Laboratory methods in equine research encompass a variety of techniques and procedures used to analyze biological samples from horses to study health, disease, genetics, and physiology. These methods include hematological analyses, biochemical assays, molecular biology techniques, and microbiological cultures. Commonly utilized laboratory techniques involve blood tests for complete blood count (CBC) and serum chemistry, polymerase chain reaction (PCR) for genetic and infectious disease studies, and enzyme-linked immunosorbent assays (ELISA) for detecting specific proteins or antibodies. These methods provide valuable data that contribute to understanding equine health and disease mechanisms. This page compiles peer-reviewed research studies and scholarly articles that explore the application, development, and outcomes of laboratory methods in the context of equine research.
Muko R, Ojima Y, Matsuda H, Toishi Y, Oikawa MA, Shin T, Sato H, Tanaka A.Previously, we demonstrated unique insertion/deletion polymorphisms of equine histidine-rich glycoprotein with five genotypes composed of 45-bp or 90-bp deletions in the histidine-rich region of in Thoroughbred horses. Although leukocytes are typically used to collect DNA for genotyping, blood sampling from animals is sometimes difficult and invasive. Moreover, the method for extracting DNA from blood leukocytes involves complicated steps and must be performed soon after blood sampling for sensitive gene analysis. In the present study, we performed genotyping using DNA, isolated from oral m...
French KM, Musiał AD, Karczewski M, Daugnora L, Shiroukhov R, Ropka-Molik K, Baranowski T, Bertašius M, Skvortsov K, Szymański P....Horse sacrifice and deposition are enigmatic features of funerary rituals identified across prehistoric Europe that persisted in the eastern Baltic. Genetic and isotopic analysis of horses in Balt cemeteries [1st to 13th centuries CE (Common Era)] dismantle prevailing narratives that locally procured stallions were exclusively selected. Strontium isotope analysis provides direct evidence for long-distance (~300 to 1500 kilometers) maritime transport of Fennoscandian horses to the eastern Baltic in the Late Viking Age (11th to 13th centuries CE). Genetic analysis proves that horses of both sexe...
Bazzano M, Marchegiani A, La Gualana F, Petriti B, Spaterna A, Laus F.Horse welfare is the product of multiple factors, including behavioral and physiological adjustments to cope with stressful situation regarding environment and housing condition. Collectively, it is supposed that a horse kept in the wild has a lower level of stress than other housing system, and the aim of the present study was to investigate the level of stress in domestic horses reared in the wild and then moved to human controlled housing, through saliva analysis. Twelve clinically healthy Catria (Italian local breed) mares, usually reared in the wild, were moved into collective paddocks fo...
Tikmehdash HT, Dehnad A, Mosavari N, Naghili Hokmabadi B, Mahmazi S.Glanders caused by Burkholderia mallei is one of the most dangerous zoonotic diseases in solipeds. Clinical diagnosis of this disease in its early stages in horses, is difficult. This study investigated serological and molecular identification of B. mallei in East Azerbaijan province. In the third and fourth quarters of 2020, throughout 2021, and in the first and second quarters of 2022, the complement fixation test (CFT) was performed on 350 horses. The malleination was used to confirm the positive CFT cases. Blood samples were taken for culture and for preparing serums to perform the enzyme-...
Green de Freitas M, Gonçalves Lino Borges D, Henrique Conde M, Takemi Muchon Nakatani M, Francieli Tutija J, Fuzaro Scaléa GO, Reckziegel GH....The aim of the present study was to validate methods of stool sample conservation for the egg hatch test (EHT). This study involved the use of a bovine naturally infected predominantly by Cooperia spp. and one equine naturally infected predominantly by cyathostomins characterized as susceptible to benzimidazoles in the EHT. Fecal samples were submitted to three treatments: aerobic methods (anaerobic storage in plastic bottles, anaerobic storage in vacuum-sealed bags or aerobic storage in plastic bags), under two temperature conditions (room temperature and refrigeration) analyzed at four diffe...
Wagner B.Host immune analyses require specific reagents to identify cellular and soluble components of the immune system. These immune reagents are often species-specific. For horses, various immunological tools have been developed and tested by different initiatives during the past decades. This article summarizes the development of well characterized monoclonal antibodies (mAbs) for equine immune cells, immunoglobulin isotypes, cytokines, and chemokines.
