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.
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...
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 ...
Fegraeus K, Riihimäki M, Nordlund J, Akula S, Wernersson S, Raine A.Single-cell RNA sequencing (scRNA-seq) is a valuable tool for investigating cellular heterogeneity in diseases such as equine asthma (EA). This study evaluates the HIVE™ scRNA-seq method, a pico-well-based technology, for processing bronchoalveolar lavage (BAL) cells from horses with EA. The HIVE method offers practical advantages, including compatibility with both field and clinical settings, as well as a gentle workflow suited for handling sensitive cells. Our results show that the major cell types in equine BAL were successfully identified; however, the proportions of T cells and macropha...
Wang J, Ren W, Li Z, Ma S, Li L, Wang R, Zeng Y, Meng J, Yao X.This study aims to analyze the whole-genome DNA methylation differences in Yili horses before and after racing, with the goal of identifying differentially methylated genes associated with racing performance and exploring the epigenetic mechanisms underlying exercise in horses. Blood samples were collected from the jugular veins of the top 3 Yili horses in a 5000 m race, which included 25 competitors, both prior to and within 5 min after the race. Genomic DNA was extracted, followed by sequencing using Whole-Genome Bisulfite Sequencing (WGBS) to assess DNA methylation levels, differentially me...
The AnalystJanuary 23, 2025
doi: 10.1039/d4an01501k
Göktaş EF, Kabil E, Söylemez Yeşilçimen E, Dirikolu L.Various technical methodologies are required to accurately detect substances of different chemical and pharmacological properties in biological samples, which are increasing in number and variety daily. Therefore, laboratories where many samples and different factors are analyzed simultaneously need methods with easy sample preparation, short analysis times and low analysis costs. In this study, the objective was to scan substances susceptible to chemical degradation, amenable to analysis without hydrolysis, and exhibiting short-term stability by employing a straightforward, expeditious, and c...
Whitlock F, van Dijk J, Hodgkinson JE, Grewar JD, Newton JR.Anthelmintic resistance (AR) threatens effective equine parasite control. Quarterly data summaries from faecal worm egg count testing (FWECT) performed by UK laboratories have appeared in Equine Quarterly Disease Surveillance Reports (EQDSR) since 2007, but have not previously been assessed. Objective: To assess strongyle FWECT methods and thresholds used by UK laboratories. To investigate factors associated with quarterly laboratory FWECT positivity rates between 2007 and 2023. Methods: Laboratory surveys and analysis of laboratory summary data. Methods: Laboratories were surveyed in Q3 2018 ...
Palomino Lago E, Ross AKC, McClellan A, Guest DJ.Bone fractures are a significant problem in Thoroughbred racehorses. The risk of fracture is influenced by both genetic and environmental factors. To determine the biological processes that are affected in genetically susceptible horses, we utilised polygenic risk scoring to establish induced pluripotent stem cells (iPSCs) from horses at high and low genetic risk. RNA-sequencing on iPSC-derived osteoblasts revealed 112 genes that were significantly differentially expressed. Forty-three of these genes have known roles in bone, 27 are not yet annotated in the equine genome and 42 currently have ...
Nemoto M, Kawanishi N, Kambayashi Y, Bannai H, Yamanaka T, Tsujimura K.Equine influenza virus (EIV) can be transmitted by inhalation of aerosolized droplets, direct contact, and contaminated fomites. However, to our knowledge, there are no reports of the recovery of EIV from the air surrounding infected horses. Here, we evaluated whether EIV can be recovered from the air in the stalls of experimentally infected horses by using an air sampler. Furthermore, we examined whether rapid molecular test kits with reaction times of less than 30 min can detect EIV from air samples for potential field application. Two horses kept in individual stalls were experimentally i...
Hidalgo M.Sperm vitrification is an alternative freezing method, which includes high cooling rates and non-permeable cryoprotectants agents. The first attempt in equids was using the spheres technique by directly dropping small volumes of the sperm into liquid nitrogen. Later, vitrification was developed using 0.25 mL straws with outer covers, which resulted in similar progressive motility when compared to conventional freezing in donkeys (44.3 ± 15.0 % vs. 44.7 ± 18.2 %) or even higher in horses (48.2 ± 2.3 % vs. 37.3 ± 2.2 %). Subsequently, the vitrification of larger volumes of sperm in 0.5 mL st...
