Developing a 670k genotyping array to tag ~2M SNPs across 24 horse breeds.

BACKGROUND: To date, genome-scale analyses in the domestic horse have been limited by suboptimal single nucleotide polymorphism (SNP) density and uneven genomic coverage of the current SNP genotyping arrays. The recent availability of whole genome sequences has created the opportunity to develop a next generation, high-density equine SNP array. RESULTS: Using whole genome sequence from 153 individuals representing 24 distinct breeds collated by the equine genomics community, we cataloged over 23 million de novo discovered genetic variants. Leveraging genotype data from individuals with both whole genome sequence, and genotypes from lower-density, legacy SNP arrays, a subset of ~5 million high-quality, high-density array candidate SNPs were selected based on breed representation and uniform spacing across the genome. Considering probe design recommendations from a commercial vendor (Affymetrix, now Thermo Fisher Scientific) a set of ~2 million SNPs were selected for a next-generation high-density SNP chip (MNEc2M). Genotype data were generated using the MNEc2M array from a cohort of 332 horses from 20 breeds and a lower-density array, consisting of ~670 thousand SNPs (MNEc670k), was designed for genotype imputation. CONCLUSIONS: Here, we document the steps taken to design both the MNEc2M and MNEc670k arrays, report genomic and technical properties of these genotyping platforms, and demonstrate the imputation capabilities of these tools for the domestic horse.

An International Genetic Survey of Breed-Specific Diseases in Working Dogs from the United States, Israel, and Poland.

Genetic diseases occur in breeds used for law enforcement. As important team members, dogs are expected to operate at peak performance for several years and are significant investments for both the initial purchase and extensive, specialized training. Previous studies have not focused on causes for retirement or euthanasia as genetic (inherited) versus acquired (environmental). We performed direct mutational analysis for breed-specific conditions on samples from 304 dogs including 267 law enforcement (122 US, 87 Israeli, and 58 Polish) and 37 search and rescue dogs. Genetic testing identified 29% (n = 89) of the dogs tested to be carriers of a genetic mutation and 6% (n = 19) to be at risk for a debilitating inherited condition that may eventually impair the dog's ability to work. At-risk dogs included Labrador Retrievers (n = 4) with exercise-induced collapse, Bloodhounds (n = 2) with degenerative myelopathy (DM), and German Shepherd dogs with DM (n = 12) or leukocyte adhesion deficiency, type III (n = 1). A substantial number of working dogs were shown to be at risk for genetic conditions that may shorten the dog's career. The loss of dogs, due to early retirement or euthanasia, as a result of preventable genetic conditions has an emotional cost to handlers and financial cost to service organizations that can be avoided with genetic screening prior to breeding, buying, or training.

Genetic ancestry of the extinct Javan and Bali tigers.

The Bali (Panthera tigris balica) and Javan (P. t. sondaica) tigers are recognized as distinct tiger subspecies that went extinct in the 1940s and 1980s, respectively. Yet their genetic ancestry and taxonomic status remain controversial. Following ancient DNA procedures, we generated concatenated 1750bp mtDNA sequences from 23 museum samples including 11 voucher specimens from Java and Bali and compared these to diagnostic mtDNA sequences from 122 specimens of living tiger subspecies and the extinct Caspian tiger. The results revealed a close genetic affinity of the 3 groups from the Sunda Islands (Bali, Javan, and Sumatran tigers P. t. sumatrae). Bali and Javan mtDNA haplotypes differ from Sumatran haplotypes by 1-2 nucleotides, and the 3 island populations define a monophyletic assemblage distinctive and equidistant from other mainland subspecies. Despite this close phylogenetic relationship, no mtDNA haplotype was shared between Sumatran and Javan/Bali tigers, indicating little or no matrilineal gene flow among the islands after they were colonized. The close phylogenetic relationship among Sunda tiger subspecies suggests either recent colonization across the islands, or else a once continuous tiger population that had subsequently isolated into different island subspecies. This supports the hypothesis that the Sumatran tiger is the closest living relative to the extinct Javan and Bali tigers.