Yi M, Asgenbaatar N, Wang X, Ulaangerel T, Shen Y, Wen X, Du M, Dong X, Dugarjav M, Bou G.DNA methyltransferases (DNMTs) are important epigenetic modification during spermatogenesis. To further evaluate the pattern of DNMTs in horse testes during development, we investigated the expression and localization of DNMT1, DNMT3a and DNMT3b at different time points. The qRT-PCR results showed that DNMT1 expression was maintained in testes tissue from 6-month-old (0.5y) to 2-year-old (2y) of age and decreased after 3-year-old (3y) (P < 0.01). The expression levels of DNMT3a and DNMT3b peaked in testes tissue at 3y (P < 0.01). At 4-year-old (4y), the expression of DNMT3a and DNMT3b was ...
Gibb Z, Aitken RJ, Sheridan AR, Holt B, Waugh S, Swegen A.The in vitro storage of stallion spermatozoa for use in artificial insemination leads to oxidative stress and imbalances in calcium homeostasis that trigger the formation of the mitochondrial permeability transition pore (mPTP), resulting in premature cell death. However, little is understood about the dynamics and the role of mPTP formation in mammalian spermatozoa. Here, we identify an important role for mPTP in stallion sperm Ca2+ homeostasis. We show that stallion spermatozoa do not exhibit "classical" features of mPTP; specifically, they are resistant to cyclosporin A-mediated inhibition...
Ramos GV, Titotto AC, da Costa GB, Ferraz GC, de Lacerda-Neto JC.The maximal lactate steady state (MLSS) is a well-known gold standard method for determining the aerobic capacity of athletic horses. Owing to its high cost and complex execution, there is a search for standardized exercise tests that can predict this value in a single session. One of the methods described for this purpose is the lactate minimum test (LMT), which could be more accurate despite being adequate to predict MLSS. This study aimed to examine the impact of training on the speed corresponding to lactate minimum speed (LMS) and to apply new mathematical methods to evaluate the fitness ...
da Silva CMB, Cano FEM, Gaitskell-Phillips G, Vega FJP.Flow cytometry is a powerful tool for the analysis of cell samples formed of multipopulations, such as spermatozoa. In recent years, multiparametric cytometers have evolved, allowing the study of different cellular characteristics, such as protein expression, DNA analysis, or mitochondrial activity. Whether using traditional fluorescent dyes or fluorophore-conjugated antibodies, each cell or cellular component is individually stained, the sample is analyzed at high velocities, and then is displayed and interpreted in a dot-plot. We hereby describe the procedure to perform a multiparametric flo...
Birckhead EM, Das S, Tidd N, Raidal SL, Raidal SR.Septic synovitis and peritonitis are routinely diagnosed in horses based on clinical examination findings and laboratory assessment of synoviocentesis and abdominocentesis samples, respectively. Diagnosis is difficult in some cases because of an overlap in laboratory results for septic and non-septic inflammation. Neutrophil extracellular trap (NET) formation is part of the innate immune response against pathogens. Identifying and quantifying NETs, which have not been explored in clinical samples from horses with septic synovitis and peritonitis, to our knowledge, may be helpful in detecting i...
Bellitto NA, Oliver FB, Pollock PJ.To evaluate the use of bidirectional knotless barbed suture material for closure of the equine linea alba. Methods: Laboratory study. Methods: Adult light horse cadavers (n = 24). Methods: A 25 cm incision was made through the linea alba, and a 200 L polyurethane bladder was positioned within the abdomen. The linea alba was closed either using USP2/EP5 bidirectional polydioxanone barbed suture or USP2/EP5 standard polydioxanone suture in a simple continuous pattern. Closure time was recorded for each suture type. The bladder was air-insufflated at 40 L/min, and the pressure at body w...
Dierberger H, Pieper L, Jung M, Schulze M.Appropriate stallion semen handling is of great importance in equine artificial insemination (AI) industry. Optimal treatment of AI doses is aiming for best sperm preservation by excluding strong environmental influences and adding media that favour sperm survival. One method widely used in stallion sperm handling is the rotation of liquid-preserved semen samples on roller benches during storage. As previous studies in boars give rise to the fact that rotation should not be considered beneficial for spermatozoa anymore, the present study investigated the influence of continuous rotation of dil...
Köhne M, Kirch F, Tönissen A, Martinsson G, Rabe U, Sieme H, Schuberth HJ.To improve accuracy in evaluating stallion ejaculates, an antibody-based, flow cytometric assay for the detection and identification of leukocyte subpopulations (CD4-, CD8-, CD21-, CD172a-positive cells) in stallion semen (n = 12) was established. For establishment of the assay, native semen was supplemented with blood leukocytes (control: 20% leukocytes, 80% sperm cells) and analysed by flow cytometry. Adding antioxidants (ascorbic acid and butylated hydroxytoluol) to semen immediately after collection inhibited rapid death of lymphoid cells in sperm leukocyte mixtures. In control set-ups...
