Using high-density SNP data to unravel the origin of the Franches-Montagnes horse breed.

BACKGROUND: The Franches-Montagnes (FM) is the last native horse breed of Switzerland, established at the end of the 19th century by cross-breeding local mares with Anglo-Norman stallions. We collected high-density SNP genotype data (Axiom™ 670 K Equine genotyping array) from 522 FM horses, including 44 old-type horses (OF), 514 European Warmblood horses (WB) from Sweden and Switzerland (including a stallion used for cross-breeding in 1990), 136 purebred Arabians (AR), 32 Shagya Arabians (SA), and 64 Thoroughbred (TB) horses, as introgressed WB stallions showed TB origin in their pedigrees. The aim of the study was to ascertain fine-scale population structures of the FM breed, including estimation of individual admixture levels and genomic inbreeding (FROH) by means of Runs of Homozygosity. RESULTS: To assess fine-scale population structures within the FM breed, we applied a three-step approach, which combined admixture, genetic contribution, and FROH of individuals into a high-resolution network visualization. Based on this approach, we were able to demonstrate that population substructures, as detected by model-based clustering, can be either associated with a different genetic origin or with the progeny of most influential sires. Within the FM breed, admixed horses explained most of the genetic variance of the current breeding population, while OF horses only accounted for a small proportion of the variance. Furthermore, we illustrated that FM horses showed high TB admixture levels and we identified inconsistencies in the origin of FM horses descending from the Arabian stallion Doktryner. With the exception of WB, FM horses were less inbred compared to the other breeds. However, the relatively few but long ROH segments suggested diversity loss in both FM subpopulations. Genes located in FM- and OF-specific ROH islands had known functions involved in conformation and behaviour, two traits that are highly valued by breeders. CONCLUSIONS: The FM remains the last native Swiss breed, clearly distinguishable from other historically introgressed breeds, but it suffered bottlenecks due to intensive selection of stallions, restrictive mating choices based on arbitrary definitions of pure breeding, and selection of rare coat colours. To preserve the genetic diversity of FM horses, future conservation managements strategies should involve a well-balanced selection of stallions (e.g., by integrating OF stallions in the FM breeding population) and avoid selection for rare coat colours.

PCR-Based Equine Gene Doping Test for the Australian Horseracing Industry.

The term 'gene doping' is used to describe the use of any unauthorized gene therapy techniques. We developed a test for five likely candidate genes for equine gene doping: EPO, FST, GH1, IGF1, and ILRN1. The test is based on real-time polymerase chain reaction (PCR) and includes separate screening and confirmation assays that detect different unique targets in each transgene. For doping material, we used nonviral (plasmid) and viral (recombinant adeno-associated virus) vectors carrying complementary DNA for the targeted genes; the vectors were accurately quantified by digital PCR. To reduce non-specific amplification from genomic DNA observed in some assays, a restriction digest step was introduced in the PCR protocol prior to cycling to cut the amplifiable targets within the endogenous genes. We made the screening stage of the test simpler and faster by multiplexing PCR assays for four transgenes (EPO, FST, IGF1, and ILRN1), while the GH1 assay is performed in simplex. Both stages of the test reliably detect at least 20 copies of each transgene in a background of genomic DNA equivalent to what is extracted from two milliliters of equine blood. The test protocol was documented and tested with equine blood samples provided by an official doping control authority. The developed tests will form the basis for screening official horseracing samples in Australia.

Congenital heart defects in Arabian horses and the prospects of genetic testing: A review.

Congenital heart defects (CHDs) can have profound and potentially life-threatening consequences on horses' health and performance capability. While CHDs are rare in the general horse population, the Arabian breed is disproportionately overrepresented and thus is widely suspected to be genetically predisposed. This review discusses the most common CHDs in Arabian horses, including ventricular septal defect (VSD), tetralogy of Fallot (TOF), patent duct arteriosus (PDA), tricuspid valve atresia (TVA) and atrial septal defect (ASD). This review also explores how future research into the genetic factors that likely underpin many CHDs can revolutionise the way these disorders are managed in Arabian horses.

Detection of Indiscriminate Genetic Manipulation in Thoroughbred Racehorses by Targeted Resequencing for Gene-Doping Control.