Molecular identification of bloodmeals from sand flies and mosquitoes collected in Israel.

In Israel, sand flies are the vectors of Leishmania Ross and mosquitoes are the vectors of West Nile Virus. In the Judean Desert and Tiberias, the sand fly Phlebotomus sergenti Parrot is the vector of Leishmania tropica (Wright) and the rock hyrax (Procavia capensis Pallas) is considered the main reservoir animal. The main vectors of West Nile Virus are Culex pipiens L. and Culex perexiguus Theobald. Bloodmeals of engorged field-caught female sand flies and mosquitoes are an important source for defining host preferences. Recent progress in DNA molecular techniques has enabled the accurate identification of blood sources within the arthropod gut. In this study, we applied molecular approach for species-specific identification based on polymerase chain reaction and nucleotide sequence analysis of polymorphic regions along two mitochondrial genes, 12S and 16S rRNA. The research was carried out on 261 engorged female sand flies collected in the Judean Desert and Tiberias and 50 engorged female mosquitoes collected in Tel-Aviv and Arava. Species identification of bloodmeals was successful in 92% of the samples. Rock hyrax was the most abundant host in bloodmeals of P. sergenti, while human blood was found in only seven (3%) females. L. tropica DNA was detected in three P. sergenti females from Tiberias that contained rock hyrax blood. Avian sequences were detected in 67% (10 of 15) of the identified bloodmeals from Cx. perexiguus and in 10% (3 of 29) of the identified meals from Cx. pipiens. Human sequences were found in 14% of the identified bloodmeals from Cx. pipiens. The successful analysis of the majority of the bloodmeals performed on wild sand flies and mosquitoes suggests that bloodmeal identification can be applied as one of the routine procedures in vector surveillance programs.

Genetic diversity of the Eurasian Otter (Lutra lutra) population in Israel.

The Israeli population of Eurasian otter (Lutra lutra) marks the Palearctic southern boundary of the species' distribution in the Levant. During the 20th century, the otter population in Israel experienced a dramatic decline due to anthropogenic habitat alterations. Currently, the otter population in Israel is estimated at about 100 individuals and defined as "Critically Endangered". The aim of this research was to characterize the Israeli otter population in order to determine its genetic diversity and fragmentation state for conservation purposes. Monitoring spraint sites during 2000-2011 along active and historic otter distribution regions indicate both stable and unstable otter subpopulations, mainly along the Jordan River. Four otter subpopulations, representing 57 individuals, were characterized by 12 microsatellites, previously used to characterize the European otter populations. The genetic results indicated three subpopulations correlating with three geographical regions: the Hula Valley, Sea of Galilee, and the Harod Valley. A moderate genetic diversity (F (st) = 0.087-0.123) was found among the subpopulations, suggesting sporadic interactions between individuals from distinct geographical locations along the Jordan Rift Valley. The Israeli otter population was found to be very small, demographically remote and genetically distinct, harboring unique alleles absent from the studied European populations. Therefore, immediate conservation actions are recommended to prevent the deterioration of the isolated, unique, and critically endangered otter population in Israel.

Fecal microbiota of the synanthropic golden jackal (Canis aureus).

The golden jackal (Canis aureus), is a medium canid carnivore widespread throughout the Mediterranean region and expanding into Europe. This species thrives near human settlements and is implicated in zoonoses such as rabies. This study explores for the first time, the golden jackal fecal microbiota. We analyzed 111 fecal samples of wild golden jackals using 16S rRNA amplicon sequencing the connection of the microbiome to animal characteristics, burden of pathogens and geographic and climate characteristics. We further compared the fecal microbiota of the golden jackal to the black-backed jackal and domestic dog. We found that the golden jackal fecal microbiota is dominated by the phyla Bacteroidota, Fusobacteriota and Firmicutes. The golden jackal fecal microbiota was associated with different variables, including geographic region, age-class, exposure to rabies oral vaccine, fecal parasites and toxoplasmosis. A remarkable variation in the relative abundance of different taxa was also found associated with different variables, such as age-class. Linear discriminant analysis effect size (LEfSe) analysis found abundance of specific taxons in each region, Megasphaera genus in group 1, Megamonas genus in group 2 and Bacteroides coprocola species in group 3. We also found a different composition between the fecal microbiota of the golden jackal, blacked-backed jackal and the domestic dog. Furthermore, LEfSe analysis found abundance of Fusobacterium and Bacteroides genera in the golden jackal, Clostridia class in blacked-backed jackal and Megamonas genus in domestic dog. The golden jackal fecal microbiota is influenced by multiple factors including host traits and pathogen burden. The characterization of the microbiota of this thriving species may aid in mapping its spread and proximity to human settlements. Moreover, understanding the jackal microbiota could inform the study of potential animal and human health risks and inform control measures.

