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.
Montes Cortés MG, Fernández-García JL, Habela Martínez-Estéllez MÁ.Equine piroplasmoses are enzootic parasitic diseases distributed worldwide with high incidence in tropical and subtropical regions. In Spain, there is insufficient epidemiological data about equine piroplasmoses. The main aim of the present study was therefore to estimate the prevalence of Theileria equi and Babesia caballi in five regions and obtain information about the risk factors. This study was conducted in the central and south-western regions of Spain, using indirect fluorescence antibody testing (IFAT) in 3,100 sera samples from apparently healthy horses of different ages, breeds, coa...
Warnken T, Brehm R, Feige K, Huber K.The aim of the study was to analyze key proteins of the equine insulin signaling cascade and their extent of phosphorylation in biopsies from muscle tissue (MT), liver tissue (LT), and nuchal AT, subcutaneous AT, and retroperitoneal adipose tissues. This was investigated under unstimulated (B1) and intravenously insulin stimulated (B2) conditions, which were achieved by injection of insulin (0.1 IU/kg bodyweight) and glucose (150 mg/kg bodyweight). Twelve warmblood horses aged 15 ± 6.8 yr (yr), weighing 559 ± 79 kg, and with a mean body condition score of 4.7 ± 1.5 were included in the s...
Mirajkar NS, Kelley MR, Gebhart CJ.Reported herein is the draft genome sequence of equine-origin Lawsonia intracellularis strain E40504, an obligate intracellular bacterium and the etiological agent of equine proliferative enteropathy. The 1.69-Mb draft genome sequence includes 1,380 protein-coding genes and 49 RNA genes, and it lacks a genomic island reported in swine-origin L. intracellularis strain PHE/MN1-00.
Mani RJ, Thachil AJ, Ramachandran A.Accurate and timely identification of infectious etiologies is of great significance in veterinary microbiology, especially for critical diseases such as strangles, a highly contagious disease of horses caused by Streptococcus equi subsp. equi. We evaluated a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) platform for use in species- and subspecies-level identification of S. equi isolates from horses and compared it with an automated biochemical system. We used 25 clinical isolates each of S. equi subsp. equi and S. equi subsp. zooepidemicus. Using ...
Aguiar FLN, Gastal GDA, Ishak GM, Gastal MO, Teixeira DIA, Feugang JM, Figueiredo JR, Gastal EL.The effect of FSH supplementation on an enriched cultured medium containing insulin (10 ng/mL) and EGF (50 ng/mL) was investigated on in vitro culture of equine ovarian biopsy tissue. Ovarian tissue fragments were collected from mares (n = 10) and distributed in the following treatments: noncultured control, cultured control, and cultured + FSH. Both treated groups were cultured for 7 or 15 days. The end points evaluated were: follicular morphology, estradiol levels in the culture medium, fluorescence intensity for TUNEL, EGFR and Ki-67 detection, and gene expression of GDF-9, BMP-15, a...
Zarski LM, High EA, Nelli RK, Bolin SR, Williams KJ, Hussey G.Equine herpesvirus 5 (EHV-5) infection is associated with pulmonary fibrosis in horses, but further studies on EHV-5 persistence in equine cells are needed to fully understand viral and host contributions to disease pathogenesis. Our aim was to develop a quantitative PCR (qPCR) assay to measure EHV-5 viral copy number in equine cell cultures, blood lymphocytes, and nasal swabs of horses. Furthermore, we used a recently developed equine primary respiratory cell culture system to study EHV-5 pathogenesis at the respiratory tract. PCR primers and a probe were designed to target gene E11 of the EH...
Sumner SM, Naskou MC, Thoresen M, Copland I, Peroni JF.Platelet preparations containing growth factors, attachment factors, and enzymes are appealing to enhance healing of injured tissues and as an alternative to xenogenic serum in cell culture media. Plateletpheresis is commonly used to collect platelets in human medicine but has not been validated in horses. Plateletpheresis to collect platelet concentrate was performed on six female, mixed breed, chemically restrained horses using commercially available apheresis equipment. Before and immediately after plateletpheresis, we performed physical examinations and collected blood for chemistry and co...