The creation of genetically modified horses is prohibited in horse racing as it falls under the banner of gene doping. In this study, we developed a test to detect gene editing based on amplicon sequencing using next-generation sequencing (NGS). We designed 1012 amplicons to target 52 genes (481 exons) and 147 single-nucleotide variants (SNVs). NGS analyses showed that 97.7% of the targeted exons were sequenced to sufficient coverage (depth > 50) for calling variants. The targets of artificial editing were defined as homozygous alternative (HomoALT) and compound heterozygous alternative (ALT1/ALT2) insertion/deletion (INDEL) mutations in this study. Four models of gene editing (three homoALT with 1-bp insertions, one REF/ALT with 77-bp deletion) were constructed by editing the myostatin gene in horse fibroblasts using CRISPR/Cas9. The edited cells and 101 samples from thoroughbred horses were screened using the developed test, which was capable of identifying the three homoALT cells containing 1-bp insertions. Furthermore, 147 SNVs were investigated for their utility in confirming biological parentage. Of these, 120 SNVs were amenable to consistent and accurate genotyping. Surrogate (nonbiological) dams were excluded by 9.8 SNVs on average, indicating that the 120 SNV could be used to detect foals that have been produced by somatic cloning or embryo transfer, two practices that are prohibited in thoroughbred racing and breeding. These results indicate that gene-editing tests that include variant calling and SNV genotyping are useful to identify genetically modified racehorses.

Low-copy transgene detection using nested digital polymerase chain reaction for gene-doping control.

Gene doping is prohibited for fair competition in human and horse sports. One style of gene doping is the administration of an exogeneous gene, called a transgene, to postnatal humans and horses. Although many transgene detection methods based on quantitative polymerase chain reaction (PCR), including real-time PCR and digital PCR, have been recently developed, it remains difficult to reliably detect low-copy transgenes. In this study, we developed and validated a nested digital PCR method to specifically detect low-copy transgenes. The nested digital PCR consists of (1) preamplification using conventional PCR and (2) droplet digital PCR detection using a hydrolysis probe. Using 5, 10, 20, 60 and 120 transgene copies as template, 496.0, 1089.7, 1820.7, 4313.3 and 7840.0 copies per microlitre, respectively, were detected using our nested digital PCR. Although high concentrations of phenol, proteinase K, ethanol, EDTA, heparin and genomic DNA all inhibited preamplification, their effects on the digital PCR detection were limited. Once preamplification was successful, even substitution of bases within the primers and probes had minimal effects on transgene detection. The nested digital PCR developed in this study successfully detected low-copy transgenes and can be used to perform a qualitative test, indicating its usefulness in the prevention of false positives and false negatives in gene-doping detection.

Invasive salmonellosis in paediatric patients in the Northern Territory, Australia, 2005-2015.

AIM: To describe the epidemiology of invasive Salmonella disease in children in the Northern Territory, Australia. METHODS: Design: A retrospective review of invasive salmonellosis cases identified by pathology records and the Northern Territory Notifiable Disease Surveillance System. Case definitions: Those aged 18 years or under, with Salmonella cultured from a usually sterile site, collected in the Northern Territory between 1 July 2005 and 30 June 2015. OUTCOME MEASURES: The primary outcome measure was the annual incidence rate of invasive salmonellosis, comparing rates between Indigenous and non-Indigenous children. RESULTS: There were 86 cases of invasive Salmonella infection in children over the 10-year period; an annual incidence of 14.1 per 100 000 population, in those aged less than 18 years. Gastrointestinal Salmonella notifications were similar between Indigenous and non-Indigenous children. In children aged less than 15 years, the rate of invasive salmonellosis was higher in Indigenous children compared to non-Indigenous children (23.4 per 100 000 compared with 11.6 per 100 000); rate ratio 2.0 (95% confidence interval 1.3-3.3, P = 0.002). Indigenous children with invasive salmonellosis had a median hospital stay of 8 days, which was compared to 5 days for non-Indigenous children (P = 0.015). The highest incidence rate of invasive salmonellosis occurred in Indigenous patients less than 12 months of age (138 per 100 000). CONCLUSION: The Northern Territory of Australia has high rates of invasive salmonellosis in children. Indigenous and non-Indigenous children experience similar rates of Salmonella gastroenteritis but Indigenous children experience higher rates of invasive salmonellosis.

