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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.
A rapid molecular method for diagnosing epidemic dermatophytosis in a racehorse facility. Identification of the species and strain of dermatophyte can play an effective role in control of disease outbreaks by establishing the source of infection. Current methods of identification are based on cultural and microscopic methods, often involving weeks before a positive identification are made. A rapid molecular diagnostic method would therefore be an important laboratory technique, but requires confirmation in equine clinical practice. Objective: To test the sensitivity and specificity of molecular diagnostic methods applied to a racehorse herd from the Korean Racehorse Authority (KRA)...
Evaluation of an in-clinic Serum Amyloid A (SAA) assay and assessment of the effects of storage on SAA samples. An in-clinic assay for equine serum amyloid A (SAA) analysis, Equinostic EVA1, was evaluated for use in a clinical setting. Stability of SAA in serum samples was determined. Methods: Intra- and inter- assay variation of the in-clinic method was determined. The in-clinic method (EVA1) results were compared to a reference method (Eiken LZ SAA) with 62 patient samples. For samples with SAA concentrations within the assay range of EVA1 (10-270 mg/L), differences between the methods were evaluated in a difference plot. Linearity under dilution was evaluated in two samples. Stability of SAA in three...
Reductive nitrosylation of ferric cyanide horse heart myoglobin is limited by cyanide dissociation. Cyanide binds to ferric heme-proteins with a very high affinity, reflecting the very low dissociation rate constant (k(off)). Since no techniques are available to estimate k(off), we report herewith a method to determine k(off) based on the irreversible reductive nitrosylation reaction to trap ferric myoglobin (Mb(III)). The k(off) value for cyanide dissociation from ferric cyanide horse heart myoglobin (Mb(III)-cyanide) was determined at pH 9.2 and 20.0 degrees C. Mixing Mb(III)-cyanide and NO solutions brings about absorption spectral changes reflecting the disappearance of Mb(III)-cyanide w...
Shared Y chromosome repetitive DNA sequences in stallion and donkey as visualized using whole-genomic comparative hybridization. The genome of stallion (Spanish breed) and donkey (Spanish endemic Zamorano-Leonés) were compared using whole comparative genomic in situ hybridization (W-CGH) technique, with special reference to the variability observed in the Y chromosome. Results show that these diverging genomes still share some highly repetitive DNA families localized in pericentromeric regions and, in the particular case of the Y chromosome, a sub-family of highly repeated DNA sequences, greatly expanded in the donkey genome, accounts for a large part of the chromatin in the stallion Y chromosome.
Atypical Dermatophilus congolensis infection in a three-year-old pony. Dermatophilosis caused by Dermatophilus congolensis causes exudative dermatitis in a variety of species. The infection is generally limited to the cutaneous tissues, where infection is acquired from the environment and bacteria penetrate into keratinized epithelium through epithelial disruption. A 3-year-old pony filly was examined for enlarging mandibular lymph nodes during the preceding 10 months. Biopsy of the node revealed mixed and granulomatous inflammation and thick, filamentous Gram-positive bacteria. Dermatophilus congolensis, confirmed by biochemical testing and sequencing of the rib...
Loop-mediated isothermal amplification assays for detection of Equid herpesvirus 1 and 4 and differentiating a gene-deleted candidate vaccine strain from wild-type Equid herpesvirus 1 strains. Loop-mediated isothermal amplification (LAMP) is a novel method for the rapid and sensitive detection of DNA without the need for expensive equipment. In the present study, LAMP assays were developed for the specific detection of Equid herpesvirus 1 and 4 (EHV-1 and EHV-4, respectively) and for the differentiation of glycoprotein E (gE)-deleted EHV-1 (DeltagE) strain, a candidate strain for a live vaccine, from field EHV-1 strains. Specific primer sets were designed for the gC and gE genes of EHV-1 and for the gC gene of EHV-4. The analytical sensitivities of the LAMP assays were compared with...
Effects of α2-adrenergic drugs on small intestinal motility in the horse: an in vitro study. The effects of selective α(2)-agonists (xylazine, detomidine and medetomidine) and antagonists (yohimbine and atipamezole) on in vitro small intestine motility in the horse were evaluated. Samples of equine jejunum were placed in isolated organ baths and drug-induced modifications of motility were measured by means of an isotonic transducer. All tested α(2)-agonists dose-dependently reduced both spontaneous and electrically-evoked phasic contractions. Conversely, α(2)-antagonists were ineffective when tested alone, and showed a heterogeneous and dose-independent ability to inhibit agonist a...
