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.
Satué K, Marcilla M, Medica P, Ferlazzo A, Fazio E.Androgens modulate maternal ovarian activity, embryo implantation and correct placental development. The objective of this study was to establish reference values for testosterone (T), androstenedione (A4) and dehydroepiandrosterone (DHEA) concentrations in pregnant mares. A total of 30 healthy Spanish Purebred mares with an age range of 9.33 ± 3.31 years, were studied during the 11 months of gestation. T, A4 and DHEA concentrations were determined using EIA validated specifically for equines. T increased in the 2nd and 3rd month (P < 0.05), showing a plateau between the 4th and 6th m...
Kalinowski M, Grądzki Z, Jarosz Ł, Adaszek Ł.Rhodococcus equi (R. hoagii) is an opportunistic pathogen commonly found in foals up to 6 months old and animal environment. The R. equi genome contains genetically stable chromosomal DNA and an 80-90 kb plasmid containing vapA gene, responsible for virulence. Most reports from around the world focus on the determination of R. equi plasmid profiles. Few studies have attempted to determine differences in nucleotide sequences between virulent strains of R. equi isolated from foals and breeding environment. The aim of the study was to perform a molecular analysis of a fragment of the chromosomal ...
Consuegra C, Crespo F, Dorado J, Ortiz I, Diaz-Jimenez M, Pereira B, Hidalgo M.Vitrification of sperm is based on high-speed freezing by direct exposure to liquid nitrogen using non-permeable cryoprotectants, mainly disaccharides; yet, the concentration of cryoprotectants has a species-specific effect on the sperm cell. The aim of this study was to assess different sucrose concentrations for stallion sperm vitrification. Semen samples (n = 9) were collected from three stallions, centrifuged and resuspended to a concentration of 50 × 10 sperm/ml in a base extender (INRA96 + 1% of bovine serum albumin) with three different sucrose concentrations (Molar): 20 mM (S...
Bernet A, Bastien A, Soulet D, Jerczynski O, Roy C, Bianchi Rodrigues Alves M, Lecours C, Tremblay MÈ, Bailey JL, Robert C, Belleannée C.Where are primary cilia (PC) organelles located during postnatal epididymal development? Our findings unveil the existence of PC sensory organelles in different epididymal cell types according to postnatal development stage. Primary cilia are sensory organelles that orchestrate major signaling pathways during organ development and homeostasis. Epididymal PC have been detected in the horses, donkey and mules but their cell-lineage specificity has never been investigated in this organ. A longitudinal study was performed by examining tissue from n = 3 to n = 10 transgenic mice at different times ...
Ghoniem SM, El Deeb AH, Aggour MG, Hussein HA.We developed a multiplex reverse-transcription real-time PCR (RT-rtPCR) assay for the simultaneous detection of the main equine respiratory viruses: equid alphaherpesviruses 1 and 4 (EHV-1, -4) and equine influenza virus (EIV; species Influenza A virus). The primers and probes amplified only the targeted viruses, and there were no inter-assay cross-amplifications or nonspecific interactions. The multiplex assay efficiencies were 92.5%, 97%, and 90% for EHV-1, EHV-4, and EIV, respectively. The R values of the monoplex and multiplex assays were ⩾0.990, and the slopes were -3.37 to -3.59. The p...
Poltep K, Tesena P, Yingchutrakul Y, Taylor J, Wongtawan T.Silent gastric ulceration occurs without evidence of clinical signs and is common in horses. There is currently no a simple and effective method to diagnose this disease. Proteomics can be used to identify serum biomarkers, but the most abundant serum protein, albumin, could conceal candidate biomarkers. Therefore, it is recommended to remove albumin before a proteomic study; however, there is no specific albumin depletion kit or standard protocol available for horse samples. The objectives of this study were to optimise a protocol to remove equine serum albumin and to use albumin-depleted ser...
Restrepo G, Rojano B, Usuga A.Cysteine-rich secretory protein-3 (CRISP-3) and some of its nonsynonymous polymorphism have been related to the fertility and freezability of stallion semen; however, the role of the CRISP-3 gene and its seminal plasma protein in the raw semen quality is still unknown. The aim of this study was to evaluate the relationship of CRISP-3 with semen quality in stallions. DNA was obtained from blood samples of 100 stallions, from which 30 stallions were randomly selected to obtain 60 ejaculates. Through PCR amplification and sequencing, the variation of four nonsynonymous SNPs from CRISP-3 was ident...
