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.
Battaglia F, Meucci V, Tognetti R, Bonelli F, Sgorbini M, Lubas G, Pretti C, Intorre L.In human medicine, procalcitonin (PCT), the precursor of calcitonin, is used for the rapid identification of the origin and severity of sepsis. In veterinary medicine, PCT has been studied in horses, cattle, and dogs, but the use of PCT in diagnostic and/or prognostic settings is not possible because of the lack of validated assays to obtain reference ranges. The aim of the present study was the investigation of commercially available ELISA kits for the detection of canine and equine PCT in plasma samples. Validation of the ELISA kits was performed by using species-specific recombinant protein...
Sukmawinata E, Sato W, Uemura R, Kanda T, Kusano K, Kambayashi Y, Sato T, Ishikawa Y, Toya R, Sueyoshi M.In this study, the occurrence of antimicrobial-resistant (AMR) enterococci was evaluated in Thoroughbred (TB) racehorses in Japan. Fecal samples were collected from 212 healthy TB racehorses at the Miho and Ritto Training Centers of the Japan Racing Association from March 2017 to August 2018. Isolation and identification were performed by enterococcus selective medium and confirmed to the species using MALDI-TOF MS. Enterococcus faecium and E. faecalis isolates were subjected to antimicrobial susceptibility test against 11 antimicrobials by minimum inhibitory concentration based on recommenda...
Gummery L, Jallow S, Raftery AG, Bennet E, Rodgers J, Sutton DGM.Infection of equids with Trypanosoma brucei (T. brucei) ssp. is of socioeconomic importance across sub-Saharan Africa as the disease often progresses to cause fatal meningoencephalitis. Loop-mediated isothermal amplification (LAMP) has been developed as a cost-effective molecular diagnostic test and is potentially applicable for use in field-based laboratories. Threshold levels for T. brucei ssp. detection by LAMP were determined using whole equine blood specimens spiked with known concentrations of parasites. Results were compared to OIE antemortem gold standard of T. brucei-PCR (TBR-PCR). Th...
Kinsley R, Pronost S, De Bock M, Temperton N, Daly JM, Paillot R, Scott S.Equine influenza is a major respiratory disease of horses that is largely controlled by vaccination in some equine populations. Virus-neutralising antibodies, the mainstay of the protective immune response, are problematic in assaying for equine influenza virus, as most strains do not replicate efficiently in cell culture. Surrogate measures of protective antibody responses include the haemagglutination inhibition (HI) test and single radial haemolysis (SRH) assay. For this study, a pseudotyped virus, bearing an envelope containing the haemagglutinin (HA) from the Florida clade 2 equine influe...
Fukuda K, Kuroda T, Tamura N, Mita H, Kasashima Y.Platelet-rich plasma (PRP) therapy has been widely applied in various medical fields including humans and horses. This study aimed to establish an optimal activation method to stably and reproducibly maximize the concentrations of platelet-derived growth factor-BB (PDGF-BB) and transforming growth factor-β1 (TGF-β1) contained in equine PRP. Autologous PRP was prepared from 11 Thoroughbreds. For the activation test, PRP was activated by either a single freeze-thaw cycle (Fr) or adding calcium and autologous serum containing thrombin (Ca). PDGF-BB and TGF-β1 concentrations in Fr, Ca, nonactiv...
Karam B, Hines S, Skipper L, Pusterla N.Serum amyloid A (SAA) is considered a major acute phase protein (APP) in horses. Serum amyloid A stall-side assays are commercially available to assess the inflammatory response of patients with various infectious and noninfectious conditions. The objective of this study was to determine the analytical performance of a new point-of-care (POC) assay for the measurement of SAA in whole blood and plasma of horses. One hundred and sixty blood samples were collected from 60 horses at various time points after immunization with an equine core vaccine. Analytical validation of the SAA POC assay inclu...
Baieli MF, Urtasun N, Hirsch DB, Miranda MV, Cascone O, Wolman FJ.Equine chorionic gonadotrophin (eCG) is a hormone widely used in superovulation protocols because of its follicle-stimulating action, which increases reproductive efficiency in animals of productive interest. It contains 45% carbohydrate, 10% of which is N-acetylneuraminic acid (sialic acid). The eCG purification procedures from equine serum or plasma are mainly based on chromatographic methods. However, before these procedures, it is necessary to follow sample pre-conditioning steps, such as several precipitation stages and/or ultrafiltration/diafiltration processes. In this work, an efficien...