Liu S, Lian S, Yang Y, Fu C, Ma H, Xiong Z, Ling Y, Zhao C.An experiment was conducted to study the association between the single nucleotide polymorphisms (SNPs) in 5'-untranslated regions (5'-UTR) of equine chorionic gonadotropin (eCG) genes and the serum eCG levels. Methods: SNPs in 5'-UTR of eCG genes were screened across 10 horse breeds, including 7 Chinese indigenous breeds and 3 imported breeds using iPLEX chemistry, and the association between the serum eCG levels of 174 pregnant Da'an mares and their serum eCG levels (determined with ELISA) was analyzed. Results: Four SNPs were identified in the 5'-UTR of the eCGα gene, and one of them was u...
Drábková Z, Schramel JP, Kabeš R.To compare two methods of Bohr-Enghoff physiological dead space to tidal volume ratio (Vd/Vt) determination using a mixing chamber and an E-CAiOVX metabolic monitor. Methods: Prospective, clinical, method-comparison study. Methods: Twenty horses anaesthetized for elective orthopaedic procedures. Methods: Horses were anaesthetized with isoflurane in oxygen and the lungs were mechanically ventilated (Vt 15±2 mL kg). Arterial blood was sampled to provide arterial partial pressure of carbon dioxide (PaCO) for dead space calculation using a metabolic monitor. Mixed expired partial pressure of ca...
Schaafstra FJWC, van Doorn DA, Schonewille JT, van Riet MMJ, Visser P, Blok MC, Hendriks WH.Methodological aspects of digestibility measurements were studied in four Welsh pony geldings consuming haylage-based diets with increasing proportions of a pelleted concentrate according to a 4×4 Latin square design experiment. Ponies were fed four experimental, iso-energetic (net energy (NE) basis) diets (i.e. 22 MJ NE/day) with increasing proportions of a pelleted concentrate (C) in relation to haylage (H). The absolute amounts of diet dry matter fed per day were 4.48 kg of H (100H), 3.36 and 0.73 kg of H and C (75H25C), 2.24 and 1.45 kg of H and C (50H50C) and 1.12 and 2.17 kg of H and C ...
Bozorgmanesh R, Sutton-Burges JW, Tablin F.Equine whole blood collection and storage methods have been evaluated to assess red blood cell viability; however, platelet (PLT) viability has not been comprehensively assessed. Objective: The purpose of the study was to compare viability of PLTs collected in whole blood into 2 different anticoagulants. Methods: Whole blood from 6 healthy adult Thoroughbred horses was collected into citrate-phosphate-dextrose-adenine (CPDA) or acid-citrate-dextrose (ACD). Platelet count, pH, and concentrations of glucose, lactate, carbon dioxide, oxygen, bicarbonate, sodium, potassium, and chloride were measu...
ELISAs are known to have a higher diagnostic sensitivity than the agar gel immunodiffusion (AGID) when employed for serological diagnosis of equine infectious anaemia (EIA). For this purpose, an "in-house" and five commercial ELISAs available in Italy were assessed by the National Reference Centre for EIA for their analytic specificity (Sp); precocity, defined as capability of detecting first antibodies produced during a new infection; precision based on repeatability and reproducibility, estimated from the coefficient of variation (CV); accuracy, estimated from multiple K and relative Sp and ...
Suganuma K, Yamasaki S, Molefe NI, Musinguzi PS, Davaasuren B, Mossaad E, Narantsatsral S, Battur B, Battsetseg B, Inoue N.Dourine is caused by Trypanosoma equiperdum via coitus with an infected horse. Although dourine is distributed in Equidae worldwide and is listed as an internationally important animal disease by the World Organization for Animal Health (OIE), no effective treatment strategies have been established. In addition, there are no reports on drug discovery, because no drug screening system exists for this parasite. A new T. equiperdum strain was recently isolated from the genital organ of a stallion that showed typical symptoms of dourine. In the present study, we adapted T. equiperdum IVM-t1 from...
Cymerys J, Słońska A, Tucholska A, Golke A, Chmielewska A, Bańbura MW.Equine herpesvirus 1 (EHV-1), like other members of the Alphaherpesvirinae subfamily, is a neurotropic virus causing latent infections in the nervous system of the natural host. In the present study, we have investigated EHV-1 replication (wild-type Jan-E strain and Rac-H laboratory strain) during long-term infection and during the passages of the virus in cultured neurons. The studies were performed on primary murine neurons, which are an excellent in vitro model for studying neurotropism and neurovirulence of EHV-1. Using real-time cell growth analysis, we have demonstrated for the first tim...
