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.
Wong CH, Tang FP, Wan TS.The authors' laboratory at one time employed four liquid chromatography/mass spectrometric (LC/MS) methods for the detection of a large variety of drugs in equine urine. Drug classes covered by these methods included anti-diabetics, anti-ulcers, cyclooxygenase-2 (COX-2) inhibitors, sedatives, corticosteroids, anabolic steroids, sulfur diuretics, xanthines, etc. With the objective to reduce labour and instrumental workload, a new ultra performance liquid chromatography/tandem mass spectrometric (UPLC/MS/MS) method has been developed, which encompasses all target analytes detected by the origina...
Virmani N, Bera BC, Shanumugasundaram K, Singh BK, Gulati BR, Singh RK, Vaid RK.India faced an epizootic of equine influenza in 2008-2009. The isolated viruses were typed as H3N8 and grouped with the clade 2 viruses of Florida sublineage on the basis of haemagglutinin (HA) gene sequence analysis. This report describes the genetic analysis and selection pressure of matrix (M) and non-structural 1 (NS1) genes of the Indian isolates. All isolates shared 98.41% and 99.54% homology with other clade 2 viruses of Asian origin for M1 and M2 amino acid (aa) sequences, respectively. There were 3 and 4 unique aa residue changes respectively in M1 and M2 proteins in all Asian isolate...
Nemoto M, Tsunemitsu H, Imagawa H, Hata H, Higuchi T, Sato S, Orita Y, Sugita S, Bannai H, Tsujimura K, Yamanaka T, Kondo T, Matsumura T.Using a total of 2018 fecal samples collected between 2003 and 2008 from foals with diarrhea, the molecular epidemiology of group A equine rotaviruses circulating in Japan was investigated by the reverse transcription-polymerase chain reaction (RT-PCR) typing and sequence analysis of the VP4 (P type) and VP7 (G type) genes. A total of 1149 samples showed positive reactions with RT-PCR, of which 462 samples (40.2%) were positive for G3 type, 502 samples (43.7%) were positive for G14 type, and 185 samples (16.1%) were positive for both G3 and G14 types. To examine P types, 59 G3 and 56 G14 posit...
DiMaio Knych HK, Arthur RM, Taylor A, Moeller BC, Stanley SD.Dantrolene is a skeletal muscle relaxant used commonly in performance horses to prevent exertional rhabdomyolysis. The goal of the study reported here was to begin to characterize cytochrome P450-mediated metabolism of dantrolene in the horse and describe the pharmacokinetics of the compound, formulated as a capsule or a compounded paste formulation, following oral administration. Dantrolene is rapidly metabolized to 5-hydroxydantrolene both in vivo and in vitro. Preliminary work with equine liver microsomes suggest that two enzymes are responsible for the metabolism of dantrolene, as evidence...
Neirinckx E, Vervaet C, Michiels J, De Smet S, Van den Broeck W, Remon JP, De Backer P, Croubels S.The aim of this study was to assess the feasibility of the Ussing chamber technique for the determination of the jejunal permeability of passively absorbed, high permeability model compounds (acetaminophen and ketoprofen) in different animal species. Additionally, electrophysiological measurements and histological examination of pre- and post-incubation tissue specimens were performed. Apparent permeability coefficients of turkey and dog jejunum were low and highly variable due to tissue fragility caused by differences in thickness of the remaining intestinal layers after stripping and resulti...
Oldenhof H, Blässe AK, Wolkers WF, Bollwein H, Sieme H.The aim of this study was to determine the osmotic tolerance limits of stallion sperm as well as the osmotic behavior of different sperm subpopulations, including viable and non-viable cells as well as viable cells of different average sizes. A flow cytometric approach was used for simultaneous assessment of cell volume and permeability of the plasma membrane for the fluorescent dye propidium iodide while exposing the cells to media with different solute concentrations. Equine spermatozoa have limited osmotic tolerance limits: exposure to hypotonic conditions below approximately 240 mOsm kg(-1...
Foster ML, Love CC, Varner DD, Brinsko SP, Hinrichs K, Teague S, Lacaze K, Blanchard TL.Sperm membrane integrity (SMI) is thought to be an important measure of stallion sperm quality. The objective was to compare three methods for evaluating SMI: flow cytometry using SYBR-14/propidium iodide (PI) stain; an automated cell counting device using PI stain; and eosin-nigrosin stain. Raw equine semen was subjected to various treatments containing 20 to 80% seminal plasma in extender, with differing sperm concentrations, to simulate spontaneous loss of SMI. The SMI was assessed immediately, and after 1 and 2 d of cooled storage. Agreement between methods was determined according to Blan...