Bonilla-Gutiérrez AF, Castillo-Franz C, López C, Álvarez ME, Giraldo CE, Carmona JU.The aim of this study was to evaluate the release of pro- and anti-inflammatory as well as anabolic mediators stimulated by a leukocyte-reduced platelet-rich gel supernatant (Lr-PRGS) and a leukocyte-reduced plasma supernatant (Lr-PL) at two concentrations (25 and 50%) on normal equine suspensory ligament explants (SLEs) and tendon explants (TEs). SLEs and TEs from six horses were independently incubated for 48 h with Lr-PRGS and Lr-PL at concentrations of 25 and 50%, respectively. Samples were collected from the incubated tissues at 1 h and 48 h, which were employed for ELISA determinat...
De-Simone SG, Nascimento HJ, Prado IC, Aguiar AS, Melgarejo AR, Pina JLS, Ferreira PF, Provance DW.The availability of purified antibodies is a prerequisite for many applications and the appropriate choice(s) for antibody-purification is crucial. Numerous methods have been developed for the purification of antibodies from different sources with affinity chromatography-based methods being the most extensively utilized. These methods are based on high specificity, easy reversibility and biological interactions between two molecules (e.g., between receptor and ligand or antibody and antigen). However, no simple techniques have yet been described to characterize and purify subclasses of immunog...
Schnabel CL, Wemette M, Babasyan S, Freer H, Baldwin C, Wagner B.Chemokines are soluble molecules directing immune cell trafficking and homing, mediating inflammation, and initiating immune responses to infection. In horses, the analysis of chemokines has been limited by the lack of specific antibodies. We generated mAbs specific for the equine C-C motif chemokine ligands (CCL) CCL2 (MCP-1), CCL3 (MIP-1α), CCL5 (RANTES) and CCL11 (eotaxin) using hybridoma technology. Antibody specificity was confirmed by intracellular staining of Chinese Hamster Ovary cells transfected with expression vectors encoding for CCL2, CCL3, CCL5, or CCL11. Transfectants were stai...
Ross KA, Kolb DS, Macedo A, Anderson M, Klein C.This study tested the hypothesis that the presence of prostaglandin E2 in seminal plasma would aid in the transport of phenolsulfonphthalein (PSP) across the uterotubal junction. Five mares in estrus were inseminated during estrus with PSP dissolved in phosphate-buffered saline and during the subsequent estrus with PSP added to a standard insemination dose. Serum and urine samples were obtained at hours 0, 1, 2, and 3 following treatment and examined for the presence of PSP. Phenolsulfonphthalein could not be detected in any of the urine samples collected from mares following either treatment....
Went HA, Scare JA, Steuer AE, Nielsen MK.Recommendations for control of equine strongylid parasites are based on regular determination of fecal egg counts to identify high strongylid shedders and to evaluate treatment efficacy. The McMaster technique has long been used as the standard egg counting technique in equine veterinary practice in most parts of the world, but recent work has found the Mini-FLOTAC technique to perform with significantly better accuracy and precision. The Mini-FLOTAC system comes with a homogenizing device, termed the Fill-FLOTAC, and it has been hypothesized that this device might have a significant impact on...
Dourine is a parasitic venereal disease of equines caused by T. equiperdum. Humoral antibodies are found in infected animals, but diagnosis of dourine must include history, clinical, and pathological findings in addition to serology. Complement Fixation Test (CFT) is the Office International des Epizooties (OIE) recommended test for international trade; however, some uninfected equines may give inconsistent or nonspecific reactions in CFT due to the anticomplementary effects of their sera. In this study an Indirect Enzyme Linked Immunosorbent Assay (iELISA) was developed. This test could be us...
Niyom S, Mama KR, King M, Contino E, Ferris D, Valdes-Martinez A, Frisbie DD, McIlwraith W, Zumbrunnen J.This study investigated the influence of changing recumbency and mode of ventilation over repeated anesthesias on the alveolar to arterial oxygen tension gradient (PO) and laboratory analytes in eight horses during a year-long imaging study. Anesthesia was induced with xylazine, diazepam or guaifenesin, and ketamine and maintained with isoflurane. Horses were positioned in right or left lateral recumbency for computed tomography. Ventilation was controlled during 47% of the anesthetics. Blood was sampled from an arterial catheter prior to (30 ± 5 min from connection to anesthetic circuit), wi...
Sumbria D, Singla LD, Kumar R, Bal MS, Kaur P.As parasitaemia is low and fluctuating during the chronic stage of infection, accurate detection of Trypanosoma evansi in blood is difficult. The primary aims of this investigation were to assess for the first time the seroprevalence of T. evansi in all agro-climatic zones of Punjab, by indirect enzyme-linked immunosorbent assay (iELISA) and card agglutination test (CATT/T. evansi), and to evaluate the risk factors associated with latent trypanosomosis. A total of 319 equine serum samples collected from 12 districts of Punjab (India) belonging to different agro-climatic zones revealed 39 (12.2...