Karatt TK, Sathiq MA, Laya S, Ajeebsanu MPM, Karakka Kal AK, Subhahar MB, Perwad Z.Recently, there has been a report suggesting that ecdysteroids can enhance sports performance, making them relevant substances in doping control. Hence, it is imperative to examine the analytical characteristics of ecdysteroids in biological samples to identify their misuse in competitive sports. Methods: To assess the doping of ecdysteroids such as ecdysone, ecdysterone, ponasterone A, turkesterone, and ajugasterone C, a fast and sensitive extraction and detection method was developed, optimized, and validated using equine urine and plasma. Different extraction techniques, namely, solid-phase...
Shakeel M, Yoon M.This study investigated the effect of heat stress on stallion testicular cells (TCs) and the effect of insulin-like growth factor (IGF)-1 on TC viability, proliferation, and apoptosis, including different stages of germ cells. TCs were divided into control or treatment groups with 0.01, 0.1, 1, 10, and 100 ng/mL of recombinant human IGF-1 (rhIGF-1) for 24 h at 34 °C and 37 °C. The population and viability were measured before and after treatment. The effects of rhIGF-1 on TC viability, proliferation, and apoptosis were determined using RT-qPCR. Proliferating cell nuclear antigen (PCNA) a...
Frohlich M, Knights K, Springer NL.Differentiating immune-mediated causes from other causes of anemia and thrombocytopenia can be challenging. Flow cytometry can detect surface-associated immunoglobulin (sIg) on red blood cells (RBC) and platelets (PLT) in dogs and horses. Sample storage parameters for ideal assay performance has not been evaluated in horses. The study objective is to identify optimal storage time and temperature of equine whole blood for the detection of RBC-sIg and PLT-sIg via flow cytometry. Both assays were performed on samples at time 0, 4, 24, 48, and 72 h post collection. RBC-sIg samples were stored at ...
Iddagoda J, Gunasekara P, Handunnetti S, Jeewandara C, Karunatilake C, Malavige GN, de Silva R, Dasanayake D.It is clinically important to identify allergens in Artocarpus heterophyllus (jackfruit), Moringa oleifera (moringa), Trianthema portulacastrum (horse purslane) and Syzygium samarangense (rose apple). This study included 7 patients who developed anaphylaxis to jackfruit (1), moringa (2), horse purslane (3) and rose apple (1). We sought to determine allergens in the edible ripening stages of jackfruit (tender, mature, and ripened jackfruit) and seeds, edible parts of moringa (seeds, seedpod, flesh inside seedpod, and leaves), horse purslane leaves and ripened rose apple fruit. The persistence o...
Bishop RC, Kemper AM, Burges JW, Jandrey KE, Wilkins PA.To evaluate a point-of-care viscoelastic coagulation monitor (VCM Vet) for use in horses by assessing variability between devices and establish reference intervals (RIs) for healthy adult horses. Methods: Prospective observational study. Methods: Two university teaching hospitals. Methods: Healthy adult horses (n = 68). Methods: None. Results: Blood collected by direct jugular venipuncture was applied directly from the syringe into 2 VCM Vet cassettes to establish coefficients of variation (CVs) and RIs for reported parameters of clotting time (CT), clot formation time (CFT), alpha angle, am...
Lightbody KL, Austin A, Lambert PA, von Samson-Himmelstjerna G, Jürgenschellert L, Krücken J, Nielsen MK, Sallé G, Reigner F, Donnelly CG....Cyathostomins are ubiquitous equine nematodes. Infection can result in larval cyathostominosis due to mass larval emergence. Although faecal egg count (FEC) tests provide estimates of egg shedding, these correlate poorly with burden and provide no information on mucosal/luminal larvae. Previous studies describe a serum IgG(T)-based ELISA (CT3) that exhibits utility for detection of mucosal/luminal cyathostomins. Here, this ELISA is optimised/validated for commercial application using sera from horses for which burden data were available. Optimisation included addition of total IgG-based calibr...
