A new Finnish flavor of feline coat coloration, "salmiak," is associated with a 95-kb deletion downstream of the KIT gene.

Cats with a distinctive white hair pattern of unknown molecular cause have been discovered in the Finnish domestic cat population. Based on the unique appearance of these cats, we have named this phenotype salmiak ("salty licorice"). The use of a commercially available panel test to genotype four salmiak-colored cats revealed the absence of all known variants associated with white-haired phenotypic loci: full White (W), Spotting (Ws) and the Birman white Gloves associated (wg) allele of the KIT proto-oncogene (KIT) gene. Whole-genome sequencing on two salmiak-colored cats was conducted to search for candidate causal variants in the KIT gene. Despite a lack of coding variants, visual inspection of the short read alignments revealed a large ~95 kb deletion located ~65 kb downstream of the KIT gene in the salmiak cats. Additional PCR genotyping of 180 domestic cats and three salmiak-colored cats confirmed the homozygous derived variant genotype fully concordant with the salmiak phenotype. We suggest the newly identified variant be designated as wsal for "w salmiak".

Biochemical and molecular characterization of Campylobacter fetus isolates from bulls subjected to bovine genital campylobacteriosis diagnosis in Spain.

BACKGROUND: Bovine genital campylobacteriosis (BGC) is caused by Campylobacter fetus subsp. venerealis (Cfv) including its biovar intermedius (Cfvi). This sexually transmitted disease induces early reproductive failure causing considerable economic losses in the cattle industry. Using a collection of well-characterized isolates (n = 13), C. fetus field isolates (n = 64) and saprophytic isolates resembling Campylobacter (n = 75) obtained from smegma samples of breeding bulls, this study evaluated the concordance of the most used phenotypic (H2S production in cysteine medium and 1% glycine tolerance) and molecular (PCR) methods for the diagnosis of BGC and assessed possible cross-reactions in the molecular diagnostic methods. RESULTS: Characterization at the subspecies level (fetus vs. venerealis) of C. fetus isolated from bull preputial samples using phenotypic and molecular (PCR targeting nahE and ISCfe1) methods showed moderate concordance (κ = 0.462; CI: 0.256-0.669). No cross-reactions were observed with other saprophytic microaerophilic species or with other Campylobacter species that can be present in preputial samples. Whole genome sequencing (WGS) of discrepant isolates showed 100% agreement with PCR identification. For the differentiation of Cfv biovars, comparison of the H2S test (at 72 h and 5 days of incubation) and a PCR targeting the L-cysteine transporter genes showed higher concordance when H2S production was assessed after 5 days (72 h; κ = 0.553, 0.329-0.778 CI vs. 5 days; κ = 0.881, 0.631-1 CI), evidencing the efficacy of a longer incubation time. CONCLUSIONS: This study confirmed the limitations of biochemical tests to correctly identify C. fetus subspecies and biovars. However, in the case of biovars, when extended incubation times for the H2S test (5 days) were used, phenotypic identification results were significantly improved, although PCR-based methods produced more accurate results. Perfect agreement of WGS with the PCR results and absence of cross-reactions with non-C. fetus saprophytic bacteria from the smegma demonstrated the usefulness of these methods. Nevertheless, the identification of new C. fetus subspecies-specific genes would help to improve BGC diagnosis.

Precision medicine using whole genome sequencing identifies a novel dystrophin (DMD) variant for X-linked muscular dystrophy in a cat.