Lee W, Ko BJ, Sim YE, Suh S, Yoon D, Kim S.Urine was most commonly used biological sample in drug test. To create a false-negative test result, some drug abusers were reported to submit animal urine instead of their own. So, the purpose of this study was to compare and differentiate human from animal urine (Rat 370, Pig 12, Horse 10, Cat 8, Dog 13, Cow 10, Monkey 10) samples through the uses of quantitative 1H-NMR and to find biomarkers that can be used for the discrimination of human urine from animal urine. The 1H-NMR spectroscopy was employed and metabolomic analysis with multivariate statistics was carried out. Human urine samples ...
Vera L, Boyen F, de Visscher A, Vandenbroucke V, Vanantwerpen G, Govaere J.The use of commercial chromogenic agar plates for the rapid, easy and correct identification of equine endometritis-causing bacteria has been proposed. Preliminary tests in our lab revealed undescribed limitations. Therefore, we tested the ability of the Brilliance UTI agar, a commercially available chromogenic agar, to accurately identify bacteria causing equine endometritis. Objective: To 1) investigate whether bacteria present in the equine uterus are able to grow on this chromogenic agar plate, 2) determine whether these bacteria belong to the genera for which these agar plates were origin...
Ji S, Na L, Ren H, Wang Y, Wang X.Human myxovirus resistance protein 2 (huMxB) has been shown to be a determinant type I interferon (IFN)-induced host factor involved in the inhibition of human immunodeficiency virus type 1 (HIV-1) as well as many other primate lentiviruses. This blocking occurs after the reverse transcription of viral RNA and ahead of integration into the host DNA, which is closely connected to the ability of the protein to bind the viral capsid. To date, Mx2s derived from nonprimate animals have shown no capacity for HIV-1 suppression. In this study, we examined the restrictive effect of equine Mx2 (eqMx2) o...
Waraksa E, Woźniak MK, Kłodzińska E, Wrzesień R, Bobrowska-Korczak B, Namieśnik J.Ibuprofen is widely used in human and veterinary medicine for the treatment of chronic pain as well as rheumatic and musculoskeletal disorders. However, the analgesic and anti-inflammatory properties of Ibuprofen have contributed to frequent drug abuse in equestrian sports. A sensitive and rapid gas chromatography with tandem mass spectrometry based method with a simple liquid-liquid extraction and derivatization requiring 200 μL volume of sample and 2 mL of extraction solvent for the simultaneous determination of ibuprofen and its metabolites was developed. The proposed procedure was optim...
Ata EB, Zaghawa A, Ghazy AA, Elsify A, Abdelrahman K, Kasem S, Nayel M.Equine herpesvirus-1 (EHV-1) is an important pathogen, which infects horses worldwide with high morbidity but low mortality rates. The respiratory disorders and abortions are the most common indicators. Ab4p (an abortigenic and paralytic virus) is one of the most important and virulent strains. The development and functional characterization of the open reading frame-68 (ORF68) negative EHV-1 Ab4p mutants and an assessment of their roles in the infection at the cellular level were the main targets of the current study. Escherichia coli DH10β containing the Ab4p bacterial artificial chromosome...
Winter RL, Seeto WJ, Tian Y, Caldwell FJ, Lipke EA, Wooldridge AA.Endothelial progenitor cells (EPCs) contribute to neovascularization and vascular repair in vivo and are attractive for clinical use in ischemic disease. Tracking of stem and progenitor cells is essential to determine engraftment after administration. Semiconductor quantum dots (QD) are promising for cell labeling due to their ease of uptake by many cell lines and their continued presence after many cell generations. The purpose of this study was to evaluate function and growth of equine EPCs after QD labeling. Additionally, this study evaluated the duration of QD label retention and mechanism...
Norris JK, Steuer AE, Gravatte HS, Slusarewicz P, Bellaw JL, Scare JA, Nielsen MK.Given the ever-increasing levels of anthelmintic resistance in livestock parasites globally, it is recommended to use parasite fecal egg counts to make treatment decisions and to evaluate treatment efficacy. The consensus in equine parasitology is to use a flotation medium with a specific gravity (SG) of ≥ 1.20 to float the main parasite egg types of interest in egg counting techniques. However, the density of common equine endoparasite eggs has been sparsely investigated. Equine tapeworm eggs are known to be particularly difficult to determine and count in fecal samples. It is unknown wheth...
Kun TJ, Wictum EJ, Penedo MCT.Degraded biological samples are a challenge for testing laboratories. Genotyping success can be improved through the use of mini-STRs, by which primers are placed adjacent to the repeat motifs to reduce amplicon size. Here, we present a genetic profiling system comprising 13 autosomal and one X-linked dinucleotide-repeat markers and the SRY gene based on the internationally accepted equine parentage panel. The markers are divided into two panels with all alleles falling at or below 182 bp. The application of this method significantly increases the ability to profile difficult samples and to p...
