Analysis of genomic copy number variation in equine recurrent airway obstruction (heaves).

We explored the involvement of genomic copy number variants (CNVs) in susceptibility to recurrent airway obstruction (RAO), or heaves-an asthmalike inflammatory disease in horses. Analysis of 16 RAO-susceptible (cases) and six RAO-resistant (control) horses on a custom-made whole-genome 400K equine tiling array identified 245 CNV regions (CNVRs), 197 previously known and 48 new, distributed on all horse autosomes and the X chromosome. Among the new CNVRs, 30 were exclusively found in RAO cases and were further analyzed by quantitative PCR, including additional cases and controls. Suggestive association (P = 0.03; corrected P = 0.06) was found between RAO and a loss on chromosome 5 involving NME7, a gene necessary for ciliary functions in lungs and involved in primary ciliary dyskinesia in humans. The CNVR could be a potential marker for RAO susceptibility but needs further study in additional RAO cohorts. Other CNVRs were not associated with RAO, although several involved genes of interest, such as SPI2/SERPINA1 from the serpin gene family, which are associated with chronic obstructive pulmonary disease and asthma in humans. The SPI2/SERPINA1 CNVR showed striking variation among horses, but it was not significantly different between RAO cases and controls. The findings provide baseline information on the relationship between CNVs and RAO susceptibility. Discovery of new CNVs and the use of a larger population of RAO-affected and control horses are needed to shed more light on their significance in modulating this complex and heterogeneous disease.

Functional modelling of an equine bronchoalveolar lavage fluid proteome provides experimental confirmation and functional annotation of equine genome sequences.

The equine genome sequence enables the use of high-throughput genomic technologies in equine research, but accurate identification of expressed gene products and interpreting their biological relevance require additional structural and functional genome annotation. Here, we employ the equine genome sequence to identify predicted and known proteins using proteomics and model these proteins into biological pathways, identifying 582 proteins in normal cell-free equine bronchoalveolar lavage fluid (BALF). We improved structural and functional annotation by directly confirming the in vivo expression of 558 (96%) proteins, which were computationally predicted previously, and adding Gene Ontology (GO) annotations for 174 proteins, 108 of which lacked functional annotation. Bronchoalveolar lavage is commonly used to investigate equine respiratory disease, leading us to model the associated proteome and its biological functions. Modelling of protein functions using Ingenuity Pathway Analysis identified carbohydrate metabolism, cell-to-cell signalling, cellular function, inflammatory response, organ morphology, lipid metabolism and cellular movement as key biological processes in normal equine BALF. Comparative modelling of protein functions in normal cell-free bronchoalveolar lavage proteomes from horse, human, and mouse, performed by grouping GO terms sharing common ancestor terms, confirms conservation of functions across species. Ninety-one of 92 human GO categories and 105 of 109 mouse GO categories were conserved in the horse. Our approach confirms the utility of the equine genome sequence to characterize protein networks without antibodies or mRNA quantification, highlights the need for continued structural and functional annotation of the equine genome and provides a framework for equine researchers to aid in the annotation effort.

A cryogenic clamping technique that facilitates ultimate tensile strength determinations in tendons and ligaments.

OBJECTIVE: To describe the use of a cryogenic clamp of novel design for tensile strength testing of tendinous and ligamentous tissues with inherently high tensile strength. METHODS: Inexpensive, easily machined steel clamps were manufactured to facilitate rapid insertion into a standard wedge-screw grip apparatus installed on a testing system with a control system attached. The deep digital flexor tendon (DDFT) of six horses was trimmed to a uniform dumbbell shape and secured in clamps using partial submersion in liquid nitrogen for approximately 45 seconds and immediately tested. Approximate time between removal from liquid nitrogen and failure of tendon was four minutes. RESULTS: Failure was achieved in all tendons tested in a region approximating a midpoint between the clamps. Ultimate failure loads of up to 6745 N were achieved without slippage of the tissue from the grips. The ultimate tensile strength of the normal equine DDFT determined in this study was 111.82 ± 11.53 N/mm2, and the stress versus grip-to-grip elongation plots for our equine DDFT were representative of a standard non-linear elastic curve obtained in similar studies. CLINICAL SIGNIFICANCE: We present a low cost device for quantifying physical properties of specimens with high connective tissue concentrations and inherent high tensile strength. Results of this study indicate that this device provides a practical alternative to other more costly methods of adequately securing larger tendons and ligaments for tensile strength testing.

