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.
Roest HI, de Bruijn CM, Picavet MT, Prins B, Parmentier D, de Zwart GM, Dijkstra YE, van Zijderveld FG.Symptoms, diagnosis and therapy of equine botulism are discussed by the presentation of two detailed reports of horses with neurological symptoms and the results of laboratory investigations over the period 2003-2008 in the Netherlands. In addition a brief summary of the available literature is presented. Prevailing symptoms of botulism in horses include paralysis of the tongue, salvation, dysphagia and paresis and paralysis of the skeletal muscles, as well as signs of colic. Symptoms and prognosis vary with the amount of botulinum neurotoxin (BoNT) involved. For early clinical diagnosis of bo...
Laurencin D, Wong A, Chrzanowski W, Knowles JC, Qiu D, Pickup DM, Newport RJ, Gan Z, Duer MJ, Smith ME.Despite the numerous studies of bone mineral, there are still many questions regarding the exact structure and composition of the mineral phase, and how the mineral crystals become organised with respect to each other and the collagen matrix. Bone mineral is commonly formulated as hydroxyapatite, albeit with numerous substitutions, and has previously been studied by (31)P and (1)H NMR, which has given considerable insight into the complexity of the mineral structure. However, to date, there has been no report of an NMR investigation of the other major component of bone mineral, calcium, nor of...
Dzieciatkowski T, Przybylski M, Cymerys J, Turowska A, Chmielewska A, Tucholska A, Banbura MW.Equine herpesvirus type 1 (EHV-1) is one of the major viral agents causing diseases in horses common worldwide. A variety of techniques, including PCR, have been used to diagnose EHV-1 infections. In this paper, an attempt of real-time PCR has been described, which uses specific fluorochrome-labeled TaqMan probes for detection of viral DNA. This method does not require post-amplification manipulations, thereby reducing the risk of cross-contamination. The assay was sensitive enough to detect EHV-1 sequences in different clinical samples, as well in mice neuronal cell cultures. The technique wa...
Jacobi S, Townsend WM, Bolin CA.To evaluate whether equine serum administered via a simulated subpalpebral lavage system (SPL) supports proliferation of Streptococcus zooepidemicus or Pseudomonas aeruginosa within the tubing. Methods: A sterile i.v. catheter with injection cap was inserted into sterilized silicone tubing (Mila). To mimic an SPL within the dorsal conjunctival fornix, the tubing was secured to an elevated platform. The tip of the tubing extended from the platform into a vial containing culture medium just inoculated with approximately 1.5 x 10(8) CFU/mL P. aeruginosa or S. zooepidemicus. To mimic administratio...
Leung GN, Tang FP, Wan TS, Wong CH, Lam KK, Stewart BD.This paper describes the application of gas chromatography-mass spectrometry (GC-MS) for in vitro and in vivo studies of 6-OXO in horses, with a special aim to identify the most appropriate target metabolite to be monitored for controlling the administration of 6-OXO in racehorses. In vitro studies of 6-OXO were performed using horse liver microsomes. The major biotransformation observed was reduction of one keto group at the C3 or C6 positions. Three in vitro metabolites, namely 6alpha-hydroxyandrost-4-ene-3,17-dione (M1), 3alpha-hydroxyandrost-4-ene-6,17-dione (M2a) and 3beta-hydroxyandrost-...
de la Rebière de Pouyade G, Franck T, Salciccia A, Deby-Dupont G, Grulke S, Heyden LV, Sandersen C, Serteyn D.Equine neutrophil elastase (NE) is a protease released in inflammatory diseases and participating in tissue destruction. To measure NE in horse plasma to assess its role in pathological conditions, we purified elastase from equine neutrophils by a double step chromatography and obtained a pure protein of 27 kDa, 4 kDa smaller than the NE 2A previously purified (Scudamore et al., 1993; Dagleish et al., 1999), which was likely to be NE 2B. We developed an ELISA by using two specific polyclonal antibodies obtained from rabbit and guinea pig. The sandwich complex was detected using a secondary ant...
Scarth JP, Spencer HA, Hudson SC, Teale P, Gray BP, Hillyer LL.In this study, the use of equine liver/lung microsomes and S9 tissue fractions were used to study the metabolism of the androgenic/anabolic steroid stanozolol as an example of the potential of in vitro technologies in sports drug surveillance. In vitro incubates were analysed qualitatively alongside urine samples originating from in vivo stanozolol administrations using LC-MS on a high-resolution accurate mass Thermo Orbitrap Discovery instrument, by LC-MS/MS on an Applied Biosystems Sciex 5500 Q Trap and by GC-MS/MS on an Agilent 7000A. Using high-resolution accurate mass full scan analysis o...