Lo Giudice R, Rizzo G, Centofanti A, Favaloro A, Rizzo D, Cervino G, Squeri R, Costa BG, La Fauci V, Lo Giudice G.The use of equine bone blocks is widely reported for bone augmentation techniques. The block must be shaped according to the form of the defect that should be regenerated. The shaping could be performed by hand before or during the surgery, in a sterile ambient, or using a CNC milling machine that could not be sterile. The aim of our study was to evaluate if a steam sterilization could provide a medical grade sterilization of the blocks and to evaluate if bone microstructure and collagen structures change after different steam sterilization protocols provided by mainstream autoclave. Methods: ...
Mawhinney I, Errington J, Stamper N, Torrens N, Engelsma MY, Roest HIJ.Sets of genital swabs are routinely taken from horses to screen for the presence of Taylorella equigenitalis, the cause of contagious equine metritis. Typically, two to four different sites are swabbed at a time and tested by culture or PCR. Objective: This study explored the feasibility of pooling these swabs for a single PCR test per animal instead of testing each swab individually. Methods: In vitro. Methods: PCR signal strengths (Ct values) from 149 historical PCR positive genital swabs, together with historical data on the number of swabs in a set expected to be positive, were used to ass...
Badial PR, Teixeira RBC, Delfiol DJZ, da Mota LSLS, Borges AS.Overo lethal white foal syndrome (OLWFS) is a genetic disorder caused by a dinucleotide mutation in the endothelin receptor type B (EDNRB) gene leading to the death of affected foals shortly after birth. The use of rapid and reliable genetic testing is imperative for the early diagnosis of the mutation avoiding, therefore, either additional suffering or the production of affected animals. In the present study, we developed and validated a high-resolution melting (HRM) genotyping assay to detect the OLWFS causative mutation, and we also determined the frequency of heterozygotes among American P...
Aleman M, Costa LRR, Crowe C, Kass PH.Neuroglycopenia refers to a shortage of glucose in the brain resulting in neuronal dysfunction and death if left untreated. Presumed neuroglycopenia has not been described in horses. Objective: To report neurological signs in horses with presumed neuroglycopenia as the result of severe hypoglycemia. Methods: Ninety horses (hours to 28 years of age) diagnosed with hypoglycemia (blood glucose concentration < 75 mg/dL [< 4.2 mmol/L]). Methods: Retrospective study. Electronic medical records were searched. Signalment, history, complaint, clinical signs, laboratory findings including CSF analysis, ...
Durán-Ferrer M, Agüero M, Zientara S, Beck C, Lecollinet S, Sailleau C, Smith S, Potgieter C, Rueda P, Sastre P, Monaco F, Villalba R....The laboratory diagnosis of African horse sickness (AHS) is important for: (a) demonstrating freedom from infection in a population, animals or products for trade (b) assessing the efficiency of eradication policies; (c) laboratory confirmation of clinical diagnosis; (d) estimating the prevalence of AHS infection; and (e) assessing postvaccination immune status of individual animals or populations. Although serological techniques play a secondary role in the confirmation of clinical cases, their use is very important for all the other purposes due to their high throughput, ease of use and good...
Ferreira-Silva JC, Basto SRL, Moura MT, Rocha JM, Freitas Neto LM, Santos Filho JP, Silva Filho ML, Oliveira MAL.The work described here aimed to verify the efficiency of different extenders for cryopreservation of equine semen using sperm motility and acrosin activity as spermatic parameters. The semen was fractioned into two equal parts and resuspended in an 11% lactose solution in a 1:1 proportion, where it remained for 20 minutes at room temperature. The semen was centrifuged at 600 g for 10 minutes, and after the second centrifugation, each pellet received the freezing extender (Merck or Zorlesco) and was loaded into 4 mL straws. Each straw was placed in liquid nitrogen vapor steam for 15 minutes ...
Ishak GM, Bashir ST, Dutra GA, Gastal GDA, Gastal MO, Cavinder CA, Feugang JM, Gastal EL.In vivo studies involving molecular markers of the follicle wall associated with follicular fluid (FF) milieu are crucial for a better understanding of follicle dynamics. The inability to obtain in vivo samples of antral follicle wall (granulosa and theca cells) without jeopardizing ovarian function has restricted advancement in knowledge of folliculogenesis in several species. The purpose of this study in mares was to develop and validate a novel, minimally invasive in vivo technique for simultaneous collection of follicle wall biopsy (FWB) and FF samples, and repeated collection from the sam...
