in Brazil, and in most reports in this country, Sarcocystis species shed by opossums have been classified as S. falcatula–like. It is unknown whether reports on S. neurona–seropositive horses in Brazil are also derived from exposure of horses to S. falcatula–like. The aim of this study was to test the sera reactivity of 409 horses in Brazil using antigens derived from a Brazilian strain of S. falcatula–like (Sarco-BA1) and from a North American strain of S. neurona (SN138). Samples were examined by immunofluorescent antibody tests (IFATs) at start dilutions of 1:20, and a selected number of samples was tested by Western blot (WB). Sera from 43/409 (10.5%) horses were reactive to S. falcatula–like and 70 of 409 (17.1%) were reactive to S. neurona antigen; sera from 25 animals (6.1%) were positive for both parasites by IFAT. A poor agreement was observed between the two employed IFATs (κ = 0.364), indicating that horses were exposed to more than one Sarcocystis species. Horse sera evaluated by WB consisted of four sera reactive to S. falcatula–like by IFAT, six sera positive to S. neurona by IFAT, two sera that tested negative to both parasites by IFAT, and a negative control horse serum from New Zealand. Proteins in the range of 16 and 30 kDa were recognized by part of IFAT-positive sera using both antigen preparations. We concluded that Brazilian horses are exposed to distinct Sarcocystis species that generate different serological responses in exposed animals. Antigens in the range of 16 and 30 kDa are probably homologous in the two parasites. Exposure of the tested horses to other Sarcocystis species, such as Sarcocystis lindsayi, Sarcocystis speeri, and Sarcocystis fayeri, or Sarcocystis bertrami cannot be excluded in the current study.

Evaluation of Protein Carbonyl Content in Healthy and Sick Hospitalized Horses.

Literature on the protein carbonyl content (PCC) evaluation in horses is scarce, thus the aims were to evaluate the PCC in healthy and SIRS (Systemic Inflammatory Response Syndrome) horses and to investigate the performances of PCC in terms of sensitivity, specificity, and likelihood ratio in identifying SIRS positive and negative horses. A total of 72 adult horses were included. All the horses underwent to a complete physical examination, blood analysis, and were evaluated for the SIRS status. Blood samples were collected once in healthy horses and at admission time, then 24, 48, 72, and 96 h after admission in sick animals. PCC was evaluated using a method previously described. Data were statistically analyzed to verify differences in PCC between healthy vs. SIRS positive or SIRS negative horses at all sampling time. The receiver operating characteristic (ROC) curve was performed to verify sensitivity and specificity of PCC in the diagnosis of SIRS-positive and SIRS negative horses. The healthy horses were standardbred mares with a median age of 8.5 years. The sick horses were 31/54 females, 16/54 geldings, and 7/54 stallions of different breeds and with a median age of 12 years old. Eight out of 54 sick horses were SIRS negative, while 46/54 were SIRS positive. Statistically significant differences were obtained between healthy and SIRS positive horses, while no differences were observed between healthy and SIRS negative horses at any sampling time. The best cutoff value of PCC to discriminate between SIRS positive, SIRS negative, and healthy horses, the sensitivity and specificity of cutoff point, the area under receiver operating characteristic curve, the 95% confidence intervals, and the likelihood ratio were reported. We found higher PCC values in sick SIRS-positive horses vs. healthy ones with a decrement over time, while no differences at admission, nor during the observational period, were obtained in sick but SIRS-negative horses. The value of 0.049 nmol/ml/mg is reported as a potential cutoff for the diagnosis of SIRS positivity vs. healthy horses with a sensibility of 74.5% and a specificity of 72.2%. In conclusion, PCC seems to be a sensitive and specific marker for SIRS in horses.

Horses feel emotions when they watch positive and negative horse-human interactions in a video and transpose what they saw to real life.

