Detection of transgenes in equine dried blood spots using digital PCR and qPCR for gene doping control.

Due to the ease of collection, transport and storage, the use of dried blood spots (DBS) offers an attractive alternative matrix for detection of the abuse of gene therapy, otherwise known as gene doping. This study evaluated the recovery, extraction efficiency and resulting detection capability of DNA from DBS by evaluating different target types, DNA extraction kits, the number of punches and blood tube preservatives. The long-term storage stability of low-copy-number transgene targets in DBS was not assessed in this study but would be noteworthy to investigate further.

Detection of adeno-associated viral DNA in equine post-administration frozen blood and plasma samples after long-term storage.

Gene doping in horses is a threat to the fairness in sport and has serious implications for animal welfare. To investigate the effect of long-term storage on the detection of AAV in plasma and whole blood, samples from an administration study using an adeno-associated virus serotype 6 expressing green fluorescence protein (AAV6-GFP) were stored at -20°C for 8 months before analysis. The AAV vector was detected in stored plasma samples, following the same detection profile as the fresh plasma samples.

Long-read sequencing assays designed to detect potential gene editing events in the myostatin gene revealed distinct haplotype signatures in the Thoroughbred horse population.

We present here the use of targeted, long-read sequencing of the myostatin (MSTN) gene as a model to detect potential gene editing events in Thoroughbred horses. MSTN is a negative regulator of muscle development, making the gene a prime candidate target for gene doping. By sequencing the complete gene in one PCR product, we can catalogue all mutations without the need to produce short-fragment libraries.

Use of mitochondrial sequencing to detect gene doping in horses via gene editing and somatic cell nuclear transfer.

Gene editing and subsequent cloning techniques offer great potential not only in genetic disease correction in domestic animals but also in livestock production by enhancement of desirable traits. The existence of the technology, however, leaves it open to potential misuse in performance-led sports such as horseracing and other equestrian events. Recent advances in equine gene editing, regarding the generation of gene-edited embryos using CRISPR/Cas9 technology and somatic cell nuclear transfer, have highlighted the need to develop tools to detect potential prohibited use of the technology.

Metagenomic investigation of the equine faecal microbiome reveals extensive taxonomic diversity.

Background: The horse plays crucial roles across the globe, including in horseracing, as a working and companion animal and as a food animal. The horse hindgut microbiome makes a key contribution in turning a high fibre diet into body mass and horsepower. However, despite its importance, the horse hindgut microbiome remains largely undefined.

Direct sequence confirmation of qPCR products for gene doping assay validation in horses.

The misuse of gene therapy by the introduction of transgenes via plasmid or viral vectors as a doping agent is an increasing concern in human and animal sports, not only in consideration to fair competition but also in potential detrimental effects to welfare. Doping events can be detected by polymerase chain reaction (PCR) amplification of a transgene-specific region of DNA. Quantitative real-time PCR (qPCR) is particularly suited to confirmatory investigations where precise limits of detection can be calculated.

Designing a field trial of an equine grass sickness vaccine: A questionnaire-based feasibility study.

Without an experimental model of equine grass sickness (EGS), a randomised controlled field trial (RCT) represents the only method of evaluating the efficacy of Clostridium botulinum type C vaccination in preventing naturally occurring disease. Clinical trial feasibility is an important aspect of preliminary work undertaken prior to initiating RCTs, estimating parameters that are important for study design. This cross-sectional study aimed to assess the feasibility of conducting a nationwide RCT of a candidate vaccine for EGS based on responses from a sample of British equine veterinary practices (n = 119/284).

Effects of preoperative administration of hypertonic saline or pentastarch solution on hematologic variables and long-term survival of surgically managed horses with colic.

Objective: To compare the effects of preoperatively administered pentastarch (10% concentration in isotonic saline [0.9% NaCl] solution) and hypertonic saline (7.2% NaCl) solutions on PCV and circulating total protein (TP) concentration in horses with colic undergoing emergency exploratory laparotomy and to assess survival rates of horses that received each treatment.

Expression of sweet receptor components in equine small intestine: relevance to intestinal glucose transport.

The heteromeric sweet taste receptor T1R2-T1R3 is expressed on the luminal membrane of certain populations of enteroendocrine cells. Sensing of sugars and other sweet compounds by this receptor activates a pathway in enteroendocrine cells, resulting in secretion of a number of gut hormones, including glucagon-like peptide 2 (GLP-2). This subsequently leads to upregulation in the expression of intestinal Na(+)/glucose cotransporter, SGLT1, and increased intestinal glucose absorption.

Alterations in microbiota and fermentation products in equine large intestine in response to dietary variation and intestinal disease.

We aimed to determine the effects of variations in dietary composition on equine gut microbiota and their fermentation products, and proposed that dietary modifications profoundly affect microbial ecosystems and their metabolites. Bacterial communities within the large intestine of three groups of horses were compared using oligonucleotide-RNA hybridisation methodology. Each group consisting of six horses was maintained on (1) a grass-only diet, (2) a concentrate diet (i.

Case control study to investigate risk factors for impaction colic in donkeys in the UK.

Impaction colic is the single most common type of colic diagnosed in a large population of donkeys (more than 2000 animals) at The Donkey Sanctuary, UK and the fatality rate from the disease is high. Here we identify risk factors for impaction colic in this population during 2006 using an unmatched case control study. There were 71 cases of impaction colic and multivariable analysis identified a number of variables associated with the disease.

