Flow cytometry and its applications in veterinary medicine

Flow cytometry is a technique for analysing and separating populations of cells (and subcellular components). The cells are generally stained with fluorescent markers (eg, fluorescent antibodies or DNA-binding dyes). Each cell is analysed individually, at high speed. Thus, assays may be performed on small samples (less than 10,000 cells). A subpopulation of interest can be separated from the remainder of the cells to a high degree of purity (up to 99 per cent). The application of flow cytometry to veterinary science is increasing and is discussed here.     

The role of flow cytometry in companion animal diagnostic medicine

Flow cytometry is a powerful tool for characterising the composition of complex cell populations. The accuracy and precision of this technology for describing and enumerating cells exceeds traditional methods. The number of diagnostic veterinary laboratories with access to a dedicated machine is increasing, and there is the potential to offer a clinical flow cytometry service. The improved availability of monoclonal antibodies (mAb) to cell markers expressed by the leukocytes of companion animals, permits the implementation of comprehensive mAb panels suitable for diagnosis of lympho- and myeloproliferative disease. Reticulated erythrocyte and platelet quantification, antiglobulin assays for immune-mediated cytopenias, lymphocyte subset analysis, and immunophenotyping of lymphoma and leukemia, have been validated for companion animal samples on the flow cytometer. It is now timely to consider the role of flow cytometry in diagnostic practice, and the requirement for quality assurance and standardization of testing procedures.     

Inheritance of equine sarcoid disease in Franches-Montagnes horses

The mode of inheritance for susceptibility to equine sarcoid disease (ES) remains unknown. The objectives of this study were to analyse a large sample of the Franches-Montagnes (FM) horse population and investigate the heritability and mode of inheritance for susceptibility to ES. Horses were clinically examined for the presence of sarcoid tumours. A standardized examination protocol and client questionnaire were used and a pedigree- and subsequent segregation-analysis for the ES trait performed. To investigate the mode of inheritance, five models were evaluated and compared in a hierarchical way.The analyses reveal that variation in susceptibility to ES is best explained by a model incorporating polygenic variation. The possible effect of a major gene, such as specific equine leukocyte antigen alleles, is unlikely, but cannot be ruled-out entirely. The heritability of the phenotype on the observation scale for the trait ‘affected with ES’ was estimated to be 8%. A corrected value for the heritability on a liability scale was estimated at 21% and it is therefore possible to estimate breeding values for ES. The arguments against the practical implementation of an estimated breeding value in a multifactorial condition like ES are discussed.     

Transvenous electrical cardioversion of equine atrial fibrillation: patient factors and clinical results in 72 treatment episodes

BACKGROUND: Transvenous electrical cardioversion (TVEC) has been developed for treatment of atrial fibrillation (AF) in horses. The relationship among patient variables, treatment response, and outcome in a typical referral population has not been evaluated. HYPOTHESIS: Patient variables such as age, sex, weight, and duration of arrhythmia affect prognosis for response to treatment and the energy level at which cardioversion occurs. ANIMALS: TVEC was applied to 72 episodes of lone AF in 63 client-owned performance horses, with the majority (54) being Standardbred racehorses. METHODS: Catheterization of the right atrium (RA) and pulmonary artery (PA) through the jugular vein was used for electrode placement before horses were placed under general anesthesia. Biphasic, truncated exponential shock waves were delivered at incremental energy levels until cardioversion was achieved or a maximum single-energy level of 300 J was reached (cumulative energy 50-1,960 J). A multivariate model was constructed to evaluate influence of patient factors on cardioversion energy. RESULTS: Cardioversion was achieved in 71 of 72 episodes (62 of 63 horses) at a mean energy of 165.43 +/- 8.75 J. Cardioversion energy was higher for females than for males, and for interaction terms, weight was negatively related to energy in females and positively related in males. Age was positively related to cardioversion energy in females. No relationship was identified between duration of arrhythmia before treatment and prognosis for response or cardioversion energy. CONCLUSIONS AND CLINICAL IMPORTANCE: TVEC is highly effective in the treatment of lone AF in horses. Although age and sex influence cardioversion energy level, duration of arrhythmia does not.     