BMC research notesApril 12, 2011
Volume 4 120 doi: 10.1186/1756-0500-4-120
Klein C, Rutllant J, Troedsson MH.Quantitative RT-PCR data are commonly normalized using a reference gene. A reference gene is a transcript which expression does not differ in the tissue of interest independent of the experimental condition. The objective of this study was to evaluate the stability of mRNA expression levels of putative reference genes in three different types of equine tissue, endometrial, testicular, and conceptus tissue. Results: The expression stability of four (uterine tissue) and six (testicular and conceptus tissue) was assessed using descriptive data analysis and the software programs Normfinder and geN...
Medina-Torres CE, Mason SL, Floyd RV, Harris PA, Mobasheri A.The aim of this study was to determine if hypoxia and the hypoxia mimetic cobalt chloride regulate the activity of matrix metalloproteinase (MMP)-2 and -9 in cultures of equine hoof keratinocytes. These effects were assessed in primary cultures of laminar keratinocytes using gelatin zymography. Incubation of keratinocytes with cobalt chloride significantly increased the levels of active MMP-2 compared to untreated controls. Hypoxia significantly increased the expression of active MMP-2 and -9 in keratinocyte cultures. This up-regulation was observed after 6h and peaked at 24h. The study findin...
Crafford JE, Guthrie AJ, Van Vuuren M, Mertens PP, Burroughs JN, Howell PG, Batten CA, Hamblin C.A polyclonal antibody-based, group-specific, competitive ELISA (C-ELISA) for the detection of antibodies to equine encephalosis virus (EEV) was developed. The assay measures the competition between a specific guinea pig antiserum and a test serum, for a pre-titrated EEV antigen. The C-ELISA detected antibodies to the seven known EEV serotypes. Reference antisera raised against other arboviruses did not cross react with EEV antigen. Negative sera from horses in the United Kingdom were used to establish the baseline for a negative population. Negative and positive populations of South African ho...
Spirito S, Campi S, Boquet M, Fernández H, Ferrari M.The aims of this study were to estimate the ability of the Feulgen reaction to identify equine sperm nuclei with different ploidy levels, to determine the frequency of haploid, diploid and polyploid sperm nuclei in the semen of fertile equines and to evaluate the relationship between the nuclear DNA content and the nuclear area. Determination of the ploidy level of Feulgen-stained spermatozoa using a scanning microspectrophotometer was very similar to the subjective estimations made with a light microscope. This indicates that the Feulgen reaction is a simple, inexpensive and reliable techniqu...
Balssa F, Fischer M, Bonnaire Y.5α-Estrane-3β,17α-diol is the major metabolite of nandrolone in horse urine. The presence of 5α-estrane-3β,17α-diol in female and gelding urines is prohibited by Racing Rules and its natural presence in male urine led regulation authorities to establish a concentration threshold of 45 ng/mL. This paper describes a rapid, simple and stereoselective synthesis of 5α-estrane-3β,17α-diol, providing horseracing laboratories with an essential reference material for their antidoping performance.
Hoffmann N, Oldenhof H, Morandini C, Rohn K, Sieme H.Cryopreserved stallion sperm displays a high degree of male-to-male variability with respect to cell viability after thawing. Animals that have semen with low viability after cryopreservation are classified as 'poor' freezers, and when post-thaw viability is high they are designated as 'good' freezers. Cryoprotective agents that are used for cryopreserving stallion sperm include glycerol, ethylene glycol, methyl formamide, and dimethylformamide, and are typically used in concentrations ranging from 1% to 4%. The aim of this study was to evaluate the osmotic stresses that stallion sperm is expo...
Liu Y, Uboh CE, Soma LR, Li X, Guan F, You Y, Rudy JA, Chen JW.Gabapentin (GPT) is an antiepileptic drug that was approved in 1993 for use in the management of neurotrophic pain and as an adjunctive therapy for refractory partial seizure in humans. It is also being tested in veterinary medicine as an adjunctive medication in the treatment of pain due to laminitis, neuropathic, or chronic pain. Gabapentin is readily available by prescription and even on the internet; therefore, it has the potential of being used in racehorses to mask pain. It is for this reason that a sensitive liquid chromatography-tandem mass spectrometry method has now been developed fo...