Okolo CC, Emejuo NT, Udeagbala NG, Emeto UE, Ezema AS, Omeje OV, Nweze NE.Sample processing methods and storage time affect the outcome of biochemical analysis. Objectives: We aimed to assess the effects of dipotassium-ethylenediaminetetraacetic acid (K2-EDTA) and lithium-heparin treatments and storage times on selected analytes in equine synovial fluid (SF). Conclusions: The analytes-except for TP-became unstable within a few hours postcollection. Lithium-heparin and K2-EDTA treatments significantly altered ALP, LDH, TNCCs, and pH but not the TP concentrations of equine SF. Studies establishing reference intervals for these analytes based on the anticoagulant used ...
Cassady KR, Minter LJ, Gruber EJ.Field veterinarians and researchers studying wild species, such as the southern white rhinoceros, often work in remote areas with limited access to standard laboratory equipment, hindering the ability to measure serum analytes.
Objectives: The first objective was to produce an inexpensive, manually operated centrifuge that could accept standard laboratory tubes by modifying a consumer-grade salad spinner with low-cost materials. The second objective was to compare biochemistry analysis results obtained from equine and southern white rhinoceros serum separated by traditional laboratory and m...
Muñoz E, Castro M, Aguila L, Contreras MJ, Fuentes F, Arias ME, Felmer R.Sperm sexing is a technology that can generate great economic benefits in the animal production sector. Techniques such as sex-sorting promise over 90% accuracy in sperm sexing. However, for the correct standardization of the technique, some laboratory methodologies are required. The present manuscript describes in detail a standardized equine sperm sex-sorting protocol using an absolute qPCR-based methodology. Furthermore, the results of absolute qPCR were implemented and validated by generating equine/bovine heterologous embryos by intracytoplasmic sperm injection (ICSI) of presumably sexed ...
Carossino M, Balasuriya UBR, Thieulent CJ, Barrandeguy ME, Vissani MA, Parreño V.Equine rotavirus A (ERVA) is the leading cause of diarrhea in foals, with G3P[12] and G14P[12] genotypes being the most prevalent. Recently, equine G3-like RVA was recognized as an emerging infection in children, and a group B equine rotavirus (ERVB) was identified as an emergent cause of foal diarrhea in the US. Thus, there is a need to adapt molecular diagnostic tools for improved detection and surveillance to identify emerging strains, understand their molecular epidemiology, and inform future vaccine development. We developed a quadruplex TaqMan RT-qPCR assay for differentiation of ERVA an...
Boyle AG, O'Shea K, Stefanovski D, Rankin SC.There is debate around the clinical significance of Streptococcus equi subsp. equi detection in low numbers using quantitative real-time PCR (qPCR). Propidium monoazide (PMA) qPCR has been used to differentiate DNA from viable and nonviable bacterial cells. The aim of this study was to evaluate the ability of PMA eqbE SEQ2190 triplex qPCR to differentiate DNA from viable and nonviable S. equi in positive and suspect positive clinical specimens. Fifty-seven stored (frozen and refrigerated) positive (36) or suspect positive (21) clinical specimens (determined via SeeI qPCR as the gold standard) ...
Pearson GB, Papa B, Mosaddegh A, Cooper H, Aprea M, Pigott J, Altier C, Cazer CL, Reesink HL.To investigate (1) variables associated with the likelihood of obtaining a positive culture, (2) commonly isolated microorganisms, and (3) antimicrobial resistance patterns of isolates from horses with presumptive synovial sepsis. Methods: Synovial fluid, synovium, and bone samples from equine cases with presumptive synovial sepsis submitted to the Cornell University Animal Health Diagnostic Center from 2000 to 2020 for microbial culture and antimicrobial sensitivity testing. Methods: Univariable and multivariable analyses were performed to determine the effect of variables on the likelihood o...
Chen Q, Wang Z, Chen H.Aromatase inhibitors (AIs) can indirectly cause increased testosterone in animals, which leads to the improvement of the athletic ability of horses. For the protection of horses and the consideration of fair competition, AIs were listed as prohibited drugs by the Federation Equestre Internationale (FEI). There were several disadvantages using traditional pretreatment methods before analyzing these drugs from biological samples. A rapid and green pretreatment method has been developed by utilizing the hydrophobic deep eutectic solvent (DES)-based vortex-assisted liquid-liquid microextraction (D...
Bergmann R, Schroedl W, Müller U, Baums CG.Streptococcus equi subsp. zooepidemicus (SEZ) is a major equine pathogen that causes pneumonia, abortion, and polyarthritis. It can also cause invasive infections in humans. SEZ expresses the M-like protein SzM, which recruits host proteins such as fibrinogen to the bacterial surface. Equine SEZ strain C2, which binds only comparably low amounts of human fibrinogen in comparison to human SEZ strain C33, was previously shown to proliferate in equine and human blood. As the expression of SzM_C2 was necessary for survival in blood, this study investigated the working hypothesis that SzM_C2 inhibi...