Thromboelastography in equine medicine: Technique and use in clinical research

Thromboelastography (TEG) is a viscoelastic, whole blood‐based assay that integrates information from both the cellular and soluble components of coagulation, providing a global evaluation of the haemostatic system. This contrasts with the conventional coagulation assays (i.e. platelet count, prothrombin time [PT], activated partial thromboplastin time [aPTT] and fibrinogen concentration [FIB]), which only provide information about one component (e.g. clotting factors in the case of PT and aPTT) of the haemostatic process, requiring the combination of several assays for a complete evaluation of haemostasis. Thromboelastography is an old technology that has been used in human medicine for over 50 years. However, it is relatively new in veterinary medicine and has only been applied to horses in the last 5 years. Clinical applications in human medicine include diagnosis and monitoring of coagulopathies. Currently, extensive research is being carried out to expand the use of TEG in dogs and cats. Therefore, it is expected that the use of this technique will also further expand in horses in the near future. To date, the available studies in the equine species have evaluated TEG in healthy horses, horses with gastrointestinal disease, septic foals, horses with exercise‐induced pulmonary haemorrhage (EIPH) and a filly with Glanzmann's thrombasthenia. The main objective of this review is to introduce the TEG technique to equine clinicians, providing information on how the TEG functions, blood sample collection and processing, variables measured and their interpretations, normal reference values and areas of potential clinical application.     

State of the art: Stem cells in equine regenerative medicine

According to Greek mythology, Prometheus' liver grew back nightly after it was removed each day by an eagle as punishment for giving mankind fire. Hence, contrary to popular belief, the concept of tissue and organ regeneration is not new. In the early 20th century, cell culture and ex vivo organ preservation studies by Alexis Carrel, some with famed aviator Charles Lindbergh, established a foundation for much of modern regenerative medicine. While early beliefs and discoveries foreshadowed significant accomplishments in regenerative medicine, advances in knowledge within numerous scientific disciplines, as well as nano‐ and micromolecular level imaging and detection technologies, have contributed to explosive advances over the last 20 years. Virtually limitless preparations, combinations and applications of the 3 major components of regenerative medicine, namely cells, biomaterials and bioactive molecules, have created a new paradigm of future therapeutic options for most species. It is increasingly clear, however, that despite significant parallels among and within species, there is no ‘one‐size‐fits‐all’ regenerative therapy. Likewise, a panacea has yet to be discovered that completely reverses the consequences of time, trauma and disease. Nonetheless, there is no question that the promise and potential of regenerative medicine have forever altered medical practices. The horse is a relative newcomer to regenerative medicine applications, yet there is already a large body of work to incorporate novel regenerative therapies into standard care. This review focuses on the current state and potential future of stem cells in equine regenerative medicine.     

Anthelmintic resistance in equine parasites - detection, potential clinical relevance and implications for control

During the past two decades anthelmintic resistance in equine parasites has been found in the group of small strongyle species (cyathostomins) and in the ascarid species Parascaris equorum. The ubiquitous nature and possible severe consequences of disease with these nematodes make them the prime targets of current worm control programmes. Traditional control strategies mainly rely on the strategic application of anthelmintics, currently represented by three major drug classes: benzimidazoles (BZ), the tetrahydropyrimidine pyrantel (PYR) and macrocyclic lactones (ML). Following decades of routine and frequent anthelmintic applications, many cyathostomin populations on horse farms in industrialised countries must be considered as resistant to BZ anthelmintics. However, to date no published cases of cyathostomin disease specifically associated with anthelmintic resistance were reported. Possibly this is due to the generally subclinical and unspecific symptoms associated with cyathostomin infections. Nevertheless, exclusive reliance on the ML drug class may increase the threat of clinical disease due to drug-resistant cyathostomins. More recently, P. equorum has been reported as having developed resistance against ivermectin and moxidectin, two representatives of the ML-class. These anthelmintics are currently the most frequently used drug class in horses. This nematode species is mainly found in foals and in younger horses due to the development of immunity following exposure to infection. Infection with P. equorum can result in clinically drastic consequences such as obstruction and/or penetration of the small intestine, the latter usually leading to death. In conclusion, on horse farms the efficacy of anthelmintic treatments should be examined routinely for each drug class. Several factors can influence the rate at which anthelmintic resistance develops; high frequency of treatment being one of the most important. Modern control strategies should therefore attempt to significantly reduce anthelmintic treatments. Several pasture and farm management practices found to be negatively associated with nematode and anthelmintic resistance prevalence will be discussed in the review presented here.     

