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.
Nolen-Walston RD, Kulp JC, Stefanovski D, van Eps AW.Hyperinsulinemia is an important and treatable risk factor for laminitis in horses. Objective: Evaluate the Tosoh AIA-360 automated fluorescence enzyme immunoassay for the measurement of serum insulin concentrations in horses, and compare it to five other immunoassays for insulin quantification. Methods: One hundred serum samples from 83 horses were submitted for insulin measurement. Methods: The Tosoh AIA-360 was assessed against a reference assay (radioactive immunoassay; RIA). Using the same samples, TOS-FEIA, ELISA, and three chemiluminescent immunoassays (CLIA) were assessed for correlati...
Hoerdemann M, Sahoo DK, Allbaugh RA, Kubai MA.To assess if an affordable, commercially available 275 nm ultraviolet C (UV-C) device can inhibit fungal pathogens associated with equine keratomycosis and to establish the optimal exposure time/dose required in vitro. Methods: Fungal inhibition zone surface areas of isolates from two fungal genera (Aspergillus and Fusarium spp.) were compared in triplicates after UV-C exposure at distances of 10, 15, and 20 mm with single or repeat doses (4 h apart) of 5, 10, 15, or 30 s duration after 24, 48, and 72 h of incubation. Inhibition zones were microscopically assessed, and regrowth chec...
Mota-Rojas D, Whittaker AL, Coria-Avila GA, Martínez-Burnes J, Mora-Medina P, Domínguez-Oliva A, Hernández-Avalos I, Olmos-Hernández A....The growing interest in managing and recognizing pain in animals has led to the search for more sensitive methods to evaluate it, especially because some species conceal any visible changes associated with pain or are not easily assessed. Research has shown that an animal's facial expression changes when exposed to painful stimuli. Thus, developing several pain scales (grimace scales) in species such as horses, cattle, pigs, sheep, donkeys, rabbits, rats, mice, and cats has helped to improve the study of pain in veterinary medicine. The possibility of using facial expression as an indicator of...
Ichikawa Y, Iinuma Y, Okagawa T, Shimbo R, Enkhtuul B, Khurtsbaatar O, Kinoshita Y, Niwa H, Aoshima K, Kobayashi A, Batbaatar V, Ohashi K, Kimura T.Glanders is a World Organization for Animal Health (WOAH)-notifiable equine disease caused by the infection of Burkholderia mallei, and is endemic in Mongolia, South Asia, Africa, and South America. While the complement fixation test (CFT) has been widely used for serodiagnosis of glanders and is considered a standard serological test, it has several limitations. These limitations include poor specificity, labor intensive techniques, variability in antigen and protocol. Consequently, indirect enzyme-linked immunosorbent assays (iELISAs) based on recombinant proteins have been developed as alte...
Bouverat VMA, Naef J, Dolf G, Lamon I, Sage SE, Gerber V.Organic dust from hay is a primary trigger of equine asthma. Objective: (1) To introduce a novel hay-shaker (HS) device for standardised hay dust generation, enabling simultaneous measurements of various particulate matter (PM) size fractions. (2) To compare these measurements with those in the horse's breathing zone (BZ) to assess the influence of environmental and individual factors. Methods: Comparative experimental study. Methods: A HS generating dust was developed. Total dust (PMT) and size fractions (PM1, PM2.5, PM4 and PM10, representing particle size in μm) were measured from 50 hay s...
Hernández-Avilés C, Varner DD, Ramírez-Agámez L, Samper JC, Love CC.In the current study, we report the effect of different commercially available semen freezing extenders utilized for the "test-freeze" procedure for 13 years (2010-2023) as part of a commercial program of stallion sperm cryopreservation. Ejaculates obtained from sexually active and healthy stallions (n = 124) were cryopreserved using up to five commercially available semen freezing extenders (Lactose-EDTA [LE], MFR5, CryoMax LE [CMLE], CryoMax MFR5 [CMMFR5] or BotuCrio [BC]). Post-thaw sperm motility (total motility - TM [%]; progressive motility - PM [%]; and curvilinear velocity - VCL [μm...
Tsujimura K, Bannai H, Kambayashi Y, Nemoto M, Ohta M.Equid alphaherpesvirus 1 (EqAHV1; Orthoherpesviridae, Varicellovirus equidalpha1) spreads by viremia to susceptible organs. Because EqAHV1 circulates in the bloodstream in a cell-associated manner, serum samples are not considered valuable for detecting EqAHV1 and have therefore not been tested by highly sensitive detection methods such as real-time PCR (rtPCR). We investigated whether EqAHV1 could be detected by this method in equine serum samples. We performed rtPCR on archived sera and peripheral blood mononuclear cells (PBMCs) collected from 3 horses experimentally inoculated with EqAHV1. ...
