Stina S. Dahlgren

Molecular characterization of five Sarcocystis species in red deer (Cervus elaphus), including Sarcocystis hjorti n. sp., reveals that these species are not intermediate host specific

Muscle tissue from 37 red deer from Norway was examined for sarcocysts. Sarcocysts from 2 reindeer were obtained for comparative studies. Cysts were excised and morphologically classified by light microscopy, scanning electron microscopy, and DNA sequence analysis. Five Sarcocystis species, Sarcocystis hjorti n. sp., Sarcocystis hardangeri, Sarcocystis ovalis, Sarcocystis rangiferi, and Sarcocystis tarandi, were found. All 5 species have previously been identified from either reindeer or moose by their sarcocyst morphology and/or ssu rRNA gene sequence. S. hjorti was the most prevalent species. Multiple variants of the ssu rRNA gene and the first internal transcribed spacer were found in S. rangiferi and S. tarandi from both red deer and reindeer. Phylogenetic analyses indicated that S. tarandi occurs in both red deer and reindeer, but it could not be clearly demonstrated whether the sequence variation within S. rangiferi between hosts was due to different paralogues or/and different species. DNA sequencing was necessary for definitive species identification, since the hair-like protrusions on the cysts of S. hjorti were not always recognizable by light microscopy and since different cervids harbour Sarcocystis species with highly similar cyst morphology of which at least some are not intermediate host specific.

Sarcocystis in moose (Alces alces): molecular identification and phylogeny of six Sarcocystis species in moose, and a morphological description of three new species.

Muscle tissues from 34 moose from Southeastern Norway and two moose from Canada were examined. Sarcocysts were excised and morphologically classified by light microscopy, and some cysts were further examined by scanning electron microscopy or DNA amplification and sequencing at the small subunit (ssu) rRNA gene. In Norwegian moose, three sarcocyst types were recognized, yet five Sarcocystis species were found by sequence analysis. New names were proposed for three species which could be characterised by both morphological and molecular methods, i.e., Sarcocystis alces, Sarcocystis ovalis, and Sarcocystis scandinavica. S. alces was the most prevalent species, whereas S. scandinavica and the two unnamed species were rare and might either use another principal intermediate host or a rare definitive host. The five species in Norwegian moose were different from Sarcocystis alceslatrans isolated from a Canadian moose. Phylogenetic analyses based on complete ssu rRNA gene sequences revealed a close relationship between the six Sarcocystis species from moose and species from reindeer and Sika deer. We conclude that molecular methods are necessary for unequivocal species identification, as different cervid hosts harbour morphologically indistinguishable sarcocysts.

The red fox ( Vulpes vulpes ) and the arctic fox ( Vulpes lagopus ) are definitive hosts of Sarcocystis alces and Sarcocystis hjorti from moose ( Alces alces )

The aim of this study was to determine whether foxes might act as definitive hosts of Sarcocystis alces in moose. In 2 experiments, 6 silver foxes (Vulpes vulpes) and 6 blue foxes (Vulpes lagopus) were fed muscle tissue from moose containing numerous sarcocysts of S. alces, and euthanazed 7-28 days post-infection (p.i.). Intestinal mucosal scrapings and faecal samples were screened microscopically for Sarcocystis oocysts/sporocysts, which were identified to species by means of species-specific primers and sequence analysis targeting the ssu rRNA gene. All foxes in both experiments became infected with Sarcocystis; the oocysts were fully sporulated by 14 days p.i., containing sporocysts measuring 14-15 x 10 microm. Molecular identification revealed that the oocysts/sporocysts belonged to 2 species, S. alces and Sarcocystis hjorti, although sarcocysts of S. hjorti were only identified in moose subsequent to the infection of foxes. In the first experiment, all 8 foxes also became infected with a Hammondia sp. derived from moose, shedding unsporulated, subspherical oocysts, measuring 10-12 microm in diameter, from 6-7 days p.i. onwards. The study proved that canids (the red fox and arctic fox) are definitive hosts for S. alces and S. hjorti, as had been inferred from the phylogenetic position of these species.