Haughan J, Jiang Z, Stefanovski D, Moss KL, Ortved KF, Robinson MA.Gene therapy promotes the expression of missing or defective genes and can be curative following administration of a single dose. Gene therapy is prohibited in equine athletes by regulatory bodies due to the high potential for abuse and novel analytical methods are needed for detection. The goal of this study was to detect the administration of an experimental gene therapy: a recombinant adeno-associated viral vector (rAAV) carrying a transgene for the anti-inflammatory cytokine IL-10 (rAAV-IL10). Twelve horses were randomly assigned to receive an intra-articular injection of rAAV-IL10 or phos...
Terhaar HM, Allbaugh RA, Mochel JP, Sebbag L.To determine total protein content (TPC) and serum albumin levels in the tears of horses with healthy or diseased eyes. Methods: Forty-two horses with healthy eyes and 11 horses with unilateral (n = 10) or bilateral (n = 1) ocular disease. Methods: Each eye underwent an ophthalmic examination including detailed conjunctivitis scoring and tear collection with Schirmer strips. TPC and serum albumin levels were quantified in tear samples and compared among healthy eyes, affected eyes, and contralateral unaffected eyes. The impact of the following variables on lacrimal protein levels were asse...
Dougherty DM, Lewis P.Using horses, we investigated three aspects of the stimulus control of lever-pressing behavior: stimulus generalization, discrimination learning, and peak shift. Nine solid black circles, ranging in size from 0.5 in. to 4.5 in. (1.3 cm to 11.4 cm) served as stimuli. Each horse was shaped, using successive approximations, to press a rat lever with its lip in the presence of a positive stimulus, the 2.5-in. (6.4-cm) circle. Shaping proceeded quickly and was comparable to that of other laboratory organisms. After responding was maintained on a variable-interval 30-s schedule, stimulus generalizat...
Eberhardt C, Amann B, Stangassinger M, Hauck SM, Deeg CA.Retinal Müller glial cells are of vital importance for maintaining a physiological environment within the retina. To this end, they provide highly specialized physiological properties to support neurons in structure, nutrition and metabolism. The purpose of this study was to isolate Müller cells from the equine retina, determine their characteristics and subsequently establish a stable equine Müller cell line (eqMC) that will provide a prerequisite for investigations on their physiological properties. Dissociated retinal cells were obtained from equine retinas by a papain digestion techniqu...
Russo D, Bombardi C, Grandis A, Furness JB, Spadari A, Bernardini C, Chiocchetti R.The distribution and chemical phenotypes of sympathetic and dorsal root ganglion (DRG) neurons innervating the equine ileocecal junction (ICJ) were studied by combining retrograde tracing and immunohistochemistry. Immunoreactivity (IR) for tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH), neuronal nitric oxide synthase (nNOS), calcitonin gene-related peptide (CGRP), substance P (SP), and neuropeptide Y (NPY) was investigated. Sympathetic neurons projecting to the ICJ were distributed within the celiac (CG), cranial mesenteric (CranMG), and caudal mesenteric (CaudMG) ganglia, as well ...
Smith CS, McLAUGHLIN A, Field HE, Edson D, Mayer D, Ossedryver S, Barrett J, Waltisbuhl D.Hendra virus (HeV) was first described in 1994 in an outbreak of acute and highly lethal disease in horses and humans in Australia. Equine cases continue to be diagnosed periodically, yet the predisposing factors for infection remain unclear. We undertook an analysis of equine submissions tested for HeV by the Queensland government veterinary reference laboratory over a 20-year period to identify and investigate any patterns. We found a marked increase in testing from July 2008, primarily reflecting a broadening of the HeV clinical case definition. Peaks in submissions for testing, and visitat...
Warner S, Hartley CA, Stevenson RA, Ficorilli N, Varrasso A, Studdert MJ, Crabb BS.Equine rhinitis A virus (ERAV) is a respiratory pathogen of horses and is classified as an Aphthovirus, the only non-Foot-and-mouth disease virus (FMDV) member of this genus. In FMDV, virion protein 1 (VP1) is a major target of protective antibodies and is responsible for viral attachment to permissive cells via an RGD motif located in a distal surface loop. Although both viruses share considerable sequence identity, ERAV VP1 does not contain an RGD motif. To investigate antibody and receptor-binding properties of ERAV VP1, we have expressed full-length ERAV VP1 in Escherichia coli as a glutat...