Tugirimana PL, De Clercq D, Holderbeke AL, Kint JA, De Cooman L, Deprez P, Delanghe JR.A turbidimetric method to determine serum C-reactive protein (CRP) concentration, based on soybean oil-phosphocholine interaction, was performed on horse serum samples to evaluate its potential diagnostic value in veterinary medicine. Intralipid 20% in 0.1 M Tris-calcium buffer (pH 7.5) was added to horse serum. After 30 min of incubation at 37 °C, the CRP-phosphocholine complexes were turbidimetrically, bichromatically (660 nm/700 nm) quantified on a commercial analyzer. Furthermore, comparison between CRP and other inflammatory markers, including white blood cell and neutrophil counts, was ...
Tanaka K, Yamada M, Tamiya-Koizumi K, Kannagi R, Aoyama T, Hara A, Kyogashima M.Free ceramides and glycosphingolipids (GSLs) are important components of the membrane microdomain and play significant roles in cell survival. Recent studies have revealed that both fatty acids and long-chain bases (LCBs) are more diverse than expected, in terms of i) alkyl chain length, ii) hydroxylation and iii) the presence or absence of double bonds. Electrospray ionization mass spectrometry and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) have been well utilized to characterize sphingolipids with high throughput, but reports to date have not ...
Tang X, Sayre LM, Tochtrop GP.Cytochrome c is a key mitochondrial respiratory protein that is particularly susceptible to modification during oxidative stress. The nature of this susceptibility is linked to the mitochondrial membrane being rich in esterified linoleic acid, which predisposes this organelle to the formation of lipid peroxidation products such as 4-hydroxy-2-(E)-nonenal (4-HNE). To better understand the nature of cytochrome c modification by 4-HNE, we initiated an in vitro study utilizing a combination of MALDI-TOF mass spectrometry, LC-ESI-MS/MS and isotope labeling to monitor 4-HNE modification of cytochrom...
Müller C, Preusser-Kunze A, Wagner K, Franzreb M.The glycoprotein hormone equine chorionic gonadotropin (eCG) is a commercial product used in animal breeding as well as in veterinary medicine. The current state of the art for the purification of eCG from serum is pH fractionation with metaphosphoric acid, two ethanol precipitation steps as well as dialysis followed by fixed-bed chromatography. Two simplified processes, including the use of magnetic microsorbents for the purification of eCG have been developed. The processes reduce or even omit the use of organic solvents and the required solid-liquid separation steps, thus making them potent...
Wong JK, Tang FP, Wan TS.The study of the metabolism of drugs, in particular steroids, by both in vitro and in vivo methods has been carried out in the authors' laboratory for many years. For in vitro metabolic studies, the microsomal fraction isolated from horse liver is often used. However, the process of isolating liver microsomes is cumbersome and tedious. In addition, centrifugation at high speeds (over 100 000 g) may lead to loss of enzymes involved in phase I metabolism, which may account for the difference often observed between in vivo and in vitro results. We have therefore investigated the feasibility of us...
Gornik K, Moore P, Figueiredo M, Vandenplas M.Human corneal cells have detectable levels of TLRs 1-10. TLRs 2 and 4 are the major corneal receptors, recognizing the PAMPs associated with fungal invasion in humans. The conjunctiva and limbus contain TLRs 2, 4, and 9. Our purpose was to determine the expression of TLRs 2, 3, 4, 6, 9, and MD-2 in the normal equine cornea, conjunctiva, and limbus. Methods: Corneal, limbal, and conjunctival tissues were collected from seven euthanized horses having no evidence of ocular disease. RNA extraction with DNase-1 digestion was performed followed by RT-PCR to determine expression of TLRs 2, 3, 4, 6, 9...
Costa MF, Carmona AK, Alves MF, Ryan TM, Davies HM, Anderson GA, Slocombe RF.Angiotensin-I converting enzyme (ACE) is a key regulator of blood pressure, electrolytes and fluid homeostasis through conversion of angiotensin I into angiotensin II. Recently, a genetic polymorphism of the ACE gene, which accounts for 47% of the variation of ACE activity in blood, has been advocated as a biomarker of athletic aptitude. Different methods of analysis and determination of ACE activity in plasma have been used in human and equine research without a consensus of a "gold standard" method. Different methods have often been used interchangeably or cited as being comparable in the ex...
De Ceulaer K, Van Ginneken C, Delesalle C, Van Brantegem L, Deprez P, Weyns A.This study aimed to evaluate the reliability of slaughterhouse-obtained small intestinal tissue as control material in equine colic research where molecular stress responses in small intestinal tissue are investigated. For this purpose, small intestinal samples from colic horses were collected during surgery or immediately after euthanasia at the oral border of strangulation resection sites and routinely processed for histopathology (i.c. rinsed with 4°C Krebs' solution, fixated overnight with 4% neutral buffered formaldehyde (FH) at room temperature). Control samples consisted of pieces of m...