A note on langerhans cells in the oesophagus epithelium of domesticated mammals. Using the zinc-iodide osmium tetroxide (ZIO) method, TEM and immunohistochemistry (for CD1a and langerin), the study demonstrates Langerhans cells in the oesophageal epithelium of domesticated mammals (herbivores: horse, cattle, goat; omnivores: pig, dog, laboratory rat; carnivores: cat), although with variations between the species. The ZIO method and TEM showed this cell type in the cat and, sporadically, in the horse; CD1a (+) Langerhans cells were demonstrated in the ovine, porcine and murine oesophagus. Positive staining for langerin was detected in single cells of the caprine, canine, mu...
Screening of drugs in equine plasma using automated on-line solid-phase extraction coupled with liquid chromatography-tandem mass spectrometry. A rapid liquid chromatography-tandem mass spectrometry (LC-MS-MS) method was developed for the simultaneous screening of 19 drugs of different classes in equine plasma using automated on-line solid-phase extraction (SPE) coupled with a triple quadrupole mass spectrometer. Plasma samples were first protein precipitated using acetonitrile. After centrifugation, the supernatant was directly injected into the on-line SPE system and analysed by a triple quadrupole LC-MS-MS in positive electrospray ionisation (+ESI) mode with selected reaction monitoring (SRM) scan function. On-line extraction and c...
[Ehrlichiosis/Anaplasmosis]. Ehrlichiosis and anaplasmosis are zoonoses caused by bacteria from the family Anaplasmataceae, including human and animal pathogens. The human pathogens are Ehrlichia chaffeensis, the causative agent of human monocytic ehrlichiosis (HME), Anaplasma phagocytophilum, the pathogen causing human granulocytic anaplasmosis (HGA), E. ewingii and Neorickettsia sennetsu, granulocytotropic and monocytotropic Ehrlichia species, respectively. Ehrlichia spp. are small, gram-negative, obligate intracellular bacteria. They replicate in the cytoplasmic vacuoles of host cells, especially granulocytes and monoc...
Cytochrome P450 1A-dependent activities in deer, cattle and horses. The objective of this study was to investigate and characterize the metabolic activities of CYP1A in deer, cattle and horses in comparison to those of rats using ethoxyresorufin O-deethylation (EROD) and methoxyresorufin O-demethylation (MROD) assays. We performed an inhibition study for these activities using anti-rat CYP1A1 antibody and identified that these activities were due to the CYP1A subfamily. Interspecies differences in the CYP1A-dependent activities were highly observed in this study. In particular, we found that the horse had the highest EROD and MROD activities among the examined...
Detection of prohibited animal products in livestock feeds by single-strand conformation polymorphism analysis. Single-strand conformation polymorphism (SSCP) analysis of amplicons produced from a mitochondrial DNA region between the tRNA(Lys) and ATPase8 genes was applied for the detection of animal product within livestock feeds. Identification of prohibited animal (cattle, elk, sheep, deer, and goat) and nonprohibited animal (pig and horse) products from North America was possible based on the differential display of the single-stranded DNA fragments for the different animal species on SSCP gels. This method allowed specific detection and identification of mixed genomic DNA from different animal spec...
Assessing the efficiency of a pharmacokinetic-based algorithm for target-controlled infusion of ketamine in ponies. The objective of this study was to assess a pharmacokinetic algorithm to predict ketamine plasma concentration and drive a target-controlled infusion (TCI) in ponies. Firstly, the algorithm was used to simulate the course of ketamine enantiomers plasma concentrations after the administration of an intravenous bolus in six ponies based on individual pharmacokinetic parameters obtained from a previous experiment. Using the same pharmacokinetic parameters, a TCI of S-ketamine was then performed over 120 min to maintain a concentration of 1 microg/mL in plasma. The actual plasma concentrations of ...
Measurement of plasma cardiac troponin I concentration by use of a point-of-care analyzer in clinically normal horses and horses with experimentally induced cardiac disease. To compare cardiac troponin I (cTnI) concentrations determined by use of a point-of-care analyzer with values determined by use of a bench-top immunoassay in plasma samples obtained from clinically normal horses with and without experimentally induced cardiac disease, and to establish a reference range for plasma equine cTnI concentration determined by use of the point-of-care analyzer. Methods: 83 clinically normal horses, 6 of which were administered monensin to induce cardiac disease. Methods: A blood sample was collected from each of the 83 clinically normal horses to provide plasma for an...
Automated counting of nucleated cells in equine synovial fluid without and with hyaluronidase pretreatment. Microscopy is usually used to obtain manual total and differential cell counts in equine synovial fluid. A faster, more precise method is desirable. Objective: The objectives were to compare an automated impedance method with a manual method for obtaining total and differential cell counts in equine synovial fluid and to evaluate the effect of pretreatment with hyaluronidase on automated results. Methods: Synovial fluid samples (n=48) were collected into EDTA and analyzed within 48 hours. Automated total and differential cell counts were evaluated using a Medonic CA620-VET hematology analyzer ...