Sumner SM, Naskou MC, Thoresen M, Copland I, Peroni JF.Platelet preparations containing growth factors, attachment factors, and enzymes are appealing to enhance healing of injured tissues and as an alternative to xenogenic serum in cell culture media. Plateletpheresis is commonly used to collect platelets in human medicine but has not been validated in horses. Plateletpheresis to collect platelet concentrate was performed on six female, mixed breed, chemically restrained horses using commercially available apheresis equipment. Before and immediately after plateletpheresis, we performed physical examinations and collected blood for chemistry and co...
Mead DG, Ramberg FB, Maré CJ.In previous experiments we have demonstrated that colonized and wild black flies are competent laboratory vectors of different Mexican and Western USA isolates of vesicular stomatitis virus, serotype New Jersey (VSV-NJ). We have recently demonstrated biological VSV-NJ transmission by black flies using animal models. In the study described here, we tested the vector competence of colonized and wild black flies for the vesicular stomatitis virus, serotype Indiana (VSV-IN). A 1998 equine isolate was used. After a 10 day incubation period, saliva from experimentally infected Simulium vittatum and ...
Hall BG, Faunce W.The genes for utilization of cellobiose are normally cryptic in both laboratory strains and natural isolates of Escherichia coli. A survey of natural isolates of E. coli reveals that functional genes for cellobiose utilization, while rare, are present. The fraction of E. coli that utilized cellobiose ranged from less than 0.01% in human fecal samples to 7% in fecal samples obtained from horses. Samples obtained from sheep, cows, dogs, and pigs contained 0.1 to 0.5% cellobiose-positive E. coli. Neither the previously identified cel genes nor the bgl genes from E. coli K-12 were expressed during...
Unger L, Fouché N, Leeb T, Gerber V, Pacholewska A.Circulating miRNAs in body fluids, particularly serum, are promising candidates for future routine biomarker profiling in various pathologic conditions in human and veterinary medicine. However, reliable standardized methods for miRNA extraction from equine serum and fresh or archived whole blood are sorely lacking. We systematically compared various miRNA extraction methods from serum and whole blood after short and long-term storage without addition of RNA stabilizing additives prior to freezing. Time of storage at room temperature prior to freezing did not affect miRNA quality in serum. Fur...
Rosiles MR, Bautista J, Fuentes VO, Ross F.Equine leukoencephalomalacia (ELEM), swine pulmonary oedema and human oesophageal cancer have been associated with fumonisine B1 (FB1) ingestion. For the first time in this study it is reported that FB1 was identified as being associated with an outbreak of ELEM at Oaxaca, Mexico. Symptoms of ELEM and Equine Venezuelan Encephalitis (EVE) are similar and a different diagnosis is obligatory. In the geographical area (Oaxaca, Mexico) where donkeys died showing a neurological syndrome, 14 corn samples were collected. With the use of TLC (Thin layer chromatography) and HPLC (High performance liquid...
Chaudhary R, Raudsepp T, Guan XY, Zhang H, Chowdhary BP.Microdissected arm specific paints (ASPs) for human (HSA) chromosomes (Chrs) 2, 5, 6, 16, and 19 were used as probes on pig (SSC) and horse (ECA) metaphase chromosomes. Regions homologous to individual human arms were delineated in the two species studied. Of the ten ASPs used, HSA6 and 16 ASPs showed complete synteny conservation of individual arms as single blocks/ arms both in pig and horse. A similar trend was, in general, also observed for HSA19 ASPs. However, contrary to these observations, synteny conservation of individual arms of HSA2 and HSA5 was not observed in pig and horse. The ar...
Schwab UE, Fulcher ML, Randell SH, Flaminio MJ, Russell DG.We describe a method for creating differentiated equine bronchial epithelial cell cultures that can be used for in vitro studies including airway disease mechanisms and pathogen-host interactions. Our method is based on the culturing of human tracheobronchial epithelial cells at an air-liquid interface (ALI) in specific serum-free, hormone-supplemented medium. Bronchial epithelial cells are isolated and grown on T-Clear® insert membranes. Within 2 to 3 wk, cells differentiate into ciliated and mucus producing cells as demonstrated by confocal and electron microscopy. Furthermore, the demonstr...
de Waal T, Liebenberg D, Venter GJ, Mienie CM, van Hamburg H.African horse sickness (AHS) is an infectious, non-contagious arthropod-borne disease of equids, caused by the African horse sickness virus (AHSV), an orbivirus of the Reoviridae family. It is endemic in sub-Saharan Africa and thought to be the most lethal viral disease of horses. This study focused on detection of AHSV in Culicoides imicola (Diptera: Ceratopogonidae) pools by the application of a RT-qPCR. Midges were fed on AHSV-infected blood. A single blood-engorged female was allocated to pools of unfed nulliparous female midges. Pool sizes varied from 1 to 200. RNA was extracted and prepa...