Schnabel CL, Babasyan S, Freer H, Wagner B.Only few quantitative reports exist about the concentrations and induction of immunoglobulin A (IgA) in mucosal secretions of horses. Despite this, it is widely assumed that IgA is the predominant immunoglobulin on mucosal surfaces in the horse. Here, two new monoclonal antibodies (mAbs) against equine IgA, clones 84-1 and 161-1, were developed and characterized in detail. Both IgA mAbs specifically bound monomeric and dimeric equine IgA in different applications, such as Western blots and fluorescent bead-based assays. Cross-reactivity with other equine immunoglobulin isotypes was not observe...
Liu Y, Uboh CE, Li X, Guan F, You Y, Maylin GA, Zhu F, Soma LR.A rapid and sensitive method for simultaneous screening, quantification and confirmation of 17 barbiturates in horse plasma using liquid chromatography-tandem mass spectrometry is described. Analytes were recovered from plasma by liquid-liquid extraction using methyl tert-butyl ether, separated on a C18 column, and analyzed in negative electrospray ionization mode. Multiple-reaction monitoring was employed for screening and quantification. Confirmation for the presence of the analytes was achieved by comparing ion intensity ratio. The ranges for limits of detection, quantification and confirma...
Loux SC, Scoggin KE, Bruemmer JE, Canisso IF, Troedsson MH, Squires EL, Ball BA.MicroRNAs (miRNAs) are small, non-coding RNAs which are produced throughout the body. Individual tissues tend to have a specific expression profile and excrete many of these miRNAs into circulation. These circulating miRNAs may be diagnostically valuable biomarkers for assessing the presence of disease while minimizing invasive testing. In women, numerous circulating miRNAs have been identified which change significantly during pregnancy-related complications (e.g. chorioamnionitis, eclampsia, recurrent pregnancy loss); however, no prior work has been done in this area in the horse. To identif...
Fragkaki AG, Kioukia-Fougia N, Kiousi P, Kioussi M, Tsivou M.The artificial increase of the physical capability of horses using drugs is well known in racing and other equine sports. Both illicit and therapeutic substances are regarded as prohibited substances in competition in most countries. Some countries make distinctions for a few, specific drugs which are, however, allowed for use in other countries. The primary objective in the case of doping control is the detection of any trace of drug exposure, either parent drug or any of its metabolites, using the most powerful analytical methods which are generally based on chromatographic/mass spectrometri...
Baskerville CL, Bamford NJ, Harris PA, Bailey SR.Insulin-like growth factor-1 (IGF-1) plays several important physiological roles, and IGF-related pathways have been implicated in developmental osteochondral disease and endocrinopathic laminitis. This factor is also a downstream marker of growth hormone activity and its peptide mimetics. Unfortunately, previously used assays for measuring equine IGF-1 (radioimmunoassays and ELISAs) are no longer commercially available, and many of the kits on the market give poor results when used on horse samples. The aim of the present study was to compare three different ELISA assays (two human and one ho...
Kuwano A, Niwa H, Arai K.To establish a new system to isolate keratolytic bacteria from the hoof wall cavity () of a racehorse, we invented the horn powder agar-translucency (HoPAT) test and horn zymography (HZ). Using routine bacteriological techniques and these methods, we isolated five strains of keratolytic soil bacteria, which were then identified by means of 16S ribosomal RNA (rRNA) gene sequencing analysis. The findings from the study on the horse suggested that Brevibacterium luteolum played the main role in the local fragility of the hoof, eventually forming a in coordination with four other strains of kerat...
Wang J, Wen S, Zhao R, Qi J, Liu Z, Li W, An J, Wood C, Wang Y.The conjugation of small ubiquitin-like modifier (SUMO) to the target protein, namely, SUMOylation, is involved in the regulation of many important biological events including host-pathogen interaction. Some viruses have evolved to exploit the host SUMOylation machinery to modify their own protein. Retroviral Gag protein plays critical roles in the viral life cycle. The HIV-1 p6 and the Moloney murine leukemia virus CA have been reported to be conjugated with SUMO. In this study, we report for the first time, to our knowledge, the covalent conjugation of equine infectious anemia virus (EIAV) G...
Gim JA, Kim HS.MicroRNAs (miRNAs) are single-stranded, small RNAs (21-23 nucleotides) that function in gene silencing and translational inhibition via the RNA interference mechanism. Most miRNAs originate from host genomic regions, such as intergenic regions, introns, exons, and transposable elements (TEs). Here, we focused on the palindromic structure of medium reiteration frequencies (MERs), which are similar to precursor miRNAs. Five MER consensus sequences (MER5A1, MER53, MER81, MER91C, and MER117) were matched with paralogous transcripts predicted to be precursor miRNAs in the horse genome (equCab2) and...
