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.
Go YY, Hazard NW, Balasuriya UBR, Chapman AM, Fitton NS, Kenéz Á, Andrews FM.Accurate quantitative analysis of equine insulin in blood samples is critical for assessing hyperinsulinemia in horses. Although there are various laboratory methods for evaluating equine serum insulin, different immunoassays show significant discrepancies between the determined insulin concentrations and are often not comparable. The aim of this study was to evaluate the Immulite® 1000 chemiluminescent immunoassay (CLIA) to establish independent laboratory and assay-specific cut values to provide an accurate diagnosis of hyperinsulinemia in horses. Thus, the analytical and clinical performan...
Mawhinney I, Bollard A.Detection of Taylorella equigenitalis (CEMO) in the horse uses genital swabs. These swabs traditionally have been put in Amies charcoal transport medium for detection by culture but are also used for PCR. We determined the suitability of swabs without transport medium (Dry swabs) for CEMO PCR compared to swabs in Amies charcoal transport medium. The experiment was a factorial design using swab type and dilution of organism in culture suspensions, done in two parts. Simulated genital swabs were prepared in the laboratory by dipping in pairs into culture suspensions containing T. equigenitalis w...
Cortez JV, Hardwicke K, Cuervo-Arango J, Grupen CG.The cloning of horses is a commercial reality, yet the availability of oocytes for cloned embryo production remains a major limitation. Immature oocytes collected from abattoir-sourced ovaries or from live mares by ovum pick-up (OPU) have both been used to generate cloned foals. However, the reported cloning efficiencies are difficult to compare due to the different somatic cell nuclear transfer (SCNT) techniques and conditions used. The objective of this retrospective study was to compare the in vitro and in vivo development of equine SCNT embryos produced using oocytes recovered from abatt...
Schenk I, Broussou D, Roques B, Lagershausen H, Machnik M, Röttgen H, Toutain PL, Thevis M.The combination of sulfadoxine (SDO) with trimethoprim (TMP) is widely used in veterinarian medicine. The aim of the present study was to compare excretion profiles and detection time windows of SDO and TMP in plasma and urine by means of a validated quantitative method. Eight horses received a single intravenous (i.v.) dose of 2.7 mg TMP and 13.4 mg SDO per kg bodyweight. Plasma and urine samples were collected up to 15 and 70 days post-administration, respectively. While urine samples underwent an enzymatic hydrolysis, plasma samples were proteolysed before further analysis. After solid-...
Junsiri W, Kamkong P, Chinkangsadarn T, Ouisuwan S, Taweethavonsawat P.Equine ocular setariasis is mainly caused by Setaria digitata, and the identification of this filarial nematode is based on morphology. However, morphological characterization alone is insufficient for the detection and differentiation of S. digitata from its congeners. In Thailand, the molecular detection of S. digitata is lacking and its genetic diversity is still unknown. This study aimed to phylogenetically characterize equine S. digitata from Thailand based on sequences derived from the mitochondrial cytochrome c oxidase subunit 1 (COI), the mitochondrial small subunit ribosomal DNA (12S ...
Hernández-Avilés C, Ramírez-Agámez L, Pearson M, Beckham AMN, Varner DD, Love CC.Analysis of sperm morphology is an important part of the stallion breeding soundness evaluation since it provides an objective measure of a stallion's sperm quality and is one of many factors that estimate a stallion's fertility potential. To describe the effect of sperm quality level on the technique (Differential Interference Contrast - DIC; Phase-contrast - PH; Dip-Quick staining - DQ; and eosin-nigrosin staining - EN; semen samples fixed in buffered-formal saline) and evaluator (three evaluators; using only DIC), stallions were categorized based on sperm quality into three categories: High...
Moellerberndt J, Hagen A, Niebert S, Büttner K, Burk J.In equine medicine, the use of regenerative therapeutics has gained growing attention, but is still a new and complex field with room for improvement. Platelet lysate (PL) can be used as therapeutic agent but is also a promising supplement for the culture of multipotent mesenchymal stromal cells. To enable a targeted use of PL both in clinic and laboratory, it is crucial to learn more details on its effective ingredients. While so far, mainly growth factor components have been analyzed in platelet-based products such as PL, the current study focuses on the content of cytokines in serum, plasma...