Peters S, Wilson J, Boyette J.The objective of this study was to determine the influence of age on expression of insulin-like growth factor 1 (IGF1), insulin-like growth factor binding protein (IGFBP5), myostatin (MSTN), and myosin (MYH1) genes which are related to growth and muscle development in the American Quarter Horse. Thus, horses (n = 10) from weanling, yearling, 2-, 3-, and 10-year-old age classes were sampled and gene expression was assessed by RT-qPCR. ΔC was calculated using the hypoxanthine-guanine phosphoribosyltransferase gene as an internal normalizer. The generalized linear model was used to determine di...
Chapman GE, Sherlock K, Hesson JC, Blagrove MSC, Lycett GJ, Archer D, Solomon T, Baylis M.There has been no evidence of transmission of mosquito-borne arboviruses of equine or human health concern to date in the UK. However, in recent years there have been a number of outbreaks of viral diseases spread by vectors in Europe. These events, in conjunction with increasing rates of globalisation and climate change, have led to concern over the future risk of mosquito-borne viral disease outbreaks in northern Europe and have highlighted the importance of being prepared for potential disease outbreaks. Here we assess several UK mosquito species for their potential to transmit arboviruses ...
Gerhard AP, Krücken J, Heitlinger E, Janssen IJI, Basiaga M, Kornaś S, Beier C, Nielsen MK, Davis RE, Wang J, von Samson-Himmelstjerna G.P-glycoproteins (Pgp) have been proposed as contributors to the widespread macrocyclic lactone (ML) resistance in several nematode species including a major pathogen of foals, Parascaris univalens. Using new and available RNA-seq data, ten different genomic loci encoding Pgps were identified and characterized by transcriptome-guided RT-PCRs and Sanger sequencing. Phylogenetic analysis revealed an ascarid-specific Pgp lineage, Pgp-18, as well as two paralogues of Pgp-11 and Pgp-16. Comparative gene expression analyses in P. univalens and Caenorhabditis elegans show that the intestine is the maj...
González-Medina S, Hyde C, Lovera I, Piercy RJ.Measurement of hypoglycin A (HGA) and its toxic metabolite, methylenecyclopropylacetic acid (MCPA), in equine serum confirms a diagnosis of atypical myopathy (AM), a pasture-associated toxic rhabdomyolysis with high mortality linked to the ingestion of Acer trees plant material. Supportive diagnostic tests include plasma acyl-carnitine profiling and urine organic acid testing, but these are not specific for AM. Previously reported HGA and MCPA analytical techniques used liquid chromatography-mass spectrometry (LC-MS) with a derivatising step, but the latter prolongs testing and increases costs...
Ishii H, Leung GN, Yamashita S, Yamada M, Kushiro A, Kasashima Y, Okada J, Kawasaki K, Kijima-Suda I.GW1516 is a peroxisome proliferator-activated receptor-δ agonist in the class of hormones and metabolic modulators. The use of GW1516 is banned in both horseracing and equestrian competitions. To the best of our knowledge, this is the first metabolic study of GW1516 in horses. Methods: After protein precipitation of pre- and post-administration plasma GW1516 samples, the supernatants were analyzed using liquid chromatography/electrospray ionization Q-Exactive high-resolution mass spectrometry to detect GW1516 and its metabolites. Monoisotopic ions of GW1516 and its metabolites were monitored ...
Cain JL, Slusarewicz P, Rutledge MH, McVey MR, Wielgus KM, Zynda HM, Wehling LM, Scare JA, Steuer AE, Nielsen MK.Fecal egg counts are the cornerstone of equine parasite control programs. Previous work led to the development of an automated, image-analysis-based parasite egg counting system. The system has been further developed to include an automated reagent dispenser unit and a custom camera (CC) unit that generates higher resolution images, as well as a particle shape analysis (PSA) algorithm and machine learning (ML) algorithm. The first aim of this study was to conduct a comprehensive comparison of method precision between the original smartphone (SP) unit with the PSA algorithm, CC/PSA, CC/ML, and ...
Tozaki T, Ohnuma A, Takasu M, Nakamura K, Kikuchi M, Ishige T, Kakoi H, Hirora KI, Tamura N, Kusano K, Nagata SI.Gene doping has raised concerns in human and equestrian sports and the horseracing industry. There are two possible types of gene doping in the sports and racing industry: (1) administration of a gene-doping substance to postnatal animals and (2) generation of genetically engineered animals by modifying eggs. In this study, we aimed to identify genetically engineered animals by whole-genome resequencing (WGR) for gene-doping control. Transgenic cell lines, in which the erythropoietin gene (EPO) cDNA form was inserted into the genome of horse fibroblasts, were constructed as a model of genetica...
