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Topic:Laboratory Methods
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.
Evolution of 17-β-estradiol, estrone and estrone-sulfate concentrations in late pregnancy of different breeds of mares using Liquid Chromatography and Mass Spectrometry. This study describes 17-β-estradiol (E2), estrone (E1) and estrone-sulfate (E1S) concentrations between 4 and 11 months in healthy equine pregnancies of two different breeds using Liquid Chromatography coupled to Mass-Spectrometry (LC-MS). In 2 stud-farms including 15 Spanish PureBred (SPB) and 11 Showjumping (SJ) types mares, combined thickness of the uterus and the placenta (CTUP) was measured and blood was sampled monthly between 4 and 11 months of gestation. Concentrations of E2, E1 and E1S were assayed with LC-MS in mares with normal CTUP. Effects of breed, day of pregnancy and mare's pa...
In vitro antifungal susceptibility of Fusarium species and Aspergillus fumigatus cultured from eleven horses with fungal keratitis. To examine the relationship between Minimum Inhibitory Concentration (MICs) and response to therapy of 6 Fusarium spp. and 5 Aspergillus fumigatus isolated from equine ulcerative keratitis cases. Methods: Fungi were identified by morphology and Internal Transcribed Spacer (ITS) polymerase chain reaction (PCR) with sequencing and evaluated at the University of Texas Fungal Testing Laboratory for susceptibility to three azole antifungals (miconazole, voriconazole, posaconazole), natamycin, and two echinocandins (anidulafungin, caspofungin). A Mann-Whitney rank sum test was used for the compariso...
Another lesson from unmapped reads: in-depth analysis of RNA-Seq reads from various horse tissues. In recent years, a vast amount of sequencing data has been generated and large improvements have been made to reference genome sequences. Despite these advances, significant portions of reads still do not map to reference genomes and these reads have been considered as junk or artificial sequences. Recent studies have shown that these reads can be useful, e.g., for refining reference genomes or detecting contaminating microorganisms present in the analyzed biological samples. A special case of this is RNA sequencing (RNA-Seq) reads that come from tissue transcriptomes. Unmapped reads from RNA-...
Optimization and implementation of four duplex quantitative polymerase chain reaction assays for gene doping control in horseracing. The concern about gene doping has remained high in horseracing and other equestrian competitions. Our laboratory has previously developed a duplex quantitative polymerase chain reaction (qPCR) assay capable of detecting in equine blood the human erythropoietin (hEPO) transgene and equine tubulin α 4a (TUBA4A) gene as an internal control the latter providing quality control over DNA extraction and qPCR. This study aimed to optimize the method for routine testing of regulatory samples. The use of an automated DNA extraction system has increased the sample throughput, consistency of DNA extracti...
Pharmacokinetics of grapiprant and effects on TNF-alpha concentrations following oral administration to horses. Grapiprant is a prostaglandin E2 receptor antagonist that has been found to be an effective anti-inflammatory in dogs and that is devoid of some of the adverse effects associated with traditional NSAIDs that elicit their effects through inhibition of PGE2 production. Previously published reports have described the pharmacokinetics of this drug in horses when administered at 2 mg/kg; however, pharmacodynamic effects in this species have yet to be described. The objective of the current study was to describe the pharmacokinetics and pharmacodynamics of grapiprant at a higher dose. Eight horses...
Serological Hendra Virus Diagnostics Using an Indirect ELISA-Based DIVA Approach with Recombinant Hendra G and N Proteins. Since the identification of Hendra virus (HeV) infections in horses in Australia in 1994, more than 80 outbreaks in horses have been reported, and four out of seven spillover infections in humans had a fatal outcome. With the availability of a subunit vaccine based on the HeV-Glycoprotein (HeV-G), there is a need to serologically ifferentiate the nfected from the accinated nimals (DIVA). We developed an indirect ELISA using HeV-G expressed in and HeV-Nucleoprotein (HeV-N) expressed in recombinant baculovirus-infected insect cells as antigens. During evaluation, we tested panels of sera from n...
Development of a Microsphere-Based Immunoassay Authenticating A2 Milk and Species Purity in the Milk Production Chain. Processed milk and milk products produced from bovine milk, commonly contain β-casein A1 (βCA1) and β-casein A2 (βCA2). Since the presence of βCA1 is linked to milk intolerance and digestion problems, A2A2 milk, which only contains βCA2, is proposed as a healthier alternative. To support this health claim, the purity of A2A2-milk has to be guaranteed. In the presented study, a multiplex immunoassay, able to distinguish between βCA2 and βCA1, was developed and real-life applicability was shown on raw milk samples from genotyped A1A1, A1A2 and A2A2 cows. Because of its ability to discrim...