Quantitative measurement of equine cytokine mRNA expression by polymerase chain reaction using target-specific standard curves.

Quantification of cytokine mRNA using reverse transcription coupled with the polymerase chain reaction (RT-PCR) has become a corner stone of the study of cytokine regulation. Quantitative competitive RT-PCR (QCRT-PCR) is commonly accepted as a reliable method for quantifying differences in mRNA levels but is both labor- and reagent-intensive. A noncompetitive polymerase chain reaction method that utilizes cytokine-specific, plasmid-derived, standard curves was developed for the quantification of equine cytokine mRNA. The assay can be performed on minute samples of cellular material, utilizes sequences identical to wild-type for the generation of standard curves, is technically facile, less reagent-and labor-intensive than competitive methods, easily accommodates high sample throughput without the use of radioactive labels, and generates replicate samples to allow statistical analysis of the data. We demonstrate the utility of the assay, which is easily adapted to any cloned mRNA sequence, using equine interleukin-10 (IL-10). Both IL-10 and beta-actin cDNA were amplified in triplicate PCR reactions from oligo-dT primed RT reactions. Dilutions of plasmid DNA encoding the respective sequence, equine IL-10 or beta-actin, were also amplified in triplicate reactions in the same run. Beta-actin cycling parameters were modified to maintain the amplification in its exponential phase by decreasing both cycle number and cDNA volume relative to the parameters used for cytokine amplification. Following amplification, aliquots of the PCR reactions were hybridized with sequence-specific tris (2,2'-bipyridine) ruthenium II chelate labeled oligonucleotide probes and quantified using the QPCR System 5000. Plasmid derived values were used to generate a standard curve for the interpolation of mRNA content in unknown cDNA samples. Beta-actin values were used to derive a factor for the relative normalization of differences among cDNA samples that are inherent in the RNA extraction and RT steps. This assay resolves at least 2-fold differences in message, is reproducible, and has a dynamic range on the order of 3 logs.

Molecular cloning and sequencing of equine interleukin 4.

We have cloned equine interleukin 4 (IL-4) cDNA using the polymerase chain reaction (PCR) and primers based on the human IL-4 sequence. The cDNA was amplified from mitogen-stimulated equine peripheral blood mononuclear cells (PBMC). The cloned PCR product shares extensive homology ith IL-4 sequences from other species.

Molecular cloning, sequencing, and expression of equine interleukin-6.

Equine interleukin-6 (IL-6) cDNA was amplified from mitogen-stimulated equine peripheral blood mononuclear cells (PBMC) using consensus sequence primers. The 727bp amplified cDNA contains the entire coding region for equine IL-6 and includes 118 bases in the 3' non-translated region. The coding sequence translates to a protein of 208 amino acids with a predicted 28 amino acid leader sequence. The mature protein of 180 amino acids has a predicted molecular mass of 20471Da without post-translational modifications. The amino acid sequence of equine IL-6 displays between 46 and 84% similarity to other mammalian IL-6 sequences. Expression of equine IL-6 in Chinese hamster ovary (CHO) cells yielded a supernatant that supported the proliferation of B9 cells in a dose-dependent manner. Treatment of B9 cells with an anti-IL-6 receptor antibody ablated the response to the recombinant equine IL-6.

Seminal vesiculitis as a cause of signs of colic in a stallion.

A 5-year-old stallion was referred because of signs of abdominal pain. During the initial examination, signs of pain were elicited when the right seminal vesicle was palpated per rectum. Signs of pain were also elicited during sexual arousal and attempts at semen collection. The right seminal vesicle was subsequently determined to be abnormal by ultrasonographic and endoscopic examination. The stallion was treated with trimethoprim/sulfamethoxazole for 6 weeks. Five months later, there had been no recurrence of the condition.

Tensile properties in collagen-rich tissues of Quarter Horses with hereditary equine regional dermal asthenia (HERDA).