Schuurman W, Gawlitta D, Klein TJ, ten Hoope W, van Rijen MH, Dhert WJ, van Weeren PR, Malda J.If chondrocytes from the superficial, middle, and deep zones of articular cartilage could maintain or regain their characteristic properties during in vitro culture, it would be feasible to create constructs comprising these distinctive zones. Objective: Zone-specific characteristics of zonal cell populations will disappear during 2-dimensional expansion but will reappear after 3-dimensional redifferentiation, independent of the culture technique used (alginate beads versus pellet culture). Methods: Controlled laboratory study. Methods: Equine articular chondrocytes from the 3 zones were expan...
Lu Z, Chambers TM, Boliar S, Branscum AJ, Sturgill TL, Timoney PJ, Reedy SE, Tudor LR, Dubovi EJ, Vickers ML, Sells S, Balasuriya UB.The objective of this study was to develop and evaluate new TaqMan real-time reverse transcription-PCR (rRT-PCR) assays by the use of the minor groove binding probe to detect a wide range of equine influenza virus (EIV) strains comprising both subtypes of the virus (H3N8 and H7N7). A total of eight rRT-PCR assays were developed, targeting the nucleoprotein (NP), matrix (M), and hemagglutinin (HA) genes of the two EIV subtypes. None of the eight assays cross-reacted with any of the other known equine respiratory viruses. Three rRT-PCR assays (EqFlu NP, M, and HA3) which can detect strains of th...
Chuang MS, Huang HH, Dixon KM, Chen KS, Mao CL, Chen CL.The pharmacokinetics of clenbuterol in equine urine and blood was investigated. Methods: Urine and blood samples were collected following 3-day multiple oral administrations. The samples were examined using enzyme-linked immunosorbent assay and further confirmed by solid phase extraction and capillary electrophoresis. Results: Urinary clenbuterol was detectable until day 14 after the last dose. The urinary excretion of clenbuterol was characterized by a biphasic pattern. The half-lives of the bi-exponential elimination (t(1/2alpha) and t(1/2beta)) for urinary clenbuterol were about 12.1 and 48...
Adaszek Ł, Winiarczyk S, Łukaszewska J.The study was aimed at determining the cause of a disease in a horse exhibiting symptoms of fever, joint effusion, weakness, and extravasations on the mucous membranes. Blood was drawn from the animal for haematological and biochemical molecular tests. The PCR technique revealed the presence of 16S RNA Ehrlichia spp. genetic material in the blood samples. DNA amplification by means of primers EHR 521 and EHR 747 gave a product with a volume of 247 bp.The sequence of the PCR product obtained showed a 97.6% similarity with a sequence of a fragment of 16S RNA Ehrlichia phagocytophila, gene number...
Ferrante M, Andreeta A, Landoni MF.Diclofenac is a hydrophilic non-steroidal anti-inflammatory drug widely used in humans and animals. Previous reports have shown that this compound has low percutaneous absorption in horses. The effect of five penetration enhancers (10% urea, 15% and 20% oleic acid and 5% and 10% d-limonene) on the percutaneous absorption of diclofenac diethylamine through horse skin was evaluated in vitro using Franz-type diffusion cells. All tested penetration enhancers induced a significant increase in diclofenac diethylamine permeation, with limonene showing the highest enhancing effect at the lowest concen...
Graham NS, Clutterbuck AL, James N, Lea RG, Mobasheri A, Broadley MR, May ST.Affymetrix GeneChip arrays are a powerful tool for transcriptome profiling and have been applied to a wide range of species. A genomic DNA (gDNA)-based probe selection method has been developed which broadens the range of species to which GeneChips may be successfully applied. This study demonstrated that gDNA-based probe selection on the Affymetrix U133+2 GeneChip array can be used to study the equine transcriptome which, to date, has received only limited attention. More than 29,000 transcripts can be detected in equine brain and liver and in primary cultures of equine articular chondrocytes...
Lake SL, Matthews JB, Kaplan RM, Hodgkinson JE.Genetic resistance against benzimidazole (BZ) anthelmintics is widespread in cyathostomins, the commonest group of intestinal parasitic nematodes of horses. Studies of BZ-resistant nematodes of sheep, particularly Haemonchus contortus, have indicated that an anthelmintic resistance-conferring T/A polymorphism, encoding an F (phenylalanine) to Y (tyrosine) substitution, in beta-tubulin isotype 1 is present at two loci, codons 167 and 200 (F167Y, F200Y). Recent studies using complementary (c) DNA derived from BZ-susceptible and -resistant cyathostomins identified statistical differences in the f...