Whole-genome resequencing using genomic DNA extracted from horsehair roots for gene-doping control in horse sports.

Gene doping is prohibited in horseracing and equestrian sports. In previous studies, we developed non-targeted transgene and genome editing detection methods based on whole genome resequencing (WGR) using genomic DNA extracted from whole blood. In this study, we aimed to develop a WGR method using DNA extracts from hair roots. Hair roots are a preferred substrate because their collection is less invasive than blood collection. Hair is also easier to store for long periods of time. Although almost all genomic DNA extracted from hair root samples stored for years at room temperature was degraded, the quality of genomic DNA from samples stored for years at refrigerated temperatures (4-8°C) was maintained. High-molecular-weight genomic DNA was isolated from hair roots using a magnetic silica beads method of extraction, enabling WGR from horsehair root extracts. Nucleotide sequencing results and numbers of single-nucleotide polymorphisms and insertions/deletions concurred with those previously reported for WGR of DNA extracted from whole blood. Therefore, we consider that storing hair samples at refrigerated temperatures prevents degradation of DNA, allowing the detection of gene doping in these samples based on WGR. It is likely this finding will also have a deterrent effect, as it is now possible to test horses with archived samples even if they or their parents are deceased. To our knowledge, this is the first report employing WGR on horsehair roots stored for a long term.

A genome-wide scan for candidate lethal variants in Thoroughbred horses.

Domestic animal populations are often characterised by high rates of inbreeding and low effective population sizes due to selective breeding practices. These practices can result in otherwise rare recessive deleterious alleles drifting to high frequencies, resulting in reduced fertility rates. This study aimed to identify potential recessive lethal haplotypes in the Thoroughbred horse breed, a closed population that has been selectively bred for racing performance. In this study, we identified a haplotype in the LY49B gene that shows strong evidence of being homozygous lethal, despite having high frequencies of heterozygotes in Thoroughbreds and other domestic horse breeds. Variant analysis of whole-genome sequence data identified two SNPs in the 3'UTR of the LY49B gene that may result in loss of function. Analysis of transcriptomic data from equine embryonic tissue revealed that LY49B is expressed in the trophoblast during placentation stage of development. These findings suggest that LY49B may have an essential, but as yet unknown function in the implantation stage of equine development. Further investigation of this region may allow for the development of a genetic test to improve fertility rates in horse populations. Identification of other lethal variants could assist in improving natural levels of fertility in horse populations.

The effects of inbreeding on covering success, gestation length and foal sex ratio in Australian thoroughbred horses.

BACKGROUND: Horses produce only one foal from an eleven-month gestation period, making the maintenance of high reproductive rates essential. Genetic bottlenecks and inbreeding can increase the frequency of deleterious variants, resulting in reduced reproductive levels in a population. In this study we examined the influence of inbreeding levels on foaling rate, gestation length and secondary sex ratio in Australian Thoroughbred mares. We also investigated the genetic change in these traits throughout the history of the breed. Phenotypic data were obtained from 27,262 breeding records of Thoroughbred mares provided by three Australian stud farms. Inbreeding was estimated using the pedigree of each individual dating back to the foundation of the breed in the eighteenth century. RESULTS: While both gestation length and foaling rate were heritable, no measurable effect of inbreeding on either trait was found. However, we did find that the genetic value for both traits had decreased within recent generations. A number of environmental factors also had significant effects on foaling rate and gestation length. Secondary sex ratio had only an extremely small paternal heritable effect and was not susceptible to environmental influences. CONCLUSIONS: In contrast to racing performance, inbreeding had no measurable effect on foaling rate or gestation length in Australian Thoroughbred horses. This could be because the level of inbreeding in the population examined is not high enough to show a discernible effect on reproductive traits. Populations that experience higher levels of inbreeding due to use of artificial reproductive technologies or extremely small population sizes may show a more pronounced reduction in natural foaling rate or gestation length. It is also possible that the intensive management techniques used in the Thoroughbred population masks any negative effects of inbreeding. The decrease in the genetic value of foaling rate is likely to be because horses with unfavourable genetic potential have not yet been selected out of the population. The change in genetic value of gestation length may be due to selective breeding favouring horses with shorter pregnancies. We also found that prioritising the mating of older mares, and avoiding out of season mating could lead to an increased breeding success.