Mousa WM, Abdel-Wahab AM, El-Gameel Sohila M, Mahdy OA.Cystic echinococcosis is an important cosmopolitan parasitic zoonosis that causes public health and economic problems in Egypt. The present study was undertaken to identify genotypes of hydatid cyst (HC) DNA isolated from different animal isolates and to identify the genotype of secondary hydatid cysts (HCs) developed in rabbits experimentally infected with camel HC for detection of any genetic mutation. In the present study, we extracted DNA from the germinal layers of 8 HCs collected from 3 camels, 1 cattle, 1 sheep and 3 donkeys in addition to 3 secondary HCs collected from rabbits experime...
van Damme CMM, van den Broek J, Sloet van Oldruitenborgh-Oosterbaan MM.In equine atopic patients intradermal testing (IDT) and immunoglobulin (Ig)E serology are used frequently. There is little evidence regarding the reproducibility of the IDT and IgE serology in horses. Objective: To compare the results of a simultaneously performed IDT on the left and right side of the neck in atopic horses, and to compare these results with allergen-specific IgE serology. Methods: Ten equine patients from a university hospital population with chronic urticaria and/or pruritus. Methods: The IDT was performed using 16 allergens and the results were evaluated after 30 min, 1, 4 ...
Fagre AC, Mayo CE, Pabilonia KL, Landolt GA.Detection of is difficult as a result of intermittent leptospiruria and brief leptospiremia. Hence, diagnosis relies heavily on serologic testing, the reference method of which is the microscopic agglutination test (MAT). In horses, clinical leptospirosis has been associated with abortion, recurrent uveitis, and sporadic cases of hepatic and renal disease. Little information exists on the seroprevalence of antibodies to in equids in the United States; past nationwide studies suggest that the seroprevalence in some areas is as high as 77% (reciprocal titer ≥ 100). We tested sera from 124 ...
Zibura AE, Cullen MA, Rutledge H, Lassalle L, Salmon JH, Gilger BC, Westermeyer HD.Determine optimal iontophoresis times for riboflavin delivery to the corneal stroma across different species and compare these to corneal injection. Methods: Ex vivo horse, dog, rabbit, and pig globes were treated with riboflavin administered with either iontophoresis for 2.5-20 minutes with or without corneal epithelium; or with purpose-designed precise corneal injection (PCI) application with intact epithelium. Immediately following riboflavin administration, samples were harvested, frozen, and sectioned. Riboflavin penetration was imaged using fluorescence microscopy. Results: Horse sample...
Chan GHM, Tang W, Curl P, Lin Y, Wan TSM, Ho ENM.Arsenic can be easily found in our surrounding environment. Because of its ubiquitous nature, horse urine and blood invariably contain low levels of arsenic. Nevertheless, inorganic arsenic, despite its general use as a tonic for horses, is an effective doping agent having a deleterious effect because of its ability to induce gastroenteritis. The misuse of arsenic in horseracing has been controlled by an international urinary threshold of total arsenic at 0.3 μg/mL. However, an equivalent threshold for total arsenic in plasma is yet to be established. In this study, an inductively coupled pla...
Kwok KY, Choi TLS, Kwok WH, Lau MY, Leung EMK, Leung GNW, Wong JKY, Wan TSM, Adrian FF, Prabhu A, Ho ENM.The use of bioactive peptides as a doping agent in both human and animal sports has become increasingly popular in recent years. As such, methods to control the misuse of bioactive peptides in equine sports have received attention. This paper describes a sensitive accurate mass method for the detection of 40 bioactive peptides and two non-peptide growth hormone secretagogues (< 2 kDa) at low pg/mL levels in horse urine using ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC/HRMS). A simple mixed-mode cation exchange solid-phase extraction (SPE) cartridge was...
Leung DKK, Wong ASY, Zhou QL, Wan TSM, Ho ENM.A non-target variable Data Independent Acquisition (vDIA) workflow based on accurate mass measurements using a Q Exactive OrbiTrap is presented for the first time for equine doping control testing. The vDIA workflow uses a combination of MS1 events (1 to 2) and multiple vDIA events to cover the analytes of interest. The workflow basically captures a digital image of a sample allowing all relevant MS1 and MS2 data to be recorded. In theory, the workflow can accommodate an unlimited number of analytes as long as they are amenable to the sample extraction protocol and fall within the mass limits ...