Comparative analysis of c-kit gene expression and c-Kit immunoreactivity in horses with and without obstructive intestinal disease.

Previous immunohistochemical studies targeting the receptor tyrosine kinase (c-Kit) have demonstrated an apparent reduction in the number of gastrointestinal pacemaker cells--the interstitial cells of Cajal (ICC)--in horses with intestinal motility disorders. This study compared the level of transcription of the c-kit gene encoding this receptor in horses with and without such motility disorders. Transcription levels of this gene were also compared to the density of ICC immunohistochemically positive for the c-Kit antigen.

Adaptive response of equine intestinal Na+/glucose co-transporter (SGLT1) to an increase in dietary soluble carbohydrate.

Experimental and epidemiological evidence suggests that consumption of hydrolyzable carbohydrate, hCHO (grain), by horses is an important risk factor for colic, a common cause of equine mortality. It is unknown whether the small intestinal capacity to digest hCHO and/or to absorb monosaccharides is limiting, or even if horses can adapt to increased carbohydrate load. We investigated changes in the brush-border membrane carbohydrate digestive enzymes and glucose absorptive capacity of horse small intestine in response to increased hCHO.

A comparison of coprological, serological and molecular methods for the diagnosis of horse infection with Anoplocephala perfoliata (Cestoda, Cyclophyllidea).

Anoplocephala perfoliata (Cestoda, Cyclophyllidea), the commonest intestinal tapeworm of horses, can cause colic, intussusceptions, ileal impactions and intestinal perforations. Common diagnostic techniques for A. perfoliata infection, i.

Intra-abdominal adhesions in horses: a retrospective evaluation of repeat laparotomy in 99 horses with acute gastrointestinal disease.

The objectives of this study were (1) to determine the prevalence of pathological abdominal adhesion formation following exploratory laparotomy; (2) to establish the site of adhesion formation and its relationship to the initial lesion; (3) to ascertain whether the development of intra-abdominal adhesions decreases long-term survival and (4) to identify risk factors for adhesion formation. Of 1014 horses treated surgically for acute gastrointestinal disease, 113 (10.1%) were subjected to repeat laparotomy, with surgical records available for 99 of these cases.

The effect of inotropic and/or vasopressor support on postoperative survival following equine colic surgery.

Objective: To determine the effects of dobutamine and phenylephrine on intra- and postoperative survival in horses undergoing emergency abdominal surgery.

Study Design: Retrospective case analysis.

Animals: A total of 637 client-owned horses undergoing colic surgery.

Epidemiology of impaction colic in donkeys in the UK.

Background: Colic (abdominal pain) is a clinical condition of serious concern affecting the welfare and survival of donkeys at the Donkey Sanctuary in the UK. One of the most commonly reported causes is due to impacted ingesta in the large intestine ("impaction colic"). However little is known about the incidence of, or risk factors for, this condition.

Is equine colic seasonal? Novel application of a model based approach.

Background: Colic is an important cause of mortality and morbidity in domesticated horses yet many questions about this condition remain to be answered. One such question is: does season have an effect on the occurrence of colic? Time-series analysis provides a rigorous statistical approach to this question but until now, to our knowledge, it has not been used in this context. Traditional time-series modelling approaches have limited applicability in the case of relatively rare diseases, such as specific types of equine colic.

Obstruction of equine small intestine associated with focal idiopathic eosinophilic enteritis: an emerging disease?

Idiopathic focal eosinophilic enteritis (IFEE) is a rare disease in the horse and few cases have been reported in the literature. The objective of this paper was to describe the clinical, surgical, histological features and post-operative progress of 12 horses with IFEE, a disease that had not been identified in the authors' hospital population prior to 2000. Diagnosis of IFEE was made at laparotomy and confirmed by histological examination of resected abnormal small intestine.

Recent developments in research into the Cyathostominae and Anoplocephala perfoliata.

Intestinal helminths are an important cause of equine disease. Of these parasites, the Cyathostominae are the commonest group that infect horses. These nematodes consist of a complex tribe of 51 species, although individual horses tend to harbour 10 or so common species, in addition to a few rarer species.

Equine welfare: risk of horses falling in the Grand National.

As in other competitive sports, the famous Grand National steeplechase, which is held at Aintree in the United Kingdom and is watched by 600 million people worldwide, sometimes results in injury. By analysing data from the past 15 Grand National races (consisting of 560 starts by horses), we are able to identify several factors that are significantly associated with failure to complete the race: no previous experience of the course and its unique obstacles, unfavourable ground conditions (too soft or too hard), a large number of runners, and the length of the odds ('starting price'). We also find that there is an increased risk of falling at the first fence and at the jump known as Becher's Brook, which has a ditch on the landing side.

Association between cribbing and entrapment of the small intestine in the epiploic foramen in horses: 68 cases (1991-2002).

Objective: To determine whether there was an association between a history of cribbing and epiploic foramen entrapment (EFE) of the small intestine in horses.

Design: Retrospective study.

Animals: 68 horses examined at the University of Illinois or the University of Liverpool veterinary teaching hospitals.

Characterisation of IgG(T) serum antibody responses to two larval antigen complexes in horses naturally- or experimentally-infected with cyathostomins.

Cyathostomins are the most common parasitic nematodes of horses. Larval stages, which inhabit the intestinal wall, are particularly pathogenic and can cause severe colitis and colic. Despite their clinical importance, diagnostic techniques for the prepatent stages do not exist.