Expression of annexin-1 in equine leucocytes and the effects of the N-terminal annexin-1 peptide, Ac2-26, on equine neutrophil superoxide production. N-terminal peptides derived from the anti-inflammatory peptide, annexin-1, inhibit neutrophil function but can also induce pro-inflammatory effects. Although equine annexin-1 has been sequenced, its cellular expression and properties have not been reported. This study has examined whether annexin-1 is present in equine leucocytes and how the N-terminal peptide, Ac2-26, affects equine neutrophil superoxide production. Annexin-1 expression in equine neutrophils and mononuclear cells and the ability of Ac2-26 to activate neutrophil p42/44 MAPK were determined by immunoblotting. Equine neutrophil ...
Induction of interleukin-4 production in neonatal IgE+ cells after crosslinking of maternal IgE. Transfer of maternal IgE antibodies to the neonate with the colostrum has been described in different mammalian species. Previous work in horses has shown that IgE bound to the surface of neonatal basophils is solely of maternal origin. However, the functional role of the maternal IgE transfer remained unclear. We hypothesized that maternal IgE mediates the onset of innate IL-4 production in equine neonatal basophils. Intracellular IL-4 production was measured in PBMC of newborn and older foals by flow cytometric analysis. A small population of IL-4(+) cells was observed in the peripheral bloo...
Identification of horse chestnut coat color genotype using SNaPshot. The Cantabrian Coast horse breeds of the Iberian Peninsula have mainly black or bay colored coats, but alleles responsible for a chestnut coat color run in these breeds and occasionally, chestnut horses are born. Chestnut coat color is caused by two recessive alleles, e and e(a), of the melanocortin-1 receptor gene, whereas the presence of the dominant, wild-type E allele produces black or bay coat horses. Because black or bay colored coats are considered as the purebred phenotype for most of the breeds from this region, it is important to have a fast and reliable method to detect alleles caus...
Development of a blocking ELISA using a recombinant glycoprotein for the detection of antibodies to vesicular stomatitis New Jersey virus. A recombinant glycoprotein (R-GP) of vesicular stomatitis New Jersey virus (VSV-NJ) was expressed in insect cells by a baculovirus system. Its utility as a diagnostic antigen in a blocking ELISA was investigated as an alternative to the current native GP extracted from VSV-NJ. With the cut-off value of 73% inhibition, the R-GP ELISA exhibited 99.1% specificity for naive sera from cattle and horses. It did not cross-react with VSV-Indiana (VSV-IN) positive sera and differentiated from foot-and-mouth disease and swine vesicular disease. Taken together, this is the first report that the R-GP has ...
Selection of reference genes for quantitative real-time PCR in equine in vivo and fresh and frozen-thawed in vitro blastocysts. Application of reverse transcription quantitative real-time polymerase chain reaction is very well suited to reveal differences in gene expression between in vivo and in vitro produced embryos. Ultimately, this may lead to optimized equine assisted reproductive techniques. However, for a correct interpretation of the real-time PCR results, all data must be normalized, which is most reliably achieved by calculating the geometric mean of the most stable reference genes. In this study a set of reliable reference genes was identified for equine in vivo and fresh and frozen-thawed in vitro embryos....
Prevalence of equine adenovirus antibodies in horses in New South Wales, Australia. There are currently two known serotypes of equine adenovirus (EAdV), equine adenovirus type 1 (EAdV1) and equine adenovirus type 2 (EAdV2); EAdV1 is predominantly associated with upper respiratory tract infections while EAdV2 appears to have a higher association with gastrointestinal infection, however, very little is known about the prevalence of these viruses in horse populations in Australia. In this study we tested 122 serum samples obtained from horses in New South Wales, Australia, using a standard serum neutralization (SN) assay and ELISA. Ninety-seven of the 122 sera displayed had mode...
Comparison of serum iohexol clearance and plasma creatinine clearance in clinically normal horses. Objective-To determine whether a limited sampling time method based on serum iohexol clearance (Cl(iohexol)) would yield estimates of glomerular filtration rate (GFR) in clinically normal horses similar to those for plasma creatinine clearance (Cl(creatinine)). Animals-10 clinically normal adult horses. Procedures-A bolus of iohexol (150 mg/kg) was administered IV, and serum samples were obtained 5, 20, 40, 60, 120, 240, and 360 minutes after injection. Urinary clearance of exogenous creatinine was measured during three 20-minute periods. The GFR determined by use of serum Cl(iohexol) and plas...