Garcia P, Pinètre J, Morel S, Jaubert M, Deruy X, Perot I, Delcourt V, Loup B, Popot MA, Bailly-Chouriberry L.Bisphosphonates are prohibited drugs according to Article 6 of the International Agreement on Breeding, Racing and Wagering of the International Federation of Horseracing Authorities (IFHA) and the International Equestrian Federation (FEI). These compounds are used for the treatment of lameness, navicular and bone diseases in horses and are divided into two groups: non-nitrogen-containing bisphosphonate drugs (e.g. clodronic acid) and nitrogen-containing bisphosphonate drugs (e.g. zoledronic acid). Their hydrophilic properties and the high affinity for the bone matrix make the control of their...
Tomlinson JE, Wolfisberg R, Fahnøe U, Sharma H, Renshaw RW, Nielsen L, Nishiuchi E, Holm C, Dubovi E, Rosenberg BR, Tennant BC, Bukh J, Kapoor A....Pegiviruses frequently cause persistent infection (as defined by >6 months), but unlike most other Flaviviridae members, no apparent clinical disease. Human pegivirus (HPgV, previously GBV-C) is detectable in 1-4% of healthy individuals and another 5-13% are seropositive. Some evidence for infection of bone marrow and spleen exists. Equine pegivirus 1 (EPgV-1) is not linked to disease, whereas another pegivirus, Theiler's disease-associated virus (TDAV), was identified in an outbreak of acute serum hepatitis (Theiler's disease) in horses. Although no subsequent reports link TDAV to disease, an...
Cruz A, Sullivan DB, Doty KF, Hess RA, Canisso IF, Reddi PP.The acrosome plays a critical role in sperm-oocyte interactions during fertilization. SP-10 is an acrosomal matrix protein, which is evolutionarily conserved among mammals. The SP-10 antibody has been shown to be useful for staging the seminiferous cycle in the mouse and human. A canonical acrosomal marker; however, has never been used for staging in the horse. The objectives of the present study were to investigate the presence of SP-10 within the horse acrosome using an anti-mouse SP-10 antibody, to classify spermatids based on the shape of the acrosome, and then to use that information to a...
Chidlow H, Giguère S, Camus M, Wells B, Berghaus R, McConachie Beasley E.Serial cerebrospinal fluid (CSF) analysis might be required in clinical neurologic disease. The effect of lumbosacral (LS) or cervical (C1-C2) centesis on subsequent CSF cytologic analyses has not been investigated in horses. Objective: To evaluate the effect of thecal puncture on subsequent CSF analyses ANIMALS: Ten healthy adult horses. Methods: Prospective study. Horses were randomly assigned to undergo CSF collection twice, 14 days apart, from either the C1-C2 or LS space. After a 4-month washout period, CSF collection was repeated from the alternate site. Continuous data were analyzed u...
Marr N, Hopkinson M, Hibbert AP, Pitsillides AA, Thorpe CT.Three-dimensional imaging modalities for optically dense connective tissues such as tendons are limited and typically have a single imaging methodological endpoint. Here, we have developed a bimodal procedure utilising fluorescence-based confocal microscopy and x-ray micro-computed tomography for the imaging of adult tendons to visualise and analyse extracellular sub-structure and cellular composition in small and large animal species. Results: Using fluorescent immunolabelling and optical clearing, we visualised the expression of the novel cross-species marker of tendon basement membrane, lam...
Göktaş EF, Kabil E, Arıöz F.Nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly used in therapeutic doses in human and veterinary medicine for the treatment of inflammation, pain, and fever. A method for the simultaneous determination of nine NSAIDs, known as therapeutic prohibited substances, in equine urine was developed and fully validated according to the European Commission Decision 2002/657/EC and Association of Official Racing Chemists criteria. The validation was performed for naproxen, flunixin, ketoprofen, diclofenac, eltenac, meclofenamic acid, phenylbutazone, vedaprofen, and carprofen in equine urine i...
Donkey placenta does not allow the passage of immunoglobulins; thus, foals are born hypogammaglobulinemic and an adequate intake of high-quality colostrum in the first 24 hours of life is crucial for the surviving. The study aims to assess the relation between colostrum immunoglobulin G (IgG) concentration evaluated by the single radial immunodiffusion (SRID) test and the Brix refractometer in donkeys to establish a cutoff value for high quality of colostrum based on Brix refractometry. Colostrum was collected at foaling, and at 6, 12, and 24 hours after foaling from the left and the right h...