Sandbaumhüter FA, Theurillat R, Bettschart-Wolfensberger R, Thormann W.The combination of ketamine and an α -receptor agonist is often used in veterinary medicine. Four different α -receptor agonists, medetomidine, detomidine, xylazine, and romifidine, which differ in their chemical structure and thus in selectivity for the α -receptor and in the sedative and analgesic potency, are typically employed during surgery of equines. Recovery following anesthesia with ketamine and an α -receptor agonist is dependent on the α -receptor agonist. This prompted us to investigate (i) the inhibition characteristics for the N-demethylation of ketamine to norketamine and (...
Chaney SB, Marsh AE, Lewis S, Carman M, Howe DK, Saville WJ, Reed SM.Equine protozoal myeloencephalitis (EPM) remains a significant central nervous system disease of horses in the American continents. Sarcocystis neurona is considered the primary causative agent and its intermediate life stages are carried by a wide host-range including raccoons (Procyon lotor) in North America. S. neurona sarcocysts mature in raccoon skeletal muscle and can produce central nervous system disease in raccoons, mirroring the clinical presentation in horses. The study aimed to develop laboratory tools whereby the life cycle and various life stages of S. neurona could be better stu...
Serafini R, Varner DD, Bissett W, Blanchard TL, Teague SR, Love CC.The effect of flash-freezing storage temperature on stallion sperm DNA has not been evaluated. Commonly, sperm are flash-frozen at various temperatures to preserve sperm DNA prior to analysis. It is unclear whether the temperature at which sperm are frozen and stored may affect the results of DNA assays. In this study, the neutral comet assay was used to evaluate the effect of flash-freezing storage temperature (freezer [-60 °C], dry ice [-78.5 °C], liquid nitrogen [-196 °C]) compared to fresh sperm DNA structure. In addition, intra- and inter-assay and intra- and inter-stallion variabil...
Heras S, Smits K, De Schauwer C, Van Soom A.Global epigenetic reprogramming is considered to be essential during embryo development to establish totipotency. In the classic model first described in the mouse, the genome-wide DNA demethylation is asymmetric between the paternal and the maternal genome. The paternal genome undergoes ten-eleven translocation (TET)-mediated active DNA demethylation, which is completed before the end of the first cell cycle. Since TET enzymes oxidize 5-methylcytosine to 5-hydroxymethylcytosine, the latter is postulated to be an intermediate stage toward DNA demethylation. The maternal genome, on the other ha...
Schnobrich MR, Gordon DL, Scoggin CF, Bradecamp EA, Canisso IF.Urine pooling, as a persistent condition, is a cause of infertility in mares due to endometrial inflammation and sperm toxicity. Identification of urometra can be challenging in mares presenting with the condition intermittently, or when urine flows into the uterus but is undetectable in the vagina. Currently, there are no reported objective methods to confirm the clinical diagnosis of urine contamination in intrauterine-fluid accumulations. Since creatinine is present in high concentrations in urine and does not diffuse across cell membranes, creatinine concentration should be increased in ma...
Steinbusch MM, Fang Y, Milner PI, Clegg PD, Young DA, Welting TJ, Peffers MJ.The development of effective treatments for the age-related disease osteoarthritis and the ability to predict disease progression has been hampered by the lack of biomarkers able to demonstrate the course of the disease. Profiling the expression patterns of small nucleolar RNAs (snoRNAs) in joint ageing and OA may provide diagnostic biomarkers and therapeutic targets. This study determined expression patterns of snoRNAs in joint ageing and OA and examined them as potential biomarkers. Using SnoRNASeq and real-time quantitative PCR (qRT-PCR) we demonstrate snoRNA expression levels in murine age...
Boelow H, Krücken J, Thomas E, Mirams G, von Samson-Himmelstjerna G.Due to high prevalence of anthelmintic resistance in equine helminths, selective treatment is increasingly promoted and in some countries a positive infection diagnosis is mandatory before treatment. Selective treatment is typically recommended when the number of worm eggs per gram faeces (epg) exceeds a particular threshold. In the present study we compared the semi-quantitative sedimentation/flotation method with the quantitative methods Mini-FLOTAC and FECPAK in terms of precision, sensitivity, inter-rater reliability and correlation of worm egg counts to improve the choice of optimal diagn...