First detection of avian influenza subtype H4N6 in Israel in a wild mallard (Anas platyrhynchos).

Avian influenza viruses (AIV) are a worldwide threat to animal and human health. As wild waterfowl circulate and spread these viruses around the world, investigations of AIV prevalence in wild populations are critical for understanding pathogen transmission, as well as predicting disease outbreaks in domestic animals and humans. Surveillance efforts in this study have isolated H4N6 for the first time in Israel from a faecal sample of a wild mallard (Anas platyrhynchos). Phylogenetic analyses of the HA and NA genes revealed that this strain is closely related to isolates from Europe and Asia. This Eurasian origin, together with Israel serving as an important migratory bottleneck of the mid Palearctic-African flyway, suggests a potential introduction of this strain by migratory birds. Additional phylogenetic analysis of the isolate's internal genes (PB1, PB2, PA, NP, M and NS) revealed high levels of phylogenetic relatedness with other AIV subtypes, indicating previous reassortment events. High reassortment rates are characteristic for H4N6 viruses, which, together with this subtype's ability to infect pigs and adaptability to the human receptor binding domain, raises the concern that it would potentially become zoonotic in the future. These results emphasize the importance of continuous AIV monitoring in migratory birds.

Evaluation of short-term safety of ultrasound-guided foetal fluid sampling in the dog (Canis lupus familiaris).

BACKGROUND: In humans, analysis of amniotic fluid is widely used for diagnostic and prognostic purposes. Amniocentesis has scarcely been used in veterinary medicine to date, despite a tremendous potential for clinical and research applications in dogs. Our study aimed to establish a safe method for foetal fluid sampling in female dogs. METHODS: Two transabdominal ultrasound-guided methods were assessed: the "free hand" and the needle-guided bracket sampling. In addition, through a subsequent routinely scheduled ovariohysterectomy, fluid was directly collected. Samples from 98 conceptuses were collected at day 46.7 ± 7.5 of pregnancy. RESULTS: The amount of fluid retrieved varied between 0.5 and 5.0 ml per collection. Macroscopic examination of the uterus and conceptuses identified 53% of the puncture sites. Neither fluid leakage nor foetal injury was detected, and six hematomas (5.8%) were visible. Ultrasound-guided foetal fluid collection was found to be potentially safe, and it can be performed by using either transabdominal method. CONCLUSION: Foetal fluid collection is possible with relative ease and low short-term risk, and may open paths for diagnostic, therapeutic and research purposes in dogs. The procedure can provide new insights into prenatal clinical medicine, including diagnostics of foetal deaths, early identification of heritable diseases and so on.

Leishmania tropica infection in golden jackals and red foxes, Israel.

During a survey of wild canids, internal transcribed spacer 1 real-time PCR and high-resolution melt analysis identified Leishmania tropica in samples from jackals and foxes. Infection was most prevalent in ear and spleen samples. Jackals and foxes may play a role in the spread of zoonotic L. tropica.

Rapid differentiation of Mycobacterium tuberculosis and M. bovis by high-resolution melt curve analysis.