Lombardi F, Palumbo P, Augello FR, Giusti I, Dolo V, Guerrini L, Cifone MG, Giuliani M, Cinque B.The ability of a collagen-based matrix to support cell proliferation, migration, and infiltration has been reported; however, the direct effect of an aqueous collagen suspension on cell cultures has not been studied yet. In this work, the effects of a high-concentration aqueous suspension of a micronized type I equine collagen (EC-I) have been evaluated on a normal mouse fibroblast cell line. Immunofluorescence analysis showed the ability of EC-I to induce a significant increase of type I and III collagen levels, parallel with overexpression of crucial proteins in collagen biosynthesis, matura...
Aitken RJ, Gregoratos D, Kutzera L, Towney E, Lin M, Wilkins A, Gibb Z.MTT is widely used in biology as a probe for cell viability by virtue of its ability to generate deposits of insoluble formazan at sites of intense oxidoreductase activity. This response is generally held to reflect mitochondrial redox activity; however, extra-mitochondrial MTT reduction has also been recorded in certain cell types. Given this background, we set out to determine the major sites of formazan deposition in mammalian spermatozoa. In the mouse, most MTT reduction took place within the extensive mitochondrial gyres, with a single minor site of formazan deposition on the sperm head. ...
Morotomi M, Yuki N, Kado Y, Kushiro A, Shimazaki T, Watanabe K, Yuyama T.Lactobacillus equi sp. nov. is described on the basis of 18 strains isolated as one of the predominant intestinal lactobacilli from horse faecal specimens. These 18 strains were isolated from 10 horses of 6 different farms out of 20 horses of 10 farms examined. They were gram-positive, facultatively anaerobic, catalase-negative, non-spore-forming, non-motile, lactic-acid-homofermentative rods. The DNA G+C content was 38.9+/-0.8 mol %. DNA-DNA hybridization failed to associate these strains closely with any of the validly described type strains used. Analysis of the 16S rRNA gene sequence of re...
Boyle AG, Rankin SC, D￮ L, Boston RC, Wheeler-Aceto H.Bacterial culture and polymerase chain reaction (PCR) assays for the detection of Streptococcus equi in nasopharyngeal washes (NPW) and guttural pouch lavage (GPL) samples have low sensitivity. In human diagnostics, processing of samples with flocked swabs has improved recovery rates of bacterial agents because of improved surface area and elution factors. Objective: For S. equi subsp. equi (S. equi) detection in NPW and GPL samples we hypothesized that: direct-PCR would be more reliable than flocked swab culture (FS culture); flocked swab PCR (FS-PCR) would be equivalent to direct-PCR; and FS...
Tung JT, Venta PJ, Caron JP.To determine the effects of recombinant equine IL-1beta and a number of antiinflammatory compounds on the expression and activity of inducible nitric oxide synthase (iNOS) in cultured equine chondrocytes. Methods: RT-PCR methods were used to amplify a portion of the equine iNOS message to prepare an RNA probe. Northern blot analysis was used to quantify the expression of iNOS in first passage cultures of equine articular chondrocytes propagated in the presence or absence of recombinant equine interleukin-1beta (reIL-1beta), dexamethasone (DEX), polysulfated glycosaminoglycan (PSGAG), hyalurona...
Prins M, van Leeuwen MW, Teske E.Modern laser-based haematology analysers such as the ADVIA 120 have species-specific software and offer the possibility of assessing new haematological parameters. These parameters have yet to be evaluated, and as these analysers are often used in referral laboratories, it is important to know whether the values of haematological parameters change during sample transport. Therefore, samples of EDTA-anticoagulated blood from nine healthy dogs and EDTA- and citrate-anticoagulated blood from six healthy horses were collected and stored at room temperature for 72 and 48 hours, respectively. In can...
Dhorne-Pollet S, Barrey E, Pollet N.Mitochondrial DNA is remarkably polymorphic. This is why animal geneticists survey mitochondrial genomes variations for fundamental and applied purposes. We present here an approach to sequence whole mitochondrial genomes using nanopore long-read sequencing. Our method relies on the selective elimination of nuclear DNA using an exonuclease treatment and on the amplification of circular mitochondrial DNA using a multiple displacement amplification step. Results: We optimized each preparative step to obtain a 100 million-fold enrichment of horse mitochondrial DNA relative to nuclear DNA. We sequ...