Catalase-Like Antioxidant Activity is Unaltered in Hypochlorous Acid Oxidized Horse Heart Myoglobin.

Activated neutrophils release myeloperoxidase that produces the potent oxidant hypochlorous acid (HOCl). Exposure of the oxygen transport protein horse heart myoglobin (hhMb) to HOCl inhibits Iron III (Fe(III))-heme reduction by cytochrome b5 to oxygen-binding Iron II (Fe(II))Mb. Pathological concentrations of HOCl yielded myoglobin oxidation products of increased electrophoretic mobility and markedly different UV/Vis absorbance. Mass analysis indicated HOCl caused successive mass increases of 16 a.m.u., consistent serial addition of molecular oxygen to the protein. By contrast, parallel analysis of protein chlorination by quantitative mass spectrometry revealed a comparatively minor increase in the 3-chlorotyrosine/tyrosine ratio. Pre-treatment of hhMb with HOCl affected the peroxidase reaction between the hemoprotein and H2O2 as judged by a HOCl dose-dependent decrease in spin-trapped tyrosyl radical detected by electron paramagnetic resonance (EPR) spectroscopy and the rate constant of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS) oxidation. By contrast, Mb catalase-like antioxidant activity remained unchanged under the same conditions. Notably, HOCl-modification of Mb decreased the rate of ferric-to-ferrous Mb reduction by a cytochrome b5 reductase system. Taken together, these data indicate oxidizing HOCl promotes Mb oxidation but not chlorination and that oxidized Mb shows altered Mb peroxidase-like activity and diminished rates of one-electron reduction by cytochrome b5 reductase, possibly affecting oxygen storage and transport however, Mb-catalase-like antioxidant activity remains unchanged.

Parent management training for conduct problems in children: Enhancing treatment to improve therapeutic change.

Background/Objective: The study evaluated two variations of Parent Management Training (PMT) for children referred to treatment for oppositional, aggressive, and antisocial behavior. The goal was to evaluate the impact of multiple enhancements to optimize common and placebo factors to augment therapeutic change. Method: The families of all children (N=138, 39 girls and 99 boys, ages 6-13) received PMT. One half of the families were assigned to receive an enhanced version with multiple additions designed to increase bonding of the parent to the therapist, professionalism of treatment and setting, credibility of the intervention, and expectancies for therapeutic change. Assessment included multiple treatment outcome measures of the child (problem behaviors, psychiatric symptoms, social competence, and adaptive functioning) and parents (depression, stress, and family relations) showed marked improvements over the course of treatment, and several process measures (therapeutic alliance, credibility of the procedures, expectancy for change). Results: The results indicated that children and parents made marked improvement in all the treatment outcome measures. The vast majority of children fell within the normative range at posttreatment on problem and prosocial behaviors. The two treatment conditions were no different in outcomes for children or parents. Conclusion: PMT led to marked changes in treatment outcome.

Antecedentes/Objetivo: Se evalúan dos versiones del Parent Management Training (PMT) para tratamiento de conducta oposicionista, agresiva y antisocial en niños. El objetivo fue evaluar el impacto de múltiples mejoras para optimizar los factores comunes y de placebo con el fin de incrementar el efecto terapéutico. Método: Las familias de los niños (N = 138, 39 niñas y 99 niños de 6 a 13 años) recibieron PMT. La mitad fue asignada a una versión mejorada con múltiples adiciones para aumentar la vinculación de los padres con el terapeuta, la profesionalidad del tratamiento y el entorno, la credibilidad de la intervención y las expectativas de cambio terapéutico. La evaluación incluyó medidas del efecto del tratamiento en el niño (problemas de comportamiento, síntomas psiquiátricos, competencia social y funcionamiento adaptativo) y los padres (depresión, estrés y relaciones familiares) y medidas del proceso (alianza terapéutica, credibilidad de los procedimientos y expectativa de cambio). Resultados: Niños y padres mejoraron notablemente en todas las medidas del efecto del tratamiento. La gran mayoría de niños se situaron dentro del rango normativo en el post-tratamiento. Las dos condiciones de tratamiento no fueron diferentes para niños ni padres. Conclusión: PMT produjo cambios significativos en el resultado del tratamiento.