Identification and characterization of the Mycobacterium tuberculosis strains are important for clinical and therapeutic management of tuberculosis. Real-time PCR with a high-resolution melt assay was found to improve the diagnostic process. The assay includes differentiation between M. tuberculosis and Mycobacterium bovis based on one single-nucleotide polymorphism (SNP) in the narGHJI and oxyR genes and determination of M. bovis based on the region of differences 1 (RD1). This assay correctly identified the 7 tested Mycobacterium reference strains and 52 clinical samples with a sensitivity of 2 pg DNA. This assay will help in prescribing adequate treatment and monitoring disease dynamics.

Mesozoic origin for West Indian insectivores.

The highly endangered solenodons, endemic to Cuba (Solenodon cubanus) and Hispaniola (S. paradoxus), comprise the only two surviving species of West Indian insectivores. Combined gene sequences (13.9 kilobases) from S. paradoxus established that solenodons diverged from other eulipotyphlan insectivores 76 million years ago in the Cretaceous period, which is consistent with vicariance, though also compatible with dispersal. A sequence of 1.6 kilobases of mitochondrial DNA from S. cubanus indicated a deep divergence of 25 million years versus the congeneric S. paradoxus, which is consistent with vicariant origins as tectonic forces separated Cuba and Hispaniola. Efforts to prevent extinction of the two surviving solenodon species would conserve an entire lineage as old or older than many mammalian orders.

DNA analysis of a 30,000-year-old Urocitellus glacialis from northeastern Siberia reveals phylogenetic relationships between ancient and present-day arctic ground squirrels.

In contrast to the abundant fossil record of arctic ground squirrels, Urocitellus parryii, from eastern Beringia, only a limited number of fossils is known from its western part. In 1946, unnamed GULAG prisoners discovered a nest with three mummified carcasses of arctic ground squirrels in the permafrost sediments of the El'ga river, Yakutia, Russia, that were later attributed to a new species, Citellus (Urocitellus) glacialis Vinogr. To verify this assignment and to explore phylogenetic relationships between ancient and present-day arctic ground squirrels, we performed 14C dating and ancient DNA analyses of one of the El'ga mummies and four contemporaneous fossils from Duvanny Yar, northeastern Yakutia. Phylogenetic reconstructions, based on complete cytochrome b gene sequences of five Late Pleistocene arctic ground squirrels and those of modern U. parryii from 21 locations across western Beringia, provided no support for earlier proposals that ancient arctic ground squirrels from Siberia constitute a distinct species. In fact, we observed genetic continuity of the glacialis mitochondrial DNA lineage in modern U. parryii of the Kamchatka peninsula. When viewed in a broader geographic perspective, our findings provide new insights into the genetic history of U. parryii in Late Pleistocene Beringia.

Before they are gone - improving gazelle protection using wildlife forensic genetics.

Throughout their habitats gazelles (genus Gazella) face immediate threats due to anthropogenic effects and natural environmental changes. Excessive poaching plays a major role in their populations decline. Three unique populations of gazelles currently live in Israel: mountain gazelle (Gazella gazella), Dorcas gazelle (Gazella Dorcas) and acacia gazelle (Gazella arabica acacia). Ongoing habitat degradation and constant pressure from illegal hunting has caused a continuous decrease in the last 10 years, stressing the need for drastic measures to prevent species extinction. Wildlife forensic science assists enforcement agencies in the escalating arms race against poachers. Wildlife forensic genetic tests being implemented in our laboratory offer both species and individual identification, which rely on two mitochondrial genes (12S rRNA and 16S rRNA) and nine nuclear Short Tandem Repeats (STR), respectively. The current study, presents a poaching case in which mitochondrial DNA-based species identification revealed the presence of mountain gazelle DNA on the seized items. Subsequently, STR markers linked the suspect to more than one gazelle, increasing the severity of the criminal charges.

Wild gazelles of the southern Levant: genetic profiling defines new conservation priorities.