Königová A, Babják M, Kuzmina TA, Burcáková Ľ, Syrota Y, Várady M.The study aimed to conduct a survey on the occurrence of benzimidazole (BZ) resistance in strongyles by in vitro egg hatch test (EHT) and larval development test (LDT) and to identify the effective indicators of early resistance detection on horse farms with associated risk factors analysis appraisal. In total, 203 horses from 8 farms underwent the fecal sampling of which 77 horses were selected for in vitro testing. Simultaneously, 18 horses were chosen to analyse the results of in vitro tests compared to the in vivo fecal egg count reduction test (FECRT). The EHT indicated the presence of re...
Gravance CG, Garner DL, Baumber J, Ball BA.The fluorescent carbocyanine dye, JC-1, labels mitochondria with high membrane potential orange and mitochondria with low membrane potential green. Evaluation of mitochondrial membrane potential with JC-1 has been used in a variety of cell types, including bull spermatozoa; however, JC-1 staining has not yet been reported for equine spermatozoa. The aim of this study was to apply JC-1 staining and assessment by flow cytometry or a fluorescence microplate reader for evaluation of mitochondrial function of equine spermatozoa. Six ejaculates from four stallions were collected and centrifuged thro...
Rizk MA, El-Sayed SA, Terkawi MA, Youssef MA, El Said el Sel S, Elsayed G, El-Khodery S, El-Ashker M, Elsify A, Omar M, Salama A, Yokoyama N....A rapid and accurate assay for evaluating antibabesial drugs on a large scale is required for the discovery of novel chemotherapeutic agents against Babesia parasites. In the current study, we evaluated the usefulness of a fluorescence-based assay for determining the efficacies of antibabesial compounds against bovine and equine hemoparasites in in vitro cultures. Three different hematocrits (HCTs; 2.5%, 5%, and 10%) were used without daily replacement of the medium. The results of a high-throughput screening assay revealed that the best HCT was 2.5% for bovine Babesia parasites and 5% for equ...
Paria N, Raudsepp T, Pearks Wilkerson AJ, O'Brien PC, Ferguson-Smith MA, Love CC, Arnold C, Rakestraw P, Murphy WJ, Chowdhary BP.Studies of the Y chromosome in primates, rodents and carnivores provide compelling evidence that the male specific region of Y (MSY) contains functional genes, many of which have specialized roles in spermatogenesis and male-fertility. Little similarity, however, has been found between the gene content and sequence of MSY in different species. This hinders the discovery of species-specific male fertility genes and limits our understanding about MSY evolution in mammals. Here, a detailed MSY gene catalogue was developed for the horse--an odd-toed ungulate. Using direct cDNA selection from horse...
Roth SP, Glauche SM, Plenge A, Erbe I, Heller S, Burk J.Decellularization of tendon tissue plays a pivotal role in current tissue engineering approaches for in vitro research as well as for translation of graft-based tendon restoration into clinics. Automation of essential decellularization steps like freeze-thawing is crucial for the development of more standardized decellularization protocols and commercial graft production under good manufacturing practice (GMP) conditions in the future. In this study, a liquid nitrogen-based controlled rate freezer was utilized for automation of repeated freeze-thawing for decellularization of equine superficia...
Smit H, Gaastra W, Kamerling JP, Vliegenthart JF, de Graaf FK.The erythrocyte receptor for Escherichia coli K99 fimbrial adhesin was isolated from equine erythrocytes and characterized as Neu5Gc-alpha(2----3)-Galp-beta(1----4)-GLcp-beta(1----1)-Ceramide. This glycolipid acted as the receptor for K99 by four different experimental approaches: inhibition of equine erythrocyte hemagglutination by preincubation of K99-positive bacteria or purified K99 fimbriae with the isolated glycolipid; inhibition of attachment of K99-positive bacteria to porcine intestinal epithelial cells in the presence of the isolated glycolipid; induction of binding of K99-positive b...
Beck C, Lowenski S, Durand B, Bahuon C, Zientara S, Lecollinet S.West Nile Fever is a zoonotic disease caused by a mosquito-borne flavivirus, WNV. By its clinical sensitivity to the disease, the horse is a useful sentinel of infection. Because of the virus' low-level, short-term viraemia in horses, the primary tools used to diagnose WNV are serological tests. Inter-laboratory proficiency tests (ILPTs) were held in 2010 and 2013 to evaluate WNV serological diagnostic tools suited for the European network of National Reference Laboratories (NRLs) for equine diseases. These ILPTs were designed to evaluate the laboratories' and methods' performances in detectin...