Animals can indirectly gather meaningful information about other individuals by eavesdropping on their third-party interactions. In particular, eavesdropping can be used to indirectly attribute a negative or positive valence to an individual and to adjust one's future behavior towards that individual. Few studies have focused on this ability in nonhuman animals, especially in nonprimate species. Here, we investigated this ability for the first time in domestic horses (Equus caballus) by projecting videos of positive and negative interactions between an unknown human experimenter (a "positive" experimenter or a "negative" experimenter) and an actor horse. The horses reacted emotionally while watching the videos, expressing behavioral (facial expressions and contact-seeking behavior) and physiological (heart rate) cues of positive emotions while watching the positive video and of negative emotions while watching the negative video. This result shows that the horses perceived the content of the videos and suggests an emotional contagion between the actor horse and the subjects. After the videos were projected, the horses took a choice test, facing the positive and negative experimenters in real life. The horses successfully used the interactions seen in the videos to discriminate between the experimenters. They touched the negative experimenter significantly more, which seems counterintuitive but can be interpreted as an appeasement attempt, based on the existing literature. This result suggests that horses can indirectly attribute a valence to a human experimenter by eavesdropping on a previous third-party interaction with a conspecific.

The Indirect ELISA Trypanosoma evansi in Equids: Optimisation and Application to a Serological Survey including Racing Horses, in Thailand.

Surra, caused by Trypanosoma evansi, is a widely distributed animal trypanosomosis; it affects both domestic and wild mammals with high economic impact. Clinical picture is moderate in bovines but severe in equids. Surra is also an important constraint for international animal trade and movements. Despite its impact, surra remains poorly diagnosed because of low sensitivity tests. To improve epidemiological knowledge of the disease and to secure international movement, efficient diagnosis tools are required. Here, we optimized and applied to equids the OIE-recommended indirect ELISA T. evansi that was validated in other species. Based on 96 positive and 1,382 negative horse reference samples from Thailand, a TG-ROC analysis was conducted to define the cutoff value. ELISA's sensitivity and specificity were estimated at 97.5% and 100%, respectively, qualifying the test to provide a reliable immune status of equids. The test was then applied on 1,961 horse samples from 18 Thai Provinces; the only scarce positives suggested that horses do not constitute a reservoir of T. evansi in Thailand. All samples from racing horses were negative. Conversely, two outbreaks of surra reported to our laboratory, originating from a bovine reservoir, exhibited high morbidity and lethality rates in horses. Finally, posttreatment follow-ups of infected animals allowed us to provide outbreak management guidelines.

Blood type and breed-associated differences in cell marker expression on equine bone marrow-derived mesenchymal stem cells including major histocompatibility complex class II antigen expression

BackgroundAs the search for an immune privileged allogeneic donor mesenchymal stem cell (MSC) line continues in equine medicine, the characterization of the cells between different sources becomes important. Our research seeks to more clearly define the MSC marker expression of different equine MSC donors.MethodsThe bone marrow-derived MSCs from two equine breeds and different blood donor-types were compared over successive culture passages to determine the differential expression of important antigens. Eighteen Thoroughbreds and 18 Standardbreds, including 8 blood donor (erythrocyte Aa, Ca, and Qa antigen negative) horses, were evaluated. Bone marrow was taken from each horse for isolation and culture of MSCs. Samples from passages 2, 4, 6, and 8 were labelled and evaluated by flow cytometry. The cell surface expression of CD11a/18, CD44, CD90 and MHC class II antigens were assessed. Trilineage assays for differentiation into adipogenic, chondrogenic and osteogenic lines were performed to verify characterization of the cells as MSCs.FindingsThere were significant differences in mesenchymal stem cell marker expression between breeds and blood antigen-type groups over time. Standardbred horses showed a significantly lower expression of MHC class II than did Thoroughbred horses at passages 2, 4 and 6. CD90 was significantly higher in universal blood donor Standardbreds as compared to non-blood donor Standardbreds over all time points. All MSC samples showed high expression of CD44 and low expression of CD11a/18.ConclusionsUniversal blood donor- type Standardbred MSCs from passages 2–4 show the most ideal antigen expression pattern of the horses and passages that we characterized for use as a single treatment of donor bone marrow-derived MSCs. Further work is needed to determine the significance of this differential expression along with the effect of the expression of MHC I on equine bone marrow-derived MSCs.