BACKGROUND: Muscular dystrophies (MDs) are a large, heterogeneous group of degenerative muscle diseases. X-linked dystrophin-deficient MD in cats is the first genetically characterized cat model for a human disease and a few novel forms have been identified. HYPOTHESIS/OBJECTIVES: Muscular dystrophy was suspected in a young male domestic shorthair cat. Clinical, molecular, and genetic techniques could provide a definitive diagnosis. ANIMALS: A 1-year-old male domestic shorthair cat presented for progressive difficulty walking, macroglossia and dysphagia beginning at 6 months of age. The tongue was thickened, protruded with constant ptyalism, and thickening and rigidity of the neck and shoulders were observed. METHODS: A complete neurological examination, baseline laboratory evaluation and biopsies of the trapezius muscle were performed with owner consent. Indirect immunofluorescence staining of muscle cryosections was performed using several monoclonal and polyclonal antibodies against dystrophy-associated proteins. DNA was isolated for genomic analyses by whole genome sequencing and comparison to DNA variants in the 99 Lives Cat Genome Sequencing dataset. RESULTS AND CLINICAL IMPORTANCE: Aspartate aminotransferase (687 IU/L) and creatine kinase (24 830 IU/L) activities were increased and mild hypokalemia (3.7 mmol/L) was present. Biopsy samples from the trapezius muscle confirmed a degenerative and regenerative myopathy and protein alterations identified by immunohistochemistry resulted in a diagnosis of a in dystrophin-deficient form of X-linked MD. A stop gain variant (c.4849C>T; p.Gln1617Ter) dystrophin was identified by genome sequencing. Precision/genomic medicine efforts for the domestic cat and in veterinary medicine support disease variant and animal model discovery and provide opportunities for targeted treatments for companion animals.

A Salmonella Pullorum outbreak with neurological signs in adult layers and outbreak investigation using whole genome sequencing.

RESEARCH HIGHLIGHTS: Cerebral granulomas are associated with nervous signs in Salmonella Pullorum outbreak.Bone marrow is also a recommended tissue for isolation of Salmonella Pullorum.Rapid plate agglutination test detects Pullorum antibodies in a vaccinated flock.Phylogenetic analysis showed clonality of isolates within the outbreak.

Precision medicine using whole genome sequencing in a cat identifies a novel COL5A1 variant for classical Ehlers-Danlos syndrome.

BACKGROUND: Ehlers-Danlos syndromes (EDS) are a heterogeneous group of heritable connective tissue disorders occurring in both human and veterinary patients. The genetics of these disorders are poorly described in small animal patients. HYPOTHESIS/OBJECTIVES: Define the clinical manifestations and genetic cause of a suspected form of EDS in a cat. ANIMALS: A 14-week-old male domestic medium hair cat was presented with skin hyperextensibility and fragility. The classic tragic facial expression was observed as well as chronic pruritus and mild hyperesthesia. METHODS: Blood samples and a skin biopsy sample were collected from the affected cat. Clinical examinations, histology, electron microscopy and whole genome sequencing were conducted to characterize the clinical presentation and identify possible pathogenic DNA variants to support a diagnosis. Criteria defining variant pathogenicity were examined including human disease variant databases. RESULTS: Histology showed sparse, disorganized collagen and an increase in cutaneous mast cells. Electron microscopy identified ultrastructural defects commonly seen in collagen type V alpha 1 chain (COL5A1) variants including flower-like collagen fibrils in cross-section. Whole genome sequencing and comparison with 413 cats in the 99 Lives Cat Genome Sequencing Consortium database identified a novel splice acceptor site variant at exon 4 in COL5A1 (c.501-2A>C). CONCLUSIONS AND CLINICAL IMPORTANCE: Our report broadens the current understanding of EDS in veterinary patients and supports the use of precision medicine techniques in clinical veterinary practice. The classification of variants for pathogenicity should be considered in companion animals.

Molecular characterization, complete genome sequencing, and pathogenicity of Novel Duck Reovirus from South Coastal Area in China.