Moeller BC, Sams RA, Guinjab-Cagmat J, Szabo NJ, Colahan P, Stanley SD.Testosterone is an anabolic androgenic steroid (AAS) that is endogenously produced by both male and female horses that also has the potential for abuse when administered exogenously to race horses. To recommend appropriate withdrawal guidelines so that veterinarians can discontinue therapeutic use prior to competition, the pharmacokinetics and elimination of testosterone were investigated. An aqueous testosterone suspension was administered intramuscularly in the neck of Thoroughbred horses (n = 20). The disposition of testosterone from this formulation was characterized by an initial, rapid a...
Fielding CL, Pusterla N, Magdesian KG, Higgins JC, Meier CA.To characterize signalment, clinical and laboratory findings, treatment, and outcome in horses with rattlesnake envenomation in northern California. Methods: Retrospective case series. Methods: 58 client-owned horses evaluated for rattlesnake envenomation at 2 referral hospitals from 1992 to 2009. Methods: Records of horses with rattlesnake envenomation were reviewed, and data concerning signalment, clinical and laboratory findings, treatment, and outcome were collected. In addition, a rattlesnake-bite severity score (RBSS) was assigned to each horse. Variables were compared between horses tha...
Cook VL, Holcombe SJ, Gandy JC, Corl CM, Sordillo LM.Monocytes are among the initial cells that interact with circulating LPS. Binding of LPS to monocyte surface receptors triggers an intracellular signaling cascade and results in the production of proinflammatory cytokines. Ethyl pyruvate, a stable derivative of pyruvate, has been effective in mitigating LPS induced alterations in isolated human monocytes. We hypothesized that ethyl pyruvate would suppress proinflammatory gene expression in LPS-stimulated equine monocytes without affecting cell viability. Equine monocytes were isolated from whole blood using a sediment-gradient centrifugation p...
Clutterbuck AL, Smith JR, Allaway D, Harris P, Liddell S, Mobasheri A.This study employed a targeted high-throughput proteomic approach to identify the major proteins present in the secretome of articular cartilage. Explants from equine metacarpophalangeal joints were incubated alone or with interleukin-1beta (IL-1β, 10ng/ml), with or without carprofen, a non-steroidal anti-inflammatory drug, for six days. After tryptic digestion of culture medium supernatants, resulting peptides were separated by HPLC and detected in a Bruker amaZon ion trap instrument. The five most abundant peptides in each MS scan were fragmented and the fragmentation patterns compared to m...
Zielińska-Dawidziak M, Błaszak R, Piasecka-Kwiatkowska D.The presented experiments show the model of expectation of equine spleen ferritin extraction in a new aqueous two-phase system which was formed by mixing polyethylene glycol (PEG) and hydroxyethyl starch (HES). The tendency of the protein to migrate in the analyzed systems was dependent on the concentrations of HES and PEG as well as PEG molecular weight. The highest concentration of ferritin in the top phase (rich in PEG) was recorded in the system composed of 6% PEG 3000 and 3% HES. The obtained concentration was 0.88 mg mL(-1). The lowest concentration was 0.42 mg mL(-1) in the system compo...
Saengseesom W, Kasempimolporn S, Akesowan S, Ouisuwan S, Sitprija V.A therapeutic anti-rabies immunoglobulin for human use has been produced mainly in horses. The presently available seroneutralization test, the rapid fluorescent focus inhibition test (RFFIT), is laborious and rather difficult to carry out in horse farms. This study was undertaken to develop a simple latex agglutination test (LAT) for determining rabies antibodies in horse sera. LAT was validated by testing a total of 468 horse serum samples characterized by RFFIT. Of these, 253 of 260 samples with antibody titers of less than 100 IU/ml had agglutination score of 1+, whereas 174 of 208 samples...
de Oliveira AM, Fonseca AA, Camargos MF, Orzil LM, Laguardia-Nascimento M, Oliveira AGG, Rodrigues JG, Sales ML, de Oliveira TFP, de Melo CB.Vesicular stomatitis is an infectious disease that occurs mainly in countries of the Western Hemisphere and affects cattle, swine and horses. The clinical symptoms in cattle and swine are similar to foot-and-mouth disease and include vesicular ulceration of the tongue and mouth. The disease requires a rapid and accurate differential diagnosis, aiming for immediate implementation of control measures. The objective of the present study was to develop and perform validation tests of multiplex RT-qPCR(s) for the detection of RNA from Alagoas vesiculovirus, considering the parameters of sensitivity...