Aho R.Hair samples from domestic and laboratory animals with suspected dermatophytosis were examined for the presence of saprophytic fungi. A nutritionally poor base medium, developed by the author, was used in the isolation and identification of the saprophytes. Three hundred and ninety-four specimens were examined of which 246 were from dogs, 75 from cats, 30 from horses, 19 from cows, 12 from guinea pigs, 5 from rats, 2 from parakeets, 2 from chinchillas and one each from a goat, a mink and a lesser panda (Ailurus fulgens). Moulds classified in 32 genera were isolated. The commonest in order of f...
Adeyefa CA, Quayle K, McCauley JW.We describe a rapid method for genetic characterisation of influenza virus genes using reverse transcription and amplification by polymerase chain reaction (RT/PCR) of all virus segments simultaneously (multiplex RT/PCR) using primers based on the conserved terminal sequences. The product has been shown to be suitable for determination of partial nucleotide sequences which can be used to search nucleotide sequence databases and rapidly map the genetic origin of each segment. We illustrate the use of the method by analysing genetic reassortment in H7N7 equine influenza viruses.
Salamon Z, Tollin G.Complex formation between horse heart cytochrome c (cyt c) and bovine cytochrome c oxidase (cco) incorporated into a supported planar egg phosphatidylcholine membrane containing varying amounts of cardiolipin (CL) (0-20 mol%) has been studied under low (10 mM) and medium (160 mM) ionic strength conditions by surface plasmon resonance (SPR) spectroscopy. Both specific and nonspecific modes of cyt c binding are observed. The dissociation constant of the specific interaction between cyt c and cco increases from approximately 6.5 microM at low ionic strength to 18 microM at medium ionic strength, ...
Moe KK, Yano T, Kuwano A, Sasaki S, Misawa N.Equine canker is a chronic pododermatitis of the hoof in horses. Although spirochetes are detectable histopathologically in the lesions, the precise etiology remains unclear. This study reports the 16S rRNA gene sequencing of randomly selected clones based on PCR with Treponema-specific primers, using the canker lesions from two horses and healthy frog and sole from a horse. A total of 114 clones were obtained from the lesions, but no clones were detected in the healthy hoof tissues. The clones from the canker lesions examined were grouped into 19 operational taxonomic units, such as treponema...
Pilling A, Davison AJ, Telford EA, Meredith DM.Glycoprotein 45 is a major envelope glycoprotein of equine herpesvirus type 1. The gene encoding this protein is located between map units 0.615 and 0.636 on the virus genome and evidence has suggested that it is encoded by gene 52, one of four genes within this region. Using PCR we have amplified gene 52 and subsequently cloned it into a mammalian expression vector under the control of the human cytomegalovirus immediate early gene promoter. The gene was expressed in COS-7 cells and its product was detected by immunofluorescence and Western blotting. The results indicate that glycoprotein 45 ...
Muñoz PM, Ferrusola CO, Lopez LA, Del Petre C, Garcia MA, de Paz Cabello P, Anel L, Peña FJ.Stallion-to-stallion variability in the quality of cryopreserved ejaculates postthaw affects the commercial acceptability of frozen semen and thus is a major constraint for the equine industry. In recent years, the molecular mechanisms associated with sperm damage during cryopreservation have become better understood. Identification of the freezability of the ejaculates before the freezing process is initiated will have a major impact on the equine industry. We studied three markers of oxidative stress in sperm, including 8-iso-PGF2alpha, 8-OH guanosine, and 4-hydroxynonenal (4-HNE); the prese...
Ballagi-Pordány A, Klingeborn B, Flensburg J, Belák S.Primers and probes were selected from the gene encoding glycoprotein 13 (gp 13) of equine herpesvirus 1 (EHV-1). The polymerase chain reaction (PCR) was run on infected and noninfected cultured cells and on 63 specimens from 29 aborted equine fetuses. The results were evaluated by electrophoresis and dot-blot hybridization using an oligonucleotide probe labeled with biotin. In the infected samples electrophoresis showed a PCR product of about 280 base pairs. The dot-blot hybridization confirmed that this product contained EHV-1 DNA sequences. PCR took 4 h and hybridization another 14 h; the re...
Biswas B, Mukherjee D, Mattingly-Napier BL, Dutta SK.Genomic amplification by the polymerase chain reaction (PCR) was used to identify a unique genomic sequence of Ehrlichia risticii directly in DNA isolated from peripheral-blood buffy coat cells of E. risticii-infected horses (Potomac horse fever) and from infected cell cultures. A specific primer pair, selected from a cloned, species-specific, 1-kb DNA fragment of the E. risticii genome as a template, was used for the amplification of the target DNA of 247 bp. The optimal number of 40 PCR cycles, determined by analyzing an amplification profile obtained with a constant Taq polymerase concentra...