First detection and genomic characterization of a new parvovirus in equines in China. Equine copivirus (EqCoPV) is a newly discovered parvovirus that infects equines. Currently, it is unclear whether this virus is prevalent in China. In the present study, serum samples were collected from equines in China and were processed for EqCoPV DNA detection by PCR. The results demonstrated that EqCoPV was circulating among the sampled equines, with a low detection rate of 0.94%. The genome sequence of one Chinese EqCoPV strain, UH26, was determined and used for genetic and phylogenetic analysis. The results demonstrated that UH26 has a close genetic relationship to EqCoPV strains from t...
Detection of Equus caballus papillomavirus-2 in equine penile/preputial papillomas and squamous cell carcinomas in southern Brazil. For approximately one decade, a novel papillomavirus termed Equus caballus papillomavirus-2 (EcPV-2) has been associated with equine penile/preputial papillomas and squamous cell carcinomas (SCCs). It is currently believed that the virus has a carcinogenic activity, being able to induce such neoplastic lesions. After being first described, EcPV-2 has been detected in many countries from North America, Europe, and Asia; however, to date, it has not been reported in Brazil. The aim of this research was to investigate the presence of EcPV-2 in penile/preputial papillomas and SCCs of Brazilian hor...
Comparison of Different Methods to Determine the Absorption of Colostral IgG in Newborn Foals. The timely diagnosis of abnormalities in the passive transfer of colostral immunoglobulins is important for the health and development of newborn foals. This study investigated three different methods for measuring immunoglobulin G concentration in neonatal foals. Comparison of a commercial SNAP assay, total protein concentration determination, and total globulin calculation by subtracting the albumin fraction from total protein as an indirect parameter was performed on a quantitative ELISA, which served as a reference method. The study included 119 samples from 148 foals between the age of 1 ...
Cortisol Concentration in Horsehair and its Relationship to Body Location, Coat Colour, and Gender. The determination of cortisol concentrations in animal hair appears to be a relatively simple and non-invasive method of assessing chronic stress. This method has advantages but it also has some drawbacks. We discovered that there are specific coat properties and individual factors in animlas that can affect corisol levels and skew the interpretation of results. We removed horsehair from 8 different areas of the body (chest, left saddle, right saddle, right thoracic fetlock, and left thoracic fetlock, the mane was also sampled at the withers, just behind the ears, and the tail strand) in 31 ho...
Comparison of FECPAKG2, a modified Mini-FLOTAC technique and combined sedimentation and flotation for the coproscopic examination of helminth eggs in horses. 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...
Allogeneic Serum and Macromolecular Crowding Maintain Native Equine Tenocyte Function in Culture. The absence of a native extracellular matrix and the use of xenogeneic sera are often associated with rapid tenocyte function losses during in vitro culture. Herein, we assessed the influence of different sera (equine serum and foetal bovine serum) on equine tenocyte morphology, viability, metabolic activity, proliferation and protein synthesis as a function of tissue-specific extracellular matrix deposition (induced via macromolecular crowding), aging (passages 3, 6, 9) and time in culture (days 3, 5, 7). In comparison to cells at passage 3, at day 3, in foetal bovine serum and without macrom...
The role of bicarbonate in the modulation of capacitation, spontaneous acrosome reaction and motility of equine fresh and frozen spermatozoa. In this study, we defined the composition of the culture medium that yield a significant percentage of alive and functional equine spermatozoa during enough time before artificial insemination. The effects of sodium bicarbonate were analyzed in fresh and frozen semen in respect to sperm viability, capacitation, spontaneous acrosome reaction and several kinetic parameters such as total motility, progressive motility, VCL, VSL, ALH, BCF, LIN. Moreover, employing Bayk-6844 and Nifedipine, the involvement of L-type voltage-gated calcium channels in the modulation of intracellular calcium concentra...
Ivermectin-induced gene expression changes in adult Parascaris univalens and Caenorhabditis elegans: a comparative approach to study anthelminthic metabolism and resistance in vitro. The nematode Parascaris univalens is one of the most prevalent parasitic pathogens infecting horses but anthelmintic resistance undermines treatment approaches. The molecular mechanisms underlying drug activity and resistance remain poorly understood in this parasite since experimental in vitro models are lacking. The aim of this study was to evaluate the use of Caenorhabditis elegans as a model for P. univalens drug metabolism/resistance studies by a comparative gene expression approach after in vitro exposure to the anthelmintic drug ivermectin (IVM). Methods: Twelve adult P. univalens worms...