Keen B, Cawley A, Reedy B, Noble G, Loy J, Fu S.The use of catechol-O-methyltransferase inhibitors may mask doping agents, primarily levodopa, administered to racehorses and prolong the stimulating effects of dopaminergic compounds such as dopamine. It is known that 3-methoxytyramine is a metabolite of dopamine and 3-methoxytyrosine is a metabolite of levodopa thus these compounds are proposed to be potential biomarkers of interest. Previous research established a urinary threshold of 4,000 ng/mL for 3-methoxytyramine to monitor misuse of dopaminergic agents. However, there is no equivalent biomarker in plasma. To address this deficiency a...
Hifumi T, Tanaka T, Sato M, Akioka K, Fujimata C, Miyoshi N.Alveolar echinococcosis in slaughtered horses remains a public health issue. This study aimed to develop a Recombinase polymerase amplification (RPA) assay targeting the mitochondrial NADH dehydrogenase subunit 5 () gene of for the rapid detection of equine alveolar echinococcosis. Thirty-six hepatic solid nodules obtained from each horse ( = 36) were evaluated based on histopathological examination and -targeted PCR and then submitted to the RPA assay. The results of the developed RPA assay were 94.4% consistent with those of PCR and Cohen's kappa coefficient value was 0.89 statistically,...
Wang J, Guo K, Li S, Liu D, Chu X, Wang Y, Guo W, Du C, Wang X, Hu Z.Salmonella enterica subsp. serovar Abortusequi is a major pathogen in horse and donkey herds, causing abortion in pregnant equids and resulting in enormous economic losses. A rapid and reliable method is urgently needed to detect . Abortusequi in herds where the disease is suspected. To achieve this goal, a TaqMan-based real-time PCR assay targeting the gene for the flagellin protein phase 2 antigen FljB was developed. This real-time PCR assay had high specificity, sensitivity, and reproducibility. The detection limit of the assay was 30 copies/μL of standard plasmid and 10 CFU/μL of bacter...
Nakken G, Kirk J, Fjordbakk CT.Autologous conditioned serum (ACS), i.e serum enriched with anti-inflammatory cytokines and growth factors, is a popular orthobiologic therapy used in equine practice. Costly specialized tubes containing glass beads are commonly used for ACS production. The objective of this in vitro study was to compare cytokine and growth factor levels in equine serum after incubation in three different tubes: commercial plastic ACS tubes (COMM); sterile 50 ml plastic centrifugation tubes (CEN); and 10 ml plastic vacutainer tubes (VAC). Blood from 15 healthy horses was incubated in the different tubes at 3...
Palmisano M, Javsicas L, McNaughten J, Gamsjäger L, Renaud DL, Gomez DE.Anecdotal evidence suggests plasma transfusions increase serum amyloid A (SAA) concentrations in healthy neonatal foals making this marker of inflammation inappropriate for therapeutic decision making in such animals. Objective: Administration of hyperimmune fresh frozen plasma (FFP) increases SAA concentration in healthy foals and in foals with failure of transfer of passive immunity (FTPI). Methods: Eighty-six healthy foals. Methods: Prospective cohort study. Foals <24 hours of age receiving plasma transfusion for treatment of FTPI (serum immunoglobulin G [IgG] concentrations 8 g/L; n...
Sánchez-Pacheco UA, Bahena-Mondragón BM, Hernández-Piedras FR, Soria-Osorio R, Meneses-Acosta A.The immunotherapy agents derived from horses are biological products that allow the neutralization of clinically relevant immunogens, such as the SARS-CoV-2 virus that causes COVID-19, or the neutralization of toxins present in the venoms of snakes, spiders, and other poisonous animals. Due to their importance, detecting adventitious viruses in equine hyperimmune serum (raw material in industrial processes) is a critical step to support the safety of products for human use, and, in consequence, it is a requirement for commercialization and distribution. The safety of the finished product is ba...