Novel Duck Reovirus (NDRV) that has been found throughout the world in waterfowl, and it has been extensively described. Here, we report the complete genome sequence of a NDRV strain isolated in China called NDRV YF10. This strain was collected from 87 samples with infected ducks in South Coastal Area. The NDRV genome consists of 23,419 bp. With the assistance of computer analysis, the promoter and terminator of each gene segment and 10 viral genes segments were identified, which encode polypeptides ranging from 98 to 1,294 amino acids. All gene fragments of this virus strain were determined and compared to previously reported strains, revealing genetic variation with similarity rates ranging from 96 to 99% for each gene segment. Each gene segment formed 2 host-associated groups, the waterfowl-derived reovirus and the avian-derived reovirus, except for the S1 gene segment, which was closely related to ARV evolution and formed a host-independent subcluster. This difference may be due to Avian Reovirus (ARV) evolving in a host-dependent manner. In order to evaluate the pathogenicity of YF10, a novel isolated strain of NDRV was tested in 2 types of ducks. It was observed that the YF10 isolated strain exhibits varying degrees of virulence, highlighting the potential risk posed to different types of ducks. In conclusion, our findings emphasize the importance of epidemiology studies, molecular characterization, and prevention of NDRV in waterfowl.

Whole-genome resequencing reveals genetic structure and introgression in Pudong White pigs.

Pudong White (PDW) pigs, historically originating from Shanghai, are the only Chinese indigenous pigs characterised by their completely white coats, with the exception of Rongchang pigs. However, there is limited information concerning their overall genetic structure or relationship with other breeds, especially the East Chinese (ECN) and European pigs. To uncover the genetic structure, selection signatures, and potential exotic introgression in PDW pigs, we sampled 15 PDW pigs using whole-genome sequencing (~20×). We then conducted in-depth population genetic analyses in 320 pigs from 27 global pig groups, namely, European wild boars, Chinese wild boars, and outgroup. Neighbour-joining tree and principal component analysis confirmed that PDW pigs belonged to the ecotype of ECN pigs. Both f3, D-statistics, and structure analysis showed that PDW pigs shared apparent alleles with Large White (LW) pigs. Three statistics, rIBD, a haplotype heat map and copy number variation, further indicated that PDW pigs shared apparent alleles with LW pigs at the KIT Proto-Oncogene, Receptor Tyrosine Kinase (KIT) and PARG-MARCHF8 loci, suggesting that the lineage of European pigs in PDW originated from LW pigs. After further detecting the KIT mutations in different pig breeds, PDW was confirmed to have the same duplication region 1, duplication region 2, and the splicing mutation on intron 17 of KIT as LW pigs that determine the white coat colour phenotype in European white pigs. We hypothesised that LW pigs were imported to China ∼110-160 years ago according to the admixture time estimate and then crossed with ECN pigs, resulting in the introgression of the KIT alleles that produce the white coat colour phenotype in the PDW pig breed. To our knowledge, this study presents the first thorough description of the genetic structure of PDW pigs via whole-genome resequencing data; moreover, the results provide a basis for the national project for the conservation of this unique Chinese local population.

A novel missense mutation of the NAT10 gene in a juvenile Schnauzer dog with chronic respiratory tract infections.

An 18-month-old intact male Schnauzer dog was evaluated for chronic, lifelong respiratory tract infections that were unresponsive to administration of a variety of antibiotics and corticosteroids. The dog developed persistent vomiting and diarrhea around 1 year of age that was minimally responsive to diet change, antibiotics, and corticosteroids. Despite supportive care, the dog was ultimately euthanized at 20 months of age due to persistent respiratory and gastrointestinal disease. Whole genome sequencing discovered a deleterious missense A/C mutation within the NAT10 gene, a gene essential for microtubule acetylation, appropriate ciliary development, and cytokinesis. Pipeline analysis of the genomes of 579 dogs from 55 breeds did not detect this mutation. Though never described in veterinary medicine, NAT10 mutation occurs in humans with ciliary aplasia, suggesting a pathophysiological mechanism for this dog and highlighting an associated mutation or possible novel genetic cause of chronic respiratory infections in dogs.

Complete genome analysis confirms that the pygmy marmoset adenovirus is a variant of the skunk adenovirus 1 - Short communication.