Gao X, Jiang CG, Han XE, Zhao LP, Shen RX, Xiang WH, Zhou JH.To elucidate the function of the S2 gene in equine infectious anemia virus (EIAV) and its role in the attenuation of the Chinese attenuated EIAV vaccine strains, the S2 in the EIAV vaccine strain EIAV (FDDV) was reverse-mutated and the in vitro replication character of the resultant virus was evaluated. Based on the sequence variation of the S2 gene between the EIAV virulent strains and vaccine strains, all the four vaccine-specific sites in the S2 of an EIAV(FDDV) infectious clone, pFDDV3-8, were reverse-mutated to the sequences of the virulent strain EIAV(DV115). The reverse-mutated molecula...
Winther L, Andersen RM, Baptiste KE, Aalbæk B, Guardabassi L.Stenotrophomonas maltophilia is being reported with increasing frequency as a human nosocomial pathogen, especially among immuno-compromised patients. To the authors' knowledge, this pathogen has not previously been associated with lower airway disease in the horse. In this paper the clinical findings, laboratory diagnosis and response to treatment of seven cases of respiratory infection with S. maltophilia in horses, presented at three equine referral hospitals in Denmark in 2007, are described. In all cases there was a clinical history of chronic coughing and abundant mucopurulent exudate wa...
Casella S, Giannetto C, Fazio F, Giudice E, Piccione G.The aim of the present study was to assess the effect of different storage conditions on prothrombin time (PT), activated partial thromboplastin time (aPTT), and fibrinogen concentration in clinical samples from healthy horses. A total of 100 healthy horses of varying breeds and gender, ranging in age from 4 to 18 years, with a mean body weight of 480 +/- 70 kg, were used. Blood was collected by jugular venipuncture, and a hemochrome-cytometric examination was conducted on all samples. All blood samples were centrifuged and divided into 4 different aliquots to assess clotting parameters by mea...
Vonaparti A, Lyris E, Panderi I, Koupparis M, Georgakopoulos C.In equine sport, salicylic acid is prohibited with a threshold level of 750 microg mL(-1) in urine; hence, doping control laboratories have to establish quantitative and qualitative methods for its determination. A simple and rapid liquid chromatographic/mass spectrometric method was developed and validated for the quantification and identification of salicylic acid. Urine samples after 900-fold dilution and addition of the internal standard (4-methylsalicylic acid) were directly injected to the liquid chromatography/quadrupole time-of-flight mass spectrometry system. Electrospray ionization i...
Endo A, Futagawa-Endo Y, Dicks LM.Lactobacillus equi, Lactobacillus hayakitensis, Lactobacillus johnsonii, and Weissella confusa/cibaria were the dominant species in 12 South African horses. The Bifidobacterium-group was detected in the feces of only one of the 12 horses. Sequencing of the nested-PCR amplicon identified the Bifidobacterium-group as Parascardovia denticolens. Cell numbers of L. equi, L. hayakitensis, and W. confusa/cibaria were consistent in all samples. P. denticolens, Bifidodobacterium pseudolongum, and a phylogenetic relative of Alloscardovia omnicolens were rarely detected. L. equigenerosi, a dominant speci...
Musilova P, Kubickova S, Horin P, Vodicka R, Rubes J.Cross-species chromosome painting has been applied to most of the species making up the numerically small family Equidae. However, comparative mapping data were still lacking in Asiatic asses kulan (Equus hemionus kulan) and kiang (E. kiang). The set of horse arm-specific probes generated by laser microdissection was hybridized onto kulan (E. hemionus kulan) and kiang (E. kiang) chromosomes in order to establish a genome-wide chromosomal correspondence between these Asiatic asses and the horse. Moreover, region-specific probes were generated to determine fusion configuration and orientation of...
Chen JW, Uboh CE, Soma LR, Li X, Guan F, You Y, Liu Y.Proper identification of racehorses competing in an official race and maintenance of defensible chain of custody are important in doping control regulations. The purpose of this study was to develop a reliable multiplex PCR method for providing genetic evidence for matching donors to test samples by using short tandem repeat (STR) loci. Amplification of 21 STR loci from blood, urine or hair root was achieved in a single tube and STR length polymorphism was analyzed using fluorescent labeled capillary electrophoresis. This novel approach showed an allele confidence interval of 0.19-0.43 bp and ...