Long S, Maleas G, Belacic ZA, Quam VG, Durgam S.To analyze the cellular, growth factor, and cytokine composition of equine sternal bone marrow aspirate (BMA) and laboratory-centrifuged BMA concentrate (BMAC). Unassigned: This was an in vitro experimental study. Cellular composition, growth factors (IGF-I, VEGF, PDGF, TGF-β1), and cytokines (IL-6, IL-10, tumor necrosis factor-α (TNF-α), and IL-1 receptor antagonist protein [IL-1Ra]) were quantified using a standard automated hematology analyzer and ELISA methods, respectively, in donor-matched BMA, BMAC (in-house centrifugation protocol), whole blood, and platelet-rich plasma (E-PET; Pall...
Frontiers in geneticsFebruary 27, 2025
Volume 16 1534461 doi: 10.3389/fgene.2025.1534461
Barber AM, Kingsley NB, Peng S, Giulotto E, Bellone RR, Finno CJ, Kalbfleisch T, Petersen JL.The Functional Annotation of Animal Genomes (FAANG) consortium aims to annotate animal genomes across species, and work in the horse has substantially contributed to that goal. As part of this initiative, chromatin immunoprecipitation with sequencing (ChIP-seq) was performed to identify histone modifications corresponding to enhancers (H3K4me1), promoters (H3K4me3), activators (H3K27ac), and repressors (H3K27me3) in eight tissues from two Thoroughbred stallions: adipose, parietal cortex, heart, lamina, liver, lung, skeletal muscle, and testis. The average genome coverage of peaks identified by...
Stefaniuk-Szmukier M, Bieniek A, Ropka-Molik K, Bellone RR.Congenital stationary night blindness (CSNB) has been connected to the leopard complex spotting phenotype (LP) in various horse breeds. CSNB associated with LP is thought to be caused by a 1378 bp insertion in TRPM1, with homozygotes being nightblind and having few to no spots of pigment in their white patterned area. This study aimed to assess the prevalence of CSNB alleles in tarant-colored horses in Poland through a three-primer system for an allele-specific Polymerase Chain Reaction (PCR). The TRPM1 gene insertion was genotyped in 221 horses belonging to Małopolska, Felin and Shetland Pon...
Lösle M, Lin CW, Beil-Wagner J, Aebi M, Buch T.Equine chorionic gonadotropin (eCG) is a widely used hormone that synchronizes the female cycle and induces estrus in livestock. eCG is a heterodimeric glycoprotein composed of non-covalently linked α- and β-chains whose glycosylation profiles determine the in vivo activity of the hormone. The commercially available eCG products are crudely purified from the serum of pregnant mares, hence called pregnant mare serum gonadotropin (PMSG). Appropriate glycosylation of the protein is crucial for the correct binding to the receptor, receptor activation, and its half-life. The exact protein composi...
MacMillan KM, Burns JJ, John E, Clancey N, Stull JW.Decreased total T4 (tT4) concentrations are frequently observed in racing Standardbred horses lacking clinical evidence of hypothyroidism. This poses a diagnostic challenge as primary hypothyroidism is reported to be rare in adult horses. Despite this, owners frequently wish to administer thyroid supplementation to horses having low tT4 concentrations. Forty racing Standardbred horses were sampled. Baseline tT4 concentrations were determined using human-based (equine) and canine-based chemiluminescent assays. Dynamic evaluation of tT4 was performed using a thyrotropin-releasing hormone (TRH) s...
Adler DMT, Nielsen AT, Olsen C, Verwilghen D, Damborg P, Olsen RH.To investigate the in vitro efficacy of antibiotics (amikacin, ceftiofur, and gentamicin) in combination with local anesthetics (LAs; bupivacaine hydrochloride, lidocaine hydrochloride, and mepivacaine hydrochloride), a combination commonly performed for IA injectate and regional limb perfusion (RLP) in horses. Unassigned: 17 equine clinical isolates were tested by the checkerboard method for their minimum inhibitory concentration (MIC) against a combination of concentrations of LAs and antibiotics from August 2020 through December 2023. Unassigned: For the majority of combinations, the antibi...
Li D, Wang X, Yao J, Chen S.Adipose tissue-derived extracellular vesicles (ATEVs) have garnered attention for their roles in intercellular communication and regulation. This study presents the first investigation of equine adipose tissue, with ATEV being extracted concurrently during the isolation of adipose-derived mesenchymal stem cells (ADSCs). Through CCK-8 cell proliferation assays and scratch migration assays, a significant promotional effect of ATEV on ADSCs was observed, which not only accelerated the proliferation rate of the stem cells but also enhanced their migratory capacity. The application of Trypan Blue e...