Murcia-Robayo RY, Jouanisson E, Beauchamp G, Diaw M.Evaluation of sperm morphology is part of the assessment of fertility in human and animal reproduction. Analyses can be performed using different techniques, including the use of staining methods In our prospective study, the morphology of equine sperm was evaluated using 3 staining methods Diff-Quik, eosin-nigrosin, and SpermBlue, the latter being a relatively new stain commonly used for human sperm. Our hypotheses were that (1) SpermBlue allows easier assessment of horse sperm morphology and facilitates better identification of sperm abnormalities, and (2) sperm morphology classification dif...
Budsuren U, Ulaangerel T, Shen Y, Liu G, Davshilt T, Yi M, Bold D, Zhang X, Bai D, Dorjgotov D, Davaakhuu G, Jambal T, Li B, Du M, Dugarjav M, Bou G.Myostatin (MSTN), a member of the transforming growth factor-β superfamily, inhibits the activation of muscle satellite cells. However, the role and regulatory network of MSTN in equine muscle cells are not well understood yet. We discovered that MSTN knockdown significantly reduces the proliferation rate of equine muscle satellite cells. In addition, after the RNA sequencing of equine satellite cells transfected with MSTN-interference plasmid and control plasmid, an analysis of the differentially expressed genes was carried out. It was revealed that MSTN regulatory networks mainly involve ge...
Cowley AB, Lukat-Rodgers GS, Rodgers KR, Benson DR.N-Acetylmicroperoxidase-8 (1) contains heme and residues 14-21 of horse mitochondrial cytochrome c (cyt c). The two thioether bonds linking protein to heme in cyt c are present in 1, and the native axial ligand His-18 remains coordinated to iron. As an approach to probing structural or functional roles played by the double covalent heme-protein linkage in cyt c, we have initiated a study in which the properties of 1 are compared with those of a synthetic mono-His coordinated heme peptide containing a single covalent linkage (2). One consequence of the greater conformational restriction imposed...
Tiralongo J, Schmid H, Thun R, Iwersen M, Schauer R.Microsomes prepared from equine submandibular glands and incubated with tritium-labelled AcCoA incorporated acid-insoluble radioactivity in a manner dependent on time, protein, membrane integrity and AcCoA concentration, with incorporation being optimal at 37 degrees C and pH 6.6. Under the experimental conditions used a K(M) of 32.1 microM for AcCoA and a V(max) of 1.2 pmol/mg protein x min was obtained. The incorporation of acid-insoluble radioactivity was also inhibited by CoA in a competitive manner (K(i)=240 microM), as well as by para-chloromercuribenzoate, 3'-dephospho-CoA, 5'-IDP, 5'-A...
Franck T, Kohnen S, Deby-Dupont G, Grulke S, Deby C, Serteyn D.An original method called SIEFED (specific immunological extraction followed by enzymatic detection) was developed for the specific detection of the activity of equine myeloperoxidase (MPO). The method consists of the extraction of MPO from aqueous solutions by immobilized anti-MPO antibodies followed by washing (to eliminate proteins and interfering molecules) and measurement of MPO activity using a detection system containing a fluorogenic substrate, hydrogen peroxide, and nitrite as reaction enhancer. The SIEFED technique was applied to study active MPO in horse biological fluids and the ef...
Ho EN, Yiu KC, Wan TS, Stewart BD, Watkins KL.Anti-diabetics such as sulfonylurea and thiazolidinedione derivatives are hypoglycemic drugs used for the treatment of diabetes. However, they can also be used as a stopper in horseracing. This paper describes a convenient method for the separation and simultaneous detection of 10 anti-diabetic drugs (namely glipizide, glibenclamide, glimepiride, gliclazide, tolazamide, tolbutamide, nateglinide, repaglinide, rosiglitazone and pioglitazone) in equine plasma and urine by LC-MS-MS. Methods: The anti-diabetics were isolated from equine plasma and urine by liquid-liquid extraction with 1,2-dichloro...
Anzai T, Eguchi M, Sekizaki T, Kamada M, Yamamoto K, Okuda T.In order to establish a rapid diagnostic method for contagious equine metritis (CEM), we developed and evaluated a polymerase chain reaction (PCR) test. Species-specific PCR primer sets were derived from the DNA sequence of a cloned DNA fragment of Taylorella equigenitalis that did not hybridize with the genome of a taxomonically related species, Oligella urethralis. Single step PCR with primer set P1-N2 and two-step semi-nested PCR with primer sets P1-N2 and P2-N2 detected as low as 100 and 10 CFU of the bacteria, respectively. Single-step PCR detected T. equigenitalis from genital swabs of e...
