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.
Parrilla Hernández S, Franck T, Munaut C, Feyereisen É, Piret J, Farnir F, Reigner F, Barrière P, Deleuze S.Myeloperoxidase (MPO), as a marker of neutrophil activation, has been associated with equine endometritis. However, in absence of inflammation, MPO is constantly detected in the uterine lumen of estrous mares. The aim of this study was to characterize MPO in the uterus of mares under physiological conditions as a first step to better understand the role of this enzyme in equine reproduction. Total and active MPO concentrations were determined, by ELISA and SIEFED assay, respectively, in low-volume lavages from mares in estrus ( = 26), diestrus ( = 18) and anestrus ( = 8) in absence of endometr...
The protein journalJanuary 18, 2023
Volume 42, Issue 1 24-36 doi: 10.1007/s10930-023-10092-x
Rodríguez MC, Mussio PE, Villarraza J, Tardivo MB, Antuña S, Fontana D, Ceaglio N, Prieto C.Equine chorionic gonadotropin (eCG) is a glycoprotein hormone widely used in timed artificial ovulation (TAI) and superovulation protocols to improve the reproductive performance in livestock. Until recently, the only eCG products available in the market for veterinary use consisted in partially purified preparations of pregnant mare serum gonadotropin (PMSG). Here, a bioactive recombinant eCG (reCG) produced in suspension CHO-K1 cells was purified employing different chromatographic methods (hydrophobic interaction chromatography and reverse-phase (RP)-HPLC) and compared with a RP-HPLC-purifi...
Loublier C, Taminiau B, Heinen J, Lecoq L, Amory H, Daube G, Cesarini C.Fecal microbiota transplantation (FMT) has been used empirically for decades in equine medicine to treat intestinal dysbiosis but evidence-based information is scarce. This in vitro study aimed at assessing the effect of a commonly used pre-FMT processing method on the bacterial composition and viability of the fecal filtrate. Three samples of fresh equine manure (T0) were processed identically: the initial manure was mixed with 1 L of lukewarm water and chopped using an immersion blender to obtain a mixture (T1), which was left uncovered during 30 min (T2) and percolated through a sieve to ob...
Knowles EJ, Hyde C, Harris PA, Elliott J, Menzies-Gow NJ.A chemiluminescent immunoassay is commonly employed to measure adrenocorticotrophic hormone (ACTH) concentrations to assist pituitary pars intermedia dysfunction diagnosis. In a previous study, seasonally-dependent assay cross-reactivity to endogenous equine corticotropin-like intermediate lobe peptide (CLIP, ACTH 18-39) was suspected. The present study aimed to demonstrate binding of endogenous equine CLIP to the capture antibody of the ACTH chemiluminescent immunoassay. Liquid chromatography - mass spectrometry (LCMS) methods were optimised to identify selected ions from synthetic human ACTH...
Bone reportsJanuary 5, 2023
Volume 18 101653 doi: 10.1016/j.bonr.2023.101653
Grissom SK, Semevolos SA, Duesterdieck-Zellmer K.The objective of this study is to better understand the pathogenesis of early equine osteochondrosis (OC) by identifying differences in gene and protein expression of extracellular matrix components and regulators in normal and diseased cartilage and bone, focusing on the osteochondral junction and cells surrounding the cartilage canals. We expected to find an upregulation of matrix metalloproteinases and a decrease in extracellular matrix constituent expression along the osteochondral junction and cells surrounding the cartilage canals in OC samples. Paraffin-embedded osteochondral samples (6...
Halvarsson P, Tydén E.Gastrointestinal nematode parasites are of major concern for horses, where Strongylus vulgaris is considered the most pathogenic among the Strongylus species. Diagnosis of S. vulgaris infections can be determined with next generation sequencing techniques, which are inherently dependent on reference sequences. The best marker for parasitic nematodes is internal transcribed spacer 2 (ITS2) and we provide the first complete ITS2 sequences from five morphologically identified S. vulgaris and additional sequences from two S. edentatus. These sequences have high similarity to already published part...
Naboulsi R, Cieślak J, Headon D, Jouni A, Negro JJ, Andersson G, Lindgren G.Gene expression differences can assist in characterizing important underlying genetic mechanisms between different phenotypic traits. However, when population-dense tissues are studied, the signals from scarce populations are diluted. Therefore, appropriately choosing a sample collection method that enriches a particular type of effector cells might yield more specific results. To address this issue, we performed a polyA-selected RNA-seq experiment of domestic horse () plucked-hair samples and skin biopsies. Then, we layered the horse gene abundance results against cell type-specific marker ge...