The mountain gazelle (Gazella gazelle), Dorcas gazelle (Gazella Dorcas) and acacia gazelle (Gazella arabica acacia) were historically abundant in the southern Levant, and more specifically in Israel. Anthropogenic and natural changes have caused a rapid decline in gazelle populations, raising concerns about their conservation status and future survival. The genetic profile of 111 wild gazelles from Israel was determined based on three regions of mitochondrial DNA (control region, Cytochrome b and 12S ribosomal RNA) and nine nuclear microsatellite markers. Genetic analysis of the mountain gazelle population, the largest known population of this rare species, revealed adequate diversity levels and gene flow between subpopulations. Nevertheless, ongoing habitat degradation and other human effects, such as poaching, suggest the need for drastic measures to prevent species extinction. Dorcas gazelles in Israel displayed inbreeding within subpopulations while still maintaining considerable genetic diversity overall. This stable population, represented by a distinctive genetic profile, is fragmented and isolated from its relatives in neighboring localities. Based on the genetic profile of a newly sampled subpopulation in Israel, we provide an alternative hypothesis for the historic dispersal of Dorcas gazelle, from the Southern Levant to northern Africa. The small acacia gazelle population was closest to gazelles from the Farasan Islands of Saudi Arabia, based on mitochondrial markers. The two populations did not share haplotypes, suggesting that these two populations may be the last remnant wild gazelles of this species worldwide. Only a dozen acacia gazelles survive in Israel, and urgent steps are needed to ensure the survival of this genetically distinctive lineage. The genetic assessments of our study recognize new conservation priorities for each gazelle species in the Southern Levant.

Evolutionary changes in the genome of Mycobacterium tuberculosis and the human genome from 9000 years BP until modern times.

The demonstration of Mycobacterium tuberculosis DNA in ancient skeletons gives researchers an insight into its evolution. Findings of the last two decades sketched the biological relationships between the various species of tubercle bacilli, the time scale involved, their possible origin and dispersal. This paper includes the available evidence and on-going research. In the submerged Eastern Mediterranean Neolithic village of Atlit Yam (9000 BP), a human lineage of M. tuberculosis, defined by the TbD1 deletion in its genome, was demonstrated. An infected infant at the site provides an example of active tuberculosis in a human with a naïve immune system. Over 4000 years later tuberculosis was found in Jericho. Urbanization increases population density encouraging M. tuberculosis/human co-evolution. As susceptible humans die of tuberculosis, survivors develop genetic resistance to disease. Thus in 18th century Hungarian mummies from Vác, 65% were positive for tuberculosis yet a 95-year-old woman had clearly survived a childhood Ghon lesion. Whole genome studies are in progress, to detect changes over the millennia both in bacterial virulence and also host susceptibility/resistance genes that determine the NRAMP protein and Killer Cell Immunoglobulin-like Receptors (KIRs). This paper surveys present evidence and includes initial findings.

Lipid biomarkers provide evolutionary signposts for the oldest known cases of tuberculosis.

Studies on the evolution of tuberculosis, and the influence of this disease on human and animal development and interaction, require the accumulation of indisputable biomarker evidence. Ideally, the determination of full genomes would provide all the necessary information, but for very old specimens DNA preservation may be compromised and only limited DNA amplification may be a possibility. Mycobacterium tuberculosis is characterised by the presence of unusual cell envelope lipids, with specific biomarker potential. Lipid biomarker recognition has been decisive in pinpointing the oldest known cases of human and animal tuberculosis; the former are a woman and child from a pre-pottery settlement at Atlit-Yam, Israel (∼9,000 ka) and the latter is an extinct Bison antiquus from Natural Trap Cave, Wyoming (∼17,000 ka). Including some new data, it is demonstrated how analysis of a combination of mycolic, mycocerosic and mycolipenic acid and phthiocerol biomarkers provide incontrovertible evidence for tuberculosis in these landmark specimens.

Tuberculosis origin: The Neolithic scenario.