Masri M, Lombardero G, Velasquillo C, Martínez V, Neri R, Villegas H, Ibarra C.The goal of this study is to evaluate the efficiency of obtaining a large number of viable cells within a construct that will not be detached by high fluid flow during arthroscopic implantation. Methods: Arthroscopic osteochondral biopsy specimens were obtained from the medial femoral trochlea of 8 horses. Chondrocytes were isolated by collagenase digestion and expanded in M199 media until confluency. After 10 to 12 days, cultures were trypsinized and cells resuspended in culture media. Then, 5 x 10(6) cells x mL(-1) were seeded on a culture dish and the same amount in a flask. Once extracellu...
Knych HK, Vidal MA, Casbeer HC, McKemie DS.The use of triamcinolone acetonide (TA) in performance horses necessitates establishing appropriate withdrawal times prior to performance. Objective: To describe the plasma pharmacokinetics of TA and time-related urine and synovial fluid concentrations following i.m. and intra-articular administration to exercised Thoroughbred horses. Methods: Block design. Methods: Twelve racing fit adult Thoroughbred horses received a single i.m. administration of TA (0.1 mg/kg bwt). After an appropriate washout period, the same horses then received a single intra-articular TA administration (9 mg) into the ...
Li LF, Guan WJ, Hua Y, Bai XJ, Ma YH.A fibroblast line was successfully established from Mongolian horse ear marginal tissue by using a primary explant technique and cell cryogenic preservation technology. Biological analysis showed the following: The cells were adherent and exhibited density-dependent inhibition of proliferation; assays of microbial contamination from bacteria, fungi, and mycoplasma were negative; the population doubling time of the cells was 33.9 h; and a 2n chromosome number of 64 at a frequency higher than 80%. A lack of cross-contamination of this cell line with other species was confirmed by isoenzyme analy...
Brandon CI, Heusner GL, Caudle AB, Fayrer-Hosken RA.The objectives of this study were to 1) identify proteins found in stallion seminal plasma utilizing two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) in conjunction with Western blot analysis; and 2) to determine if any of these individual proteins were correlated with stallion fertility utilizing regression analysis. Fertility was quantified by assigning a breeding score for each stallion. Each score was calculated by dividing the number of conceptions by the number of breedings for each stallion for four successive breeding seasons (1992-1995). Ejaculates from stallions of known ...
Kirsch K, Detilleux J, Serteyn D, Sandersen C.Portable blood gas analyzers are used to facilitate diagnosis and treatment of disorders related to disturbances of acid-base and electrolyte balance in the ambulatory care of equine patients. The aim of this study was to determine whether 2 portable analyzers produce results in agreement with a stationary analyzer. Blood samples from 23 horses hospitalized for various medical reasons were included in this prospective study. Blood gas analysis and electrolyte concentrations measured by the portable analyzers VetStat and epoc were compared to those produced by the cobas b 123 analyzer via conco...
Melnick JL, Schmidt NJ, Hampil B, Ho HH.This paper describes the preparation of seven combination pools of equine antisera, designated J though P, for identification of 19 coxsackievirus A immunotypes. Each pool is composed of 4 to 6 antisera; the serotypes included are A1-6, 8, 10-15, and 17-22. These pools, unlike the previously prepared A-H enterovirus pools, were lyophilized from volumes of 0.5 ml dispensed into 5-ml vials, and when rehydrated with 5 ml of diluent provide 50-antibody-unit material ready for use in identification tests without further dilution. Procedures for using the antiserum pools are given, and guidance is p...
De Santis T, Casavola V, Reshkin SJ, Guerra L, Ambruosi B, Fiandanese N, Dalbies-Tran R, Goudet G, Dell'Aquila ME.The extracellular calcium-sensing receptor (CASR) plays an important role in cells involved in calcium (Ca2+) homeostasis by directly sensing changes in the extracellular Ca2+ ion concentration. We previously reported the localization and quantitative expression of CASR protein in human oocytes. In this study, we examined the expression and the functional role of CASR during oocyte meiotic maturation in a large mammal animal model, the horse. As in humans, CASR protein was found to be expressed in equine oocytes and cumulus cells. Western-blot analysis revealed a single 130 kDa band in denuded...