Skurková L, Matulníková L, Peťková B, Florian M, Slivková M, Lešková L, Mesarčová L, Kottferová J.This study aimed to assess the seasonal changes of the cortisol concentrations in horsehair (HCC), as an indicator of long-term or chronic stress. Hair samples were taken four times throughout one year from 12 horses (aged from 1.5 to 30 years), and analysed by the ELISA method. The sampling interval (interval between two collections of the samples representing two different seasons) ranged between 102-115 days. Seasonal dynamics have been confirmed with a pattern: August ˃ November ˃ January ˃ April (F (3.93) = 50.28, P < 0.001). The lowest cortisol concentrations were detected in A...
Faulk C.Sequencing a genome by students has now become practical as we demonstrated with our recent publication of the Przewalski's horse (Equus ferus przewalskii) genome. In this review, I describe my experience teaching genome assembly in the classroom. In my course, students sequenced, assembled, and published a high-quality genome for Przewalski's horse using Oxford Nanopore long-read sequencing with only $4000 of materials. Along with the genome, we assembled the mitochondrial genome, sequence variants, predicted gene annotations, and DNA methylation levels. Our genome statistics far exceeded the...
Littiere TO, Costa GB, Sales NAA, Carvalho JRG, Rodrigues IDM, Ramos GV, Ferraz GC.In humans, there is an ongoing academic discussion about measuring plasma lactate concentration ([La]) during an incremental exercise test (IET) to establish thresholds that can predict the maximal lactate steady state (MLSS). Training studies on horses have utilized the onset of [La] (OBLA), also known as V, the velocity at which the [La] of 4 mM is reached. This study assesses the effectiveness of four methods for estimating the MLSS in teaching horses. Ten teaching horses underwent a single IET to determine four exercise intensity thresholds for predicting MLSS and performed several constan...
Alim S, Cheppali SK, Pawar SS, Swamy MJ.Fibronectin type-II (FnII) proteins are major constituents in the seminal plasma of many mammals and play a crucial role in sperm capacitation. Additionally, the seminal FnII proteins from bull and horse exhibit chaperone-like activity (CLA), by acting as small heat shock proteins (shsps). The present work demonstrates that the major FnII protein of donkey seminal plasma, DSP-1 exhibits CLA with broad specificity and protects various client proteins such as alcohol dehydrogenase, lactate dehydrogenase and enolase against thermal and oxidative stress. Binding of phosphorylcholine (PrC) - the he...
Byambaragchaa M, Park SH, Park MH, Kang MH, Min KS.Equine chorionic gonadotropin (eCG) hormone, comprising highly glycosylated α- and β-subunits, elicits responses similar to follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in non-equid species. This study aimed to establish a mass production of recombinant eCG (rec-eCG) using CHO DG44 cells. Single-chain rec-eCG β/α was expressed in CHO DG44 cells. FSH- and LH-like activities were evaluated in CHO-K1 and HEK 293 cells expressing the equine LH/CG receptor (eLH/CGR), rat LH/CGR (rLH/CGR), and rFSHR. pERK1/2 activation and β-arrestin 2 recruitment were assessed in PathHunter ...
Simões BP, Cerri FM, Takahira RK, Borges AS, Oliveira-Filho JP, Amorim RM.Chlorfenapyr is a pesticide that interferes with mitochondrial function, leading to cell death and mortality. Although poisoning is primarily documented in humans and dogs, it can result in severe clinical signs, including sweating, respiratory distress, and neurological dysfunction. This report describes the clinical and laboratory findings of a horse with suspected accidental intoxication with chlorfenapyr. A 4-year-old Quarter Horse mare was referred to the veterinary hospital with signs of excessive sweating, hyperthermia, tachypnea, muscle fasciculation, and fearful facial expression. The...
Johnston TJ, Stewart AJ, Dryburgh EL, Bertin FR.Pituitary pars intermedia dysfunction (PPID) is diagnosed by measuring the ACTH concentrations. Due to the reported instability of ACTH, it is recommended to transfer centrifuged plasma into cryovials; however, in practice, cryovials are infrequently used, and serum (red-top) tubes are used instead. This study investigated whether this procedure affects ACTH concentrations and the diagnosis of PPID. Unassigned: This was a cohort study. Blood was collected into EDTA tubes from 9 horses with PPID and 7 controls. After centrifugation, plasma was either aliquoted into a cryovial or into a serum tu...
Facile V, Magliocca M, Dini FM, Imposimato I, Mariella J, Freccero F, Urbani L, Rinnovati R, Sel E, Gallina L, Castagnetti C, Galuppi R, Battilani M....Equine piroplasmosis is a tick-borne disease caused by and species. Despite its presence in Europe, no laboratory testing is required for animal movement, even though some countries remain free of this disease. Differentiating between species and genotypes is crucial to determine the most effective treatment, as dosage, active compounds, and duration vary. However, diagnosis is often challenging due to genetic variability and the limited sensitivity of molecular methods. The aims of this study were to compare the performances of different molecular diagnostic tests to identify the most effec...