Boorman S, Hanson RR, Velloso Alvarez A, Zhong K, Hofmeister E, Boone LH.To determine the effect of concurrent versus delayed treatment with corticosteroid on equine articular tissues also treated with local anesthetic in vitro in the presence of inflammatory mediators. Methods: Controlled laboratory study. Methods: Five geldings, one mare (aged 3-18 years). Methods: From each horse, 24 synovial and 12 osteochondral explants were cultured in a 12-well plate (2 wells/group, 2 synovial and 1 osteochondral explant/well, total 216 explants in the study). Explants were stimulated in culture medium with 10 μg/ml recombinant equine interleukin-1β and 10 μg/ml tumor...
Liu L, Zhang Y, Ma H, Cao H, Liu W.Horse Milk has important quantitative characteristics and high economic value. However, the DNA methylation regulators involved in horse milk traits have not been clarified. To explore the important role of genome-wide DNA methylation in regulating equine milk yield, this study systematically investigated the genome-wide DNA methylation profiles of Kazakh horse blood by comparing a high-production group (HP, average daily milk yield of 7.5 kg) and low-production group (LP, average daily milk yield of 3.2 kg) using deep whole-genome bisulfite sequencing. First, both groups showed similar prop...
Alghamdi AS, Fedorka CE, Scoggin KE, Esteller-Vico A, Beatty K, Davolli G, Ball BA, Troedsson MHT.Sperm-neutrophil binding is an important facet of breeding and significantly impacts fertility. While a specific seminal plasma protein has been found to reduce this binding and improve fertility (CRISP-3), additional molecule(s) appear to promote binding between defective sperm and neutrophils. Recent work has suggested one of these proteins is lactoferrin (LF), an 80 kDa iron-binding protein found throughout the body, but the purity of the protein was not confirmed. It is unknown if LF binds to sperm selectively based on viability, and if receptors for LF are located on equine sperm. To eval...
In this study, a new sandwich-type immunoenzymatic assay, based on a molecularly imprinted polymer (MIP) as an artificial antibody (pseudo-ELISA), was developed for the determination of procalcitonin (PCT) in veterinary species. The quantification of PCT in human medicine represents the state of the art for the diagnosis of sepsis; instead the clinical studies on the relevance of PCT as a sepsis predictor in veterinary patients are few, likely due to the total absence of validated assays. MIPs have been widely used as antibody mimics for important applications, and MIP-based sandwich assays ha...
Wagner GE, Berner A, Lipp M, Kohler C, Assig K, Lichtenegger S, Saqib M, Müller E, Trinh TT, Gad AM, Söffing HH, Ehricht R, Laroucau K, Steinmetz I.Burkholderia mallei, the causative agent of glanders, is a clonal descendant of Burkholderia pseudomallei, the causative agent of melioidosis, which has lost its environmental reservoir and has a restricted host range. Despite limitations in terms of sensitivity and specificity, complement fixation is still the official diagnostic test for glanders. Therefore, new tools are needed for diagnostics and to study the B. mallei epidemiology. We recently developed a highly sensitive serodiagnostic microarray test for human melioidosis based on the multiplex detection of B. pseudomallei proteins. In ...
Yang G, Zhou B, Chen K, Hu Z, Guo W, Wang X, Du C. () and () are the causative pathogens of Equine piroplasmosis (EP), a disease that has brought huge economic losses and great restrictions to the global equine industry. Rapid and accurate diagnostic methods are critical for the effective monitoring of the disease. In this study, we developed novel competitive ELISA methods and western blot assays based on the EMA1 or Bc48 proteins to detect antibodies against or , respectively. In the novel cELISA, horseradish peroxidase (HRP)-labeled monoclonal antibodies are used in place of enzyme-conjugated secondary antibodies, in order to speed up th...
Garner C, Stephen C, Pant SD, Ghorashi SA.Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) is one of the causative agents of equine endometritis. In this study, a panel of different bacterial species, and colonies derived from bacteriological cultures of 38 clinical samples, were subjected to Loop-Mediated Isothermal Amplification (LAMP) assay and PCR, followed by high-resolution melt (HRM) curve analysis. All clinical samples were genotyped into three distinct groups based on HRM curve analysis. Differences in melting curve profiles were a reflection of DNA variation in sorD gene which was confirmed by DNA sequencing. A mat...
Vigolo V, Gautier C, Falomo ME, Aurich C.The use of microfluidic technology is increasing in artificial reproduction technologies: With a small amount of semen, it allows for the selection of sperm with the best characteristics of kinetics, morphology and chromatin integrity. The ZyMot Multi (850 μl) is the most popular device of ZyMot Fertility Inc. To date, it was proven to be a valid instrument for sperm selection for in vitro fertilization and intracytoplasmic sperm injection in men. The aim of this study was to test the efficacy of the ZyMot Multi (850 μl) for stallion semen. Frozen-thawed semen from 15 stallions that were...