Verheij HM, Volwerk JJ, Jansen EH, Puyk WC, Dijkstra BW, Drenth J, de Haas GH.It is known that His-48 is part of the active center in pancreatic phospholipase. To further elucidate the role of histidine-48 in the active center of pancreatic phospholipase A2, we have modified the enzyme with a number of bromo ketones and methyl benzenesulfonates. Rapid methylation occurred with methyl p-nitrobenzenesulfonate. Methylated phospholipase shows total loss of enzymatic activity whereas binding of substrate and the cofactor Ca2+ remains intact. Amino acid analysis of methylated equine phospholipase showed the loss of the single molecule of histidine and the formation of one mol...
Poissant J, Gavriliuc S, Bellaw J, Redman EM, Avramenko RW, Robinson D, Workentine ML, Shury TK, Jenkins EJ, McLoughlin PD, Nielsen MK, Gilleard JS.Horses are ubiquitously infected by a diversity of gastro-intestinal parasitic helminths. Of particular importance are nematodes of the family Strongylidae, which can significantly impact horse health and performance. However, knowledge about equine strongyles remains limited due to our inability to identify most species non-invasively using traditional morphological techniques. We developed a new internal transcribed spacer 2 (ITS2) DNA metabarcoding 'nemabiome' assay to characterise mixed strongyle infections in horses and assessed its performance by applying it to pools of infective larvae ...
Scepek S, Coorssen JR, Lindau M.Using the patch-clamp technique, we studied the role of protein phosphorylation and dephosphorylation on the exocytotic fusion of secretory granules with the plasma membrane in horse eosinophils. Phorbol 12-myristate 13-acetate (PMA) had no effect on the amplitude and dynamics of degranulation, indicating that the formation of fusion pores is insensitive to activation of protein kinase C (PKC). Fusion pore expansion, however, was accelerated approximately 2-fold by PMA, and this effect was abolished by staurosporine. Elevating intracellular Ca2+ to 1.5 microM also resulted in a 2-fold accelera...
Dobbe JC, van der Meer Y, Spaan WJ, Snijder EJ.The recent development of arterivirus full-length cDNA clones makes possible the construction of chimeric arteriviruses for fundamental and applied studies. Using an equine arteritis virus (EAV) infectious cDNA clone, we have engineered chimeras in which the ectodomains of the two major envelope proteins, the glycoprotein GP(5) and the membrane protein M, were replaced by sequences from envelope proteins of related and unrelated RNA viruses. Using immunofluorescence microscopy, we monitored the transport of the hybrid GP(5) and M proteins to the Golgi complex, which depends on their heterodime...
Nikiforov TT, Rendle RB, Goelet P, Rogers YH, Kotewicz ML, Anderson S, Trainor GL, Knapp MR.A new method for typing single nucleotide polymorphisms in DNA is described. In this method, specific fragments of genomic DNA containing the polymorphic site(s) are first amplified by the polymerase chain reaction (PCR) using one regular and one phosphorothioate-modified primer. The double-stranded PCR product is rendered single-stranded by treatment with the enzyme T7 gene 6 exonuclease, and captured onto individual wells of a 96 well polystyrene plate by hybridization to an immobilized oligonucleotide primer. This primer is designed to hybridize to the single-stranded target DNA immediately...
Di Gennaro A, Lorusso A, Casaccia C, Conte A, Monaco F, Savini G.A serum neutralization assay (SN) was compared with the official plaque reduction neutralization test for the quantitation of West Nile virus antibodies. A total of 1,348 samples from equid sera and 38 from human sera were tested by these two methods. Statistically significant differences were not observed, thus supporting the use of SN for routine purposes.
Nally JE, Artiushin S, Timoney JF.Leptospira interrogans is a mammalian pathogen which must adapt to a range of new environmental conditions including temperature change when it infects new hosts. In vitro studies of organisms cultured at 30 degrees C and shifted to 37 degrees C for 5 to 7 days have confirmed that synthesis of several proteins involved in equine infection is regulated in response to temperature change (J. E. Nally, J. F. Timoney, and B. Stevenson, Infect. Immun. 69:400-404, 2001). In order to specifically identify antigenic proteins upregulated at 37 degrees C, groups of three ponies were immunized with organi...
Alhassan A, Thekisoe OM, Yokoyama N, Inoue N, Motloang MY, Mbati PA, Yin H, Katayama Y, Anzai T, Sugimoto C, Igarashi I.Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid method whereby DNA is amplified with high specificity, efficiency, and rapidity under isothermal conditions using a set of four specifically designed primers and a DNA polymerase with strand displacement activity. In this study, we used LAMP primer sets designed from EMA-1 and Bc 48 genes for detection of Theileria equi and Babesia caballi infections, respectively. These primer sets specifically amplified DNA of the respective parasites. Both primer sets amplified T. equi and B. caballi up to 10(-6) dilution of 10-fold seria...