Cheung HW, Wong KS, Lin VYC, Wan TSM, Ho ENM.The misuse of genetic manipulation technology to enhance athletic performance is termed gene doping which is prohibited in human sports, horseracing, and equestrian sports. Although many qPCR assays have been developed, most assays employ genomic DNA (gDNA) from humans, non-human primates, and mice as a background and they may not be applicable for testing horse samples. This study aimed to develop a qPCR assay for the detection of human erythropoietin (hEPO) transgene in horse blood cells where the viral vectors used in gene therapy can reside for months. For the detection of hEPO transgene, ...
Zhang W, Cui S, Cheng XL, Wei F, Ma S.In this study, probe/primers of high specificity and sensitivity were selected to analyze donkey-hide gelatin for donkey DNA and to look for horse, ox, and pig DNA as possible adulterants. The mitochondrial CO I genes in donkey, horse, and ox were selected as target sequences for design and synthesis of three pairs of specific probes and primers. In addition, eight pairs of probe/primers were obtained via literature search. Out of these eleven groups of probe/primers, those with the highest specificity and sensitivity were selected, which was fulfilled by the screening firstly with animal hide...
Surasi K, Cummings KJ, Hanson C, Morris MK, Salas M, Seftel D, Ortiz L, Thilakaratne R, Stainken C, Wadford DA.The Abbott BinaxNOW rapid antigen test is cheaper and faster than real-time reverse transcription PCR (rRT-PCR) for detecting severe acute respiratory syndrome coronavirus 2. We compared BinaxNOW with rRT-PCR in 769 paired specimens from 342 persons during a coronavirus disease outbreak among horse racetrack workers in California, USA. We found positive percent agreement was 43.3% (95% CI 34.6%-52.4%), negative percent agreement 100% (95% CI 99.4%-100%), positive predictive value 100% (95% CI 93.5%-100%), and negative predictive value 89.9% (95% CI 87.5%-92.0%). Among 127 rRT-PCR-positive ...
Milinovich GJ, Burrell PC, Pollitt CC, Bouvet A, Trott DJ.Four Gram-positive, catalase-negative, coccoid-shaped isolates were obtained from the caecum and rectum of horses with oligofructose-induced equine laminitis. Phenotypic and phylogenetic studies were performed on these isolates. Initial biochemical profiling assigned two of the isolates to Streptococcus bovis. The other two isolates, however, could not be assigned conclusively on the basis of their biochemical profiles. Gene sequence analysis demonstrated that the four new isolates were related most closely to Streptococcus suis based on the 16S rRNA gene and to Streptococcus orisratti based o...
Chapman MR, Kearney MT, Klei TR.Historically, surveys of equine parasites either are not quantitative in regard to prevalence and intensities of cyathostome species, or if quantitative, are estimates based on the identification of a very small sample of the population. Commonly 100-200 worms are identified. In the current study cyathostomes from 10 ponies were counted and identified to species in subsets of approximately 200 worms each from 5% aliquots of the large intestine contents until all worms in the aliquot were examined. A mean of 10.9+/-4.3 species were identified by examining 200 cyathostomes from each animal. This...
Stewart JM, Blakely JA, Johnson MD.Non-covalent modification of cytochrome c may have implications for electron transport and energy metabolism. We examined the interaction of various fatty acids (FAs), their coenzyme A and carnitine esters, and fatty alcohols with horse heart ferrocytochrome c. A comparison of FAs indicated a minimum chain length of 14 carbons was required for significant effect on the ferroheme chromophore and major changes in electronic spectra. Coenzyme A and carnitine esters interacted less strongly than FAs whereas long-chain alcohols did not interact with the protein. We found a single, saturable FA bind...
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...
Voelter-Ratson K, Monod M, Unger L, Spiess BM, Pot SA.To characterize the conjunctival fungal flora and to determine the susceptibility of 2 isolated molds to antifungal drugs in samples of 64 healthy horses from The National Stud in Switzerland. Methods: Conjunctival cytobrush samples were collected from both eyes of 64 ophthalmologically normal horses in August 2012 and subsequently cultured on Sabouraud's agar medium. Growing fungi were identified and counted. Etests or broth microdilution tests for Aspergillus fumigatus and Eurotium amstelodami were carried out to determine antifungal drug sensitivity. These species had previously been detect...