Sedykh SE, Purvinsh LV, Burkova EE, Dmitrenok PS, Ryabchikova EI, Nevinsky GA.Exosomes are nanovesicles with a 40-150 nm diameter and are essential for communication between cells. Literature data suggest that exosomes obtained from different sources (cell cultures, blood plasma, urea, saliva, tears, spinal fluid, milk) using a series of centrifugations and ultracentrifugations contain hundreds and thousands of different protein and nucleic acid molecules. However, most of these proteins are not an intrinsic part of exosomes; instead, they co-isolate with exosomes. Using consecutive ultracentrifugation, gel filtration, and affinity chromatography on anti-CD9- and anti-C...
Kabil E, Göktaş EF, Güneş E, Yatanaslan L, Zor TA, Tektaş MH, İnceman B, Tufan M.A recent trend in the use of high-resolution accurate mass screening (HRAMS) for doping control testing in both human and animal sports has emerged owing to significant improvement in high-resolution mass spectrometry in terms of sensitivity, mass accuracy, mass resolution and mass stability. Several HRAMS methods have been reported for the detection of multidrug residues in human or equine urine. These improved analytical technologies have led to changes in the use of prohibited substances, and the administration of more than one substance at low concentrations as a "cocktail" has become one ...
Drozdzewska K, Gehlen H.The diagnosis of internal neoplasia in horses is challenging. Increased production of hormones physiologic for adult animals (e.g., adrenocorticotropin, norepinephrine, and erythropoietin) or typical for the foetal phase (alpha-fetoprotein, anti-Müllerian hormone, and parathyroid-hormone-related protein) might aid in tumour diagnostics. Thymidine kinase-1 and alkaline phosphatase are examples of intracellular enzymes, whose activity in the blood may increase in some neoplasia cases. Furthermore, inappropriate production of abnormal monoclonal or autologous antibodies can accompany lymphoma an...
Sandbaumhüter FA, Aerts JT, Theurillat R, Andrén PE, Thormann W, Jansson ET.The chiral drug ketamine has long-lasting antidepressant effects with a fast onset and is also suitable to treat patients with therapy-resistant depression. The metabolite hydroxynorketamine (HNK) plays an important role in the antidepressant mechanism of action. Hydroxylation at the cyclohexanone ring occurs at positions 4, 5, and 6 and produces a total of 12 stereoisomers. Among those, the four 6HNK stereoisomers have the strongest antidepressant effects. Capillary electrophoresis with highly sulfated γ-cyclodextrin (CD) as a chiral selector in combination with mass spectrometry (MS) was us...
Berryhill EH, Urbina NS, Marton S, Vernau W, Alonso FH.The Wellness Ready Test (WRT) is a lateral flow, stall-side assay that measures equine insulin in whole blood and requires validation before recommending clinical use. We evaluated intra- and inter-assay precision and linearity and compared the WRT with a radioimmunoassay (RIA). Tested concentrations ranged from 695 pmol/L (100 μIU/mL). For 20 replicates at each insulin level, intra-assay CVs of the WRT for insulin were 13.3%, 12.9%, and 15.3% at low (139-278 pmol/L; 20-40 μIU/mL), intermediate (278-417 pmol/L; 40-60 μIU/mL), and high (>417 >60 μIU/mL) concentrations...
Taesuji M, Rattanamas K, Kulthonggate U, Mamom T, Ruenphet S.The immune responses of animals infected with African horse sickness (AHS) virus are determined by enzyme-linked immunosorbent assay (ELISA), complement fixation, and virus neutralization test. During the outbreaks of AHS in Thailand, the immune response after vaccination has been monitored using commercial test kits such as blocking ELISA, which are expensive imported products unavailable commercially in Thailand. This study aimed to assess the sensitivity and specificity of anti-AHS virus antibodies using dot blotting based on monovalent and polyvalent strains of live attenuated AHS vaccine....
Dordas-Perpinyà M, Yanez-Ortiz I, Sergeant N, Mevel V, Bruyas JF, Catalán J, Delehedde M, Briand-Amirat L, Miró J.ProAKAP4 is the precursor of AKAP4 (A-kinase Anchor protein 4), the main structural protein of the fibrous sheath of sperm. The amount of proAKAP4 reflects the ability of spermatozoa to maintain the flagellum activity and functionality up to the site of fertilization and is positively correlated with progressive motility in several mammalian species. The aim of this study was to investigate the relationship between proAKAP4 concentration with horse sperm motility descriptors and spermatic motile subpopulations. For this purpose, a total of 48 ejaculates from 13 different stallions were analyze...