Takahashi T, Yawata M, Raudsepp T, Lear TL, Chowdhary BP, Antczak DF, Kasahara M.In rodents, the Ly49 family encodes natural killer (NK) receptors interacting with classical MHC class I molecules, whereas the corresponding receptors in primates are members of the killer cell immunoglobulin-like receptor (KIR) family. Recent evidence indicates that the cattle, domestic cat, dog, and pig have a single LY49 and multiple KIR genes, suggesting that predominant NK receptors in most non-rodent mammals might be KIR. Here, we show that the horse has at least six LY49 genes, five with an immunoreceptor tyrosine-based inhibition motif (ITIM) and one with arginine in the transmembrane...
Slusarewicz P, Pagano S, Mills C, Popa G, Chow KM, Mendenhall M, Rodgers DW, Nielsen MK.Intestinal parasites are a concern in veterinary medicine worldwide and for human health in the developing world. Infections are identified by microscopic visualisation of parasite eggs in faeces, which is time-consuming, requires technical expertise and is impractical for use on-site. For these reasons, recommendations for parasite surveillance are not widely adopted and parasite control is based on administration of rote prophylactic treatments with anthelmintic drugs. This approach is known to promote anthelmintic resistance, so there is a pronounced need for a convenient egg counting assay...
Zhen H, Han T, Fennell DE, Mainelis G.We report here that stress experienced by bacteria due to aerosolization and air sampling can result in severe membrane impairment, leading to the release of DNA as free molecules. Escherichia coli and Bacillus atrophaeus bacteria were aerosolized and then either collected directly into liquid or collected using other collection media and then transferred into liquid. The amount of DNA released was quantified as the cell membrane damage index (ID), i.e., the number of 16S rRNA gene copies in the supernatant liquid relative to the total number in the bioaerosol sample. During aerosolization by ...
Kim CM, Blanco LB, Alhassan A, Iseki H, Yokoyama N, Xuan X, Igarashi I.We developed a TaqMan real-time polymerase chain reaction (PCR) assay for the quantitative detection of Theileria equi from the in vitro-cultured parasite and field blood samples collected from horses living in Ghana and Brazil. The detection limit for the assay was determined to be 1.5 parasites/microl per sample, and the quantitative capacity was demonstrated using the in vitro-cultured parasite. For field applications, the real-time PCR assay was compared to a previously established nested PCR assay used as the gold standard for the real-time PCR assay. Of 65 field blood samples, 46 samples...
Richardson DW, Dodge GR.To determine the effects of interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) on expression and regulation of several matrix-related genes by equine articular chondrocytes. Methods: Articular cartilage harvested from grossly normal joints of 8 foals, 6 yearling horses, and 8 adult horses. Methods: Chondrocytes maintained in suspension cultures were treated with various doses of human recombinant IL-1beta or TNF-alpha. Northern blots of total RNA from untreated and treated chondrocytes were probed with equine complementary DNA (cDNA) probes for cartilage matrix-related ge...
McPartlin LA, Suarez SS, Czaya CA, Hinrichs K, Bedford-Guaus SJ.Capacitation is a complex and not well-understood process that encompasses all the molecular changes sperm must undergo to successfully fertilize an oocyte. In vitro fertilization has remained elusive in the horse, as evidenced by low in vitro fertilization (IVF) rates (0%-33%); moreover, only two foals have ever been produced using IVF. Incubation of stallion sperm in modified Whittens supplemented with bovine serum albumin and sodium bicarbonate yielded significant rates of time-dependent protein tyrosine phosphorylation and induced acrosomal exocytosis, consistent with capacitation. The obj...
Rudolph J, O'Callaghan DJ, Osterrieder N.The genome of equine herpesvirus type 1 (EHV-1) strain RacL11, a highly virulent isolate obtained from an aborted foal, and that of the modified live vaccine strain KyA, were cloned as bacterial artificial chromosomes (BAC) in Eseherichia coli. Mini F plasmid sequences were inserted into the viral genomes by homologous recombination instead of the gene 71 (EUS4) open reading frame after co-transfection of viral DNA and recombinant plasmid pdelta71-pHA2 into RK13 cells. After isolation of recombinant viruses by three rounds of plaque purification, viral DNA was isolated from RK13 cells infected...