Petry S, Breuil MF, Duquesne F.Contagious equine metritis (CEM) detection by PCR is recognized by the European Union according to Commission Implementing Regulation (EU) No 846/2014, and real-time PCR is now recommended by the World Organisation for Animal Health Terrestrial Manual at the same level as the culture method. The present study highlights the creation of an efficient network of approved laboratories in France in 2017 for CEM detection by real-time PCR. The network currently consists of 20 laboratories. A first proficiency test (PT) was organized by the national reference laboratory for CEM in 2017 to evaluate th...
You Y, Proctor RM, Haughan J, Missanelli JR, Robinson MA.Fentanyl, a powerful synthetic mu opioid receptor agonist, is banned in equine sports by the Association of Racing Commissioners International and the Fédération Équestre Internationale. The presence of fentanyl in equine blood has been confirmed during routine post-race screening for doping substances in the authors' laboratory. While fentanyl can be detected and confirmed in blood, it is rapidly metabolized, and screening for the metabolite N-[1-(2-phenethy-4-piperidinyl)] maloanilinic acid (PMA) in equine urine is expected to allow for a longer detection time. In this study, a quantitati...
Assumpção TI, Lançoni R, Foschini M, Vieira CS.This study aimed to select high-quality spermatozoa by sperm separation by magnetic activation of the fresh equine semen, compared to density gradient centrifugation and evaluating cell quality after selection. The semen of 10 stallions was collected by the artificial vagina technique. The samples analyzed were: (1) fresh semen; (2) density gradient centrifugation (DGC); (3) separation by magnetic activation (MASS) (nonapoptotic portion NAP); (4) separation by MASS (apoptotic portion-APT). Was analyzed: motility (light microscopy), concentration (Neubauer chamber), semen morphology (humid cham...
Fukushi N, Badr Y, Fukushi H.Equine herpesvirus type 1 (EHV-1) UL11 is a 74-amino-acid (aa) protein encoded by ORF51. UL11 is modified by acylation including myristoylation and palmitoylation. Myristoylation of EHV-1 UL11 is assumed to occur on the N-terminal glycine, while palmitoylation is assumed to occur on the seventh and ninth cysteines. ORF51, which encodes the first 24 aa, overlaps ORF50 encoding UL12. We previously demonstrated that UL11 was essential for EHV-1 replication in cultured cells and that UL11 was localized at the Golgi apparatus where herpesviruses obtain their final envelope. It is unclear whether th...
Sanmartí J, Robles-Guirado JA, Jose-Cunilleras E, Bassols A.The determination of iCa and iMg is important in veterinary medicine, but their immediate determination in whole blood is not always possible. Their stability in other sample types and the existence of interferences must be evaluated before its use. Objective: We aimed to analyze the effects of storage time on the stability of iCa, iMg, and other analytes in whole blood, plasma, and serum samples in horses and assess the interference of heparin in these measurements. Methods: Whole blood, heparin-plasma, and serum samples from 10 horses were stored at 4°C and analyzed 1, 2, 3, 4, 5, 6, 7, 8, ...
Lewis N, Canesin H, Choi YH, Foss R, Felix M, Rader K, Hinrichs K.Information on factors associated with developmental competence of equine in vitro -produced (IVP) blastocysts is lacking. Objective: To determine the relationships of stage, grade, day of development, and specific morphological parameters of equine IVP blastocysts, to pregnancy and foaling rates. Methods: Photomicrographs of 316 IVP embryos with known pregnancy outcomes were scrutinised individually by four observers. Inter-observer variation was assessed, and pregnancy outcome evaluated in relation to day of blastocyst development and assigned grade and stage. Individual component analysis w...
Davis MS, Barrett MR.Mitochondrial function-oxidative phosphorylation and the generation of reactive oxygen species-is critical in both health and disease. Thus, measuring mitochondrial function is fundamental in biomedical research. Skeletal muscle is a robust source of mitochondria, particularly in animals with a very high aerobic capacity, such as horses, making them ideal subjects for studying mitochondrial physiology. This article demonstrates the use of high-resolution respirometry with concurrent fluorometry, with freshly harvested skeletal muscle mitochondria, to quantify the capacity to oxidize substrates...