Ferrer MS, Hurley DJ, Norton N, Ellerbrock RE.The objectives of this study were to evaluate the ability of five diagnostic tests to detect polymorphonuclear cells (PMNs) in stallion semen, and to determine the concentration of PMNs that affects sperm motility. We hypothesized that all tests have diagnostic value, and even low concentrations of PMNs affect motility. One ejaculate was obtained from six stallions. Aliquots of 50 × 10 purified sperm were incubated, in triplicate, with six concentrations of purified PMNs: 1) no PMNs, 2) 0.25 × 10 PMN/ml, 3) 0.5 × 10 PMN/ml, 4) 2.5 × 10 PMN/ml, 5) 5 × 10 PMN/ml, 6) 10 × 10 PMN...
Morse-Wolfe B, Bleach E, Kershaw C.Stallion spermatozoa are typically cryopreserved at 200 to 300 million sperm/ml; however recent advances such as intracytoplasmic sperm injection (ICSI) requires only one spermatozoon, wasting many, after thawing a whole straw. Cryopreserving at concentrations less than the current standard or refreezing thawed spermatozoa could maximize the use of genetically valuable animals and reduce waste. This investigation aimed to identify if lowering the sperm concentration for cryopreservation affected post-thaw quality after one and two freeze-thaw cycles. Nine ejaculates were collected from three f...
Li J, Zhang X, Bai B, Zhang M, Ma W, Lin Y, Wang X, Wang XF.All lentiviruses encode a post-transcriptional transactivator, Rev, which mediates the export of viral mRNA from the nucleus to the cytoplasm and which is required for viral gene expression and viral replication. In the current study, we demonstrate that equine infectious anemia virus (EIAV), an equine lentivirus, encodes a second post-transcriptional transactivator that we designate Grev. Grev is encoded by a novel transcript with a single splicing event that was identified using reverse transcription-PCR (RT-PCR) and RNA-seq in EIAV-infected horse tissues and cells. Grev is about 18 kDa in...
Stoeckle SD, Timmermann D, Merle R, Gehlen H.Pituitary pars intermedia dysfunction is one of the most common diseases of aged horses and ponies. In Parkinson's disease, which is, similar to PPID, a disease that involves oxidative damage to dopaminergic pathways but with different clinical signs, alterations to the serum amino acid profile have been reported. To examine changes in the plasma amino acid profile in horses with PPID, EDTA plasma of horses that were presented for various reasons that required laboratory examinations of blood anticoagulated with EDTA was collected. With this plasma, the basal ACTH concentration as well as the ...
Goni JI, Hendrix K, Kritchevsky J.It is important to determine if a horse is shedding Salmonella spp., but a complete culture series can be cost prohibitive. Objective: Determine the optimal pooling technique to maintain high sensitivity of Salmonella spp. culture using spiked samples, and then demonstrate the efficacy of this protocol on clinical submissions. Objective: Pooled fecal samples are as sensitive as 5 individual cultures for the detection of Salmonella shedding. Methods: A single Salmonella-negative horse from the university herd, and 19 hospitalized horses. Methods: Salmonella-free fecal samples were spiked with d...
Akköse M, Karabulut E, İnal Ş, Dik GÇ, Özbeyaz C, Kaya U, Çam M, Topal B.The aim of this study was to evaluate changes in the serum immunoglobulin G (IgG) and serum total protein (STP) concentrations and serum Brix percentages of neonatal Arabian foals during first 3 weeks of life. Blood samples were collected from 12 apparently healthy foals by jugular venipuncture at birth and at 12-hours, 24-hours, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 and 21 days of age. Serum IgG and STP concentrations and Brix percentages were measured by the radial immunodiffusion assay, and digital STP and Brix refractometers, respectively. Based on the serum IgG concentrations measured at 24 h, ...
Philip M, Karakka Kal AK, Subhahar MB, Karatt TK, Mathew B, Perwad Z, Graiban FM, Caveney MR, Sayed R, Kadry AM.According to previous research, aminorex is the major metabolite of levamisole; however, in the screening of levamisole-positive racehorse urine and plasma samples, aminorex could only be detected in trace amounts or not at all. In forensic laboratories, hydroxy levamisole and its phase II conjugates make it easier to confirm levamisole misuse and to differentiate between the abuse of levamisole and aminorex. This study aimed to identify the major levamisole metabolites that can be detected along with the parent drug. Methods: The study describes levamisole and its metabolites in thoroughbred ...