Hereditary equine regional dermal asthenia (HERDA) in Quarter Horses: A review of clinical signs,genetics and research

Hereditary equine regional dermal asthenia (HERDA) results from a genetic mutation which affects the skin and other tissues of Quarter Horses and horses with Quarter Horse lineage. The disease HERDA has an autosomal recessive mode of inheritance and has become a significant concern in the Quarter Horse industry due to the high frequency of heterozygote carriers. Affected homozygous horses appear normal at birth; however, within the first 2 years of life they usually acquire loose, hyperextensible skin and wounds which result in disfiguring scars either spontaneously or from minor trauma. Some severely affected horses also develop haematomas and seromas. Consequently, most affected horses are subjected to euthanasia at an early age. No treatment options other than palliative therapy currently exist. As part of a five panel test ( http://www.aqha.com/News/News-Articles/2013/April/04292013-Genetic-Testing.aspx ) the American Quarter Horse Association presently requires DNA testing for HERDA on all breeding stallions. There are currently no restrictions on registration of horses heterozygous or homozygous for the HERDA mutation. Due to the autosomal recessive nature of the disease, Quarter Horse mares and horses of all breeds from HERDA‐associated bloodlines should also be tested.     

Flow cytometry in Spermatology: A bright future ahead

Techniques such as mass spectrometry have led to unprecedented knowledge of the proteins that are present in the spermatozoa of humans and other mammals. However, in spite of their high‐throughput and fractioning techniques, most of the techniques in use only offer average values for the entire sperm population. Yet, ejaculate is very heterogeneous, and average values may mask relevant biological information.The application of flow cytometry may overcome this disadvantage, allowing proteomic analysis at the single‐cell level. Moreover, recent advances in cytometry, allowing multiple analyses within a single cell combined with powerful statistical tools, as an expanding subfield in spermatology, are described. The increased use of advanced flow cytometers in andrology laboratories will allow the rapid development of multiparametric, multicolour flow cytometry in andrology that will expand the clinical applications and research possibilities of flow cytometry‐based proteomic approaches, especially in the subfields of clinical andrology and sperm biotechnology.     

Pathological studies of cheek teeth apical infections in the horse: 2. Quantitative measurements in normal equine dentine

Measurements of primary, regular and irregular secondary dentine and pulp dimensions were made on transverse, sub-occlusal and mid-tooth sections, of 40 maxillary and 42 mandibular control equine cheek teeth (CT) of different ages. Maxillary and mandibular CT primary dentine in different age groups had a mean thickness of 922–1065 μm and 1099–1179 μm, respectively, on the lateral aspects, and 1574–2035 μm and 1155–1330 μm, respectively, on the medial aspects of pulp horns. Surprisingly, some increase in thickness was found in some mandibular CT primary enamel in the first few years following eruption.Regular secondary dentine thickness increased with age, for example at mid-tooth level in mandibular CT from 124 μm at 3 years dental age to 290 μm at >7 years dental age on the lateral aspect of pulp horns, and from 166 μm to 509 μm on the medial aspects of pulp horns, indicating a deposition rate of 0.5–10 μm/day. This type of dentine was thicker sub-occlusally than in the mid-tooth region. Maxillary dentinal dimensions showed a similar age-related increase in thickness. Maxillary CT dentine was significantly thicker (72% in primary, 43% in regular secondary dentine) on the medial compared to the lateral aspects of pulp horns, but mandibular CT dentine was just 15% and 14% thicker in primary and regular secondary dentine thickness, respectively, on the their medial as compared to their lateral aspects. Dentinal and pulp dimensions varied between individual pulp horns, Triadan tooth position, and dental age, with complex interactions between these variables for some parameters.     