Sandersen C, Dmitrovic P, Dupont J, Cesarini C, Guyot H, Serteyn D, Kirsch K.Different blood gas analyzers are used in equine practice. Every machine needs to be validated, as they have not been designed for use in horses. The aim of this study was to compare the newly marketed GEM5000 machine to the formerly validated epoc machine for blood gas analysis in horses. In this prospective, non-blinded, comparative laboratory analyzer study, 43 equine blood samples were analyzed on both analyzers and values were compared between the two machines via Lin's concordance analysis, Passing-Bablok regression analysis and Bland-Altman plots. Duplicate measurements were conducted o...
Quattrini C, Scalco R, Vernau W, Dini P, Aleman M.Cerebrospinal fluid (CSF) is highly labile and delayed processing might alter results of analysis. Objective: To determine the effects of time and addition of autologous serum on cytological evaluation of CSF. Methods: Ten client-owned adult horses requiring euthanasia. Methods: Prospective study. Serum and CSF were collected from each horse before and within 10 minutes after euthanasia. CSF samples were divided into 15 aliquots (2 mL each); 1 aliquot was submitted for routine CSF analysis within 60 minutes of collection. Four drops of autologous serum were added to 7 of the aliquots, and ...
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...
Cocco MJ, Lecomte JT.A proton nuclear magnetic resonance spectroscopic study of horse apomyoglobin was undertaken in order to define the regions of myoglobin that are and that are not structurally affected by the binding of the prosthetic group. It was found that, in spite of the poor spectral resolution, a number of spin systems could be identified by using standard correlated methods. Four clusters consisting mostly of hydrophobic residues were detected by nuclear Overhauser spectroscopy, two of which involved the tryptophan side chains. Extensive similarities to nuclear Overhauser spectroscopy data collected on...
Harvey JW.Deficiencies of enzymes involved in erythrocyte metabolism can have significant effects on erythrocyte function and survival. Animals with pyruvate kinase (PK) or phosphofructokinase (PFK) deficiencies have shortened erythrocyte life spans and regenerative anemia. PK-deficient dogs (but not PK-deficient cats) develop progressive myelofibrosis and osteosclerosis of bone marrow and hemochromatosis and cirrhosis of the liver. PFK-deficient dogs have sporadic episodes of hyperventilation-induced intravascular hemolysis and hemoglobinuria. Cytochrome b5 reductase (Cb5R) deficiency in dogs and cats ...
Zheng YH, Nakaya T, Sentsui H, Kameoka M, Kishi M, Hagiwara K, Takahashi H, Kono Y, Ikuta K.We have studied a horse which exhibited typical clinical signs of disease when experimentally infected with a non-adapted virulent strain of equine infectious anaemia virus (EIAV), designated V70. Five viruses (F1V, F2V, F3V, F4V and F5V) were recovered during periodic febrile episodes. Cross-neutralization tests revealed that all of these variants and the parental V70 were antigenically distinct. Sequencing of their full-length env gp90 genes and gp45 5' sequences revealed novel mutations at a limited number of nucleotide positions, consisting of insertions and duplications in the gp90 princi...
Foss R, Ortis H, Hinrichs K.Intracytoplasmic sperm injection (ICSI) is used to produce foals from otherwise infertile mares and from stallions with limited sperm stores, but requires expensive equipment and is technically demanding. Methods to transport oocytes to ICSI laboratories would allow collection of oocytes by the referring veterinarian and enable greater application of this technique. Objective: This study was conducted to evaluate protocols that could be used to transport immature and maturing oocytes for ICSI. Methods: In vitro experiment. Methods: Oocytes were recovered by transvaginal ultrasound-guided folli...
Kovaleva EG, Plapp BV.Binding of NAD+ to wild-type horse liver alcohol dehydrogenase is strongly pH-dependent and is limited by a unimolecular step, which may be related to a conformational change of the enzyme-NAD+ complex. Deprotonation during binding of NAD+ and inhibitors that trap the enzyme-NAD+ complex was examined by transient kinetics with pH indicators, and formation of complexes was monitored by absorbance and protein fluorescence. Reactions with pyrazole and trifluoroethanol had biphasic proton release, whereas reaction with caprate showed proton release followed by proton uptake. Proton release (200-55...