Goldberg M, Molon Noblot M, Septier D.The effect of 2 methods of demineralization on the preservation of proteoglycans and glycoproteins was studied in the intertubular and peritubular dentine of the horse. The specimens embedded in Epon were demineralized with a 2% acid formic solution (Bonucci and Gheradi, 1975). Other fragments were treated with an organic solution of EDTA alkylammonium salt (Scott and Kyffin, 1979). These methods preserved in a satisfactory way these labile organic components. In the intertubular dentine, glycoproteins and proteoglycans were also identified, either associated with collagen fibres as a glue and...
Taylor KC, Vitello LB, Erman JE.The ionization of 4-nitroimidazole to 4-nitroimidazolate was investigated as a function of ionic strength. The apparent pKa varies from 8.99 to 9.50 between 0.001 and 1.0 M ionic strength, respectively, at 25 degrees C. The ionic strength dependence of this ionization is anomalous. The binding of 4-nitroimidazole by horse metmyoglobin was studied between pH 5.0 and 11.5 and as a function of ionic strength between 0.01 and 1.0 M. The association rate constant is pH-dependent, varying from 24 M(-1)s(-1) at pH 5 to a maximum value of 280 M(-1)s(-1) at pH 9.5 and then decreasing to 10 M(-1)s(-1) a...
Li N, Dou S, Feng L, Zhu Q, Lu N.In matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), the analyte is usually distributed unevenly throughout the sample spot. The area with aggregated analyte molecules contributing abundant signal, is termed as "sweet spot", which results in poor detection reproducibility and makes it impossible to quantify analytes without internal standards. We proposed a strategy to eliminate sweet spot in MALDI-MS by using a hydrophobic ordered structure as target. The target is fabricated by creating a hydrophobic silicon nanopillar array and subsequently decorating it uniformly wi...
Patton S, Huston GE, Jenness R, Vaucher Y.Milk fat globules are secreted by envelopment in plasma membrane of the lactating cell. SDS-gel electrophoresis of proteins from this membrane has revealed differences between milk donors in two mucin-like glycoproteins. One of these glycoproteins resolves in 3% acrylamide stacking gel and the other in 4% running gel. The proteins vary in number of bands (one or two) and band mobilities. This polymorphism arises, at least in part, from expression of hypervariable genes. In this study, gel electrophoretic evidence of similar polymorphism in glycoproteins from cow, chimpanzee, horse and human mi...
Scopel e Silva D, de Castro CC, da Silva e Silva F, Sant'anna V, Vargas GD, de Lima M, Fischer G, Brandelli A, da Motta Ade S, Hübner Sde O.P34 is an antimicrobial peptide produced by a Bacillus sp. strain isolated from the intestinal contents of a fish in the Brazilian Amazon basin with reported antibacterial activity. The aim of this work was to evaluate the peptide P34 for its in vitro antiviral properties against canine adenovirus type 2 (CAV-2), canine coronavirus (CCoV), canine distemper virus (CDV), canine parvovirus type 2 (CPV-2), equine arteritis virus (EAV), equine influenza virus (EIV), feline calicivirus (FCV) and feline herpesvirus type 1 (FHV-1). The results showed that the peptide P34 exhibited antiviral activity a...
Grady JK, Zang J, Laue TM, Arosio P, Chasteen ND.Sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) was used to characterize the H- and L-subunit ratios of several mammalian ferritins and one bacterioferritin. Traditionally, SDS-PAGE has been used to characterize the H- and L-subunit ratios in ferritin; however, this technique is relatively slow and requires staining, destaining, and scanning before the data can be processed. In addition, the H- and L-subunits of ferritin are fairly close in molecular weight (approximately 21,000 and approximately 20,000, respectively) and are often difficult to resolve in SDS-PAGE slab gels. In ...
Shin SJ, Lein DH, Aronson AL, Nusbaum SR.A total of 19 pathogenic bacterial species was isolated from uterine swabs of 498 out of 1539 mares over 4 years. The swabs were taken by 5 veterinary clinicians using 2 different techniques. Bacterial contamination during swabbing was minimized by scrupulous attention to cleansing of the external genitalia and the perineal area, and in the handling of the culture specimen. The most prevalent organisms isolated were beta-haemolytic streptococcus (39%), Escherichia coli (27%) and Klebsiella pneumoniae (7%). Interpretation of microbiological findings correlated well with clinical findings when n...