Zahradnik E, Sander I, Lotz A, Liebers V, Thullner I, Tacke S, Raulf M.The study aimed to determine the allergen, endotoxin and β-(1,3)-glucan concentrations at various areas on a university campus of veterinary medicine. Methods: Dust samples were collected four times a year for three years using electrostatic dust collectors (EDC) at 25 different locations on a campus of veterinary medicine and in laboratories of inorganic chemistry as a control area representing animal-free environment. Major animal allergens from dog, cat, horse, cattle and mouse, domestic mite (DM) allergens, and β-(1,3)-glucan were measured using enzyme immunoassays and endotoxin using th...
Roberts MM, Oroszlan S.Capsids of equine infectious anemia virus have been isolated as cone-shaped particles 60 x 120 nm in size. Detergent treatment of whole virus followed by two cycles of rate-zonal centrifugation in Ficoll produces these capsids in a yield of approximately 10%. The major protein components are the gag-encoded p11 nucleocapsid protein and p26 capsid protein, which are present in equimolar amounts. Substantial cleavage of p11 to p6 and p4 can be observed under conditions where the viral protease packaged in the capsid is enzymatically active.
Kinoshita Y, Niwa H, Uchida-Fujii E, Nukada T.Microbial communities are commonly studied by using amplicon sequencing of part of the 16S rRNA gene. Sequencing of the full-length 16S rRNA gene can provide higher taxonomic resolution and accuracy. To obtain even higher taxonomic resolution, with as few false-positives as possible, we assessed a method using long amplicon sequencing targeting the rRNA operon combined with a CCMetagen pipeline. Taxonomic assignment had > 90% accuracy at the species level in a mock sample and at the family level in equine fecal samples, generating similar taxonomic composition as shotgun sequencing. The...
Brandt S, Haralambus R, Schoster A, Kirnbauer R, Stanek C.Bovine papillomaviruses of types 1 and 2 (BPV-1 and -2) chiefly contribute to equine sarcoid pathogenesis. However, the mode of virus transmission and the presence of latent infections are largely unknown. This study established a PCR protocol allowing detection of <or=10 copies of the BPV-1/-2 genes E5 and L1. Subsequent screening of peripheral blood mononuclear cell (PBMC) DNA derived from horses with and without BPV-1/2-induced skin lesions demonstrated the exclusive presence of E5, but not L1, in PBMCs of BPV-1/2-infected equines. To validate this result, a blind PCR was performed from ...
Nautscher N, Bauer A, Steffl M, Amselgruber WM.This study described corneal morphology in different domestic animals using histological and immunohistochemical methods. Specifically, we evaluated the species-specific distribution pattern of cytokeratins (CKs) and aquaporins (AQPs) to assess their contribution to the strength and transparency of the cornea, respectively. Methods: Corneal sections (2 μm thick) were obtained from 28 pigs, 11 cows, two goats, six horses, four dogs, and five cats and stained with hematoxylin-eosin and periodic acid-Schiff (PAS) reaction. Immunohistochemistry was also performed using CK1 to 3 and AQP1 and 5 an...
Nazari F, Parham A, Maleki AF.Quantitative real time reverse transcription PCR (qRT-PCR) is one of the most important techniques for gene-expression analysis in molecular based studies. Selecting a proper internal control gene for normalizing data is a crucial step in gene expression analysis via this method. The expression levels of reference genes should be remained constant among cells in different tissues. However, it seems that the location of cells in different tissues might influence their expression. The purpose of this study was to determine whether the source of mesenchymal stem cells (MSCs) has any effect on exp...
Fouchécourt S, Métayer S, Locatelli A, Dacheux F, Dacheux JL.Proteins present in and secreted into the lumen of various regions of the stallion epididymis were characterized qualitatively and quantitatively by two-dimensional electrophoresis. Using this proteomic approach, 201 proteins were found in the lumen and 117 were found that were secreted by the epithelium in various parts of the organ. Eighteen proteins made up 92.6% of the total epididymal secretory activity, lactoferrin (41.2%) and clusterin (24.8%) being the most abundant. Procathepsin D, HE1/CTP (cholesterol transfer protein), GPX (glutathione peroxidase), beta-N-acetyl-hexosaminidase, and ...