Hoogewijs MK, de Vliegher SP, Govaere JL, de Schauwer C, de Kruif A, van Soom A.Sperm motility is considered to be one of the key features of semen analysis. Assessment of motility is frequently performed using computer-assisted sperm analysis (CASA). Nevertheless, no uniform standards are present to analyse a semen sample using CASA. Objective: We hypothesised that the type of counting chamber used might influence the results of analysis and aimed to study the effect of chamber type on estimated concentration and motility of an equine semen sample assessed using CASA. Methods: Commonly used disposable Leja chambers of different depths were compared with disposable and re...
Lilliehöök I, Tvedten H.The Sysmex XT-2000iV is a laser-based, flow cytometric hematology system that stains nucleic acids in leukocytes with a fluorescent dye. A 4-part differential is obtained using side fluorescence light and laser side scatter. Objective: The purpose of this study was to validate the Sysmex XT-2000iV for determining differential leukocyte counts in blood from ill dogs, cats, and horses. Methods: Blood samples from diseased animals (133 dogs, 65 cats, and 73 horses) were analyzed with the Sysmex XT-2000iV (Auto-diff) and the CELL-DYN 3500. Manual differentials were obtained by counting 100 leukocy...
Hagen A, Lehmann H, Aurich S, Bauer N, Melzer M, Moellerberndt J, Patané V, Schnabel CL, Burk J.Translation of multipotent mesenchymal stromal cell (MSC)-based therapies is advancing in human and veterinary medicine. One critical issue is the culture of MSC before clinical use. Using fetal bovine serum (FBS) as supplement to the basal medium is still the gold standard for cultivation of many cell types including equine MSC. Alternatives are being explored, with substantial success using platelet lysate-supplemented media for human MSC. However, progress lags behind in the veterinary field. The aim of this study was to establish a scalable protocol for equine platelet lysate (ePL) produc...
Axon JE, Carrick JB, Barton MD, Collins NM, Russell CM, Kiehne J, Coombs G.To evaluate if methicillin-resistant Staphylococcus aureus (MRSA) is present in the horse population in Australia. Methods: A two-part retrospective study of laboratory submissions of microbial culture results from horses. Methods: Part A: medical records of 216 horses that had MRSA screening performed on nasal swabs collected over a 30-day period at admission to the Scone Equine Hospital Clovelly Intensive Care Unit were retrieved. Part B: laboratory records from 2004 to 2009 of culture submissions to the Scone Veterinary Laboratory were reviewed and cultures that grew MRSA were identified. T...
Hübert PH, Birkenmaier S, Rziha HJ, Osterrieder N.The equine herpesvirus type-1 modified live-vaccine strain RacH (256th passage on porcine embryonic kidney cells) was investigated by restriction-enzyme analysis and compared to representative plaque isolates of the 12th passage (RacL11, RacL22) and 185th passage (RacM24, RacM36). The restriction patterns of all Rac plaque isolates differed compared with reference strain Ab4. The left UL terminus was shortened by 0.1 kbp and a missing BamHI site led to the fusion of the f and t fragments. In some Rac derivatives, losses of restriction sites without deletions were observed: 1. One BamHI site lo...
Nebbia C, Dacasto M, Rossetto Giaccherino A, Giuliano Albo A, Carletti M.The apoprotein expression and the catalytic activities of cytochrome P450s involved in the biotransformation of xenobiotics were investigated in horse liver microsomes and compared with those of food producing (cattle, pigs, broiler chicks, and rabbits) and laboratory species (rats). Western blot analysis revealed the presence of proteins immunorelated to rat CYP 1A, CYP 2B, CYP 2E, and CYP 3A subfamilies in hepatic microsomes from horses and from any other examined species. With the exception of the N-demethylation of N-nitrosodimethylamine in broiler chicks, all the recorded interspecies dif...