Scheike AS, Plomp S, Fugazzola MC, Meurot C, Berenbaum F, van Weeren PR, Tryfonidou MA, von Hegedus JH.This study investigates the anti-inflammatory properties of liraglutide, a glucagon-like peptide 1 receptor agonists, in equine in vitro models and in an in vivo acute synovitis model in Shetland ponies. The anti-inflammatory effect of liraglutide was assessed by measuring concentrations of inflammatory biomarker C-C Motif Chemokine Ligand 2 (CCL2) in culture media of equine whole blood, peripheral blood mononuclear cells (PBMCs), chondrocytes, and synoviocytes, with or without lipopolysaccharide (LPS) or interleukin-1β. In the in vivo experiment, acute synovitis was bilaterally induced with ...
Barot D, Usimaki A, Linardi RL, Arensberg CM, Ortved KF.Quantify the concentration of α2-macroglobulin (A2M), immunomodulatory cytokines, and TGF-β1 factors in 4 commercially available autologous blood-based products including conditioned A2M (CA2M; Alpha2EQ; Astaria Global), autologous protein solution (APS; Pro-Stride; Zoetis), platelet-rich plasma (PRP; Restigen; Zoetis), and autologous conditioned plasma (ACP; Arthrex ACP). We hypothesized that CA2M would have higher concentrations of A2M and lower concentrations of cytokines and growth factors compared to APS, PRP, and ACP. Unassigned: Blood was obtained from 6 healthy, adult horses and proc...
Barbazanges P, Couroucé A, Le Digarcher G, Cardwell JM, Schmitt E, Toquet MP, Lemonnier LC, Richard EA.Fungi are ubiquitous in horses' environment. Their contribution to the pathophysiology of severe asthma (SA) is acknowledged, while controversies remain for mild-moderate asthma (MA). Objective: We hypothesised that fungi are a risk factor for asthma. Our objective was to compare different combinations of analytical methods (cytology, culture) and sampling sites (tracheal wash (TW), bronchoalveolar lavage fluid (BALF)) in relation to clinical status (control, MA, SA). Methods: Prospective cross-sectional study. Methods: The study population included asymptomatic racing horses in the field and ...
Finno CJ.In the field of equine muscle disorders, many conditions have a genetic basis. Therefore, genetic testing is an important part of the diagnostic evaluation. Validated genetic tests are currently available for 5 equine muscle disorders: hyperkalemic periodic paralysis, malignant hyperthermia, glycogen branching enzyme disease, type 1 polysaccharide storage myopathy, and myosin heavy chain myopathy. These diseases should be tested for in the appropriate breeds with clinical signs of disease or as part of breeding management. Genetic testing in veterinary medicine is not regulated, and therefore,...
Pusterla N, Barnum S, Lawton K, Craig B, James K.Equid alphaherpesvirus 4 (EqAHV4; Orthoherpesviridae, Varicellovirus equidalpha4; equine rhinopneumonitis virus) has seldom been associated with complications such as abortion and myeloencephalopathy, given the low tendency of this virus to induce viremia. We investigated the frequency of EqAHV4 viremia in horses with fever and respiratory signs. Case selection included all equids with EqAHV4 quantitative real-time PCR (qPCR)-positive nasal secretions (defined as EqAHV4 qPCR-positive cases) submitted to a diagnostic laboratory. Controls consisted of each case submitted before and after each Eq...
Halo M, Kirchner R, Tirpák F, Slanina T, Tokárová K, Kováčik A, Miškeje M, Greń A, Formicki G, Halo M, Madeddu R, Massányi P.The abundance of chemical elements in the blood of horses can indicate the physiological balance, health of animal as well as can be taken as an indicator of environmental pollution. The aim of this work was to analyse haematological, biochemical parameters, TOS, FRAP, SOD, Gpx, TAS and their correlations with concentrations of essential and risk elements in blood of horses stabled in two different locations: The National Stud Farm Topoľčianky (n = 11; 11 stallions, consisting of the breeds 6 Lipizzan, 3 Slovak warmblood, 2 Holsteiner) and Experimental Centre at Institute of Animal Husba...
Flood JA, Collins NM, Russell CM, Cuming RS, Carrick JB, Cudmore LA.Sepsis is a leading cause of morbidity and mortality in neonatal foals. Administration of appropriate antimicrobials and early aggressive supportive treatment is central to the efficacious treatment of neonatal sepsis and has proven to positively influence outcomes. The primary aim of our study was to evaluate microorganism results and antimicrobial sensitivities of neonatal foals with a positive blood culture submitted to our intensive care unit (ICU) during 2005-2022. The secondary aim was to compare the results to those of a similar previous study performed at the same ICU during 1999-2004....