Bacigalupo MA, Meroni G, Secundo F, Scalera C, Quici S.Highly luminescent Eu(3+) and Tb(3+) complexes of 10-[4-(3-isothiocyanatopropoxy)benzoylmethyl]-1,4,7,10-tetraazacyclododecane-1,4,7 triacetic acid Eu(3+) is a subset of 1 and Tb(3+) is a subset of 1 were conjugated with a goat anti-rabbit IgG and a rabbit anti-mouse IgG, respectively, and applied as markers in a time resolved immunoassay for simultaneous quantitative determination of anabolic compounds clenbuterol (CL) and hydrocortisone (HC). The assay was performed in horse urine, using a monoclonal antibody specific to CL and a rabbit polyclonal antibody specific to the free HC. These lant...
Epstein KL, Brainard BM, Lopes MA, Barton MH, Moore JN.To develop a standardized technique for thrombelastography (TEG) analysis in healthy adult horses, with and without the ex vivo addition of tissue factor (TF) as an activator. To determine reference intervals for TEG parameters in the horse, and to determine if traditional coagulation tests correlate with TEG. Methods: Prospective, observational. Methods: Veterinary teaching hospital. Methods: Twenty-six healthy adult horses. Methods: None. Results: Thrombelastography with (TF-TEG) and without (TEG) the addition of TF performed by 4 operators. Coagulation profiles (prothrombin time, activated ...
Tompkins D, Hudgens E, Horohov D, Baldwin CL.This report describes the initial cloning and characterization of the equine interleukin-17 (IL-17) expressed gene sequence from mRNA obtained from equine intestinal tissue and interleukin-23 (IL-23) expressed gene sequence from mRNA obtained from equine peripheral blood mononuclear cells. Equine IL-17 has 462 nucleotides in the translated region, determined by homology with known human and mouse sequences, and shares 84% and 75% identity, respectively. For the deduced amino acid sequences, the identity with human and mouse is 76% and 70%. Equine IL-23 has 579 nucleotides in the translated reg...
Casasola A, Ramos-Cerrillo B, de Roodt AR, Carbajal Saucedo A, Chippaux JP, Alagón A, Stock RP.Venoms of snakes belonging to the same Genera tend to share biochemical, toxinological and antigenic characteristics. Accordingly, paraspecific neutralization of venom lethality by experimental antisera and commercial antivenoms has been reported. We studied the spectrum of neutralization of lethality of an experimental monovalent equine antiserum against the strongly neurotoxic African forest cobra (Naja melanoleuca) when tested against venoms of most species of African Naja, both neuro and cytotoxic as described by some authors. We report a comparison of the median lethal doses (LD50) of the...
Frampton AR, Uchida H, von Einem J, Goins WF, Grandi P, Cohen JB, Osterrieder N, Glorioso JC.To initiate infection, equine herpesvirus type 1 (EHV-1) attaches to heparan sulfate on cell surfaces and then interacts with a putative glycoprotein D receptor(s). After attachment, virus entry occurs either by direct fusion of the virus envelope with the plasma membrane or via endocytosis followed by fusion between the virus envelope and an endosomal membrane. Upon fusion, de-enveloped virus particles are deposited into the cytoplasm and travel to the nucleus for viral replication. In this report, we examined the mechanism of EHV-1 intracellular trafficking and investigated the ability of EH...
Lin M, Zhang C, Gibson K, Rikihisa Y.Neorickettsia risticii is an obligate intracellular bacterium of the trematodes and mammals. Horses develop Potomac horse fever (PHF) when they ingest aquatic insects containing encysted N. risticii-infected trematodes. The complete genome sequence of N. risticii Illinois consists of a single circular chromosome of 879 977 bp and encodes 38 RNA species and 898 proteins. Although N. risticii has limited ability to synthesize amino acids and lacks many metabolic pathways, it is capable of making major vitamins, cofactors and nucleotides. Comparison with its closely related human pathogen N. senn...
Schmitz A, Theurillat R, Lassahn PG, Mevissen M, Thormann W.CE with multiple isomer sulfated-CD as selector was used for the simultaneous analysis of the stereoisomers of ketamine, norketamine, 5,6-dehydronorketamine and hydroxylated metabolites of norketamine in liquid/liquid extracts of (i) in vitro incubations with ketamine or norketamine and equine liver microsomes and (ii) plasma and urine of ponies receiving a target-controlled infusion of ketamine under isoflurane anesthesia. Hydroxynorketamine metabolites with the hydroxy group at the cyclohexanone ring could be shown to be formed stereoselectively both in vitro and in vivo. Due to the lack of ...