Langemeier JL, Cook SJ, Cook RF, Rushlow KE, Montelaro RC, Issel CJ.Control of equine infectious anemia (EIA) is currently based on detection of anti-EIA virus (EIAV) antibodies. However, serologic diagnostic methods may give false-negative results in infected horses that fail to respond adequately or are in the early stages of infection. We developed a reverse transcriptase nested PCR (RT-nPCR) assay for the detection of viral gag gene sequences in plasma from EIAV-infected horses. The ability of RT-nPCR to detect field strains of EIAV was investigated by assaying plasma samples from 71 horses stabled on EIA quarantine ranches. Positive PCR signals were detec...
Gross MK, Toscano DG, Toscano WA.Calmodulin (CaM), the calcium binding protein that modulates the activity of a number of key regulatory enzymes, is present at high levels in sperm. To determine whether CaM regulates adenylate cyclase in mammalian sperm, the actions of EGTA and selected CaM antagonists on a solubilized adenylate cyclase from mature equine sperm were examined. The activity of equine sperm adenylate cyclase was inhibited by EGTA in a concentration-dependent manner with a half-maximal inhibitory concentration (IC50) of 2 mM. Equine sperm adenylate cyclase was also inhibited in a concentration-dependent manner by...
Zientara S, Sailleau C, Moulay S, Cruciere C.A single tube reverse transcription-polymerase chain reaction (RT-PCR) method for detection of African horse sickness virus (AHSV) in splenic tissues from infected horses is described. Double stranded RNA was extracted from infected organs of horses and used to produce complementary DNA (cDNA) with the two primers selected for the PCR. The 1179 bp amplified product (the segment 7 which encodes for VP 7), detected by electrophoresis on agarose gel and ethidium bromide staining, was hydrolysed with eight restriction endonucleases for characterization of the AHSV. The sensitivity of this method i...
Miglio A, Morelli C, Maresca C, Felici A, Di Gianbattista A, Antognoni MT.Serum protein electrophoresis (SPE) reference intervals (RIs) have been evaluated in different horses, but no specific values are shown for equine breeds as previously described in other species (dogs, cats), and no studies have been performed on SPE in draft horses. Objective: This study aimed to determine RIs for SPE in heavy draft horses (Italian Heavy Draft Horse-IHDH) living in central Italy. A comparison between different physiologic states (pregnancy and no pregnancy) and ages (foals and adults) was executed. Methods: Blood samples were collected from 215 apparently healthy horses (mare...
Kitai Y, Kondo T, Konishi E.A complement-dependent cytotoxicity (CDC) assay was established to measure antibodies to the West Nile virus (WNV) nonstructural protein 1 (NS1) in horses. Sera collected from a WNV-infected horse mediated lysis of WNV NS1-expressing cells in a dose-dependent manner at higher percentages than sera from a Japanese encephalitis virus (JEV)-infected horse. The percentages of specific lysis for sera diluted 1:10 to 1:80 were <19.8% (assay cutoff) for almost all of the 100 JEV-infected or uninfected horses tested, in contrast to 55 to 76% in WNV-infected horses. Experimental infection revealed t...
Torgeman A, Mador N, Dorozko M, Lifshitz A, Eschar N, White MD, Wolf DG, Epstein E.Assuring viral safety of horse plasma-derived products is fundamental for ethical and regulatory reasons. We previously demonstrated the ability of pepsin digestion at low pH to inactivate West Nile and Sindbis viruses in horse plasma. The present study further examined the efficiency of pepsin digestion to inactivate four additional viruses: HSV-1 and BVDV (lipid-enveloped), BPV and Reo-3 (nonenveloped). These viruses were spiked into hyperimmunized horse plasma against botulinum toxin and subjected to low pH (3.2) alone or combined with pepsin digestion (1200 units/ml). Peptic digestion inac...
Sato F, Kanno T, Nagasawa S, Yanaihara N, Ishida N, Hasegawa T, Iwanaga T.We investigated the existence of chromogranin A (CgA) in salivary glands of the horse by Western blotting and enzyme immunoassay (EIA) using an antiserum against a peptide sequence of equine CgA. We also compared its cellular distribution between the horse and rat salivary glands with a tyramide signal amplification immunofluorescence technique. Western blotting gave three significant immunoreactive bands (74, 56 and 48 kDa) in adrenal medulla and three major salivary glands of horses. Immunoreactivities for CgA measured by EIA in horses were 154.05 +/- 41.46, 20.32 +/- 5.59 and 4.43 +/- 2.23 ...