Novel Quantitative PCR for Rhodococcus equi and Macrolide Resistance Detection in Equine Respiratory Samples. is an important veterinary pathogen that takes the lives of many foals every year. With the emergence and spread of MDR to current antimicrobial treatment, new tools that can provide a fast and accurate diagnosis of the disease and antimicrobial resistance profile are needed. Here, we have developed and analytically validated a multiplex qPCR for the simultaneous detection of and related macrolide resistance genes in equine respiratory samples. The three sets of oligos designed in this study to identify housekeeping gene and macrolide resistance genes (46) and (51) showed high analytic se...
Assessing the impact of USER-treatment on hyRAD capture applied to ancient DNA. Ancient DNA preservation in subfossil specimens provides a unique opportunity to retrieve genetic information from the past. As ancient DNA extracts are generally dominated by molecules originating from environmental microbes, capture techniques are often used to economically retrieve orthologous sequence data at the population scale. Post-mortem DNA damage, especially the deamination of cytosine residues into uracils, also considerably inflates sequence error rates unless ancient DNA extracts are treated with the USER enzymatic mix prior to library construction. While both approaches have rec...
The potential of three whole blood microRNAs to predict outcome and monitor treatment response in sarcoid-bearing equids. MicroRNAs (miRNAs) have been proposed as biomarkers for equine sarcoid (ES) disease. In this study, the suitability of three whole blood miRNAs to diagnose ES and to predict and monitor the outcome of therapy was explored. Using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), expression levels of eca-miR-127, eca-miR-379, and eca-miR-432 in whole blood of ES-affected equids before and at least one year after therapy were compared to those of unaffected control equids. Associations of age, sex, species, diagnosis, and therapy outcome with miRNA expression levels were exa...
Quantitative analysis of the serum proteome during early pregnancy in mares. Equine pregnancy is currently diagnosed by rectal palpation, ultrasonographic examination, or by measuring changes in hormones in the blood. In the present study, we identified proteins that are differentially expressed in the sera of early pregnant and non-pregnant mares in order to develop a novel method for diagnosing equine pregnancy. Serum samples were obtained from 18 adult mares, pregnancy at day 32 after ovulation (n = 9) and in diestrus (n = 9). Proteomic analysis of the samples was conducted using liquid chromatography-electrospray ionization-tandem mass spectrometry. We iden...
Laboratory diagnosis of Clostridioides (Clostridium) difficile infection in domestic animals: A short review. Despite the known importance of Clostridioides (Clostridium) difficile infection (CDI) in animals, there are no published guidelines for the diagnosis of CDI. The performance of the available commercial methods, all standardized for human stool samples, can vary according to the animal species. Thus, the aim of the present study was to review the literature on the detection of C. difficile in pigs, horses, and dogs. The detection of toxins A and B using enzyme immunoassays seems to have low performance in piglet and dog samples, while it shows high sensitivity for the diagnosis of CDI in foal...
The effect of pergolide mesylate on adrenocorticotrophic hormone responses to exogenous thyrotropin releasing hormone in horses. Thyrotropin releasing hormone (TRH) stimulation testing is often used to support a diagnosis of pituitary pars intermedia dysfunction (PPID) in horses although it is unclear whether or not repeat TRH stimulation testing post-treatment is a valid means of assessing response to medical therapy. Laboratory submissions from 64 suspected equine PPID cases were examined including the initial pre-treatment TRH stimulation test and a follow up test within 100 days of starting medical therapy with pergolide. In a subset of cases, further follow-up tests were examined beyond 100 days of starting treatme...
A quantitative method for the simultaneous detection of SR9009 and SR9011 in equine plasma using liquid chromatography-electrospray ionization-tandem mass spectrometry. SR9009 and SR9011 are metabolic modulators pharmacologically targeting REV-ERB receptors as synthetic agonists. A liquid chromatography-tandem mass spectrometry method for the detection of SR9009 and SR9011 in equine plasma was developed and validated. Plasma samples were pretreated by protein precipitation with methanol and were loaded onto an ACQUITY ultra performance liquid chromatography high-strength silica C18 column (2.1 × 150 mm, 1.8 μm) for chromatographic separation. The mobile phase consisted of 5-mM ammonium formate (pH 3.0) in distilled water and 0.1% formic acid in ac...
CD154 Expression Indicates T Cell Activation Following Tetanus Toxoid Vaccination of Horses. Despite the relevance of adaptive immunity against equine pathogens antigen-specific T cell responses of horses are not well characterized and the lack of insight into T cell responses hampers the understanding of the pathogeneses of important diseases. In this study we used tetanus toxoid (TT) as a well-defined antigen to characterize antigen-reactive T cells. Six healthy adult horses received a routine booster against tetanus with an immune stimulating complex (ISCOM)-based vaccine and were followed for 28 days. TT-specific serum antibodies were quantified by ELISA and increased in all horse...