Kublicka A, Lorek D, Mikołajczyk-Martinez A, Chodaczek G, Chwirot A, Bażanów B, Matczuk AK.The process of viral entry into host cells is crucial for the establishment of infection and the determination of viral pathogenicity. A comprehensive understanding of entry pathways is fundamental for the development of novel therapeutic strategies. Standard techniques for investigating viral entry include confocal microscopy and flow cytometry, both of which provide complementary qualitative and quantitative data. Imaging flow cytometry, which integrates the advantages of both methodologies, offers significant potential in virological studies. In this investigation, we employed imaging flow ...
Wang W, Wang S, Hou C, Xing Y, Cao J, Wu K, Liu C, Zhang D, Zhang L, Zhang Y, Zhou H.Recent studies have found that copy number variations (CNVs) are widespread in human and animal genomes. CNVs are a significant source of genetic variation, and have been shown to be associated with phenotypic diversity. However, the effect of CNVs on genetic variation in horses is not well understood. In the present study, CNVs in 6 different breeds of mare horses, Mongolia horse, Abaga horse, Hequ horse and Kazakh horse (all plateau breeds) and Debao pony and Thoroughbred, were determined using aCGH. In total, seven hundred CNVs were identified ranging in size from 6.1 Kb to 0.57 Mb across a...
Henson FM, Bowe EA, Davies ME.To identify the effect of fibroblastic growth factor-2 (FGF-2) on the intrinsic damage-repair response in articular cartilage in vitro. Methods: Articular equine cartilage explants, without subchondral bone, had a single impact load of 500 g applied from a height of 2.5 cm. Explants were then cultured in 0, 12, 25, 50 or 100 ng/ml FGF-2 for up to 28 days. Unimpacted discs served as controls for each time-point. Histological and immunohistochemical techniques were used to quantify and characterise the response of putative chondrocyte progenitor cells (CPC) to damage and FGF-2 treatment. Results...
Whalley JM, Robertson GR, Scott NA, Hudson GC, Bell CW, Woodworth LM.A gene in equine herpesvirus 1 (EHV-1; equine abortion virus) equivalent to the gB glycoprotein gene of herpes simplex virus (HSV) has been identified by DNA hybridization and nucleotide sequencing. A 4.3 kbp EHV-1 PstI-ClaI sequence (0.40 to 0.43 map units) contained an open reading frame flanked by appropriate control elements and was capable of encoding a polypeptide of 980 amino acids. This had 50 to 60% identity over a 617 amino acid conserved region with the gB gene products of HSV and three other alphaherpesviruses, and 20 to 30% identity with those of human cytomegalovirus and Epstein-...
Weiss M, Horzinek MC.In equine dermis cells infected with Berne virus particles were first detected 10 h after infection. Virions were encountered in all parts of the Golgi system and, infrequently, in the rough endoplasmic reticulum. A unique form of budding of preassembled rigid tubular nucleocapsids was demonstrated. Masses of tubular nucleocapsids of a lesser diameter and electron density were prominent in the cytoplasm and the nucleus of infected cells. Within the Golgi system and cytoplasmic cisternae virions appeared as straight or slightly curved rods. Extremely long, aberrant virions (250 nm) were occasio...
Hackett ES, McCue PM.Glucose assessment and regulation are important factors in the treatment of hospitalized horses and foals. Objective: The purpose of this study was to compare glucose measurement by a veterinary glucometer, adjusted by code for use in horses and foals, to a reference chemistry analyzer. It was hypothesized that the veterinary glucometer and reference analyzer would yield similar results and that interpretation of glucose values obtained from a veterinary glucometer would result in clinically appropriate decisions. Methods: Fifty blood samples from adult horses and 50 blood samples from neonata...
Contreras-Aguilar MD, Tvarijonaviciute A, Monkeviciene I, Martín-Cuervo M, González-Arostegui LG, Franco-Martínez L, Cerón JJ, Tecles F....Measurement of adenosine deaminase (ADA) can provide information about cell-mediated immunity. This report's objective was to study the enzymatic activity of total ADA (tADA) and its isoenzymes ADA1 and ADA2 in canine, equine, porcine, and bovine serum and saliva and their changes in different inflammatory situations in each species. Besides, an automated method for ADA2 measurement was developed and validated. Results: tADA was present in serum and saliva of healthy animals of the four species. Erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) concentration of 0.47 mM was needed for ADA1 inhibit...
Bhoora R, Quan M, Franssen L, Butler CM, van der Kolk JH, Guthrie AJ, Zweygarth E, Jongejan F, Collins NE.A quantitative real-time polymerase chain reaction (qPCR) assay using a TaqMan minor groove binder (MGB) probe was developed for the detection of Babesia caballi infection in equids from South Africa. Nine previously published sequences of the V4 hypervariable region of the B. caballi 18S rRNA gene were used to design primers and probes to target unique, conserved regions. The B. caballi TaqMan MGB qPCR assay was shown to be efficient and specific. The detection limit, defined as the concentration at which 95% of positive samples can be detected, was determined to be 0.000114% parasitized eryt...