White SD, Foley JE, Spiegel IB, Ihrke PJ.Equine sarcoidosis is a rare, multisystemic, noncaseating, granulomatous and lymphoplasmacytic disease of unknown etiology. A recent report described a horse with granulomatous skin disease displaying histologic, electron microscopic, and polymerase chain reaction (PCR) findings consistent with equine herpesvirus 2 (EHV-2). Objective: To investigate the presence of EHV-2 and equine herpesvirus 1 (EHV-1) in 8 horses with sarcoidosis. Methods: Eight horses with sarcoidosis, reported previously. Methods: Retrospective study. PCR assays of the tissues were performed to detect DNA associated with E...
Bauquier JR, Tennent-Brown BS, Tudor E, Bailey SR.Polymyxin-B is used to treat equine systemic inflammation. Bacterial toxins other than lipopolysaccharide (LPS) contribute to systemic inflammation but the effects of polymyxin-B on these are poorly defined. Whole blood aliquots from six healthy horses diluted 1:1 with RPMI were incubated for 21 hr with 1 μg/ml of LPS, lipoteichoic acid (LTA) or peptidoglycan (PGN) in the presence of increasing concentrations of polymyxin-B (10-3000 μg/ml). A murine L929 fibroblast bioassay was used to measure TNF-α activity. Polymyxin-B significantly inhibited the effects of all three bacterial toxins. ...
Boyer S, Garcia P, Popot MA, Steiner V, Lesieur M.A sensitive and specific method has been developed to detect semi-quantitatively testosterone in horse hair samples. The method involved a washing step with sodium dodecylsulfate aqueous solution. The mane and tail hair samples (100mg) were dissolved in 1 mL of sodium hydroxide for 15 min at 95 degrees C in the presence of d3-boldenone used as internal standard. The next three steps involved diethyl ether extraction and a solid phase extraction on Isolute C18 (EC) cartridges eluted with methanol. The residue was derivatized by adding 100 microL of acetonitrile and 30 microL of PFPA then incuba...
Andersson L, Juneja RK, Sandberg K.Genetic linkage between the equine loci for phosphohexose isomerase (PHI) and serum Xk protein was demonstrated by means of segregation data from three sire families. The recombination frequency was estimated from pooled data to be 0.23 +/- 0.02; a significant heterogeneity between sires for estimates of the recombination frequency was observed. No indication of linkage was detected between Xk and 14 other blood marker loci. Linkage between the Xk locus and the locus for soluble malic enzyme (ME1) has recently been reported in horses. An equine linkage group designated LG IV comprising the thr...
Giese A, Jude R, Kuiper H, Piumi F, Schambony A, Guérin G, Distl O, Töpfer-Petersen E, Leeb T.Acidic epididymal glycoprotein 1 (AEG1), also called cysteine-rich secretory protein 1 (CRISP1), is a member of the CRISP protein family which is characterized by 16 conserved cysteine residues at the C-terminus. The CRISP proteins are expressed in the male genital tract and are thought to be involved in sperm-egg fusion. Therefore, their genes are of interest as candidate genes for inherited male fertility dysfunctions and as putative quantitative trait loci for male fertility traits. In this report, the cloning and DNA sequence of 90 kb of horse genomic DNA from equine chromosome 20q22 conta...
Scofield D, Black J, Wittenburg L, Gustafson D, Ferris R, Hatzel J, Traub-Dargatz J, McCue P.Systemic administration of ceftiofur crystalline free acid (CCFA) may be a potential treatment for infectious endometritis caused by Streptococcus equi ssp. zooepidemicus (S. zooepidemicus) and other susceptible bacterial organisms in the mare. Objective: To determine if i.m. administration of CCFA at the label dose will exceed the minimum inhibitory concentration (MIC) of S. zooepidemicus in the endometrium following single administration and multiple administration protocols. Methods: Experimental pharmacokinetic study. Methods: Three mares (Group 1) were administered a single i.m. dos...
Dunnett M, Lees P.Analysis of human hair for drug residues is being used increasingly as a diagnostic tool in the investigation of drug use and abuse. Hair analysis is complementary to urine/blood testing in that it can provide an extensive historical record of drug use, is noninvasive, impersonal and can facilitate retesting. However, the technique has not been studied in horses. Objective: That the systemic administration of drugs in horses could be identified by the detection of drug residues in hair. Objective: To evaluate hair analysis as a potential retrospective diagnostic test for drug administration in...
Crump A, Donaldson WL, Miller J, Kydd JH, Allen WR, Antczak DF.Antibodies to fetal major histocompatibility complex (MHC) antigens are routinely detected in the serum of pregnant mares some 2-4 weeks after formation of the endometrial cups at Day 36-38 after ovulation. Several experimental approaches were taken to determine whether paternal MHC antigens are expressed on horse placental tissues. First, absorption of anti-paternal MHC antisera with a large volume of endometrial cup cells removed antibody activity in only 2 of 4 experiments. Second, repeated immunization of horses with endometrial cup tissue recovered from a mare on Day 47 of pregnancy faile...