Parent management training for conduct problems in children: Enhancing treatment to improve therapeutic change / Capacitación en entrenamiento de padres para problemas de conducta en niños: mejorar el tratamiento para mejorar el cambio terapéutico

BACKGROUND/OBJECTIVE: The study evaluated two variations of Parent Management Training (PMT) for children referred to treatment for oppositional, aggressive, and antisocial behavior. The goal was to evaluate the impact of multiple enhancements to optimize common and placebo factors to augment therapeutic change. METHOD: The families of all children (N=138, 39 girls and 99 boys, ages 6-13) received PMT. One half of the families were assigned to receive an enhanced version with multiple additions designed to increase bonding of the parent to the therapist, professionalism of treatment and setting, credibility of the intervention, and expectancies for therapeutic change. Assessment included multiple treatment outcome measures of the child (problem behaviors, psychiatric symptoms, social competence, and adaptive functioning) and parents (depression, stress, and family relations) showed marked improvements over the course of treatment, and several process measures (therapeutic alliance, credibility of the procedures, expectancy for change). RESULTS: The results indicated that children and parents made marked improvement in all the treatment outcome measures. The vast majority of children fell within the normative range at posttreatment on problem and prosocial behaviors. The two treatment conditions were no different in outcomes for children or parents. CONCLUSION: PMT led to marked changes in treatment outcom

ANTECEDENTES/OBJETIVO: Se evalúan dos versiones del Parent Management Training (PMT) para tratamiento de conducta oposicionista, agresiva y antisocial en niños. El objetivo fue evaluar el impacto de múltiples mejoras para optimizar los factores comunes y de placebo con el fin de incrementar el efecto terapéutico. MÉTODO: Las familias de los niños (N = 138, 39 niñas y 99 niños de 6 a 13 años) recibieron PMT. La mitad fue asignada a una versión mejorada con múltiples adiciones para aumentar la vinculación de los padres con el terapeuta, la profesionalidad del tratamiento y el entorno, la credibilidad de la intervención y las expectativas de cambio terapéutico. La evaluación incluyó medidas del efecto del tratamiento en el niño (problemas de comportamiento, síntomas psiquiátricos, competencia social y funcionamiento adaptativo) y los padres (depresión, estrés y relaciones familiares) y medidas del proceso (alianza terapéutica, credibilidad de los procedimientos y expectativa de cambio). RESULTADOS: Niños y padres mejoraron notablemente en todas las medidas del efecto del tratamiento. La gran mayoría de niños se situaron dentro del rango normativo en el post-tratamiento. Las dos condiciones de tratamiento no fueron diferentes para niños ni padres. CONCLUSIÓN: PMT produjo cambios significativos en el resultado del tratamiento

A potential regulatory region near the EDN3 gene may control both harness racing performance and coat color variation in horses.

The Swedish-Norwegian Coldblooded trotter and the heavier North-Swedish draught horse both descend from the North-Swedish horse, but the Coldblooded trotters have been selected for racing performance while the North-Swedish draught horse is mainly used for agricultural and forestry work. By comparing the genomes of Coldblooded trotters, North-Swedish draught horses and Standardbreds for a large number of single-nucleotide polymorphisms (SNPs), the aim of the study was to identify genetic regions that may be under selection for racing performance. We hypothesized that the selection for racing performance, in combination with unauthorized crossbreeding of Coldblooded trotters and Standardbreds, has created regions in the genome where the Coldblooded trotters and Standardbreds are similar, but differ from the North-Swedish draught horse. A fixation index (Fst) analysis was performed and sliding window Delta Fst values were calculated across the three breeds. Five windows, where the average Fst between Coldblooded trotters and Standardbreds was low and the average Fst between Coldblooded trotters and North-Swedish draught horses was high, were selected for further investigation. Associations between the most highly ranked SNPs and harness racing performance were analyzed in 400 raced Coldblooded trotters with race records. One SNP showed a significant association with racing performance, with the CC genotype appearing to be negatively associated. The SNP identified was genotyped in 1915 horses of 18 different breeds. The frequency of the TT genotype was high in breeds typically used for racing and show jumping while the frequency of the CC genotype was high in most pony breeds and draught horses. The closest gene in this region was the Endothelin3 gene (EDN3), a gene mainly involved in melanocyte and enteric neuron development. Both functional genetic and physiological studies are needed to fully understand the possible impacts of the gene on racing performance.