Indirect immunofluorescence test using polyclonal antibodies for the detection of Taylorella equigenitalis. Contagious equine metritis is a horse disease that causes endometrial inflammation due to Taylorella equigenitalis. Since Taylorella asinigenitalis was characterized, genital swab culture has proved to be an insufficient method for distinguishing between the two Taylorella species. Here, we developed an indirect immunofluorescence (IIF) test using polyclonal antibodies. Specificity, sensitivity, and detection limit were assessed using isolated bacteria (55 T. equigenitalis strains, 46 T. asinigenitalis strains and 18 other bacterial species), experimental and genital swabs in comparison to bac...
A comparison between the 2N and 4N HCl acid-insoluble ash methods for digestibility trials in horses. The digestibility of horse feeds and rations can be determined using different techniques such as calculations based on the chemical composition, in vivo or in vitro methods. The marker methods overcome difficulties like discomfort for the animals and longer experimental times encountered using the ingesta/egesta method. In field conditions, a natural indigestible marker like acid-insoluble ash (AIA), with no changes in the normal ration, could be a very useful tool for digestibility trials. A group of six standardbred horses was used in a set of seven apparent digestibility trials. The diets ...
Characterization of a thymidine kinase-deficient mutant of equine herpesvirus 4 and in vitro susceptibility of the virus to antiviral agents. Equine herpesvirus 4 (EHV-4) is an important equine pathogen that causes respiratory tract disease among horses worldwide. A thymidine kinase (TK)-deletion mutant has been generated by using bacterial artificial chromosome (BAC) technology to investigate the role of TK in pathogenesis. Deletion of TK had virtually no effect on the growth characteristics of WA79DeltaTK in cell culture when compared to the parent virus. Also, virus titers and plaque formation were unaffected in the absence of the TK gene. The sensitivity of EHV-4 to inhibition by acyclovir (ACV) and ganciclovir (GCV) was studied...
Detection of treponemes in canker lesions of horses by 16S rRNA clonal sequencing analysis. Equine canker is a chronic pododermatitis of the hoof in horses. Although spirochetes are detectable histopathologically in the lesions, the precise etiology remains unclear. This study reports the 16S rRNA gene sequencing of randomly selected clones based on PCR with Treponema-specific primers, using the canker lesions from two horses and healthy frog and sole from a horse. A total of 114 clones were obtained from the lesions, but no clones were detected in the healthy hoof tissues. The clones from the canker lesions examined were grouped into 19 operational taxonomic units, such as treponema...
Development of a novel equine whole transcript oligonucleotide GeneChip microarray and its use in gene expression profiling of normal articular-epiphyseal cartilage. No large scale equine microarray is available commercially to allow genomic and transcriptional profiling of the majority of genes that would define the genetic basis of equine disease. Objective: To generate a whole transcript target labelled GeneChip to interrogate the equine transcriptome and validate chip performance using RNA samples derived from organs, articular cells and normal cartilage. Methods: Equine mRNA and selected equine gene sequences derived from perfect cross-hybridisation of equine RNA on human microarray GeneChips, were used to design a custom equine gene microarray. Seque...
Establishing a reproducible method for the culture of primary equine corneal cells. To establish a reproducible method for the culture of primary equine corneal epithelial cells, keratocytes, and endothelial cells and to describe each cell's morphologic characteristics, immunocytochemical staining properties and conditions required for cryopreservation. Methods: Corneas from eight horses recently euthanized for reasons unrelated to this study were collected aseptically and enzymatically separated into three individual layers for cell isolation. The cells were plated, grown in culture, and continued for several passages. Each cell type was characterized by morphology and immun...
The distal pocket histidine residue in horse heart myoglobin directs the O-binding mode of nitrite to the heme iron. It is now well-established that mammalian heme proteins are reactive with various nitrogen oxide species and that these reactions may play significant roles in mammalian physiology. For example, the ferrous heme protein myoglobin (Mb) has been shown to reduce nitrite (NO(2)(-)) to nitric oxide (NO) under hypoxic conditions. We demonstrate here that the distal pocket histidine residue (His64) of horse heart metMb(III) (i.e., ferric Mb(III)) has marked effects on the mode of nitrite ion coordination to the iron center. X-ray crystal structures were determined for the mutant proteins metMb(III) H...
Cationic and neutral amino acid transporter transcript abundances are differentially expressed in the equine intestinal tract. To test the hypothesis that AA transporter transcripts are present in the large intestine and similarly expressed along the intestinal tract, mRNA abundance of candidate AA transporter genes solute carrier (SLC) family 7, member 9 (SLC7A9), SLC7A1, SLC7A8, and SLC43A1 encoding for b(0,+)-type AA transporter (b(0,+)AT), cationic AA transporter-1 (CAT-1), L-type AA transporter-2 (LAT-2), and L-type AA transporter-3 (LAT-3), respectively, was determined in small and large intestinal segments of the horse. Mucosa was collected from the equine small (jejunum and ileum) and large intestine (cecum, l...