The use of progestins in equine medicine: A review

Progesterone is in constant fluctuation throughout the lifespan of the horse. As a key regulator of the hypothalamic–pituitary–gonadal axis, progesterone is involved in numerous aspects of reproduction. Synthetic analogs of progesterone, deemed progestins, are widely available to industry to act as progestogenic within the reproductive tract, although few are bioactive in the horse. Utilised to suppress oestrous, delay ovulation, maintain pregnancy, and for behavioural modifications, progestins are a commonly administered class of pharmaceuticals in veterinary medicine. In this review, we discuss the progestins available to equine industry, in addition to assessing efficacy, pharmacokinetics, and potential side effects following administration of each.     

Stem cells: classifications, controversies, and clinical applications

The application of stem cells in regenerative and reparative therapies is emerging in surgery. Published information can lead to an over simplified view of stem cells with respect to their definitions, tissues of origin, abilities to differentiate into tissue lineages, and their capacity for functional tissue regeneration. The goals of this review article are to define embryonic and adult stem cells, compare differences between them, and summarize their potential clinical applications.     

Equine Multinodular Pulmonary Fibrosis in association with asinine herpesvirus type 5 and equine herpesvirus type 5: a case report

A standardbred gelding with a history of 10 days pyrexia and lethargy was referred to the Equine Hospital at the Swedish University of Agricultural Sciences in Uppsala, Sweden.The horse had tachypnea with increased respiratory effort and was in thin body condition. Laboratory findings included leukocytosis, hyperfibrinogenemia and hypoxemia. Thoracic radiographs showed signs of pneumonia with a multifocal nodular pattern, which in combination with lung biopsy findings indicated Equine Multinodular Pulmonary Fibrosis (EMPF). EMPF is a recently described disease in adult horses with clinical signs of fever, weight loss and respiratory problems. The pathological findings include loss of functional pulmonary parenchyma due to extensive nodular interstitial fibrosis which has been related to infection with the equine herpesvirus type 5 (EHV-5). In this case, lung biopsy and tracheal wash samples tested positive for both asinine herpesvirus type 5 (AHV-5) and EHV-5 using PCR assays. The horse failed to respond to treatment and was euthanized for humane reasons. Postmortem examination confirmed the diagnosis of EMPF. This case suggests that not only EHV-5 alone should be considered in association with the development of this disease.     

Respirable dust concentrations in equine stables. Part 2: the benefits of soaking hay and optimising the environment in a neighbouring stable

The effect of soaking hay to minimise equine breathing zone respirable dust concentration (RDC) is unknown, as is the duration of soaking required. Additionally, the influence of the bedding and forage used in one stable on the mean and maximum RDC in a neighbouring stable within a common airspace is unknown. Consequently, in the management of equine environmental respiratory disease uncertainty remains about the necessity for optimising conditions in neighbouring stables. Investigations using a real-time continuous particle monitor revealed that when feeding hay, horses' mean breathing zone RDC was significantly reduced if the hay was immersed or soaked for a prolonged time, prior to feeding. There was no advantage in soaking for an extended time period. Implementing management changes in one stable (changing from straw bedding and hay feeding to wood shavings bedding and haylage feeding) significantly reduced mean and maximum background RDC in a neighbouring stable within a common airspace.     

Multinodular Malignant Cutaneous Mast Cell Tumor in a Horse With Generalized Pruritus and Reactive Fibrosis: A Case Report