Payne SL, La Celle K, Pei XF, Qi XM, Shao H, Steagall WK, Perry S, Fuller F.The Wyoming strain of equine infectious anaemia virus (EIAV) is a highly virulent field strain that replicates to high titre in vitro only in primary equine monocyte-derived macrophages. In contrast, Wyoming-derived fibroblast-adapted EIAV strains (Malmquist virus) replicate in primary foetal equine kidney and equine dermis cells as well as in the cell lines FEA and Cf2Th. Wyoming and Malmquist viruses differ extensively both in long terminal repeat (LTR) and envelope region sequences. We have compared the promoter activities of the Wyoming LTR with those of LTRs derived from fibroblast-adapte...
Coultous RM, Phipps P, Dalley C, Lewis J, Hammond TA, Shiels BR, Weir W, Sutton DGM.Equine piroplasmosis (EP) has historically been of minor concern to UK equine practitioners, primarily due to a lack of competent tick vectors. However, increased detection of EP tick vector species in the UK has been reported recently. EP screening is not currently required for equine importation, and when combined with recent relaxations in movement regulations, there is an increased risk regarding disease incursion and establishment into the UK. This study evaluated the prevalence of EP by both serology and PCR among 1242 UK equine samples submitted for EP screening between February and Dec...
de Winter NJ, Snoeck C, Claeys P.The study of stable isotopes in fossil bioapatite has yielded useful results and has shown that bioapatites are able to faithfully record paleo-environmental and paleo-climatic parameters from archeological to geological timescales. In an effort to establish new proxies for the study of bioapatites, intra-tooth records of enamel carbonate stable isotope ratios from a modern horse are compared with trace element profiles measured using laboratory micro X-Ray Fluorescence scanning. Using known patterns of tooth eruption and the relationship between stable oxygen isotopes and local temperature se...
Barrey E, Jayr L, Mucher E, Gospodnetic S, Joly F, Benech P, Alibert O, Gidrol X, Mata X, Vaiman A, Guérin G.Recurrent exertional rhabdomyolysis (RER) is frequently observed in race horses like trotters. Some predisposing genetic factors have been described in epidemiological studies. However, the exact aetiology is still unknown. A calcium homeostasis disruption was suspected in previous experimental studies, and we suggested that a transcriptome analysis of RER muscles would be a possible way to investigate the pathway disorder. The purpose of this study was to compare the gene expression profile of RER vs. control muscles in the French Trotter to determine any metabolic or structural disruption. T...
Hidalgo M, Rodríguez I, Dorado J, Soler C.This work used semen samples collected from 12 stallions and assessed for sperm morphometry by the Sperm Class Analyzer (SCA) computer-assisted system. A discriminant analysis was performed on the morphometric data from that sperm to obtain a classification matrix for sperm head shape. Thereafter, we defined six types of sperm head shape. Classification of sperm head by this method obtained a globally correct assignment of 90.1%. Moreover, significant differences (p<0.05) were found between animals for all the sperm head morphometric parameters assessed.
Parker RA, Clegg PD, Taylor SE.To investigate the effects of commonly used antibiotics on cell viability and gene expression of equine bone marrow-derived mesenchymal stromal cells (MSC) in vitro. Methods: Bone marrow-derived MSC were cultured in media containing gentamicin, amikacin, penicillin, enrofloxacin or ceftiofur at concentrations of 50, 100, 200 and 500 µg/ml. The alamarBlue fluorescence assay was used to assess cell viability over 48 h. After 5 days the cells were released and lysed prior to RNA extraction and reverse transcription. RNA levels were assessed using spectrophotometry and quantitative PCR was used...
Tassone S, Renna M, Barbera S, Valle E, Fortina R.Successful studies on in vitro digestibility measurement of feedstuffs with fecal inoculum have been reported for horses, whereas data on donkeys are currently lacking. In this study, we evaluated the use of the Daisy Incubator for in vitro digestibility measurement of feedstuffs using donkey feces as source of microbial inoculum. The method was tested using seven feedstuffs commonly used in donkey diets (alfalfa, bromegrass, ryegrass, and timothy hays; wheat bran and wheat straw; barley grains). Feces were obtained from four female donkeys, and incubations were carried out at 1-week interva...