Pavulraj S, Bera BC, Joshi A, Anand T, Virmani M, Vaid RK, Shanmugasundaram K, Gulati BR, Rajukumar K, Singh R, Misri J, Singh RK, Tripathi BN....Equine influenza viruses (EIV)-H3N8 continue to circulate in equine population throughout the world. They evolve by the process of antigenic drift that leads to substantial change in the antigenicity of the virus, thereby necessitating substitution of virus strain in the vaccines. This requires frequent testing of the new vaccines in the in vivo system; however, lack of an appropriate laboratory animal challenge model for testing protective efficacy of equine influenza vaccine candidates hinders the screening of new vaccines and other therapeutic approaches. In the present investigation, BALB/...
Wills JM, Watson G, Lusher M, Mair TS, Wood D, Richmond SJ.This paper describes the isolation and characterisation of a strain of Chlamydia psittaci obtained from a nasal swab taken from a horse with serous nasal discharge. Initial isolation was achieved in cycloheximide-treated McCoy cell monolayers. Chlamydial inclusions stained by immunofluorescence either with a rabbit antiserum raised against C. psittaci or with a monoclonal antibody directed against the genus-specific lipopolysaccharide antigen were single and compact. They did not stain with iodine or with a monoclonal antibody reactive against Chlamydia trachomatis. The agent was re-isolated i...
Dong JB, Zhu W, Cook FR, Goto Y, Horii Y, Haga T.Equine infectious anemia (EIA) has posed a major challenge and caused significant losses to the equine industry worldwide. PCR detection methods have considerable potential as an adjunct to conventional serological diagnostic techniques. However, most published PCR methods, including that recommended by the OIE, were designed using laboratory-adapted virus strains and do not function with field isolates of EIA virus (EIAV). In the present study, a nested PCR assay for detection of EIAV proviral DNA in peripheral blood cells of naturally infected horses was developed. Primer sets were designed ...
Raeside JI, Christie HL, Renaud RL, Waelchli RO, Betteridge KJ.Because estradiol (E(2)) production by the early equine conceptus is considered crucial to the establishment of pregnancy, the amounts of E(2), estrone (E(1)), and their sulfates (E(2)S, E(1)S) were measured by RIA in yolk-sac fluid of 63 conceptuses collected by transcervical lavage over the period of 11-26 days after ovulation. Amounts increased significantly with age of conceptus, especially for E(1)S. Then, the metabolism of E(2), which may be highly relevant for its action, was examined in the conceptus and endometrium over the period when the conceptus ceases to migrate within the uterus...
Love CC, Brinsko SP, Rigby SL, Thompson JA, Blanchard TL, Varner DD.The relationship between seminal plasma level (0, 10, or 20%) and extender type [Kenney type (EZ-Mixin-CST) or Kenney-modified Tyrodes-KMT] to the susceptibility of sperm DNA to denaturation and sperm motility measures were investigated in cooled (5 degrees C) stallion sperm. Three ejaculates from each of three fertile stallions were collected in an artificial vagina and processed as follows: diluted one part uncentrifuged semen with four parts of extender to a final concentration of 20% seminal plasma in either CST or KMT (20% CST; 20% KMT); diluted to a final concentration of 25 million sper...
Renzi S, Lombardo T, Dotti S, Dessì SS, De Blasio P, Ferrari M.The advent of stem cells and stem cell-based therapies for specific diseases requires particular knowledge of laboratory procedures, which not only guarantee the continuous production of cells, but also provide them an identity and integrity as close as possible to their origin. Their cryopreservation at temperatures below -80°C and typically below -140°C is of paramount importance. This target can be achieved by incorporating high molar concentrations of cryoprotectant mixtures that preserve cells from deleterious ice crystal formation. Usually, dimethyl sulfoxide (DMSO) and animal proteins...
Connard SS, Linardi RL, Even KM, Berglund AK, Schnabel LV, Ortved KF.The immunomodulatory properties of mesenchymal stem cells (MSCs) have been studied extensively due to their increasing clinical application for tissue regeneration and repair following culture expansion. We have studied the effect of continuous passage on the immunomodulatory capacity of equine bone marrow-derived MSCs (BM-MSCs). Equine BM-MSCs were isolated and culture expanded to passage three, six, and nine (P3, P6, P9). Immunomodulatory properties of each passage were assessed using a T cell proliferation assay and cytokine synthesis following stimulation with interferon gamma (IFN-γ). Re...