The complete genomic sequence along with phylogenetic analyses of an adenovirus (AdV), isolated from a dead captive pygmy marmoset (Callithrix pygmaea) from a Hungarian zoo is reported. Earlier, based on the phylogenetic analysis of the sequence of a PCR-amplified fragment from the DNA polymerase gene, the pygmy marmoset AdV (PMAdV) has been reported to cluster closest to certain chiropteran AdVs. In the following years similar AdVs were discovered in additional mammalian hosts, including a skunk (Mephitis mephitis), African pygmy hedgehogs (Atelerix albiventris), North American porcupines (Erethizon dorsatum) and grey fox (Urocyon cinereoargenteus). After the full genome analysis of the skunk adenovirus (SkAdV-1), a novel species Skunk mastadenovirus A (SkAdV-A) has been established. The AdVs, originating from the African pygmy hedgehogs, have been found to belong to virus species SkAdV-A. Partial gene sequences from the porcupine AdVs have also implied their very close genetic relatedness to SkAdV-A. The complete genomic sequence of PMAdV, examined in this study, was found to share 99.83% nucleotide identity with SkAdV-1, thus unequivocally represents a genomic variant of SkAdV-1. The observation that viruses classifiable as SkAdV-A are able to infect and cause diseases in several, distantly related mammals seems to deserve further studies to elucidate the infection biology of this intriguing AdV.

Molecular and Biological Characterization of a Cervidpoxvirus Isolated From Moose with Necrotizing Dermatitis.

Cervidpoxvirus is one of the more recently designated genera within the subfamily Chordopoxvirinae, with Deerpox virus (DPV) as the only recognized species to date. In this study, the authors describe spontaneous disease and infection in the North American moose (Alces americanus) by a novel Cervidpoxvirus, here named Moosepox virus (MPV). Three 4-month-old moose calves developed a multifocal subacute-to-chronic, necrotizing, suppurative-to-granulomatous dermatitis that affected the face and the extremities. Ultrastructurally, all stages of MPV morphogenesis-that is, crescents, spherical immature particles, mature particles, and enveloped mature virus-were observed in skin tissue. In vitro infection with MPV confirmed that its morphogenesis was similar to that of the prototype vaccinia virus. The entire coding region, including 170 putative genes of this MPV, was sequenced and annotated. The sequence length was 164,258 bp with 98.5% nucleotide identity with DPV (strain W-1170-84) based on the whole genome. The genome of the study virus was distinct from that of the reference strain (W-1170-84) in certain genes, including the CD30-like protein (83.9% nucleotide, 81.6% amino acid), the endothelin precursor (73.2% nucleotide including some indels, 51.4% amino acid), and major histocompatibility class (MHC) class I-like protein (81.0% nucleotide, 68.2% amino acid). This study provides biological characterization of a new Cervidpoxvirus attained through in vivo and in vitro ultrastructural analyses. It also demonstrates the importance of whole-genome sequencing in the molecular characterization of poxviruses identified in taxonomically related hosts.

A de novo germline mutation of KIT in a white-spotted Brown Swiss cow.

White-spotting coat colour phenotypes in cattle are either fixed characteristics of specific cattle breeds or occur sporadically owing to germline genetic variation of solid-coloured parents. A Brown Swiss cow showing a piebald pattern resembling colour-sidedness was referred for genetic evaluation. Both parents were normal solid-brown-coloured cattle. The cow was tested negative for the three known DNA variants in KIT, MITF and TWIST2 associated with different depigmentation phenotypes in Brown Swiss cattle. Whole-genome sequencing of the cow was performed and a heterozygous variant affecting the coding sequence of the bovine KIT gene was identified on chromosome 6. The variant is a 40 bp deletion in exon 9, NM_001166484.1:c.1390_1429del, and leads to a frameshift that is predicted to produce a novel 50 amino acid-long C-terminus replacing almost 50% of the wt KIT protein, including the functionally important intracellular tyrosine kinase domain (NP_001159956.1:p.(Asn464AlafsTer50)). Interestingly, among three available offspring, two solid-coloured daughters were genotyped as homozygous wt whereas a single son showing a slightly milder but still obvious depigmentation phenotype inherited a copy of the novel variant allele. The genetic findings provide strong evidence that the identified loss-of-function KIT variant most likely represents a de novo germline mutation that is causative owing to haploinsufficiency.