Certoma A, Lunt RA, Vosloo W, Smith I, Colling A, Williams DT, Tran T, Blacksell SD.Australian bat lyssavirus (ABLV) is closely related to the classical rabies virus and has been associated with three human fatalities and two equine fatalities in Australia. ABLV infection in humans causes encephalomyelitis, resulting in fatal disease, but has no effective therapy. The virus is maintained in enzootic circulation within fruit bats ( spp.) and at least one insectivorous bat variety ( ). Most frequently, laboratory testing is conducted on pteropodid bat brains, either following a potential human exposure through bites, scratches and other direct contacts with bats, or as opportun...
Cook RF, Gann SJ, Mumford JA.An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of serum antibodies to equine arteritis virus (EAV). Results from this assay produced a good correlation with results from virus neutralisation tests in horses which had not been regularly vaccinated with commercially available mammalian tissue culture-derived viral vaccines. Vaccination of some horses with tissue culture-derived vaccines induced the formation of antibodies to bovine serum. These antibodies reacted with the bovine protein contaminants in the EAV ELISA antigen, producing false-positive results. Non-vir...
Centromeres exert an inhibitory effect on meiotic recombination, but the possible contribution of satellite DNA to this "centromere effect" is under debate. In the horse, satellite DNA is present at all centromeres with the exception of the one from chromosome 11. This organization of centromeres allowed us to investigate the role of satellite DNA on recombination suppression in horse spermatocytes at the stage of pachytene. To this aim we analysed the distribution of the MLH1 protein, marker of recombination foci, relative to CENP-A, marker of centromeric function. We demonstrated that the sa...
Bromfield EG, Aitken RJ, Gibb Z, Lambourne SR, Nixon B.While IVF has been widely successful in many domesticated species, the development of a robust IVF system for the horse remains an elusive and highly valued goal. A major impediment to the development of equine IVF is the fact that optimised conditions for the capacitation of equine spermatozoa are yet to be developed. Conversely, it is known that stallion spermatozoa are particularly susceptible to damage arising as a consequence of capacitation-like changes induced prematurely in response to semen handling and transport conditions. To address these limitations, this study sought to develop a...
Ozaki H, Sugiura T, Sugita S, Imagawa H, Kida H.Antibodies to the nonstructural protein (NS1) of A/equine/Miami/1/63 (H3N8) influenza virus were detected exclusively in the sera of mice experimentally infected with A/Aichi/2/68 (H3N2) and horses infected with A/equine/Kentucky/1/81 (H3N8) or A/equine/La Plata/1/93 (H3N8), but not in those of the animals immunized with the inactivated viruses, by enzyme-linked immunosorbent assay (ELISA) using a recombinant NS1 as antigen. The results indicate that the present method is useful for serological diagnosis to distinguish horses infected with equine H3 influenza viruses from those immunized with ...
Ortega-Ferrusola C, Martin Muñoz P, Ortiz-Rodriguez JM, Anel-López L, Balao da Silva C, Álvarez M, de Paz P, Tapia JA, Anel L, Silva-Rodríguez A....We hypothesized that thiols and particularly glutathione (GSH) are essential for the regulation of stallion sperm functionality. To test this hypothesis, we initially investigated the relationship between sperm function and GSH content, revealing highly significant correlations between GSH, sperm viability, motility, and velocity parameters (P < 0.001). Furthermore, the deleterious effects of GSH depletion using menadione and 1,3 dimethoxy 1,4, naphtoquinone (DMNQ) were able to be prevented by the addition of cysteine, but no other antioxidant. Pre-incubation with cysteine prevented menad...
Pusterla N, Madigan JE, Leutenegger CM.The focus of rapid diagnosis of infectious disease of horses in the last decade has shifted from the conventional laboratory techniques of antigen detection, microscopy, and culture to molecular diagnosis of infectious agents. Equine practitioners must be able to interpret the use, limitations, and results of molecular diagnostic techniques, as they are increasingly integrated into routine microbiology laboratory protocols. Polymerase chain reaction (PCR) is the best-known and most successfully implemented diagnostic molecular technology to date. It can detect slow-growing, difficult-to-cultiv...
Hamada D, Kuroda Y, Kataoka M, Aimoto S, Yoshimura T, Goto Y.One unique aspect of cytochrome c folding concerns the involvement of the covalently attached heme group and its axial ligands. To elucidate the role of the ligands in stabilizing the native and molten globule states, we studied the conformational and thermodynamic features of the iron-free derivative of horse cyctochrome c (porphyrin-cytochrome c). At neutral pH, far-UV circular dichroism suggested that porphyrin-cytochrome c has native-like alpha-helices, whereas near-UV CD suggested that the side-chains are flexible. Its stability against heat or denaturants was much less than that of the i...