Derse D, Carvalho M, Carroll R, Peterlin BM.Transcriptional regulatory mechanisms found in lentiviruses employ RNA enhancer elements called trans-activation responsive (TAR) elements. These nascent RNA stem-loops are cis-acting targets of virally encoded Tat effectors. Interactions between Tat and TAR increase the processivity of transcription complexes and lead to efficient copying of viral genomes. To study essential elements of this trans activation, peptide motifs from Tats of two distantly related lentiviruses, equine infectious anemia virus (EIAV) and human immunodeficiency virus type 1 (HIV-1), were fused to the coat protein of b...
Janssen IJ, Krücken J, Demeler J, von Samson-Himmelstjerna G.P-glycoproteins (Pgps) are suspected to mediate drug extrusion in nematodes contributing to macrocyclic lactone resistance. This association was recently shown for Parascaris Pgp-11. Ivermectin resistance was correlated with the presence of three pgp-11 single nucleotide polymorphisms and/or increased pgp-11 mRNA levels. In the present study, the ability of Pgp-11 to modulate ivermectin susceptibility was investigated by its expression in a pgp-11-deficient Caenorhabditis elegans strain. Expression of Parascaris pgp-11 in two transgenic lines significantly decreased ivermectin susceptibility i...
Blommaert D, Sergeant N, Delehedde M, Jouy N, Mitchell V, Franck T, Donnay I, Lejeune JP, Serteyn D.A-kinase anchor protein 4 (AKAP4) is playing a central role in flagellar structure, chemotaxis, capacitation and sperm motility. In mammals, AKAP4 is expressed during spermatogenesis. AKAP4 is synthesized as a precursor, proAKAP4, which is cleaved into mature AKAP4 during fibrous sheath assembly. The proAKAP4 is a good indicator of sperm quality in humans and boars. The aims of this work were to study the expression, the localization and the concentration of proAKAP4 and AKAP4 in equine semen, and to evaluate the possible correlation between the total and progressive motility and the concentra...
Lange-Consiglio A, Lazzari B, Perrini C, Pizzi F, Stella A, Cremonesi F, Capra E.Cell-derived microvesicles (MVs) are a recently discovered mechanism of cell-to-cell communication. Our previous data show that MVs secreted by equine amniotic mesenchymal-derived cells (AMCs) are involved in downregulation of proinflammatory genes in lipopolysaccharide-stressed equine tendon and endometrial cells. The aim of the present study was to evaluate whether AMC-MVs contain selected microRNAs (miRNAs) involved in inflammation. Two pools of cells, derived from 3 amniotic membranes each, and their respective MVs were collected. Small RNAs were extracted and deep sequenced, followed by m...
Kleinheinz J, Jung S, Wermker K, Fischer C, Joos U.Current approaches in bone regeneration combine osteoconductive scaffolds with bioactive cytokines like BMP or VEGF. The idea of our in-vitro trial was to apply VEGF165 in gradient concentrations to an equine collagen carrier and to study pharmacological and morphological characteristics of the complex in a circulation model. Methods: Release kinetics of VEGF165 complexed in different quantities in a collagen matrix were determined in a circulation model by quantifying protein concentration with ELISA over a period of 5 days. The structural changes of the collagen matrix were assessed with lig...
Grooters AM, Gee MK.Pythium insidiosum is an important cause of cutaneous and gastrointestinal disease in horses and dogs in the southeastern United States. Culture-based diagnosis of pythiosis is rarely definitive because production and identification of reproductive structures is difficult. The purpose of this study was to develop a polymerase chain reaction (PCR)-based assay for the identification of P insidiosum. Genomic DNA was extracted from 3 clinical isolates of P insidiosum and I isolate each of Pythium graminicola and Pythium arrhenomanes. The ITS I region of the ribosomal RNA gene of each isolate was a...
Celi P, Sullivan M, Evans D.Increasing interest in the role of oxidative stress (OS) in equine medicine has highlighted the need to develop reliable methods to quantify it. In this study we describe the effect of refrigeration (at 4 degrees C) on the stability of the reactive oxygen metabolites (d-ROMs) and biological antioxidant potential (BAP) tests carried out on 15 healthy horses. Blood samples, collected from the jugular vein, were immediately placed on ice and analysed using both the d-ROMs and BAP tests. Samples were also refrigerated at 4 degrees C and tested after 3, 7 and 24 h. The average results were similar ...
Ciscotto P, Machado de Avila RA, Coelho EA, Oliveira J, Diniz CG, Farías LM, de Carvalho MA, Maria WS, Sanchez EF, Borges A, Chávez-Olórtegui C.Venoms from the bee Apis mellifera, the caterpillar Lonomia achelous, the spiders Lycosa sp. and Phoneutria nigriventer, the scorpions Tityus bahiensis and Tityus serrulatus, and the snakes Bothrops alternatus, Bothrops jararaca, Bothrops jararacussu, Bothrops moojeni, Bothrops neuwiedi, Crotalus durissus terrificus, and Lachesis muta were assayed (800mug/mL) for activity against Staphylococcus aureus. Venoms from B. jararaca and B. jararacussu showed the highest S. aureus growth inhibition and also against other Gram-positive and Gram-negative bacteria. To characterize the microbicidal compon...