Mast cell tumor (MCT) has long been considered as an uncommon neoplasm in horses. Cytological and behavioral evidence of its malignancy is usually lacking, and only a few reports have described MCT displaying malignant behavior. An 18-year-old Friesian stallion presented with a one-year history of intermittent and progressive skin lesions on the left forelimb associated with intense, generalized pruritus and apathy temporarily responsive to glucocorticoids and antibiotics. The horse was alert and responsive with poor body condition and marked generalized pruritus. The left forelimb was markedly enlarged and surrounded by numerous firm 2- to 20-cm masses that were ulcerated and focally necrotic. A 7-cm round firm mass was observed on the left dorsal neck. Dermatological examination revealed generalized moth-eaten alopecia and scaling with erosions and ulcers secondary to pruritus. A direct skin smear from the affected leg showed severe eosinophilic inflammation and neutrophilic inflammation with pleomorphic bacteria. Histopathology of the skin and biopsies of the underlying tissues revealed an abundant population of atypical mast cells consistent with a malignant MCT. The horse was euthanized and necropsy revealed a marked fibrous reaction on longitudinal sections of the affected limb, and the tumor could be detected on only a few histological slides. Diagnosis of equine MCT can be challenging because of the massive accompanying fibrous reaction. Mast cell tumor should be suspected in the presence of eosinophilic infiltration of the affected tissue and in cases of generalized pruritus not attributable to other causes.     

Sex Preselection in Swine: Altered Sex Ratios in Offspring following Surgical Insemination of Flow Sorted X- and Y-Bearing Sperm

Flow cytometric sorting of X and Y chromosome-bearing sperm into separate populations, followed by surgical insemination into the isthmus of the oviduct of mature gilts, produced litters with phenotypic sex ratios skewed in the direction of male or female according to the predicted sex of the sperm population inseminated. The skewing of the sex ratio of the offspring was predicted from flow cytometric DNA reanalysis of flow sorted sperm populations. Gilts inseminated with intact, viable flow-sorted X-bearing sperm produced litters of off spring that were 74% female (P < .01). Gilts inseminated with intact, viable flow-sorted Y-bearing sperm gave off spring that were 68% male (P < .04). These results validate the use of DNA as a marker for pre-selecting the sex of offspring when used in conjunction with flow cytometric sorting of sperm .     

Flow cytometry: An aid in monitoring lymphocyte transformation in whole blood cultures

A combination of whole blood cultures, hypotonic fluorescent staining of nucleated cells, and flow cytometry resulted in a greatly improved technique for measuring lymphocyte blastogenesis. Sample preparation was reduced to two steps with no centrifugation. The technique was successfully used to analyze blood cultures derived from experimentally and naturally infected cows. The technique was also shown to be ideally suited for analyzing lymphocytes cultured in whole blood cultures and (1) avoids the problems of tritiated thymidine analysis and (2) provides a direct analysis of the number of cells responding to antigenic stimulation.     

Quality of equine veterinary care. Part 2: Client satisfaction in equine top sports medicine in The Netherlands

The aim of this study was to evaluate systematically the quality of equine veterinary top sports medicine in The Netherlands and the degree to which the expectations in the field are met. Focus was on structure, process and outcome of care. The structure of care is generally satisfactory but there is insufficient transfer of applicable scientific knowledge to the equine community and a lack of regular checks by team vets. The process of care is hampered by the failure in communication between the veterinary and paraveterinary members of a team. For continuation of the excellent international results of Dutch riders and drivers a more collaborative approach, abandoning the often secretive character of equine medicine in relation to sport horses, is necessary.     

Respirable dust concentrations in equine stables. Part 1: validation of equipment and effect of various management systems

Traditional methods of measuring airborne dust concentrations (ADC) in animal housing have included the collection of dust onto pre-weighed filters permitting the calculation of mean, not maximum, ADC. However real-time continuous particle monitors are advantageous in identifying short duration elevations in ADC which may be detrimental to equine respiratory health in the face of a relatively low mean ADC. These monitors have not previously been used to measure ADC in equine stables. Comparisons of a filter-based sampler and a real-time continuous particle monitor revealed no significant difference (P=0.079) and good agreement (>or=95% of the points fell within two standard deviations of the mean of the differences and the mean of the differences approximated zero) between the devices, with respect to mean respirable dust concentration (RDC) measurements. Investigations of the influence of various equine management systems on RDC revealed that both mean and maximum breathing zone RDC were significantly reduced (P<0.05) in equine stables by changing the environment from hay feed and straw bedding, to haylage feed and wood shavings bedding (reduction in mean - 0.0867mg/m(3) to 0.0260mg/m(3); reduction in maximum - 4.0758mg/m(3) to 0.2182mg/m(3), respectively).