Lindquist CD, Evans JJ, Wictum EJ.Accurate DNA quantification is essential for optimizing DNA testing and minimizing sample consumption. Real-time quantitative polymerase chain reaction (qPCR) assays have been published for human and canine nuclear DNA, and the need for quantifying other forensically important species was evident. Following the strategy employed for the canine qPCR assay, we developed individual assays to accurately quantify feline, bovine, equine, and cervid nuclear DNA. Each TaqMan-based assay incorporates a genus-specific probe targeting the Melanocortin-1 Receptor gene and includes a piece of synthetic DNA...
Gaitskell-Phillips G, Martín-Cano FE, Ortiz-Rodríguez JM, da Silva-Álvarez E, Masot J, Redondo E, Gil MC, Ortega-Ferrusola C, Peña FJ.Seminal plasma proteins have important roles in sperm functionality, and different mechanisms including micro-vesicle transport of proteins are involved in the regulation of sperm biology. Due to the role of seminal plasma, we hypothesized that specific proteins present in seminal plasma may be used as discriminant variables with potential to identify stallions producing different quality ejaculates; 10 fertile stallions, with different motility and velocity values (although within normal ranges) were used in this study. Motilities and velocities were studied using computer assisted sperm anal...
Weiss DJ, Kraemer R, Schmit K.A simple discontinuous Percoll density-gradient technique was adapted for isolation of granulocytes and mononuclear cells from cats, dogs, horses and cattle. Separation was accomplished at low speeds using a standard tabletop centrifuge. Cell purity was 100% for both granulocytes and mononuclear cells and cell viability exceeded 95%. Percent recovery of leukocytes ranged from 69 to 83%.
Al-Izzi SA, Al-Bassam LS.Pseudomonas mallei was isolated from pus samples obtained from 34 mallein-positive horses. The isolates were subjected to in vitro sensitivity test using 16 different antimicrobial discs. All isolates (34) were sensitive to sulfamethizole, gentamycin, tetracycline, sulfathiazole, kanamycin, tobramycin, streptomycin and a combination of trimethoprim and sulfamethoxazole while none of them were sensitive to cephalothin, colistin, ampicillin, penicillin and nitrofurantoin. Rifapicin, chloramphenicol and carbenicillin were effective against 32, 26 and 18 isolates respectively. The minimum inhibito...
Wise LM, Bodaan CJ, Mercer AA, Riley CB, Theoret CL.Wounds in horses often exhibit sustained inflammation and inefficient vascularization, leading to excessive fibrosis and clinical complications such as "proud flesh". Orf virus-derived proteins, vascular endothelial growth factor (VEGF)-E and interleukin (ovIL)-10, enhance angiogenesis and control inflammation and fibrosis in skin wounds of laboratory animals. Objective: The study aimed to determine if equine dermal cells respond to VEGF-E and ovIL-10. Equine dermal cells are expected to express VEGF and IL-10 receptors, so viral protein treatment is likely to alter cellular gene expression an...
Scare JA, Dini P, Norris JK, Steuer AE, Scoggin K, Gravatte HS, Howe DK, Slusarewicz P, Nielsen MK.Ascarid parasites infect a variety of hosts and regular anthelmintic treatment is recommended for all species. Parascaris spp. is the only ascarid species with widespread anthelmintic resistance, which allows for the study of resistance mechanisms. The purpose of this study was to establish an in vitro drug exposure protocol for adult anthelmintic-naïve Parascaris spp. and report a preliminary transcriptomic analysis in response to drug exposure. Live worms were harvested from foal necropsies and maintained in RPMI-1640 at 37 °C. Serial dilutions of oxibendazole (OBZ) and ivermectin (IVM) we...
Lepage N, St-Laurent G, Carman S, Archambault D.The genetic variation in equine arteritis virus (EAV) Gs protein encoding gene was investigated. Nucleic and deduced amino acid sequences from eight different EAV isolates (one European, two American and five Canadian isolates) were compared with those of the Bucyrus reference strain. Nucleotide and amino acid identities between these isolates and the Bucyrus reference strain ranged from 92.3 to 96.4%, and 93.2 to 95.5%, respectively. However, phylogenetic tree analysis and estimation of genetic distances based on the Gs protein encoding gene sequences showed that the European prototype Vienna...