Fornstedt B, Rosengren E, Carlsson A.The 5-S-cysteinyl derivatives of dopamine, dopa (3,4-dihydroxyphenylalanine) and dopac (3,4-dihydroxyphenylacetic acid) were synthesized and used as reference compounds in high performance liquid chromatography analyses of extracts from various brain regions of eight mammalian species. All three metabolites were detected in the brains of all the species studied. The regional distribution of the metabolites was similar to that of dopamine; the metabolite concentrations ranged from less than 0.1 percent to more than 1 percent of the dopamine level, the highest ratios generally being found in sub...
Haber M, Cao Z, Panjwani N, Bedenice D, Li WW, Provost PJ.The physiologic mechanisms involving growth factors, including PDGF-BB, EGF, and TGF-beta 1, as potent mediators of fibroblasts and epithelial cells in corneal wound healing remain unknown. The goal of this study was to determine culture methods for equine epithelial cells and keratocytes and to investigate how exogenous growth factors influence proliferation of both cell types. Methods: Cell cultures were established from healthy corneas harvested from horses immediately following euthanasia and maintained using standard tissue culture protocols. To determine the effects of PDGF-BB, EGF, TGF-...
Nielsen MK, Peterson DS, Monrad J, Thamsborg SM, Olsen SN, Kaplan RM.Strongylus vulgaris is an important strongyle nematode with high pathogenic potential infecting horses world-wide. Several decades of intensive anthelmintic use has virtually eliminated clinical disease caused by S. vulgaris, but has also caused high levels of anthelmintic resistance in equine small strongyle (cyathostomin) nematodes. Recommendations aimed at limiting the development of anthelmintic resistance by reducing treatment intensity raises a simultaneous demand for reliable and accurate diagnostic tools for detecting important parasitic pathogens. Presently, the only means available t...
Kolodziejek J, Dürrwald R, Herzog S, Ehrensperger F, Lussy H, Nowotny N.The aim of this study was to gain more detailed insights into the genetic evolution and variability of Borna disease virus (BDV). Phylogenetic analyses were performed on field viruses originating from naturally infected animals, the BDV vaccine strain 'Dessau', four widely used laboratory strains and the novel BDV subtype No/98. Four regions of the BDV genome were analysed: the complete p40, p10 and p24 genes and the 5'-untranslated region of the X/P transcript. BDV isolates from the same geographical area exhibited a clearly higher degree of identity to each other than to BDV isolates from ot...
Zhao Y, Holden VR, Smith RH, O'Callaghan DJ.The UL3 protein of equine herpesvirus 1 (EHV-1) KyA strain is a homolog of the ICP27 alpha regulatory protein of herpes simplex virus type 1 (HSV-1) and the ORF 4 protein of varicella-zoster virus. To characterize the regulatory function of the UL3 gene product, a UL3 gene expression vector (pSVUL3) and a vector expressing a truncated version of the UL3 gene (pSVUL3P) were generated. These effector plasmids, in combination with an EHV-1 immediate-early (IE) gene expression vector (pSVIE) and chimeric EHV-1 promoter-chloramphenicol acetyltransferase (CAT) reporter constructs, were used in trans...
Wang ZW, Nara M, Wang YX, Kotlikoff MI.The effects of sulfhydryl reduction/oxidation on the gating of large-conductance, Ca(2+)-activated K+ (maxi-K) channels were examined in excised patches from tracheal myocytes. Channel activity was modified by sulfhydryl redox agents applied to the cytosolic surface, but not the extracellular surface, of membrane patches. Sulfhydryl reducing agents dithiothreitol, beta-mercaptoethanol, and GSH augmented, whereas sulfhydryl oxidizing agents diamide, thimerosal, and 2,2'-dithiodipyridine inhibited, channel activity in a concentration-dependent manner. Channel stimulation by reduction and inhibit...
Campbell AJ, Gasser RB, Chilton NB.In the current study, molecular techniques were evaluated for the species identification of individual strongyle eggs. Adult worms of Strongylus edentatus, S. equinus and S. vulgaris were collected at necropsy from horses from Australia and the U.S.A. Genomic DNA was isolated and a ribosomal transcribed spacer (ITS-2) amplified and sequenced using polymerase chain reaction (PCR) techniques. The length of the ITS-2 sequence of S. edentatus, S. equinus and S. vulgaris ranged between 217 and 235 nucleotides. Extensive sequence analysis demonstrated a low degree (0-0.9%) of intraspecific variation...