This paper follows the dramatic changes in scientific research during the last 20 years regarding the relationship between the Mycobacterium tuberculosis complex and its hosts - bovids and/or humans. Once the M. tuberculosis and Mycobacterium bovis genomes were sequenced, it became obvious that the old story of M. bovis evolving into the human pathogen should be reversed, as M. tuberculosis is more ancestral than M. bovis. Nevertheless, the timescale and geographical origin remained an enigma. In the current study human and cattle bone samples were examined for evidence of tuberculosis from the site of Atlit-Yam in the Eastern Mediterranean, dating from 9250 to 8160 (calibrated) years ago. Strict precautions were used to prevent contamination in the DNA analysis, and independent centers used to confirm authenticity of findings. DNA from five M. tuberculosis genetic loci was detected and had characteristics consistent with extant genetic lineages. High performance liquid chromatography was used as an independent method of verification and it directly detected mycolic acid lipid biomarkers, specific for the M. tuberculosis complex. These, together with pathological changes detected in some of the bones, confirm the presence of the disease in the Levantine populations during the Pre-pottery Neolithic C period, more than 8000 years ago.

Evolutionary Genomics and Conservation of the Endangered Przewalski's Horse.

Przewalski's horses (PHs, Equus ferus ssp. przewalskii) were discovered in the Asian steppes in the 1870s and represent the last remaining true wild horses. PHs became extinct in the wild in the 1960s but survived in captivity, thanks to major conservation efforts. The current population is still endangered, with just 2,109 individuals, one-quarter of which are in Chinese and Mongolian reintroduction reserves [1]. These horses descend from a founding population of 12 wild-caught PHs and possibly up to four domesticated individuals [2-4]. With a stocky build, an erect mane, and stripped and short legs, they are phenotypically and behaviorally distinct from domesticated horses (DHs, Equus caballus). Here, we sequenced the complete genomes of 11 PHs, representing all founding lineages, and five historical specimens dated to 1878-1929 CE, including the Holotype. These were compared to the hitherto-most-extensive genome dataset characterized for horses, comprising 21 new genomes. We found that loci showing the most genetic differentiation with DHs were enriched in genes involved in metabolism, cardiac disorders, muscle contraction, reproduction, behavior, and signaling pathways. We also show that DH and PH populations split ∼45,000 years ago and have remained connected by gene-flow thereafter. Finally, we monitor the genomic impact of ∼110 years of captivity, revealing reduced heterozygosity, increased inbreeding, and variable introgression of domestic alleles, ranging from non-detectable to as much as 31.1%. This, together with the identification of ancestry informative markers and corrections to the International Studbook, establishes a framework for evaluating the persistence of genetic variation in future reintroduced populations.

Tuberculosis: from prehistory to Robert Koch, as revealed by ancient DNA.

During the past 10 years palaeomicrobiology, a new scientific discipline, has developed. The study of ancient pathogens by direct detection of their DNA has answered several historical questions and shown changes to pathogens over time. However, ancient DNA (aDNA) continues to be controversial and great care is needed to provide valid data. Here we review the most successful application of the technology, which is the study of tuberculosis. This has provided direct support for the current theory of Mycobacterium tuberculosis evolution, and suggests areas of investigation for the interaction of M tuberculosis with its host.

A possible case of cherubism in a 17th-century Korean mummy.

Cherubism is a benign fibro-osseous disease of childhood limited specifically to the maxilla and mandible. The progressive replacement of the jaw bones with expansile multilocular cystic lesions causes eventual prominence of the lower face, and hence the classic "cherubic" phenotype reflecting variable extents of jaw hypertrophy. Histologically, this condition has been characterized as replacement of the normal bone matrix with multicystic pockets of fibrous stroma and osteoclastic giant cells. Because of radiographic features common to both, primarily the presence of multiloculated lucencies with heterogeneous "ground-glass" sclerosis on CT imaging, cherubism was long mistaken for a craniofacial subtype of fibrous dysplasia. In 1999, however, the distinct genetic basis for cherubism was mapped to chromosome 4p16.3 and the SH-3 binding protein SH3BP2. But while there are already three suspected cases of fibrous dysplasia amongst archaeological populations, no definitive cases of cherubism have yet been reported in historical populations. In the current study we describe micro- and macro-structural changes in the face of a 17th century Joseon Dynasty Korean mummy which may coincide with the clinic-pathologic and radiologic features of cherubism.