Peng S, Magdesian KG, Dowd J, Blea J, Carpenter R, Ho W, Finno CJ.Increases in serum gamma-glutamyltransferase (GGT) activity have been reported in Thoroughbred (TB) racehorses and associated with maladaptation to training but the underlying etiology remains unknown. Objective: Classify the etiology of high GGT syndrome in racing TBs by assessment of pancreatic enzymes, vitamin E concentrations, and both a candidate gene and whole genome association study. We hypothesized that a genetic variant resulting in antioxidant insufficiency or pancreatic dysfunction would be responsible for high GGT syndrome in TBs. Methods: A total of 138 California racing TBs. Amy...
Meyer J, Delay J, Bienzle D.Clinical, laboratory and tissue findings from 37 horses with lymphoma were investigated. Horses ranged in age from 0.3 to 20.5 years (median 5.0 years) and included 18 females and 19 males. Weight loss (n = 25) and ventral edema (n = 21) were the most common historical and physical abnormalities. The most common laboratory abnormalities were hyperfibrinogenemia (n = 26), hypoalbuminemia (n = 19), anemia (n = 19), leukemia (n = 14), hyperglobulinemia (n = 13), and thrombocytopenia (n = 13). Thirty-four tumors involved multiple lymphoid tissues and abdominal or thoracic organs, and 3 tumors were...
Jiménez-Aristizabal RF, López C, Álvarez ME, Giraldo C, Prades M, Carmona JU.To compare the temporal release (over three weeks) of tumor necrosis factor alpha (TNF-α), interleukin 4 (IL-4), IL-1 receptor antagonist (IL-1ra), platelet-derived growth factor BB (PDGF-BB) and transforming growth factor beta-1 (TGF-β1) from two platelet-rich fibrin (PRF) preparations from equine blood obtained at either 240g/8min or 416g/10min. Whole blood from 10 horses was used to obtain PRF clots by two different centrifugation protocols. After 1h of rest, PRF clots were deposited in wells with culture medium, which was changed at 6h, 24h and then every 48h to 21days. Cytokines and GFs...
Morrell JM, Johannisson A, Dalin AM, Rodriguez-Martinez H.The objective was to investigate whether it is possible to improve the quality of stallion semen, with respect to sperm morphology and chromatin integrity, both of which have been linked to fertility, using either density gradient centrifugation (DGC) or a new method, hereby named single layer centrifugation (SLC). The two methods of colloidal centrifugation were evaluated using 38 ejaculates from 10 stallions. Sperm morphology, subjective motility and sperm chromatin integrity were compared in uncentrifuged samples and in centrifuged sperm preparations. The proportion of morphologically norma...
Ainsworth DM, Wagner B, Franchini M, Grünig G, Erb HN, Tan JY.To evaluate time-dependent alterations in gene expression of chemokines in bronchial epithelium of recurrent airway obstruction (RAO)-affected horses and whether alterations resulted from increases in gene expression of interleukin (IL)-17 in cells isolated from bronchoalveolar lavage fluid (BALF). Methods: 8 RAO-susceptible horses and 9 control horses. Methods: In 2 experiments, both groups of horses were evaluated after being maintained on pasture and after being stabled and fed dusty hay for 1, 14, 35, and 49 days (experiment 1) or 14 and 28 days (experiment 2). In experiment 1, gene expres...
Côté GP, Smillie LB.Whereas skeletal muscle tropomyosin binds strongly to muscle F-actin in a buffer containing 30 mM KCl and 1-2 mM free Mg2+, equine platelet tropomyosin only binds stoichiometrically (1 tropomyosin molecule per 6 actin monomers) at higher Mg2+ concentrations (7-8 mM free Mg2+). At low free Mg2+ concentrations (1.5 mM) the binding of the platelet protein is only marginally increased by raising the KCl concentration to an optimal value (0.10-0.20 M). This weaker binding can be attributed to the relatively poor head-to-tail polymerization of platelet tropomyosin and its fewer actin-binding sites. ...
Pezzanite LM, Chow L, Johnson V, Griffenhagen GM, Goodrich L, Dow S.To evaluate effects of Toll-like and nucleotide-binding oligomerization domain (NOD)-like receptor (TLR, NLR) ligand stimulation of equine mesenchymal stromal cells (MSCs) on antibacterial and immunomodulatory properties in vitro. Methods: Controlled laboratory study. Methods: Equine bone-marrow-derived MSCs (three horses). Methods: MSCs were stimulated with TLR (polyinosinic:polycytidylic acid [pIC] and lipopolysaccharide [LPS]) and NLR agonists (γ-d-Glu-mDAP [IE-DAP]) for 2 h, and plated at 1 × 105 cells/well 24 h. MSC-conditioned media (MSC-CM) were collected and assessed for antimicr...