Wagner HM, Balasuriya UB, James MacLachlan N.In an effort to further characterize the humoral immune response of horses to equine arteritis virus (EAV), direct and competitive enzyme-linked immunosorbent assays (c-ELISAs) were developed using monoclonal and polyclonal anti-sera to structural (G(L), N and M) and non-structural (nsp1) viral proteins. A nsp1-specific monoclonal antibody was produced to facilitate development of a c-ELISA to this protein. Data obtained using the various c-ELISAs confirm that the M protein is a major target of the antibody response of horses to EAV. However, none of the c-ELISAs that were developed were as se...
Bellone RR, Forsyth G, Leeb T, Archer S, Sigurdsson S, Imsland F, Mauceli E, Engensteiner M, Bailey E, Sandmeyer L, Grahn B, Lindblad-Toh K, Wade CM.Leopard Complex spotting occurs in several breeds of horses and is caused by an incompletely dominant allele (LP). Homozygosity for LP is also associated with congenital stationary night blindness (CSNB) in Appaloosa horses. Previously, LP was mapped to a 6 cm region on ECA1 containing the candidate gene TRPM1 (Transient Receptor Potential Cation Channel, Subfamily M, Member 1) and decreased expression of this gene, measured by qRT-PCR, was identified as the likely cause of both spotting and ocular phenotypes. This study describes investigations for a mutation causing or associated with the Le...
Pacholewska A, Drögemüller M, Klukowska-Rötzler J, Lanz S, Hamza E, Dermitzakis ET, Marti E, Gerber V, Leeb T, Jagannathan V.Complete transcriptomic data at high resolution are available only for a few model organisms with medical importance. The gene structures of non-model organisms are mostly computationally predicted based on comparative genomics with other species. As a result, more than half of the horse gene models are known only by projection. Experimental data supporting these gene models are scarce. Moreover, most of the annotated equine genes are single-transcript genes. Utilizing RNA sequencing (RNA-seq) the experimental validation of predicted transcriptomes has become accessible at reasonable costs. To...
Guthrie AJ, Maclachlan NJ, Joone C, Lourens CW, Weyer CT, Quan M, Monyai MS, Gardner IA.Blood samples collected from 503 suspect cases of African horse sickness (AHS) and another 503 from uninfected, unvaccinated South African horses, as well as 98 samples from horses from an AHS free country, were tested with an AHS virus (AHSV) specific duplex real-time reverse transcription quantitative PCR (RT-qPCR) assay and virus isolation (VI). The diagnostic sensitivity and specificity of this AHSV RT-qPCR assay and VI were estimated using a 2-test 2-population Bayesian latent class model which made no assumptions about the true infection status of the tested animals and allowed for the p...
Dunbar LK, Mielnicki KA, Dembek KA, Toribio RE, Burns TA.Several tests have been evaluated in horses for quantifying insulin dysregulation to support a diagnosis of equine metabolic syndrome. Comparing the performance of these tests in the same horses will provide clarification of their accuracy in the diagnosis of equine insulin dysregulation. Objective: The aim of this study was to evaluate the agreement between basal serum insulin concentrations (BIC), the oral sugar test (OST), the combined glucose-insulin test (CGIT), and the frequently sampled insulin-modified intravenous glucose tolerance test (FSIGTT). Methods: Twelve healthy, light-breed ho...
Pringle J, Roberts C, Kohl M, Lekeux P.The effects of epidermal pigmentation and hair covering on the relative transparency of various animal tissues to near infrared (NIR) light were examined, and the pathlengths of NIR light through tissues at four wavelengths in the NIR range were subsequently determined. Black hair covering and black or dark-coloured hooves prevented NIR light from penetration sufficient for conduction of pathlength or NIR spectroscopy measurements. Non-pigmented hair covering of the head did not appear to be a barrier to successful NIR light transmission. Tissues sufficiently transparent to NIR light had the d...