Olver CS, Webb TL, Long LJ, Scherman H, Prenni JE.Disease-specific biomarkers hold diagnostic promise in both human and veterinary medicine, but serum biomarkers in low concentrations may be masked by the presence of abundant proteins, mostly albumin and IgG. Methods to deplete albumin and IgG exist, but efficacy of these methods for depleting equine serum of these proteins has not been established. Objective: The aim of this study was to determine if albumin and IgG could be depleted from equine serum using several commercially available kits and procedures. Methods: One-dimensional gel electrophoresis followed by densitometry was used to de...
de Kock SS, Rodgers JP, Swanepoel BC.Previous studies have shown that insulin-like growth factor 1 (IGF-1) is a promising marker for the detection of growth hormone (GH) abuse in the horse. The significant increases observed with GH administration in comparison to natural levels imply the possibility of setting a threshold level for IGF-1 that would be indicative of GH abuse. Although an immunoradiometric assay (IRMA) has been identified as a reliable screening method, a more specific IGF-1 quantification method needs to be developed for the prosecution of GH abuse by horseracing authorities. This study describes such an HPLC ele...
Simeonova GP, Krastev SZ, Simeonov RS.The pathogenic mechanism of equine recurrent uveitis (ERU) is still poorly defined and many variations between experimental animal models and spontaneous disease exist. Objective: The aim of our study was to investigate if Th17 cell-mediated response plays role in the pathogenesis of the used experimental model in horses and to reveal its pathological findings. Methods: Experimental uveitis was induced in 6 healthy horses. The concentrations of retinal autoantigen CRALBP and IL-17 were measured using ELISA in aqueous humor and vitreous body of the 12 inflamed eyes as well as in 12 control non-...
Yeargan MR, Howe DK.Equine protozoal myeloencephalitis (EPM) is a common neurologic disease of horses that is caused by the apicomplexan pathogen Sarcocystis neurona. To help improve serologic diagnosis of S. neurona infection, we have modified existing enzyme-linked immunosorbent assays (ELISAs) based on the immunogenic parasite surface antigens SnSAG2, SnSAG3, and SnSAG4 to make the assays polyvalent, thereby circumventing difficulties associated with parasite antigenic variants and diversity in equine immune responses. Two approaches were utilized to achieve polyvalence: (1) mixtures of the individual recombin...
Anis E, Ilha MRS, Engiles JB, Wilkes RP.Equine infectious disease outbreaks may have profound economic impact, resulting in losses of millions of dollars of revenue as a result of horse loss, quarantine, and cancelled events. Early and accurate diagnosis is essential to limit the spread of infectious diseases. However, laboratory detection of infectious agents, especially the simultaneous detection of multiple agents, can be challenging to the clinician and diagnostic laboratory. Next-generation sequencing (NGS), which allows millions of DNA templates to be sequenced simultaneously in a single reaction, is an ideal technology for co...
Bell K, Patterson S, Pollitt CC.The plasma protease inhibitor system (Pi) of Standardbred horses was studied by thin-layer, high-voltage, acid polyacrylamide gel electrophoresis (pH 4.6) followed by protein staining and staining for trypsin and chymotrypsin inhibition. In addition to the eight Thoroughbred alleles (PiF, G, I, L, N, S1, S2, U), another 10 alleles, designated PiH, J, K, O, P, Q, R, V, X, Z, were postulated to account for the 98 Pi types which were observed in Standardbreds. Detailed inhibitory spectra of the 'new' alleles were determined and further exceptions to the Pi1, Pi2 classification of Juneja et al. (1...
Wilgus H, Stellwagen E.Changes in the visible absorbance spectra of complexes of horse heart cytochrome c hemopeptide 1-65, peptide 66-104, and their guanidinated counterparts are compared with those characteristic of native and fully guanidinated ferricytochrome c over the pH range 7 to 11. Upon raising the pH, the methionine ligand in the guanidinated hemopeptide 1-65.peptide 66-104 complex is replaced by a strong field ligand. By contrast, the methionine ligand in the hemopeptide 1-65.guanidinated peptide 66-104 is replaced by a weak field ligand. These results demonstrate that lysine 13 does not ligate with the ...
Pedersen HG, Watson ED, Telfer EE.Follicular atresia has been examined previously by various biochemical and histological methods. The aim of this study was to compare, for the first time, detection of granulosa cell apoptosis by biochemical DNA analysis and microscopic examination of fresh granulosa cell morphology with the established method of detecting atresia by histology in equine follicles. DNA extracted from granulosa cells was examined by staining with ethidium bromide and end-labelling with [(32)P]dideoxy-ATP, which labels the free 3'-end of DNA fragments. In 25 of 26 follicles (96%) there was agreement between end-l...