Ellis SA, Martin AJ, Holmes EC, Morrison WI.Nineteen horse MHC class I specificities have been serologically identified previously at a single locus (ELA-A), and two other specificities appear to be coded at other loci. Biochemical studies indicate that there are at least two expressed loci. In order to establish the number of transcribed horse MHC class I genes, we made a cDNA library from a heterozygous animal (ELA-A3/A7), and screened for positive clones using a bovine class I probe. More than 200 class I clones were isolated in this way, and so far seven unique full length sequences have been identified. All of the sequences are pre...
Donnelly KS, Giuliano EA, Sharma A, Tandon A, Rodier JT, Mohan RR.To explore (i) the potential of polyethylenimine (PEI) nanoparticles as a vector for delivering genes into equine corneal fibroblasts (ECFs) using green fluorescent protein (GFP) marker gene, (ii) whether PEI nanoparticle-mediated decorin (DCN) gene therapy could be used to inhibit fibrosis in the equine cornea using an in vitro model. Methods: Polyethylenimine-DNA nanoparticles were prepared at nitrogen-to-phosphate (N-P) ratio of 15 by mixing 22 kDa linear PEI and a plasmid encoding either GFP or DCN. ECFs were generated from donor corneas as previously described. Initially, GFP was introduc...
Magnarelli LA, Ijdo JW, Van Andel AE, Wu C, Padula SJ, Fikrig E.To determine whether horses living in tick-infested areas of northeastern United States with clinical signs of borreliosis or granulocytic ehrlichiosis had detectable serum antibodies to both Borrelia burgdorferi and Ehrlichia equi. Methods: Prospective study. Methods: Serum samples from 51 clinically normal horses, 14 horses with clinical signs of borreliosis, and 17 horses with clinical signs of granulocytic ehrlichiosis. Methods: Serum B burgdorferi or E equi antibodies were measured by use of an ELISA, immunoblot analysis, or indirect fluorescent antibody (IFA) staining. Results: Of the 82...
Burnaugh L, Sabeur K, Ball BA.Low-level production of the superoxide anion (O2*-) is an important signal transduction event in sperm function including capacitation; however, excessive production of O2*- can be detrimental to sperm function. The objective of this study was to assess dihydroethidium (DHE) as a probe for O2*- in equine spermatozoa. Ejaculated spermatozoa were separated by centrifugation over a Percoll gradient (40:80), and loaded with DHE (2.0 microM) as well as with calcein-acetoxymethylester (CAM, 7.8 nM) to determine cell viability. In Experiment 1, cells were incubated with the xanthine-xanthine oxidase ...
Mumford JA, Rossdale PD, Jessett DM, Gann SJ, Ousey J, Cook RF.An extensive outbreak of EHV-1 abortions occurred on a stud farm in England in 1985. Of the 67 pregnant mares present on the stud farm, 31 were challenged with EHV-1, resulting in the loss of 22 fetuses or foals. Laboratory investigations revealed that the spread of the virus closely followed movement of apparently healthy mares (during the incubation period of the infection). During the outbreak mares were challenged 1-4 months before the expected foaling date. There was no relationship between the gestational age at the time of challenge and the subsequent outcome of infection in terms of ab...
Kurtz BM, Singletary LB, Kelly SD, Frampton AR.In this study, Equus caballus major histocompatibility complex class I (MHC-I) was identified as a cellular entry receptor for the alphaherpesvirus equine herpesvirus type 1 (EHV-1). This novel EHV-1 receptor was discovered using a cDNA library from equine macrophages. cDNAs from this EHV-1-susceptible cell type were inserted into EHV-1-resistant B78H1 murine melanoma cells, these cells were infected with an EHV-1 lacZ reporter virus, and cells that supported virus infection were identified by X-Gal (5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside) staining. Positive cells were subjected t...
Smith KC.Abortion or neonatal disease may follow infection with several alpha, beta and gamma-herpesviruses. The alpha-herpesvirus, equid herpesvirus-1 (EHV-1), causes single or epizootic abortions or neonatal deaths in equids, and the closely related virus EHV-4 causes sporadic equine abortions. In cattle, the alpha-herpesviruses, bovine herpesvirus-1 (infectious bovine rhinotracheitis virus) and bovine herpesvirus-5 (bovine encephalitis virus), and a gamma-herpesvirus, bovine herpesvirus-4, have all been implicated as causes of abortion. In pigs, suid herpesvirus-1 (SHV-1: pseudorabies virus), an alp...