Rhodococcus equiU19 strain harbors a nonmobilizable virulence plasmid. Rhodococcus equiis the causative agent of pyogenic pneumonia in foals, and a virulence-associated protein A (VapA) encoded on the pVAPA virulence plasmid is important for its pathogenicity. In this study, we analyzed the virulence of R. equi strain U19, originally isolated in the Netherlands in 1997 and the genetic characteristics of the pVAPA_U19 plasmid. U19 expressed VapA that was regulated by temperature and pH and underwent significant intracellular proliferation in macrophages. The restriction fragment length polymorphism of pVAPA_U19 digested with EcoRI was similar to that of pREAT701 (...
Comparison between the techniques of inclusion in glycol methacrylate (GMA)-based plastic resin and paraffin for evaluation intestinal morphometry in horses. To determine the effect of the inclusion method on the histomorphometric evaluation of the gastrointestinal mucosa of horses, jejunum samples were collected using flank laparotomy. Sixteen mixed breed healthy adult horses, including four males and 12 females, aged 4-14 years with an average body weight of 248.40 ± 2.28 kg, were used. Jejunal biopsies were collected and analyzed by light microscopy using two methods: group 1 comprised biopsies fixed using 10% neutral formalin and embedded in paraffin; biopsies in group 2 were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer pH 7.2, follo...
Effects of cyclooxygenase and soluble epoxide hydrolase inhibitors on apoptosis of cultured primary equine chondrocytes. Apoptosis is an important mechanism underlying chondrocyte loss in osteoarthritis that could be affected by modulation of lipid signaling via inhibition of cyclooxygenases (COX) and soluble epoxide hydrolase (sEH). Objective: To determine the impact of inhibiting COX and sEH alone or in combination on apoptosis of equine chondrocytes. Methods: Cultured primary equine chondrocytes were subjected to serum deprivation or incubation with 1 μg/ml tunicamycin for 24 h to induce apoptosis via caspase activation and endoplasmic reticulum (ER) stress, respectively. Cells were treated with the non-se...
Molecular Dynamics and Evolution of Centromeres in the Genus Equus. The centromere is the chromosomal locus essential for proper chromosome segregation. While the centromeric function is well conserved and epigenetically specified, centromeric DNA sequences are typically composed of satellite DNA and represent the most rapidly evolving sequences in eukaryotic genomes. The presence of satellite sequences at centromeres hampered the comprehensive molecular analysis of these enigmatic loci. The discovery of functional centromeres completely devoid of satellite repetitions and fixed in some animal and plant species represented a turning point in centromere biology...
Assessment of BPV-1 Mediated Matrix Metalloproteinase Genes Deregulation in the In Vivo and In Vitro Models Designed to Explore Molecular Nature of Equine Sarcoids. Matrix metalloproteinases (s) represent a family of enzymes capable of biocatalytically breaking down the structural and functional proteins responsible for extracellular matrix (ECM) integrity. This capability is widely used in physiological processes; however, imbalanced MMP activity can trigger the onset and progression of various pathological changes, including the neoplasmic transformation of different cell types. We sought to uncover molecular mechanisms underlying alterations in transcriptional profiles of genes coding for s, which were comprehensively identified in equine adult dermal ...
Development of a 17-Plex of Penta- and Tetra-Nucleotide Microsatellites for DNA Profiling and Paternity Testing in Horses. Tetranucleotide and pentanucleotide short tandem repeat (hereafter termed tetraSTR and pentaSTR) polymorphisms have properties that make them desirable for DNA profiling and paternity testing. However, certain species, such as the horse, have far fewer tetraSTRs than other species and for this reason dinucleotide STRs (diSTRs) have become the standard for DNA profiling in horses, despite being less desirable for technical reasons. During our testing of a series of candidate genes as potentially underlying a heritable condition characterized by megaesophagus in the Friesian horse breed, we foun...
Biological Potential of Polyphenols in the Context of Metabolic Syndrome: An Analysis of Studies on Animal Models. Metabolic syndrome (MetS) is a disease that has a complex etiology. It is defined as the co-occurrence of several pathophysiological disorders, including obesity, hyperglycemia, hypertension, and dyslipidemia. MetS is currently a severe problem in the public health care system. As its prevalence increases every year, it is now considered a global problem among adults and young populations. The treatment of choice comprises lifestyle changes based mainly on diet and physical activity. Therefore, researchers have been attempting to discover new substances that could help reduce or even reverse t...