Pöschke A, Krähling B, Failing K, Staszyk C.The equine periodontal ligament (PDL) is a fibrous connective tissue that covers the intra-alveolar parts of the tooth and anchors it to the alveolar bone-it, therefore, provides a similar function to a tendinous structure. While several studies have considered the formation and structure of tendons, there is insufficient information particularly on the molecular composition of the PDL. Especially for the equine PDL, there is limited knowledge concerning the expression of genes commonly regarded as typical for tendon tissue. In this study, the gene expression of, e.g., (, and fibrocartilage m...
Sun L, Gong Z, Oberst EJ, Betancourt A, Adams AA, Horohov DW.While born with a limited production, foals' interferon-gamma (IFN-γ) expression increases after birth. The underlying mechanisms remain unknown. DNA methylation is considered to be involved. Therefore, the DNA methylation status of the Ifng promoter in CD4(+) cells from neonatal foal was determined using a methylation-specific PCR (MSP), and its relevance to IFN-γ mRNA expression was estimated. The effect of environment on the DNA methylation was also evaluated by comparing ponies that were kept in a barn versus those on pasture. The DNA in the Ifng promoter was hypermethylated and its deme...
Leschke DH, Muir GS, Hodgson JK, Coyle M, Horn R, Bertin FR.Diseases associated with insulin dysregulation (ID), such as equine metabolic syndrome and pituitary pars intermedia dysfunction, are of interest to practitioners because of their association with laminitis. Accurate insulin concentration assessment is critical in diagnosing and managing these diseases. Objective: To determine the effect of time, temperature, and collection tube type on insulin concentrations in horses at risk of ID. Methods: Eight adult horses with body condition score >6/9. Methods: In this prospective study, subjects underwent an infeed oral glucose test 2 hours before...
Polkes AC, Giguère S, Lester GD, Bain FT.Neonatal foals with isoerythrolysis (NI) often die, but the risk factors for death have not been identified. Objective: To identify factors associated with outcome in foals with NI and to identify factors associated with death from liver failure or kernicterus in the same population. Methods: Seventy-two foals with NI examined at referral institutions. Methods: Retrospective case series. Information on signalment, clinical examination findings, laboratory testing, treatment, complications, outcome, and necropsy results were obtained. Results: The overall survival rate was 75% (54 of 72). Liver...
Jensen TK, Boye M, Bille-Hansen V.Fluorescent in situ hybridization, immunohistochemistry, and Grocott's methenamine-silver nitrate staining were compared as diagnostic methods for Pneumocystis carinii pneumonia in formalin-fixed lung tissue from foals and pigs. An oligonucleotide probe targeting 18S ribosomal RNA of P. carinii was designed for in situ hybridization, and a commercially available monoclonal antibody was used for immunohistochemistry. Samples from six foals and 10 pigs with P. carinii pneumonia, as verified by Grocott's methenamine-silver nitrate staining, were examined concurrently with samples from seven anima...
Rebolledo-Mendez J, Hestand MS, Coleman SJ, Zeng Z, Orlando L, MacLeod JN, Kalbfleisch T.The reference assembly for the domestic horse, EquCab2, published in 2009, was built using approximately 30 million Sanger reads from a Thoroughbred mare named Twilight. Contiguity in the assembly was facilitated using nearly 315 thousand BAC end sequences from Twilight's half brother Bravo. Since then, it has served as the foundation for many genome-wide analyses that include not only the modern horse, but ancient horses and other equid species as well. As data mapped to this reference has accumulated, consistent variation between mapped datasets and the reference, in terms of regions with no...
Crespo F, Gosalvez J, Gutiérrez-Cepeda L, Serres C, Johnston SD.Stallion spermatozoa recovered and examined immediately after colloidal centrifugation resulted in a higher straight-line velocity (VSL) than sperm processed using direct conventional centrifugation (p = 0.000), but there was no differences in the progressive motility or sperm DNA fragmentation (SDF) as determined by the sperm chromatin dispersion assay. However, when centrifuged spermatozoa were incubated at 37 °C for 24 h to determine the rate of SDF (r-SDF), a lower r-SDF (p = 0.0011) was observed in those sperm recovered after colloidal separation (0.5 ± 0.1%/h) compared to direct (1.2 ...