Rivero JL, Serrano AL, Barrey E, Valette JP, Jouglin M.Combined methodologies of enzyme-linked immunosorbent assay (ELISA), sodium dodecyl sulphate polyacrilamide gel electrophoresis (SDS-PAGE), immunoblotting, traditional myofibrillar ATPase (mATPase) histochemistry and immunocytochemistry of whole biopsied samples were used to study myosin heavy chain (MHC) isoforms in the equine gluteus medius muscle. The ELISA technique allowed the quantification of the three MHC isoforms known to be present in different horse muscles: slow (MHC-I) and two fast (termed MHC-IIA and MCH-IIX). The SDS-PAGE method resolved MHCs in three bands: MHC-I, MHC-IIX and M...
Baxi MK, Efstathiou S, Lawrence G, Whalley JM, Slater JD, Field HJ.Neural tissues from specific pathogen-free ponies that had been experimentally infected with equine herpesvirus 1 (EHV-1) were analysed by in situ hybridization. Digoxigenin-labelled EHV-1 BamHI fragments spanning almost the entire EHV-1 genome were hybridized to RNA in tissue sections from latently infected trigeminal ganglia. The BamHI E fragment detected EHV-1 RNA antisense to gene 63 (HSV-1 homologue ICP0) in a small number of neurons. Sixteen other BamHI fragments gave negative results in 20 sections tested with each fragment. Latency associated transcripts (LATs) were localized to the ne...
Thorne PS, Perry SS, Saito R, O'Shaughnessy PT, Mehaffy J, Metwali N, Keefe T, Donham KJ, Reynolds SJ.As a potent inflammatory agent, endotoxin is a key analyte of interest for studies of lung ailments in domestic environments and occupational settings with organic dust. A relatively unexplored advance in endotoxin exposure assessment is the use of recombinant factor C (rFC) from the Limulus pathway in a fluorometric assay. In this study, we compared airborne endotoxin concentrations in laboratory- and field-collected parallel air samples using the kinetic Limulus amebocyte lysate (LAL) assay and the rFC assay. Air sampling was performed using paired Institute of Occupational Medicine (IOM) sa...
Shahar R, Zaslansky P, Barak M, Friesem AA, Currey JD, Weiner S.Young's modulus and Poisson's ratios of 6mm-sized cubes of equine cortical bone were measured in compression using a micro-mechanical loading device. Surface displacements were determined by electronic speckle pattern-correlation interferometry. This method allows for non-destructive testing of very small samples in water. Analyses of standard materials showed that the method is accurate and precise for determining both Young's modulus and Poisson's ratio. Material properties were determined concurrently in three orthogonal anatomic directions (axial, radial and transverse). Young's modulus va...
Kojima K, Taniguchi K, Kobayashi N.We have sequenced gene 5 encoding NSP1 for three human, two porcine, two bovine, one feline, and five equine rotavirus strains, and compared the nucleotide and deduced amino acid sequences with the published sequences for other various strains. Subgroup I human strains L26, 69M, and DS-1 were found to have a similar NSP1 sequence despite their different G serotypes, VP4 genotypes, and RNA patterns. The NSP1 sequence of the human strain K8 showed a high degree of homology to those of porcine strains OSU and YM. A high degree of homology was found among three equine strains (H2, FI-14, and FI23)...
Moro LN, Viale DL, Bastón JI, Arnold V, Suvá M, Wiedenmann E, Olguín M, Miriuka S, Vichera G.The application of new technologies for gene editing in horses may allow the generation of improved sportive individuals. Here, we aimed to knock out the myostatin gene (MSTN), a negative regulator of muscle mass development, using CRISPR/Cas9 and to generate edited embryos for the first time in horses. We nucleofected horse fetal fibroblasts with 1, 2 or 5 µg of 2 different gRNA/Cas9 plasmids targeting the first exon of MSTN. We observed that increasing plasmid concentrations improved mutation efficiency. The average efficiency was 63.6% for gRNA1 (14/22 edited clonal cell lines) and 96.2% ...
Provitera P, Bouamr F, Murray D, Carter C, Scarlata S.Human immunodeficiency virus (HIV) and equine infectious anemia virus (EIAV) are closely related lentiviruses that infect immune cells, but their pathogenesis differ. Localization to the cytosolic leaflet of the plasma membrane is critical for replication of both viruses. This localization is accomplished through the matrix (MA) domain of the Gag precursor protein. In HIV-1, association of MA to anionic membranes appears to be primarily driven by a linear cluster of basic residues in the MA domain and an N-myristoylation signal. Interestingly, the MA protein of EIAV does not contain either of ...