Tozaki T, Kakoi H, Mashima S, Hirota K, Hasegawa T, Ishida N, Miura N, Choi-Miura NH, Tomita M.Microsatellite 15 TKY System was characterized for parentage verification of horse registry. The Microsatellite 15 TKY System was constructed by using 15 microsatellites, TKY279, TKY287, TKY294, TKY297, TKY301, TKY312, TKY321, TKY325, TKY333, TKY337, TKY341, TKY343, TKY344, TKY374, and TKY394, to provide stringent PCR-based microsatellite typing specifically optimized for multicolor fluorescence detection. The Microsatellite 15 TKY System showed good resolutions for 250 unrelated Thoroughbred horses, and the probability of exclusion (PE) at each microsatellite ranged from 0.437 to 0.621, resul...
Södersten F, Ekman S, Schmitz M, Paulsson M, Zaucke F.Injuries of the equine superficial digital flexor tendon are common in racing horses. Knowledge of the tendon matrix composition is crucial to understand physiological and pathological processes in the tendon. The aim of this study was to analyze TSP-4 expressed in equine tendon. Equine tendons were extracted with 10 mM EDTA-containing buffer and TSP-4 purified with ion-exchange chromatography followed by heparin affinity chromatography. The purified TSP-4 was analyzed by one- and two-dimensional SDS-PAGE, immunoblotting, and MALDI-TOF mass spectrometry. Purified TSP-4 gave bands reacting with...
Breckenridge WC, Kuksis A.The triglyceride compositions of the milk fats of man, dog, guinea pig, cow, sheep, goat, and horse were compared by gas-liquid chromatography of the intact triglycerides and of the butyl esters of the component fatty acids. The milk fats of man, dog, and guinea pig, which were largely made up of long-chain fatty acids, showed a common pattern with major contributions made by the glycerides with 48-54 acyl carbon atoms. The milk fats of cow, sheep, and goat, which were rich in short-chain acids, showed significant proportions of triglycerides with 28-54 acyl carbon atoms. Horse milk, which con...
Goodrich LR, Choi VW, Carbone BA, McIlwraith CW, Samulski RJ.Cell transplantation for the treatment of joint disease is an important clinical tool. Genetic modification of cells before transplantation has shown enhanced healing. Ex vivo genetic modification of joint tissue cells with various adeno-associated virus (AAV) serotypes has not been investigated. The transduction efficiencies of self-complementary AAV serotypes (1-6 and 8) were determined in joint tissue containing chondrocytes and synoviocytes isolated from equine models. When comparing scAAV serotypes for efficient transduction ex vivo, in chondrocytes versus synoviocytes, serotypes 6 and 2,...
Baskurt OK, Farley RA, Meiselman HJ.Horse blood has a higher tendency to form red blood cell (RBC) aggregates compared with human blood, with this enhanced aggregation previously attributed to differences in plasma factors. Our results confirm this observation and further indicate that washed horse RBC also have a significantly higher aggregation tendency in dextran 70 solutions (i.e., horse RBC have a higher "aggregability"). In contrast, the aggregation tendency of rat RBC, both in autologous plasma and in dextran 70, is significantly less compared with human and horse RBC. Other rheological findings for horse and rat RBC incl...
Borroto-Esoda K, Boone LR.The endogenous reverse transcriptase reaction of equine infectious anemia virus (EIAV) has been studied, and conditions allowing synthesis of full-length minus-strand DNA have been determined. In contrast to results reported for other retroviruses, synthesis of EIAV full-length minus-strand DNA was not impaired by high concentrations of Nonidet P-40, a nonionic detergent used to make the virion envelope permeable. All components of the reaction were titrated for maximum synthesis of complete minus strands, and a time course under the standardized conditions was determined. Minor subgenomic ban...
Scholtzek AD, Hanke D, Walther B, Eichhorn I, Stöckle SD, Klein KS, Gehlen H, Lübke-Becker A, Schwarz S, Feßler AT.The detection of borderline oxacillin-resistant (BORSA) represents a challenge to both, veterinary and human laboratories. Between 2015 and 2017, 19 equine with elevated minimal inhibitory concentrations for oxacillin were detected in routine diagnostics. The aim of this study was to characterize these isolates to identify factors possibly associated with the BORSA phenotype. All were subjected to antimicrobial susceptibility testing and whole genome sequencing (WGS). A quantifiable β-lactamase activity assay was performed for a representative subset of 13 isolates. The WGS data analysis o...