Barrier Battut I, Kempfer A, Becker J, Lebailly L, Camugli S, Chevrier L.Several laboratories routinely use flow cytometry to evaluate stallion semen quality. However, objective and practical tools for the on-field interpretation of data concerning fertilizing potential are scarce. A panel of nine tests, evaluating a large number of compartments or functions of the spermatozoa: motility, morphology, viability, mitochondrial activity, oxidation level, acrosome integrity, DNA integrity, "organization" of the plasma membrane, and hypoosmotic resistance, was applied to a population of 43 stallions, 33 of which showing widely differing fertilities (19%-84% pregnancy rat...
Bosco A, Maurelli MP, Ianniello D, Morgoglione ME, Amadesi A, Coles GC, Cringoli G, Rinaldi L.Nematode infections in horses are widespread across the world. Increasing levels of anthelmintic resistance, reported worldwide in equine parasites, have led to the creation of programs for the control of nematodes based on faecal egg counts (FEC). To improve nematode egg counting in equine faecal samples and establish whether the matrix of equine faeces or the eggs affect the counts, the analytical sensitivity, accuracy and precision of Mini-FLOTAC (combined with Fill-FLOTAC), McMaster and Cornell-Wisconsin techniques were compared. Known numbers of eggs extracted from equine or ovine faeces ...
Rodríguez-Lázaro D, Lewis DA, Ocampo-Sosa AA, Fogarty U, Makrai L, Navas J, Scortti M, Hernández M, Vázquez-Boland JA.We developed a novel quantitative real-time PCR (Q-PCR) method for the soil actinomycete Rhodococcus equi, an important horse pathogen and emerging human pathogen. Species-specific quantification was achieved by targeting the chromosomal monocopy gene choE, universally conserved in R. equi. The choE Q-PCR included an internal amplification control (IAC) for identification of false negatives. A second Q-PCR targeted the virulence plasmid gene vapA, carried by most horse isolates but infrequently found in isolates from other sources. The choE-IAC and vapA assays were 100% sensitive and specific ...
Gerhard AP, Krücken J, Heitlinger E, Janssen IJI, Basiaga M, Kornaś S, Beier C, Nielsen MK, Davis RE, Wang J, von Samson-Himmelstjerna G.P-glycoproteins (Pgp) have been proposed as contributors to the widespread macrocyclic lactone (ML) resistance in several nematode species including a major pathogen of foals, Parascaris univalens. Using new and available RNA-seq data, ten different genomic loci encoding Pgps were identified and characterized by transcriptome-guided RT-PCRs and Sanger sequencing. Phylogenetic analysis revealed an ascarid-specific Pgp lineage, Pgp-18, as well as two paralogues of Pgp-11 and Pgp-16. Comparative gene expression analyses in P. univalens and Caenorhabditis elegans show that the intestine is the maj...
Karges HE, Funk KA, Ronneberger H.Using diagnostics for the determination of clotting factors and fibrinolytic parameters in human plasma, samples from rat, guinea pig, rabbit, cat, dog, sheep, cattle, horse, pig, and monkey were analysed. The human system was employed even for standard curves and controls. Results obtained in this way are relative values in relation to pooled fresh human plasma of healthy donors which is defined to contain 100% of the norm or 1 unit of each factor per 1 ml. Under these conditions, marked differences between the human clotting system and those of different animal species appear. Thus, rabbits ...
Barrey E, Bonnamy B, Barrey EJ, Mata X, Chaffaux S, Guerin G.MicroRNAs (miRNA) are small endogenous noncoding interfering RNA molecules (18-25 nucleotides) regarded as major regulators in eukaryotic gene expression. They play a role in developmental timing, cellular differentiation, signalling and apoptosis pathways. Because of the central function of miRNAs in the proliferation and differentiation of the myoblasts demonstrated in mouse and man, it is assumed that they could be present in equine muscles and their expression profile may be related to the muscle status. Objective: To identify miRNA candidates in the muscles of control and affected horses ...
Constable PD.The strong ion approach provides a quantitative physicochemical method for describing the mechanism for an acid-base disturbance. The approach requires species-specific values for the total concentration of plasma nonvolatile buffers (A(tot)) and the effective dissociation constant for plasma nonvolatile buffers (K(a)), but these values have not been determined for human plasma. Accordingly, the purpose of this study was to calculate accurate A(tot) and K(a) values using data obtained from in vitro strong ion titration and CO(2) tonometry. The calculated values for A(tot) (24.1 mmol/l) and K(a...