Kachrimanidou M, Tzika E, Filioussis G. is ubiquitous in the environment and is also considered as a bacterium of great importance in diarrhea-associated disease for humans and different animal species. Food animals and household pets are frequently found positive for toxigenic without exposing clinical signs of infection. Humans and animals share common ribotypes (RTs) suggesting potential zoonotic transmission. However, the role of animals for the development of human infection due to remains unclear. One major public health issue is the existence of asymptomatic animals that carry and shed the bacterium to the environment, an...
Nagata K, Tsukita S, Tamura T, Sone N.A Helicobacter pylori membrane fraction oxidized yeast and equine cytochrome c, and N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD). When ascorbate was used as reductant, the Vmax and apparent Km values were 612 nmol electron min-1 (mg protein)-1 and 14 microM for yeast, and 419 nmol electron min-1 (mg protein)-1 and 19 microM for equine cytochrome c, respectively. For TMPD oxidation, the Vmax and Km values were 640 nmol electron min-1 (mg protein)-1 and 182 microM, respectively. These oxidase activities showed a high affinity for oxygen. Inhibition of both cytochrome-c and TMPD oxidase activi...
Couto MA, Harwig SS, Lehrer RI.Equine neutrophil antimicrobial peptide 2 (eNAP-2), a recently described antimicrobial peptide isolated from equine neutrophils, was found to selectively inactivate microbial serine proteases (subtilisin A and proteinase K) without inhibiting mammalian serine proteases (human neutrophil elastase, human cathepsin G, and bovine pancreatic trypsin). Although the primary structure of eNAP-2 resembled that of several known antiproteases that belong to the 4-disulfide core peptide family, this pattern of selectivity is unique. eNAP-2 formed a noncovalent complex with native subtilisin A or proteinas...
Cullinane AA, Rixon FJ, Davison AJ.The genome structure of equine herpesvirus 1 (EHV-1) subtype 2 was shown by electron microscopic studies and restriction endonuclease site mapping to comprise two covalently linked segments (L, 109 kbp; S, 35 kbp). The S segment contains a unique sequence (US) flanked by a substantial inverted repeat (TRS/IRS). Thus, the genome structure of EHV-1 subtype 2 is similar to that published previously for EHV-1 subtype 1, but the two subtypes differ in the occurrences of EcoRI and BamHI restriction sites. Hybridization studies using cloned EHV-1 DNA showed that the genome of EHV-1 subtype 2 is colin...
Gohari IM, Arroyo L, Macinnes JI, Timoney JF, Parreira VR, Prescott JF.Up to 60% of cases of equine colitis have no known cause. To improve understanding of the causes of acute colitis in horses, we hypothesized that Clostridium perfringens producing enterotoxin (CPE) and/or beta2 toxin (CPB2) are common and important causes of severe colitis in horses and/or that C. perfringens producing an as-yet-undescribed cytotoxin may also cause colitis in horses. Fecal samples from 55 horses (43 adults, 12 foals) with clinical evidence of colitis were evaluated by culture for the presence of Clostridium difficile, C. perfringens, and Salmonella. Feces were also examined by...
McGuire TC, Van Hoosier GL, Henson JB.The role of non-complement-fixing anti-equine infectious anemia (EIA) antibody in the conversion of complement fixation (CF) tests from positive to negative in EIA-infected horses was investigated. Complement-fixation inhibition (CFI) tests demonstrated antibodies in sera that were CF negative. These antibodies would bind to antigen, but would not fix complement. The inhibiting antibodies were isolated and shown to be IgG(T) by immunoelectrophoresis and immunodiffusion against monospecific anti-IgG(T) antisera. Separation of immunoglobulins from affected horse sera by DEAE cellulose chromatogr...
Proudman CJ, Edwards GB.A centrifugation/flotation technique for the identification of equine tapeworm eggs is described. It was validated by using faeces samples from 80 horses of known tapeworm status, and had a sensitivity of 61 per cent and a specificity of 98 per cent. The exclusion of false negative results in animals with less than 20 tapeworms increased the sensitivity to 92 per cent. No significant correlation was found between the number of eggs observed and the number of tapeworms present in the horses.
Jolly RD, Palmer DN, Dalefield RR.Analytical studies of three lipopigments show that much can be achieved. Lipopigment from ovine ceroid-lipofuscinosis is composed of discrete protein and lipid molecules in orderly arrays and lipid peroxidation is not involved in its formation. Subunit c of mitochondrial ATP synthase accounts for approximately 50% of accumulated material and is specific to the disease process in this and other forms of the disease. Lipofuscin from bovine heart was mostly soluble and also contained discrete proteins, lipids and metals. Equine thyroid lipofuscin was less soluble but also had a relatively high pr...