Hidalgo M, Ortiz I, Dorado J, Morrell JM, Gosálvez J, Consuegra C, Diaz-Jimenez M, Pereira B, Crespo F.The aims of this study were to: 1) develop a new method for stallion sperm selection using a modified swim-up procedure through a colloid and 2) evaluate its impact in good quality ejaculates from bad freezers in comparison to methods involving centrifugation such as single layer centrifugation and sperm washing. Ejaculates were processed before freezing using three different procedures: sperm washing (SW), colloid single layer centrifugation (SLC) and a modified colloid swim-up (SU). After semen processing, sperm recovery rates were measured and sperm were frozen. Post-thaw sperm motility (as...
Isani G, Ferlizza E, Cuoghi A, Bellei E, Monari E, Bianchin Butina B, Castagnetti C.Characterisation of the physiologic equine amniotic fluid (AF) proteome is a prerequisite to study its changes during diseases and discover new biomarkers. The aim of this study was to identify by a proteomic approach the most abundant proteins of equine AF. AF samples were collected at parturition from 24 healthy mares that delivered healthy foals. All samples were subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) on 4-12% gels. A pool of the 24 samples, after SDS-PAGE, was cut in 25 slices, trypsin-digested and analysed by mass spectrometry (MS) for protein i...
Peters LM, Demmel S, Pusch G, Buters JT, Thormann W, Zielinski J, Leeb T, Mevissen M, Schmitz A.Ketamine is an anesthetic and analgesic regularly used in veterinary patients. As ketamine is almost always administered in combination with other drugs, interactions between ketamine and other drugs bear the risk of either adverse effects or diminished efficacy. Since cytochrome P450 enzymes (CYPs) play a pivotal role in the phase I metabolism of the majority of all marketed drugs, drug-drug interactions often occur at the active site of these enzymes. CYPs have been thoroughly examined in humans and laboratory animals, but little is known about equine CYPs. The characterization of equine CYP...
Grosenbaugh DA, Hood DM.In this study, we described water-insoluble proteins extracted from the germinative regions (stratum internum and coronary band epithelium) and the cornified outer surface (stratum medium) of the equine hoof wall. Two major types of polypeptides were identified: the intermediate filaments (IF) and the IF-associated proteins. The IF, including keratins, composed a major portion of this fraction, had electrophoretic mobilities on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the range of 40 to 80 kDa, and reacted with acidic or basic keratin-specific monoclonal antibodies. Differe...
Bleumink-Pluym NM, ter Laak EA, Houwers DJ, van der Zeijst BA.The ability of Taylorella equigenitalis, the causative agent of contagious equine metritis, to invade and replicate in equine derm cells was studied. The kinetics of invasion and replication were determined for four T. equigenitalis strains. On the basis of these experiments, a simpler assay in which the invasive as well as the replicative properties of a particular strain could be determined was developed. This assay was used to characterize 32 strains, which had previously been typed by field inversion gel electrophoresis of genomic restriction fragments. The invasiveness of T. equigenitalis...
Khan FA, Chenier TS, Murrant CL, Foster RA, Hewson J, Scholtz EL.Nitric oxide (NO) may have a role in persistent breeding-induced endometritis in mares through an inhibitory effect on uterine contractility. The objectives of this study were to test the effect of NO on spontaneous uterine contractility in-vitro and to evaluate whether this effect varied between the longitudinal and circular muscle layers of the uterus. Reproductive tracts were collected from eight euthanized non-pregnant mares (age 4-19 years; body weight 405-530 kg). Transrectal examination of the reproductive tract was performed before euthanasia to evaluate stage of the estrous cycle and...
Kankavi O, Roberts MS.Once considered unique to the lung, surfactant proteins have been clearly identified in the intestine and peritoneum and are suggested to exist in several other organs. In the lung, surfactant proteins assist in the formation of a monolayer of surface-active phospholipid at the liquid-air interface of the alveolar lining, reducing the surface tension at this surface. In contrast, surface-active phospholipid adsorbed to articular surfaces has been identified as the load-bearing boundary lubricant of the joint. This raises the question of whether surfactant proteins in synovial fluid (SF) are re...
Bugno M, Słota E, Pieńkowska-Schelling A, Schelling C.Fluorescent in situ hybridisation (FISH) using a panel of molecular probes for all chromosome pairs obtained by chromosome microdissection of the domestic horse ( Equus caballus ) was used to diagnose karyotype abnormalities in 35 horses (32 mares, 2 stallions and 1 intersex), which were selected for the study due to infertility (23 horses), reduced fertility (10 horses) and developmental anomalies (2 horses). The use of the FISH technique with probes for each horse chromosome pair enabled the diagnosis of many different chromosome aberrations in this population. Among the horses analysed, 21 ...