Duck Tembusu virus detection and characterization from mosquitoes in duck farms, Thailand.

Duck Tembusu virus (DTMUV), an emerging infectious disease in ducks, belongs to the Flavivirus genus and Flaviviridae family. The transmission of DTUMV involves mosquito vectors; however, the exact role of mosquitoes in the ecology of DTMUV in Thailand remains unclear. This study was conducted to examine DTMUV detection and characterization from mosquitoes in duck farms in central Thailand. Mosquitoes were collected from two duck farms in Sing Buri Province and two duck farms in Ang Thong Province from September 2015 to July 2016 using four CDC-light traps. A total of 30,841 mosquitoes were collected and identified to seven species (Anopheles (An.) barbirostris, An. stephensi, Culex (Cx.) gelidus, Cx. quinquefasciatus, Cx. tritaeniorhynchus, Mansonia (Ma.) annulifera and Ma. uniformis). The most common collected species from each duck farm and each collection time was Cx. tritaeniorhynchus. Mosquitoes were pooled according to species, location, and collection time and then examined for DTMUV by RT-PCR. A total of 273 mosquito pools were examined, with only one pool of Cx. tritaeniorhynchus collected from Sing Buri Province in November 2015 testing positive for DTMUV. Phylogenetic analysis of the polyprotein genes demonstrated that a mosquito-derived Thai DTMUV was grouped into subcluster 2.1 and most closely related to the 2013 Thai DTMUVs. Thus, this study indicated that Cx. tritaeniorhynchus may play a role as a vector in the transmission of DTMUV in Thailand. However, additional studies concerning the vector competence of this mosquito for DTMUV are needed.

Fowl Adenovirus (FAdV) Recombination with Intertypic Crossovers in Genomes of FAdV-D and FAdV-E, Displaying Hybrid Serological Phenotypes.

After analyzing 27 new genomes from fowl adenovirus (FAdV) field isolates and so-far unsequenced prototypes, we report the first evidence for recombination in FAdVs. Recombination was confined to species FAdV-D and FAdV-E, accommodating the largest number of, and the intraspecies-wise most differentiated, types. The majority of detected events occurred in FAdV-E, involving segments with parental origin of all constitutive types. Together with the diversity of breakpoints, this suggests widespread recombination in this species. With possible constraints through species-specific genes and diversification patterns, the recombinogenic potential of FAdVs attains particular interest for inclusion body hepatitis (IBH), an important disease in chickens, caused by types from the recombination-prone species. Autonomously evolving, recombinant segments were associated with major sites under positive selection, among them the capsid protein hexon and fiber genes, the right-terminal ORFs 19, 25, and the ORF20/20A family. The observed mosaicism in genes indicated as targets of adaptive pressures points toward an immune evasion strategy. Intertypic hexon/fiber-recombinants demonstrated hybrid neutralization profiles, retrospectively explaining reported controversies on reference strains B3-A, T8-A, and X11-A. Furthermore, cross-neutralization supported sequence-based evidence for interdomain recombination in fiber and contributed to a tentatively new type. Overall, our findings challenge the purported uniformity of types responsible for IBH, urging more complete identification strategies for FAdVs. Finally, important consequences arise for in vivo studies investigating cross-protection against IBH.

Precision medicine validation: identifying the MYBPC3 A31P variant with whole-genome sequencing in two Maine Coon cats with hypertrophic cardiomyopathy.