Penedo MC, Millon LV, Bernoco D, Bailey E, Binns M, Cholewinski G, Ellis N, Flynn J, Gralak B, Guthrie A, Hasegawa T, Lindgren G, Lyons LA, Røed KH....A comprehensive male linkage map was generated by adding 359 new, informative microsatellites to the International Equine Gene Map half-sibling reference families and by combining genotype data from three independent mapping resources: a full sibling family created at the Animal Health Trust in Newmarket, United Kingdom, eight half-sibling families from Sweden and two half-sibling families from the University of California, Davis. Because the combined data were derived primarily from half-sibling families, only autosomal markers were analyzed. The map was constructed from a total of 766 marker...
Turtinen LW, Allen GP, Darlington RW, Bryans JT.The molecular and serologic relatedness of 2 recent respiratory tract isolates of equine herpesvirus type 1, designated T1 and T2, were compared with the Army 183, Kentucky-A hamster-adapted (KyA-ha), and L-M cell-adapted (KyA-LM) strains. Electrophoresis in polyacrylamide gels revealed differences in virion structural proteins among 4 purified strains. Seven envelope glycoproteins (molecular weight of 93,000, 65,000, 62,000, 60,000, 36,000, 20,000, and 18,000) corresponding to virion proteins 13, 16, 17, 18, 23, 25, and 26a, respectively, found in both the Army 183 and KyA-ha strains had slig...
Isa P, Wood AR, Netherwood T, Ciarlet M, Imagawa H, Snodgrass DR.DIG-labelled ssRNA probes were prepared from variable regions of VP4 and VP7 cognate genes, and used in hybridization assays for P and G genotyping of group A cell culture-adapted equine rotaviruses and fecal samples collected from foals with and without diarrhea. The probes confirmed known P and G serotypes of sixteen cell culture-adapted strains. From one-hundred and twenty-one rotavirus-positive samples, 83 reacted when tested for their P and G genotype specific probes. From these, 71 were found to contain G3 P12 genotypes, and 11 G14 P12 genotypes. No sample reacted with H1 or L338 P and G...
Ochoa M, Bárcena J, de la Luna S, Melero JA, Douglas AR, Nieto A, Ortín J, Skehel JJ, Portela A.Characterization of the epitopes recognized by 21 monoclonal antibodies (MAbs) specific for the influenza A virus PA (13 MAbs) and PB2 (8 MAbs) polypeptides (Bárcena et al. (1994) J. Virol. 68, 6900-6909) raised against denatured polypeptides produced in E. coli is described. MAbs were characterized by: (1) competitive binding ELISAs; (2) mapping of the protein regions that specify their binding sites; and (3) analyses of their ability to recognize the corresponding viral protein in a number of viral isolates. Five and three non-overlapping antigenic areas were defined by the anti-PA and anti...
Raudsepp T, Chowdhary BP.A pilot study comparing horse and donkey karyotypes on a molecular basis was initiated using the chromosomal microdissection approach. All equine meta- and submetacentric chromosomes, viz. ECA1 to ECA13 and the X and Y chromosomes, were microdissected. The DNA was PCR amplified, non-radioactively labelled and used as probes on equine metaphase chromosomes to confirm their origin. Once tested, the paints were used as probes on donkey metaphase chromosomes to detect homologous chromosomal segments between the two species. The results not only detected conservation of whole chromosome and/or arm ...
Kurowski PB, Traub-Dargatz JL, Morley PS, Gentry-Weeks CR.To use real-time polymerase chain reaction (PCR) technology to develop a highly sensitive and specific diagnostic assay for the detection of Salmonella spp in fecal specimens. Methods: 299 fecal specimens from cattle, horses, and dogs. Methods: Enrichment of fecal specimens was followed by genomic DNA extraction by use of commercially available isolation kits. Real-time PCR assay was performed to target a Salmonella spp-specific DNA segment. Results of real-time PCR assay were compared with bacterial culture results to determine relative sensitivity and specificity. Results: Use of the spaQ pr...