Engvall EO, Pettersson B, Persson M, Artursson K, Johansson KE.A PCR-based assay was developed for detecting DNA of granulocytic ehrlichiae in blood samples from dogs, horses, and cattle, Primers were designed from 16S rRNA sequence information to specifically amplify DNA from a newly identified Swedish Ehrlichia species. The 16S rRNA nucleotide sequence of this Swedish species differs in only two and three positions from the sequences of Ehrlichia phagocytophila and Ehrlichia equi, respectively, which were also amplified by this PCR system. For evaluation, PCR results were compared with microscopic examination of stained blood smears for the detection of...
Scare JA, Slusarewicz P, Noel ML, Wielgus KM, Nielsen MK.Fecal egg counts are emphasized for guiding equine helminth parasite control regimens due to the rise of anthelmintic resistance. This, however, poses further challenges, since egg counting results are prone to issues such as operator dependency, method variability, equipment requirements, and time commitment. The use of image analysis software for performing fecal egg counts is promoted in recent studies to reduce the operator dependency associated with manual counts. In an attempt to remove operator dependency associated with current methods, we developed a diagnostic system that utilizes a ...
Botton SA, Pereira DI, Costa MM, Azevedo MI, Argenta JS, Jesus FP, Alves SH, Santurio JM.Pythium insidiosum is a fungus-like organism present in subtropical and tropical areas, such as Brazil, known to infect humans and various animal species. P. insidiosum is the etiological agent of pythiosis, an emerging and granulomatous disease characterized mainly by cutaneous and subcutaneous lesions in horses, the principal species affected. Accurate diagnosis of pythiosis and identification of its causal agent by microbiological and serological tests can be often difficult and inconclusive principally for horses and humans. The aim of this study was to evaluate the application of the prev...
Lazary S, Antczak DF, Bailey E, Bell TK, Bernoco D, Byrns G, McClure JJ.Six laboratories participated in the Fifth International Workshop on Lymphocyte Alloantigens of the Horse, testing 132 alloantisera against lymphocytes of 880 horses chosen to represent different families and breeds. Most of the alloantisera were produced by lymphocyte immunization between horses matched at the ELA-A locus. All horses were also tested with antisera contributed to the workshop by participating laboratories which identified ELA specificities A1-A10 and W12-W21. Previously identified workshop specificities ELA-W14, W15 and W19 were accepted as products of the ELA-A locus based on...
Egberink HF, Ederveen J, Montelaro RC, Pedersen NC, Horzinek MC, Koolen MJ.Feline immunodeficiency virus (FIV) grown in cat lymphocyte and thymocyte cultures was labelled with L-[35S]methionine or [3H]glucosamine and virus-coded proteins were identified using immunoprecipitation. Polypeptides with apparent Mr values of 15K, 24K, 43K, 50K, 120K and 160K were detected. An additional polypeptide of 10K was detected by Western blot analysis. The two highest Mr species sometimes appeared as one band, of which only the 120K polypeptide was glycosylated. In the presence of tunicamycin gp120 was no longer detectable and a non-glycosylated precursor of 75K was found instead. ...
Zhou M, Wang Q, Sun J, Li X, Xu L, Yang H, Shi H, Ning S, Chen L, Li Y, He T, Zheng Y.The importance of microRNAs at the post-transcriptional regulation level has recently been recognized in both animals and plants. We used the simple but effective sequential method of first Blasting known animal miRNAs against the horse genome and then using the located candidates to search for novel miRNAs by RNA folding method in the vicinity (+ -500 bp) of the candidates. Here, a total of 407 novel horse miRNA genes including 354 mature miRNAs were identified, of these, 75 miRNAs were grouped into 32 families based on seed sequence identity. MiRNA genes tend to be present as clusters in som...
Welch HM, Bridges CG, Lyon AM, Griffiths L, Edington N.The polymerase chain reaction (PCR) and co-cultivation were used to identify the lymphoreticular system as the site of latency of equid herpesvirus I (EHV-1). Primers for PCR were designed from aligned nucleotide sequences of the glycoprotein gB genes to amplify the same region of both the EHV-1 and EHV-4 genomes. Subsequent restriction digests using specific enzymes distinguished the amplified fragments of the EHV-1 genome from those of the EHV-4 genome. Ten weeks following an experimental infection of five ponies with EHV-1, latent virus was detected by PCR and recovered by co-cultivation, p...
Nielsen JM.The objective of this study was to compare results from endometrial culture swabs with results from culturing of endometrial biopsies. The culture results were related to cytological findings (polymorphonuclear; PMN-cells) and histological observations (PMN-cells). Biopsy and swab samples were smeared on the surface of a blood agar petri dish, and examined for growth of bacteria. Cytology samples were obtained from endometrial biopsies, stained and examined under microscopy for the presence of PMN-cells. Endometrial biopsies were examined for the presence of PMN-infiltration of the endometrial...