Development of an immunoblotting assay for serodiagnosis of Burkholderia mallei infection: the whole-cell proteome-based paradigm

Burkholderia mallei is the leading cause of glanders, a highly transmittable and an OIE-notifiable disease of equidae. Despite the importance of B. mallei, little is known about serodiagnosis of glanders. The present study aimed to develop an immunoblotting assay based on whole-cell proteome of B. mallei to enable accurate serodiagnosis of glanders. Three farm horses were subcutaneously immunized with a crude suspension (106 cfu/ml) of heat-inactivated B. mallei formulated with incomplete Freund's adjuvant (IFA) to achieve a hyperimmune sera panel. The immunization was done for 1, 14 and 28 days with 1 dose of 1 ml antigen containing 106 cfu/ml. The hyperimmunity of sera was confirmed by CFT. B. mallei whole-cell proteome was prepared through sonication and the protein content was visualized by SDS-PAGE and quantified by Western blot using HRP-conjugated rabbit anti-horse IgG. A comprehensive set of positive and negative horse sera validated the test. A ladder pattern of the B. mallei immunoreactive antigens was seen within the region of 20-90 kDa clearly and the immunoblot was scored positive, while no reaction was seen for the negative sera. The Western blot assay indicated a noticeably higher diagnostic specificity for positive or negative sera of glanders. The whole-cell proteome-based immunoblot proved reliable and straightforward in our study. The prepared antigen was adaptable for application in immunoblotting. We assumed this improved immunoblotting system provides appropriate sensitivity and also specificity expected in serodiagnosis of glanders in endemic areas and typically in less-developed countries.

Immunological response in horses following West Nile virus vaccination with inactivated or recombinant vaccine.

To evaluate the immunological response following vaccination, 40 WNV serologically negative horses were selected and divided in two groups of 20 animals. One group was vaccinated (booster after 28 days) with a whole inactivated viral strain and the second group with a live recombinant canarypox virus expressing the genes coding for the WNV preM/E viral proteins. IgM, IgG and neutralizing antibodies were monitored by class specific ELISAs and serum neutralization assay for 360 days. In both groups, IgM antibodies were first detected 7 days post vaccination (dpv). However, in the group vaccinated with inactivated vaccine, IgM antibodies were detected until day 42 pv, whereas in the group vaccinated with the recombinant vaccine, they were detected up to day 52 pv. A similar (P > 0.05) proportion of horses showed IgM antibodies after vaccination with either recombinant [30%; 95% confidence interval (CI): 14.59%-52.18%] or inactivated (32%; 95% CI: 15.39-54.28%) vaccine. Both vaccines induced in vaccinated horses a detectable IgG antibody response starting from day 7 pv and lasting till the end of the trial. Analogously, both products elicited WNV specific neutralizing antibodies. The response induced by the live canarypox virus-vectored vaccine was higher (mean titres 1:298 vs 1:18.9) and lasted longer than did that induced by the killed-virus vaccines. In fact, one year after the vaccination, neutralizing antibodies were still detectable in the horses which received the canarypox virus-based vaccine but not in the group vaccinated with the killed product. The use of vaccines is a valuable tool to prevent WNV disease in horses and the availability of different products facilitates the control of the disease in endemic areas.

Validation of an immunoblot assay employing an objective reading system and used as a confirmatory test in equine infectious anaemia surveillance programs

Equine infectious anaemia (EIA) is a blood borne disease that is listed among the notifiable diseases of the World Organisation for Animal Health (OIE). EIA is also regulated by the OIE for the international trading provisions and is generally subject to control programmes. Since 2011, Italy has been conducting a surveillance plan based on a three-tier diagnostic system, using a serological ELISA as screening test, an agar gel immunodiffusion test (AGIDT) as a confirmatory method, and an immunoblot (IB) as an alternative confirmatory assay for discordant results between the first two tests. As for the in-house competitive ELISA (c-ELISA) and the AGIDT, the Italian National Reference Laboratory for EIA (NRL) validated the IB according to the OIE guidelines, employing eight panels containing positive sera, including those from EIA virus (EIAV) proven infected horses, and negative horse, mule and donkey sera collected from different geographical areas. In addition, two international reference image panels were employed for the optimization and the validation of the digital image reading system adopted that allows an impartial measurement of the serum reactivity in the IB assay.The immunological reactivity to EIAV antigens, p26, gp45 and gp90 adsorbed on the IB membrane, determines the serological status of the animal and for EIA, a p26 positive band together with at least one of the other antigen defines a subject as serologically positive for EIAV.For validation, the parameters assessed were threshold values, analytical and diagnostic sensitivity and specificity, repeatability and reproducibility. These parameters were evaluated for each antigen as well as in combination, according to the diagnostic algorithm established above.The validation data defined the IB as having a satisfactory sensitivity, specificity, repeatability and reproducibility for all antigens and species tested.An instrumental recording of the results improves the confidence in using IB as a confirmatory test for EIAV, differently from the AGIDT that is read by an operator.The advantages of using the IB are its higher sensitivity, to that of the AGIDT, which allows an earlier detection of infection that reduces the risk of transmission and therefore the incidence of the EIA, and its higher specificity to that of the ELISA which is based on the discrimination of subjects reacting only against the p26, the antigen used by all ELISAs available, which are not considered as infected by EIAV.In particular, when this assay is used in outbreaks it can detect new cases earlier than the AGIDT, and therefore reduce the restriction period with an economic benefit for the animal owners and the public veterinary sanitary system.