Zaayman D, Human S, Venter M.In recent years, West Nile virus has been responsible for outbreaks in regions where it has not previously been found. Five genetic lineages with specific geographic distributions exist. Recent outbreaks of WNV associated with the introduction of lineage 1 strains into the western hemisphere, together with the emergence of lineage 2 WNV in Central Europe, has highlighted the potential for spread of pathogenic WNV strains beyond their expected geographical boundaries. Therefore, genotyping of WNV strains may have important applications in surveillance and epidemiology. We report here the develo...
Perez de Souza L, Naake T, Tohge T, Fernie AR.The grand challenge currently facing metabolomics is the expansion of the coverage of the metabolome from a minor percentage of the metabolic complement of the cell toward the level of coverage afforded by other post-genomic technologies such as transcriptomics and proteomics. In plants, this problem is exacerbated by the sheer diversity of chemicals that constitute the metabolome, with the number of metabolites in the plant kingdom generally considered to be in excess of 200 000. In this review, we focus on web resources that can be exploited in order to improve analyte and ultimately metabol...
Chauret N, Gauthier A, Martin J, Nicoll-Griffith DA.As domestic animals such as cat, horse, and dog increasingly become the clinical targets for drug discovery programs, the need to understand how these animals metabolize xenobiotics becomes more important. In the present study, substrates and inhibitors that were reported to be selective for particular P450 isozymes were used as probes to study in vitro metabolism in horse, dog, cat, and human liver microsomes. Seven selective catalytic activity markers for cytochrome P450-mediated reactions were measured: phenacetin O-deethylase (P4501A1/2), coumarin 7-hydroxylase (P4502A6), tolbutamide hydro...
Klein C, Troedsson MH.Establishment and maintenance of pregnancy are critically dependent on embryo-maternal communication during the preimplantation period. The horse is one of the few domestic species in which the conceptus-derived pregnancy recognition signal has not been identified. To gain new insights into the factors released by the equine conceptus, transcriptional profiling analyses of conceptuses retrieved 8, 10, 12, and 14 days after ovulation were performed using a whole-genome microarray. Selected array data were confirmed using quantitative PCR, and the expression of proteins of interest was confirmed...
Iwatani S, Zendo T, Yoneyama F, Nakayama J, Sonomoto K.A novel bacteriocin, lacticin Z, produced by Lactococcus lactis QU 14 isolated from a horse's intestinal tract was identified. Lacticin Z was purified through a three step procedure comprised of hydrophobic-interaction, cation-exchange chromatography, and reverse-phase HPLC. ESI-TOF MS determined the molecular mass of lacticin Z to be 5,968.9 Da. The primary structure of lacticin Z was found to consist of 53 amino acid residues without any leader sequence or signal peptide. Lacticin Z showed homology to lacticin Q from L. lactis QU 5, aureocin A53 from Staphylococcus aureus A53, and mutacin BH...
Ljungberg K, Whitmore AC, Fluet ME, Moran TP, Shabman RS, Collier ML, Kraus AA, Thompson JM, Montefiori DC, Beard C, Johnston RE.A novel genetic vaccine that is based on a Venezuelan equine encephalitis virus (VEE) replicon launched from plasmid DNA is described. The plasmid encodes a VEE replicon under the transcriptional control of the cytomegalovirus immediate-early promoter (VEE DNA). The VEE DNA consistently expressed 3- to 15-fold more green fluorescent protein in vitro than did a conventional DNA vaccine. Furthermore, transfection with the DNA-launched VEE replicon induced apoptosis and type I interferon production. Inoculation of mice with VEE DNA encoding human immunodeficiency virus type 1 gp160 significantly ...
Al Jassim RA, Scott PT, Trebbin AL, Trott D, Pollitt CC.Seventy-two lactic acid producing bacterial isolates (excluding streptococci) were cultured from the gastrointestinal tract of six horses. Two of the horses were orally dosed with raftilose to induce lactic acidosis and laminitis while the remaining four were maintained on a roughage diet. Near complete 16S rDNA was amplified by PCR from the genomic DNA of each isolate. Following RFLP analysis with the restriction enzymes MboI, HhaI and HinfI, the PCR products from the 18 isolates that produced L- and/or D-lactate were subsequently cloned and sequenced. DNA sequence analysis indicated that the...
Kitamoto N, Ramig RF, Matson DO, Estes MK.The production of viral antigen after infection of MA104, HepG2 (derived from human liver), and CaCo-2 (derived from human colon) cells with various cultivatable human and animal rotavirus strains was compared using immunofluorescence tests. All rotavirus strains examined expressed antigen in CaCo-2 cells and MA104 cells, but only some virus strains, namely, SA11-Cl3 (simian), RRV (simian), CU-1 (canine), and Ty1 (turkey), produced antigen in numbers of infected HepG2 cells comparable to infections in MA104 and CaCo-2 cells. Fl-14 (equine), OSU (porcine), NCDV (bovine), and Ch2 (chicken) strai...