McGuire TC, Crawford TB.Immunoglobulin A (IgA) was demonstrated in equine serum and secretions. This immunoglobulin had a molecular weight extending from 150,000 to 700,000 and reacted with specific antihuman alpha-chain antiserum. Antigenic determinants specific for secretory IgA were demonstrated and found to be absent on serum IgA. Antigen binding activity was detected in IgA from tears. Purified IgA was antigenically distinct from equine IgG, IgM, IgG(T), and aggregating immunoglobulin. Quantitative studies demonstrated that IgA was the predominant immunoglobulin in tears and milk but not in colostrum. The electr...
Duquesne F, Pronost S, Laugier C, Petry S.A direct-PCR assay was developed for the rapid detection of Taylorella equigenitalis, a Gram-negative bacterium responsible for contagious equine metritis (CEM) in Equidae. The bacteria may be detected in equine genital swabs without need for a preliminary step of DNA extraction or bacterial isolation. Specificity was determined with 125 isolates of T. equigenitalis, 24 isolates of Taylorella asinigenitalis, five commensal bacteria of the genital tract and a facultative intracellular pathogen of foals found in large concentration in soil. Our PCR is specific and amplified a 413-bp 16S ribosoma...
Bai B, Wang XF, Zhang M, Na L, Zhang X, Zhang H, Yang Z, Wang X.Tetherin is an interferon-inducible type II transmembrane glycoprotein which inhibits the release of viruses, including retroviruses, through a "physical tethering" model. However, the role that the glycosylation of tetherin plays in its antiviral activity remains controversial. In this study, we found that mutation of N-glycosylation sites resulted in an attenuation of the antiviral activity of equine tetherin (eqTHN), as well as a reduction in the expression of eqTHN at the plasma membrane (PM). In addition, eqTHN N-glycosylation mutants colocalize obviously with ER, CD63, LAMP1 and endosome...
Moyaert H, Decostere A, Vandamme P, Debruyne L, Mast J, Baele M, Ceelen L, Ducatelle R, Haesebrouck F.Gram-negative, curved, motile bacteria (strains EqF1T and EqF2) were isolated from faecal samples from two clinically healthy horses. Both strains possessed a single, monopolar, sheathed flagellum and were urease-negative. The novel strains grew at 37 degrees C under microaerobic conditions and were positive for oxidase, catalase and alkaline phosphatase activities. The isolates reduced nitrate to nitrite, but gamma-glutamyl transpeptidase activity was not detected. The novel isolates did not grow at 42 degrees C or on media containing 1 % glycine. They were resistant to cephalotin and nalidix...
Furr M, Howe D, Reed S, Yeargan M.Diagnosis of equine protozoal myeloencephalitis (EPM) remains a challenge for equine practitioners. Current utilized methods have inadequate sensitivity and specificity, because of a high number of false positive results. Objective: Evaluation of antibody indices to Sarcocystis neurona should provide high sensitivity and specificity for diagnosis of EPM. Methods: Archived samples from 29 clinical patients. Methods: Archived serum and cerebrospinal fluid (CSF) samples from clinical patients with either EPM (14) or cervical vertebral compressive myelopathy (CVM) (15) were examined and tested for...
Königová A, Várady M, Corba J.The objective of the study was to compare the in vitro egg hatch test (EHT), larval development test (LDT) and in vivo faecal egg count reduction test (FECR test) for the detection of benzimidazole resistance in equine strongyles. The presence of resistant or susceptible strongyle populations was determined in 25 stud farms using the in vivo FECR test and in vitro EHT. On the basis of the FECR values, resistance to fenbendazole was detected on 15 of the 25 farms (60%). The ED50 value (anthelmintic concentration producing 50% inhibition of hatching) for suspected resistant populations varied fr...
Palmer JL, Bertone AL, McClain H.A modification of a colorimetric assay was used to determine synovial fluid total and individual sulphated-glycosaminoglycan concentration in various clinical presentations of joint disease in horses. Concentrations of synovial fluid and serum sulphated-glycosaminoglycan (GAG) were measured by the 1,9-dimethylmethylene blue (DMMB) dye assay in normal horses (n = 49), horses with acute (n = 26) or chronic (n = 27) joint disease (defined by clinical, radiographic, and clinicopathological parameters), and horses with cartilaginous lesions at diagnostic arthroscopy, but with normal radiographs and...
Samper JC, Morris CA.Various factors affect the success of AI with frozen-thawed semen in horses. Stallion variability is thought to be one of the major factors, but semen processing and evaluation techniques, thawing protocols, packaging systems and timing of insemination are far from standardized among laboratories. Our objective was to survey current methods for stallion semen cryopreservation used commercially around the world. From the answers to the questions in the survey, we attempted to provide an overview of procedures that are standard as well as those that are used by only few laboratories and to revie...