REASONS FOR PERFORMING STUDY: Hereditary equine regional dermal asthenia (HERDA) is an autosomal recessive disorder of Quarter Horses characterised by skin fragility. Horses with HERDA have a missense mutation in peptidyl-prolyl cis-trans isomerase B (PPIB), which encodes cyclophilin B and alters folding and post translational modifications of fibrillar collagen. OBJECTIVES: The study aimed to test the hypothesis that tendons, ligaments and great vessels, which, like skin, are rich in fibrillar collagen, will also have abnormal biomechanical properties in horses with HERDA. STUDY DESIGN: Ex vivo biomechanical study comparing horses with and without a diagnosis of HERDA. METHODS: Forelimb suspensory ligament, superficial and deep digital flexor tendons; withers, forelimb and abdominal skin; the main pulmonary artery and the aortic arch were harvested from 6 horses with HERDA and 6 control horses without the HERDA allele. Tissues were distracted to failure. Tensile strength (TS), elastic modulus (EM) and energy to failure (ETF) were compared. RESULTS: Horses with HERDA had significantly lower TS and EM in tendinoligamentous tissues and great vessels, respectively. The TS, EM and ETF were significantly lower in skin from horses with HERDA. Differences in TS and ETF were more extreme at the withers than at the forelimb or abdomen. CONCLUSIONS: Tendinoligamentous tissue, great vessels and skin are significantly weaker in horses with HERDA than in horses lacking the PPIB mutation, substantiating that diverse tissues with high fibrillar collagen content are abnormal in HERDA and that the HERDA phenotype is not limited to the integument.

The equine cervical spine: comparing MRI and contrast-enhanced CT images with anatomic slices in the sagittal, dorsal, and transverse plane.

BACKGROUND: The impact of cervical pathology on performance is of great importance to the horse industry. Accurate diagnosis of cervical disease with imaging modalities, including computed tomography (CT) and magnetic resonance imaging (MRI), requires thorough appreciation of normal cervical anatomy. OBJECTIVES: (1) To describe in detail the anatomy of the equine cervical spine by comparing anatomical sections with corresponding MR and contrast-enhanced CT images in the sagittal, dorsal, and transverse plane. (2) To discuss the ability of MR and contrast-enhanced CT imaging to visualize anatomical structures in the cervical spine. ANIMALS AND METHODS: Three cervical spines of young adults (3-8 years), collected immediately after humane euthanasia, were used. The spine was stabilized on a frame in a natural flexed position with an angle of 20°. MR and contrast-enhanced CT imaging was performed within six hours after euthanasia. Anatomical sections of 1 cm were made in the sagittal, dorsal, and transverse plane and compared with corresponding CT and MR images. The intervertebral disk thickness, facet joint angle, sagittal dural space diameter and ventromedial facet joint projection were quantified. RESULTS: The anatomic location of clinically important structures including the facet joints, spinal cord, cervical nerve roots and intervertebral disks were reliably identified in the anatomical sections and their corresponding MR images. Contrast-enhanced CT images depicted all osseous borders, whereas MR images were superior for soft tissue structures. CONCLUSION AND CLINICAL IMPORTANCE: This study enhances our understanding of normal cervical spine anatomy and the diagnostic usefulness of cervical MRI and contrast-enhanced CT in the horse.

Hypersensitivity disorders in horses.

Hypersensitivity is an exaggerated immunologic response to a foreign agent that results in inflammation and organ dysfunction. Hypersensitivity disorders are broadly divided into antibody-mediated and T-cell-mediated reactions. The inflammatory pathways that result in disease are initiated in an antigen-specific manner through Fab portions of antibodies or the T-cell receptor, causing the up-regulation of effector mechanisms designed to clear the offending agent. Effector mechanisms include the generation of inflammatory chemicals such as cytokines and chemokines and the attraction of leukocytes and potentiation of their function. This article reviews current concepts in the immunopathogenesis of hypersensitivity disorders and demonstrates these mechanisms as they apply to equine disease.

Immunodiagnostic assays.

The immune system is a complex interactive network. Defects in its function can be characterized broadly as being the result of actual deficiencies in the network or misdirection of normal immunologic functions. The assays that are available to detect deficiencies in the immunologic network barely scrape the surface of the possibilities. These assays primarily evaluate humoral immune function, but undetected defects in innate and cellular immunity are sure to exist. Although assays of humoral immunity have allowed the characterization of a number of immunodeficiency syndromes in horses, closer evaluation of the equine immune system using newer assays described in this text, as well as future assays yet to be developed are sure to determine new syndromes. Assays of misdirected immunologic functions have been limited to detection of misdirected antibody responses, but the dependence of antibody production on help from T cells could reflect an underlying defect of cellular immunity. Similar to immunodeficiency syndromes, misdirected responses of the innate and adaptive arms of immunity are sure to occur but will only be detected by more diligent surveillance of diseased horses and application of new immunodiagnostic technologies.