Founder-specific inbreeding depression affects racing performance in Thoroughbred horses.

The Thoroughbred horse has played an important role in both sporting and economic aspects of society since the establishment of the breed in the 1700s. The extensive pedigree and phenotypic information available for the Thoroughbred horse population provides a unique opportunity to examine the effects of 300 years of selective breeding on genetic load. By analysing the relationship between inbreeding and racing performance of 135,572 individuals, we found that selective breeding has not efficiently alleviated the Australian Thoroughbred population of its genetic load. However, we found evidence for purging in the population that might have improved racing performance over time. Over 80% of inbreeding in the contemporary population is accounted for by a small number of ancestors from the foundation of the breed. Inbreeding to these ancestors has variable effects on fitness, demonstrating that an understanding of the distribution of genetic load is important in improving the phenotypic value of a population in the future. Our findings hold value not only for Thoroughbred and other domestic breeds, but also for small and endangered populations where such comprehensive information is not available.

Equine performance genes and the future of doping in horseracing.

A horse's success on the racetrack is determined by genetics, training and nutrition, and their translation into physical traits such as speed, endurance and muscle strength. Advances in genetic technologies are slowly explaining the roles of specific genes in equine performance, and offering new insights into the development of novel therapies for diseases and musculoskeletal injuries that cause early retirement of many racehorses. Gene therapy approaches may also soon provide new means to artificially enhance the physical performance of racehorses. Gene doping, the misuse of gene therapies for performance enhancement, is predicted to be the next phase of doping faced by horseracing. The risk of gene doping to human sports has been recognised for almost 15 years, and the introduction of the first gene doping detection tests for doping control in human athletes is imminent. Gene doping is also a threat to horseracing, but there are currently no methods to detect it. Efficient and accurate detection methods need to be developed to deter those looking to use gene doping in horses and to maintain the integrity of the sport. Methods developed for human athletes could offer an avenue for detection in racehorses. Development of an equine equivalent test will first require identification of equine genes that will likely be targeted by gene doping attempts. This review focuses on genes that have been linked to athletic performance in horses and, therefore, could be targeted for genetic manipulation. The risks associated with gene doping and approaches to detect gene doping are also discussed. Copyright © 2017 John Wiley & Sons, Ltd.

Impact of the Balanced School Day on Glycemic Control in Children with Type 1 Diabetes.

OBJECTIVE: The balanced school day (BSD) is an alternative elementary school schedule whereby children have 2 20-minute eating periods instead of 1 20-minute lunch, as is found in the traditional schedule (TS). We assessed the glycated hemoglobin (A1C) levels of children with type 1 diabetes in the TS vs. the BSD because 2 eating periods have the potential to impact blood glucose control. METHODS: A1C levels representative of the summer months (SumA1C) and A1C levels occurring at least 3 months after the start of the school year (SchA1C) were obtained retrospectively. A parental survey of perceptions of lunch planning, activity levels and diabetes management at school was also completed. RESULTS: Our sample included 97 students (TS=42, BSD=55). The mean age ± SD was 10.9±2.6 and 10.1±2.8 years in the TS and BSD, respectively (p=0.12). Sex distribution was not statistically different; 54% were female in TS vs. 36% in BSD; p=0.08. SumA1C was similar in the 2 groups (TS: 8.3±1.1% vs. BSD: 8.0±0.8%; p=0.08). There was a significant within-group increase from SumA1C to SchA1C in the BSD group only (p=0.001), with mean A1C values increasing from 8.0%±0.8% to 8.5%±1.0% in the BSD group compared to no significant increase in the TS group. Parental perceptions of lunch planning, physical activity and diabetes management were similar, regardless of school schedule. CONCLUSIONS: Children with type 1 diabetes in the BSD appear to have worse diabetes control during the school year compared to the summer, which is not evident in children in the TS. Additional school supports may assist students in the BSD.