Ljungberg K, Whitmore AC, Fluet ME, Moran TP, Shabman RS, Collier ML, Kraus AA, Thompson JM, Montefiori DC, Beard C, Johnston RE.A novel genetic vaccine that is based on a Venezuelan equine encephalitis virus (VEE) replicon launched from plasmid DNA is described. The plasmid encodes a VEE replicon under the transcriptional control of the cytomegalovirus immediate-early promoter (VEE DNA). The VEE DNA consistently expressed 3- to 15-fold more green fluorescent protein in vitro than did a conventional DNA vaccine. Furthermore, transfection with the DNA-launched VEE replicon induced apoptosis and type I interferon production. Inoculation of mice with VEE DNA encoding human immunodeficiency virus type 1 gp160 significantly ...
Grooters AM, Gee MK.Pythium insidiosum is an important cause of cutaneous and gastrointestinal disease in horses and dogs in the southeastern United States. Culture-based diagnosis of pythiosis is rarely definitive because production and identification of reproductive structures is difficult. The purpose of this study was to develop a polymerase chain reaction (PCR)-based assay for the identification of P insidiosum. Genomic DNA was extracted from 3 clinical isolates of P insidiosum and I isolate each of Pythium graminicola and Pythium arrhenomanes. The ITS I region of the ribosomal RNA gene of each isolate was a...
Das PJ, Paria N, Gustafson-Seabury A, Vishnoi M, Chaki SP, Love CC, Varner DD, Chowdhary BP, Raudsepp T.Sperm mRNA transcriptional profiles can be used to evaluate male fertility, yet differences between species in sperm attributes and packaging require adjustments in sperm RNA isolation protocols. The objective was to optimize RNA isolation methodology for fresh, frozen, and extended ejaculates, and epididymal sperm of stallions. Additionally, a protocol for RNA isolation from testis biopsies was established. Separation of sperm from somatic cells was critical for assuring the isolation of sperm-specific RNA. The highest purity was obtained by centrifuging ejaculates and epididymal sperm at 200...
Yang F, Fu B, O'Brien PC, Robinson TJ, Ryder OA, Ferguson-Smith MA.Complete sets of chromosome-specific painting probes, derived from flow-sorted chromosomes of human (HSA), Equus caballus (ECA) and Equus burchelli (EBU) were used to delineate conserved chromosomal segments between human and Equus burchelli, and among four equid species, E. przewalskii (EPR), E. caballus, E. burchelli and E. zebra hartmannae (EZH) by cross-species chromosome painting. Genome-wide comparative maps between these species have been established. Twenty-two human autosomal probes revealed 48 conserved segments in E. burchelli. The adjacent segment combinations HSA3/21, 7/16p, 16q/1...
Salmonella enterica subsp. enterica serovar Abortusequi is frequently reported as a cause of abortion in mares and neonatal septicemia and polyarthritis in Asian and African countries, but only sporadically in Europe and the United States. We report an outbreak of S. Abortusequi in foals in Italy, characterized by high mortality. In a herd of Murgese horses, 10 of 34 newborns died at birth and a further 7 died, after developing severe clinical signs, during the first 10 d of life. Tissue specimens from different organs of 2 dead foals, synovial fluids from 4 sick foals, and vaginal and rectal ...
Konishi E, Shoda M, Ajiro N, Kondo T.Antibodies to Japanese encephalitis virus (JEV) nonstructural 1 (NS1) protein constitute a marker of natural JEV infection among populations vaccinated with inactivated JE vaccine. In Japan, with few recent human JE cases, the natural infection rate is critical to evaluate the necessity of continuing JE vaccination. A sensitive immunochemical staining method for detecting NS1 antibodies in individuals naturally and subclinically infected with JEV was previously established. Here, an enzyme-linked immunosorbent assay (ELISA) to detect NS1 antibodies in equine sera was developed and evaluated as...
Wagner B, Freer H, Rollins A, Erb HN, Lu Z, Gröhn Y.Lyme disease is a zoonotic, vector-borne disease and occurs in mammals including horses. The disease is induced by infection with spirochetes of the Borrelia burgdorferi sensu lato group. Infection of mammalian hosts requires transmission of spirochetes by infected ticks during tick bites. Lyme disease diagnosis is based on clinical signs, possible exposure to infected ticks, and antibody testing which is traditionally performed by ELISA and Western blotting (WB). This report describes the development and validation of a new fluorescent bead-based multiplex assay for the detection of antibodie...