Janda J, Plattet P, Torsteinsdottir S, Jonsdottir S, Zurbriggen A, Marti E.Allergic horses react to innocuous environmental substances by activation of Th2 cells and production of allergen-specific IgE antibodies. The mechanisms leading to Th2 differentiation are not well understood. In humans and mice, epithelial cell-derived thymic stromal lymphopoietin (TSLP) plays a central role in this process. Little is known about equine TSLP (eqTSLP) and its role in allergic diseases and our current knowledge is limited to the assessment of TSLP mRNA expression. In order to be able to study eqTSLP at the protein level, the aim of the present study was to produce recombinant e...
Musilova P, Kubickova S, Horin P, Vodicka R, Rubes J.Cross-species chromosome painting has been applied to most of the species making up the numerically small family Equidae. However, comparative mapping data were still lacking in Asiatic asses kulan (Equus hemionus kulan) and kiang (E. kiang). The set of horse arm-specific probes generated by laser microdissection was hybridized onto kulan (E. hemionus kulan) and kiang (E. kiang) chromosomes in order to establish a genome-wide chromosomal correspondence between these Asiatic asses and the horse. Moreover, region-specific probes were generated to determine fusion configuration and orientation of...
Muscatello G, Browning GF.Selective agar media have been used for many years to facilitate the isolation of Rhodococcus equi from environmental and clinical samples. However, characterisation of R. equi still requires the use of immunochemical or polymerase chain reaction (PCR) analysis to differentiate between virulent and avirulent isolates. Here, we describe a novel method to detect and differentiate between R. equi isolates using colony blotting and DNA hybridization. Radiolabelled PCR product derived from the R. equi rrnA gene and specific hybridization conditions enabled differentiation of colonies of R. equi fro...
Odoi A, Carter CN, Riley JW, Smith JL, Dwyer RM.To develop an early-warning automated surveillance-data-analysis system for early outbreak detection and reporting and to assess its performance on an abortion outbreak in mares in Kentucky. Methods: 426 data sets of abortions in mares in Kentucky during December 2000 to July 2001. Methods: A custom software system was developed to automatically extract and analyze data from a Laboratory Information Management System database. The software system was tested on data on abortions in mares in Kentucky reported between December 1, 2000, and July 31, 2001. The prospective space-time permutations sc...
Permyakov SE, Khokhlova TI, Uversky VN, Permyakov EA.The triggering of Ca(2+) signaling pathways relies on Ca(2+)/Mg(2+) specificity of proteins mediating these pathways. Two homologous milk Ca(2+)-binding proteins, bovine alpha-lactalbumin (bLA) and equine lysozyme (EQL), were analyzed using the simplest "four-state" scheme of metal- and temperature-induced structural changes in a protein. The association of Ca(2+)/Mg(2+) by native proteins is entropy-driven. Both proteins exhibit strong temperature dependences of apparent affinities to Ca(2+) and Mg(2+), due to low thermal stabilities of their apo-forms and relatively high unfavorable enthalpi...
Nasir L, Gault E, Morgan IM, Chambers G, Ellsmore V, Campo MS.BPV-1 DNA is the predominant viral type detected in equine sarcoids and represents the only reported natural cross species infection of papillomaviruses. In this study, nucleotide variations in the LCR and the E2 regions of equine sarcoid-associated BPV-1 were characterised by sequence analysis. Variants particular to sarcoid BPV-1 were identified in both the LCR and E2 sequence. The functionality of the most common LCR variant was examined in equine and bovine cells. These studies showed that the activity of the variant LCR was higher in equine cells than bovine cells; the activity of the var...
von Samson-Himmelstjerna G, Janssen IJI, Ramünke S, Goday C, Borges FA, Koudela B, Niedźwiedź A, Tomczuk K, Studzińska MB, Kornas S, Krücken J.Equines were over decades considered to be infected by two morphologically virtually indistinguishable ascarid species, Parascaris univalens and Parascaris equorum. Reliable species discrimination is only possible using enzyme isoelectric focussing and karyotyping with P. univalens having one and P. equorum two chromosome pairs. However, presumably the complexity of both methods prevented their routine use in nearly all previous studies about prevalence and drug resistance of Parascaris spp. These have barely been performed on the species level although most studies stated presence of one or t...
van Gennip RGP, van de Water SGP, Potgieter CA, van Rijn PA.The Reoviridae family consists of nonenveloped multilayered viruses with a double-stranded RNA genome consisting of 9 to 12 genome segments. The Orbivirus genus of the Reoviridae family contains African horse sickness virus (AHSV), bluetongue virus, and epizootic hemorrhagic disease virus, which cause notifiable diseases and are spread by biting Culicoides species. Here, we used reverse genetics for AHSV to study the role of outer capsid protein VP2, encoded by genome segment 2 (Seg-2). Expansion of a previously found deletion in Seg-2 indicates that structural protein VP2 of AHSV is not essen...