OBJECTIVES: The objective of this study was to perform a proof-of-concept experiment that validates a precision medicine approach to identify variants associated with hypertrophic cardiomyopathy (HCM). We hypothesized that whole-genome sequencing would identify variant(s) associated with HCM in two affected Maine Coon/Maine Coon cross cats when compared with 79 controls of various breeds. METHODS: Two affected and two control Maine Coon/Maine Coon cross cats had whole-genome sequencing performed at approximately × 30 coverage. Variants were called in these four cats and 77 cats of various breeds as part of the 99 Lives Cat Genome Sequencing Initiative ( http://felinegenetics.missouri.edu/99lives ) using Platypus v0.7.9.1, annotated with dbSNP ID, and variants' effect predicted by SnpEff. Strict filtering criteria (alternate allele frequency >49%) were applied to identify homozygous-alternate or heterozygous variants in the two HCM-affected samples when compared with 79 controls of various breeds. RESULTS: A total of four variants were identified in the two Maine Coon/Maine Coon cross cats with HCM when compared with 79 controls after strict filtering. Three of the variants identified in genes MFSD12, BTN1A1 and SLITRK5 did not segregate with disease in a separate cohort of seven HCM-affected and five control Maine Coon/Maine Coon cross cats. The remaining variant MYBPC3 segregated with disease status. Furthermore, this gene was previously associated with heart disease and encodes for a protein with sarcomeric function. CONCLUSIONS AND RELEVANCE: This proof-of-concept experiment identified the previously reported MYBPC3 A31P Maine Coon variant in two HCM-affected cases. This result validates and highlights the power of whole-genome sequencing for feline precision medicine.

Evaluation of genes associated with human myxomatous mitral valve disease in dogs with familial myxomatous mitral valve degeneration.

Myxomatous mitral valve disease (MMVD) is the most common heart disease in the dog. It is believed to be heritable in Cavalier King Charles spaniels (CKCS) and Dachshunds. Myxomatous mitral valve disease is a familial disease in human beings as well and genetic mutations have been associated with its development. We hypothesized that a genetic mutation associated with the development of the human form of MMVD was associated with the development of canine MMVD. DNA was isolated from blood samples from 10 CKCS and 10 Dachshunds diagnosed with MMVD, and whole genome sequences from each animal were obtained. Variant calling from whole genome sequencing data was performed using a standardized bioinformatics pipeline for all samples. After filtering, the canine genes orthologous to the human genes known to be associated with MMVD were identified and variants were assessed for likely pathogenic implications. No variant was found in any of the genes evaluated that was present in least eight of 10 affected CKCS or Dachshunds. Although mitral valve disease in the CKCS and Dachshund is a familial disease, we did not identify genetic cause in the genes responsible for the human disease in the dogs studied here.

A de novo mutation in the EXT2 gene associated with osteochondromatosis in a litter of American Staffordshire Terriers.

BACKGROUND: We aimed to identify mutations associated with osteochondromatosis in a litter of American Staffordshire Terrier puppies. HYPOTHESIS: We hypothesized that the associated mutation would be located in a gene that causes osteochondromatosis in humans. ANIMALS: A litter of 9 American Staffordshire puppies, their sire and dam, 3 of 4 grandparents, 26 healthy unrelated American Staffordshire Terriers, and 154 dogs of 27 different breeds. METHODS: Whole genome sequencing was performed on the proband, and variants were compared against polymorphisms derived from 154 additional dogs across 27 breeds, as well as single nucleotide polymorphism database 146. One variant was selected for follow-up sequencing. Parentage and genetic mosaicism were evaluated across the litter. RESULTS: We found 56,301 genetic variants unique to the proband. Eleven variants were located in or near the gene exostosin 2 (EXT2), which is strongly associated with osteochondromatosis in humans. One heterozygous variant (c.969C > A) is predicted to result in a stop codon in exon 5 of the gene. Sanger sequencing identified the identical mutation in all affected offspring. The mutation was absent in the unaffected offspring, both parents, all available grandparents, and 26 healthy unrelated American Staffordshire Terriers. CONCLUSIONS AND CLINICAL IMPORTANCE: These findings represent the first reported mutation associated with osteochondromatosis in dogs. Because this mutation arose de novo, the identical mutation is unlikely to be the cause of osteochondromatosis in other dogs. However, de novo mutations in EXT2 are common in humans with osteochondromatosis, and by extension, it is possible that dogs with osteochondromatosis could be identified by sequencing the entire EXT2 gene.