Bowling AT, Eggleston-Stott ML, Byrns G, Clark RS, Dileanis S, Wictum E.A parallel testing of 4803 routine Quarter Horse parentage cases, using 15 loci of blood group and protein polymorphisms (blood typing) and 11 loci of dinucleotide repeat microsatellites (DNA typing), validated DNA markers for horse pedigree verification. For the 26 loci, taken together, the theoretical effectiveness of detecting incorrect parentage was 99.999%, making it extremely unlikely that false parentage would fail to be recognized. The tests identified incorrect parentage assignment for 95 offspring (2% of cases). Despite fewer loci, DNA typing was as effective as blood typing and, in ...
Linardi RL, Dodson ME, Moss KL, King WJ, Ortved KF.Cartilage injury occurs commonly in equine athletes, often precipitating posttraumatic osteoarthritis (PTOA). Orthobiologics such as autologous conditioned serum (ACS) and autologous protein solution (APS) may be useful in decreasing posttraumatic inflammation, thereby preventing PTOA. The objective of this study was to quantify cytokine concentrations in ACS and APS and evaluate the protective effects of ACS and APS on inflamed chondrocytes cultured . We hypothesized that the combination of platelet-derived growth factors (PDGF) and anti-inflammatory cytokines present in APS would be superior...
González-Fernández L, Macías-García B, Loux SC, Varner DD, Hinrichs K.Protein tyrosine phosphorylation (PY) is a hallmark of sperm capacitation. In stallion sperm, calcium inhibits PY at pH 7.8). These results show for the first time a role for calcium/calmodulin-dependent kinases in PRKA-dependent sperm PY; a non-PRKA-dependent pathway regulating sperm PY; and the apparent involvement of the FAK family of protein tyrosine kinases downstream in both pathways.
MacLeod JN, Burton-Wurster N, Gu DN, Lust G.Fibronectin is an extracellular matrix glycoprotein encoded by a single gene. Alternative RNA splicing has been reported at three sites, ED (extra type III domain)-A, ED-B, and the variable or V region. Articular cartilage fibronectin monomers are rarely (ED-A)+, but approximately 25% are (ED-B)+. RNA gel electrophoresis and Northern blot analysis identified two (ED-B)+ and two (ED-B)- fibronectin transcripts in cartilage, each pair differing by approximately 750 bases. This difference results from a previously unreported RNA splicing pattern that eliminates not only the V region but also nucl...
O'Callaghan DJ, Hyde JM, Gentry GA, Randall CC.Infection of exponential-phase suspension cultures of mouse fibroblast cells (L-M) with equine abortion virus (EAV) resulted in inhibition of cell growth and marked alterations in host metabolic processes. The synthesis of deoxyribonucleic acid (DNA) and ribonucleic acid was inhibited within 4 hr after infection and was suppressed by more than 90% by the time of maximal virus replication (14 to 18 hr). The overall rate of protein synthesis, however, was similar in uninfected and virus-producing cells as determined by measurements of net protein and isotope incorporation. The time course of vir...
Cleton NB, van Maanen K, Bergervoet SA, Bon N, Beck C, Godeke GJ, Lecollinet S, Bowen R, Lelli D, Nowotny N, Koopmans MPG, Reusken CBEM.The genus Flavivirus in the family Flaviviridae includes some of the most important examples of emerging zoonotic arboviruses that are rapidly spreading across the globe. Japanese encephalitis virus (JEV), West Nile virus (WNV), St. Louis encephalitis virus (SLEV) and Usutu virus (USUV) are mosquito-borne members of the JEV serological group. Although most infections in humans are asymptomatic or present with mild flu-like symptoms, clinical manifestations of JEV, WNV, SLEV, USUV and tick-borne encephalitis virus (TBEV) can include severe neurological disease and death. In horses, infection wi...
Fernandez NJ, Hecker KG, Gilroy CV, Warren AL, Léguillette R.Reliable enumeration of mast cells and eosinophils in equine bronchoalveolar lavage (BAL) fluid is important because small increases in the percentages of these cells support the clinical diagnosis of inflammatory airway disease (IAD). Increases in BAL neutrophils also occur with IAD but are not specific due to overlap between IAD and recurrent airway obstruction (RAO). Objective: The objectives of this study were to evaluate the reliability of a standard 400-cell leukocyte differential count and an alternate method evaluating 5 microscopic fields at 500× magnification in equine BAL fluid cyt...