Granier T, Comberton G, Gallois B, d'Estaintot BL, Dautant A, Crichton RR, Précigoux G.We refined the structure of the tetragonal form of recombinant horse L-chain apoferritin to 2.0 A and we compared it with that of the cubic form previously refined to the same resolution. The major differences between the two structures concern the cadmium ions bound to the residues E130 at the threefold axes of the molecule. Taking advantage of the significant anomalous signal (f" = 3.6 e-) of cadmium at 1.375 A, the wavelength used here, we performed anomalous Fourier difference maps with the refined model phases. These maps reveal the positions of anomalous scatterers at different locations...
Considering the course of the current SARS-CoV-2 pandemic, it is important to have serological tests for monitoring humoral immune response against SARS-CoV-2 infection and vaccination. Herein we describe a novel bridge enzyme-linked immunosorbent assay (b-ELISA) for SARS-CoV-2 antibodies detection in human and other species, employing recombinant Spike protein as a unique antigen, which is produced at high scale in insect larvae. Eighty two human control sera/plasmas and 169 COVID-19 patients' sera/plasmas, confirmed by rRT-PCR, were analyzed by the b-ELISA assay. In addition, a total of 27 a...
Reepmeyer JC, Brower JF, Ye H.Equilin-3-sulfate and delta8,9-dehydroestrone-3-sulfate are two isomers found in equine conjugated estrogens that differ in structure only by the position of a double bond in the steroid B-ring. These geometric isomers were not resolved on a C18 column during the analysis of conjugated estrogen drug products by LC-MS using acetonitrile-ammonium acetate buffer as the mobile phase. While no separations of these two isomers were observed on C18 or other alkyl-bonded silica based phases using a variety of mobile phase conditions, partial separations were achieved on phenyl bonded silica phases wit...
Oldenhof H, Schütze S, Wolkers WF, Sieme H.Sperm chromatin structure and condensation determine accessibility for damage, and hence success of fertilization and development. The aim of this study was to reveal characteristic spectral features coinciding with abnormal sperm chromatin packing (i.e., DNA-protein interactions) and decreased fertility, using Fourier transform infrared spectroscopy. Chromatin structure in spermatozoa obtained from different stallions was investigated. Furthermore, spermatozoa were exposed to oxidative stress, or treated with thiol-oxidizing and disulfide-reducing agents, to alter chromatin structure and pack...
Thomasy SM, Moeller BC, Stanley SD.To compare the density and binding characteristics of opioid receptor subtypes in horse, rat, and guinea pig cerebral cortex and cerebellum. Methods: Prospective receptor binding study. Methods: Whole brains were obtained from four neurologically normal adult horses during necropsy. Rat and guinea pig brains were obtained commercially. Methods: The cerebellum and cerebral cortex were dissected from each brain, and tissue homogenates prepared. A radioligand binding technique with the highly selective ligands [(3)H]-DAMGO, [(3)H]-U69593, and [(3)H]-DPDPE was used to identify the mu- (mu), kappa-...
Finno CJ, Packham AE, David Wilson W, Gardner IA, Conrad PA, Pusterla N.The purpose of this study was to determine the effect of blood contamination of cerebrospinal fluid (CSF) on the results of indirect fluorescent antibody tests (IFATs) for Sarcocystis neurona and Neospora hughesi. The in vitro study used antibody-negative CSF collected from non-neurologic horses immediately after euthanasia and blood samples from 40 healthy horses that had a range of IFAT antibody titers against S. neurona and N. hughesi. Serial dilutions of whole blood were made in seronegative CSF to generate blood-contaminated CSF with red blood cell (RBC) concentrations ranging from 10 to ...
Healy PJ.Activities of lysosomal hydrolases were measured in the leucocytes of cattle, sheep, goats, horses and pigs. There was high activity of arylsulphatase in leucocytes from cattle, high activities of alpha-fucosidase and beta-glucuronidase in leucocytes from horses and high activity of acid phosphatase in granulocytes from pigs. Within species, arylsulphatase and beta-galactosidase activities were higher in granulocytes than in mononuclear cells, but beta-glucuronidase, phosphodiesterase and alpha-galactosidase activities were higher in mononuclear cells than in granulocytes. Eosinophils of cattl...