Hammondia triffittae n. comb. of foxes (Vulpes spp.): biological and molecular characteristics and differentiation from Hammondia heydorni of dogs

Genomic DNAs from 3 oocyst isolates of Hammondia sp. from foxes (Vulpes vulpes and V. lagopus) and 1 oocyst isolate of Hammondia heydorni from a dog, were examined by PCR and sequence analysis of 6 loci in order to determine whether the isolates were conspecific. Consistent genetic differences were found between the fox and dog isolates, respectively, at the ITS-2 region, the lsu rRNA gene, the alpha tubulin gene and the HSP70 gene, but not at the ssu rRNA gene or ITS-1 locus. Infection experiments established that dogs were unsuitable as definitive hosts for Hammondia sp. of foxes; hence this species is regarded as separate from H. heydorni of wolf-like canids, but probably identical with Isospora triffittae (syn. Isospora triffitti) previously reported from foxes. This species has therefore been named Hammondia triffittae n. comb. Reindeer, moose, sheep, goats, foxes and rabbits may act as intermediate hosts for H. triffittae. Muscle tissues of inoculated intermediate hosts were infectious for foxes from 16 days post-infection. Oocysts of H. triffittae were subspherical, averaging 12.5 × 10.9 μm in size. The lsu rRNA gene and the alpha tubulin gene seem to be suitable genetic markers for differentiating between H. triffittae and H. heydorni.

Sarcocystis in Norwegian roe deer (Capreolus capreolus): molecular and morphological identification of Sarcocystis oviformis n. sp. and Sarcocystis gracilis and their phylogenetic relationship with other Sarcocystis species

Fresh muscle tissue from six roe deer from Southeastern Norway was examined for sarcocysts. Cysts were excised and morphologically classified by light microscopy, and some cysts were further examined by scanning electron microscopy or DNA amplification and sequencing of the small subunit (ssu) rRNA gene. Two Sarcocystis species, Sarcocystis gracilis and Sarcocystis oviformis n. sp., were found and described by morphological and molecular methods. S. gracilis was found in all animals, whereas S. oviformis was found in only one roe deer. Polymerase chain reaction identification was necessary for definitive species identification, since cysts of S. gracilis varied in surface structure and since cysts of both S. gracilis and S. oviformis were morphologically indistinguishable from sarcocysts in other cervidae. Phylogenetic analyses based on complete ssu rRNA gene sequences revealed a close relationship between S. gracilis and other canine-transmitted Sarcocystis species, whereas S. oviformis formed a well-supported group with Sarcocystis hardangeri of reindeer and Sarcocystis ovalis of moose.

A Multi-Breed Genome-Wide Association Analysis for Canine Hypothyroidism Identifies a Shared Major Risk Locus on CFA12

Hypothyroidism is a complex clinical condition found in both humans and dogs, thought to be caused by a combination of genetic and environmental factors. In this study we present a multi-breed analysis of predisposing genetic risk factors for hypothyroidism in dogs using three high-risk breeds—the Gordon Setter, Hovawart and the Rhodesian Ridgeback. Using a genome-wide association approach and meta-analysis, we identified a major hypothyroidism risk locus shared by these breeds on chromosome 12 (p = 2.1x10-11). Further characterisation of the candidate region revealed a shared ~167 kb risk haplotype (4,915,018–5,081,823 bp), tagged by two SNPs in almost complete linkage disequilibrium. This breed-shared risk haplotype includes three genes (LHFPL5, SRPK1 and SLC26A8) and does not extend to the dog leukocyte antigen (DLA) class II gene cluster located in the vicinity. These three genes have not been identified as candidate genes for hypothyroid disease previously, but have functions that could potentially contribute to the development of the disease. Our results implicate the potential involvement of novel genes and pathways for the development of canine hypothyroidism, raising new possibilities for screening, breeding programmes and treatments in dogs. This study may also contribute to our understanding of the genetic etiology of human hypothyroid disease, which is one of the most common endocrine disorders in humans.

A genome-wide association study identifies a region strongly associated with symmetrical onychomadesis on chromosome 12 in dogs.

Symmetrical onychomadesis causes periodic loss of claws in otherwise healthy dogs. Genome-wide association analysis in 225 Gordon Setters identified a single region associated with symmetrical onychomadesis on chromosome 12 (spanning about 3.3 mb). A meta-analysis including also English Setters indicated that this genomic region predisposes for symmetrical onychomadesis in English Setters as well. The associated region spans most of the major histocompatibility complex and nearly 1 Mb downstream. Like many other autoimmune diseases, associations of symmetrical onychomadesis with DLA class II alleles have been reported. In this study, no associated markers were revealed within any of the DLA-DRB1, -DQA1 or -DQB1 genes, and the odds for symmetrical onychomadesis in the Gordon Setters were much higher, carrying significant single nucleotide polymorphisms compared to the odds of any of the recorded DLA-DRB1/DQA1/DQB1 haplotypes. We noticed that some of the associated DLA haplotypes were different between the English Setters and the Gordon Setters. Interestingly, associated SNP chip markers showed a more consistent pattern of allelic variants related to cases or controls regardless of breed. In conclusion, the associated genetic markers identified in this study hold the potential to aid in selection of breeding animals to reduce the frequency of symmetrical onychomadesis in the dog.