Development and validation of a duplex real-time PCR assay for the diagnosis of equine piroplasmosis

BackgroundEquine piroplasmosis (EP) is an economically significant infection of horses and other equine species caused by the tick-borne protozoa Theileria equi and Babesia caballi. The long-term carrier state in infected animals makes importation of such subclinical cases a major risk factor for the introduction of EP into non-enzootic areas. Regulatory testing for EP relies on screening of equines by serological methods. The definitive diagnosis of EP infection in individual animals will benefit from the availability of sensitive direct detection methods, for example, when used as confirmatory assays for non-negative serological test results. The objectives of this study were to develop a real-time quantitative polymerase chain reaction (qPCR) assay for simultaneous detection of both agents of EP, perform comprehensive evaluation of its performance and assess the assay’s utility for regulatory testing.ResultsWe developed a duplex qPCR targeting the ema-1 gene of T. equi and the 18S rRNA gene of B. caballi and demonstrated that the assay has high analytical sensitivities for both piroplasm species. Validation of the duplex qPCR on samples from 362 competitive enzyme-linked immunosorbent assay (cELISA)-negative horses from Canada and the United States yielded no false-positive reactions. The assay’s performance was further evaluated using samples collected from 430 horses of unknown EP status from a highly endemic area in Brazil. This set of samples was also tested by a single-target 18S rRNA qPCR for T. equi developed at the OIE reference laboratory for EP in Japan, and a previously published single-target 18S rRNA qPCR for B. caballi whose oligonucleotides we adopted for use in the duplex qPCR. Matching serum samples were tested for antibodies to these parasites using cELISA. By the duplex qPCR, T. equi-specific 18S rRNA qPCR and cELISA, infections with T. equi were detected in 87.9% (95% confidence interval, CI: 84.5–90.7%), 90.5% (95% CI: 87.3–92.3%) and 87.4% (95% CI: 84.0–90.2%) of the horses, respectively. The B. caballi prevalence estimates were 9.3% (95% CI: 6.9–12.4%) by the duplex qPCR and 7.9% (95% CI: 5.7–10.9%) by the respective single-target qPCR assay. These values were markedly lower compared to the seroprevalence of 58.6% (95% CI: 53.9–63.2%) obtained by B. caballi-specific cELISA. The relative diagnostic sensitivity of the duplex qPCR for T. equi was 95.5%, as 359 of the 376 horses with exposure to T. equi confirmed by cELISA had parasitemia levels above the detection limit of the molecular assay. In contrast, only 39 (15.5%) of the 252 horses with detectable B. caballi-specific antibodies were positive for this piroplasm species by the duplex qPCR.ConclusionsThe duplex qPCR described here performed comparably to the existing single-target qPCR assays for T. equi and B. caballi and will be more cost-effective in terms of results turnaround time and reagent costs when both pathogens are being targeted for disease control and epidemiological investigations. These validation data also support the reliability of the ema-1 gene-specific oligonucleotides developed in this study for confirmatory testing of non-negative serological test results for T. equi by qPCR. However, the B. caballi-specific qPCR cannot be similarly recommended as a confirmatory assay for routine regulatory testing due to the low level of agreement with serological test results demonstrated in this study. Further studies are needed to determine the transmission risk posed by PCR-negative equines with detectable antibodies to B. caballi.