Genomic and Transcriptomic Characterization of Atypical Recurrent Flank Alopecia in the Cesky Fousek.

Non-inflammatory alopecia is a frequent skin problem in dogs, causing damaged coat integrity and compromised appearance of affected individuals. In this study, we examined the Cesky Fousek breed, which displays atypical recurrent flank alopecia (aRFA) at a high frequency. This type of alopecia can be quite severe and is characterized by seasonal episodes of well demarcated alopecic areas without hyperpigmentation. The genetic component responsible for aRFA remains unknown. Thus, here we aimed to identify variants involved in aRFA using a combination of histological, genomic, and transcriptomic data. We showed that aRFA is histologically similar to recurrent flank alopecia, characterized by a lack of anagen hair follicles and the presence of severely shortened telogen or kenogen hair follicles. We performed a genome-wide association study (GWAS) using 216 dogs phenotyped for aRFA and identified associations on chromosomes 19, 8, 30, 36, and 21, highlighting 144 candidate genes, which suggests a polygenic basis for aRFA. By comparing the skin cell transcription pattern of six aRFA and five control dogs, we identified 236 strongly differentially expressed genes (DEGs). We showed that the GWAS genes associated with aRFA are often predicted to interact with DEGs, suggesting their joint contribution to the development of the disease. Together, these genes affect four major metabolic pathways connected to aRFA: collagen formation, muscle structure/contraction, lipid metabolism, and the immune system.

Actinomycosis in a gray four-eyed opossum (Philander opossum) caused by a novel species of Schaalia.

BACKGROUND: Infective lesions of the jaws and adjacent tissues (lumpy jaw disease, LJD) have been recognized as one major cause of death of captive macropods. Fusobacterium necrophorum and Actinomyces species serve as the main source of LJD in kangaroos and wallabies. Currently, little is reported about LJD or similar diseases in opossums. CASE PRESENTATION: Here we report a case of actinomycosis resembling the entity lumpy jaw disease in a gray four-eyed opossum, caused by a novel species of Schaalia. A 2.8 year old male Philander opossum was presented with unilateral swelling of the right mandible. After an initial treatment with marbofloxacin, the opossum was found dead the following day and the carcass was submitted for necropsy. Postmortem examination revealed severe mandibular skin and underlying soft tissue infection with subsequent septicemia as the cause of death. Histological examination demonstrated Splendore-Hoeppli phenomenon, typically seen in classical cases of actinomycosis. Bacteriology of liver and mandibular mass yielded a previously undescribed species of Schaalia, whose 16 S rRNA gene sequence was 97.0 % identical to Schaalia canis. Whole genome sequencing of the opossum isolate and calculation of average nucleotide identity confirmed a novel species of Schaalia, for which no whole genome sequence is yet available. CONCLUSIONS: The herewith reported Schaalia infection in the gray four-eyed opossum resembling classical actinomycosis gives a novel insight into new exotic animal bacterial diseases. Schaalia species may belong to the normal oral microbiome, as in macropods, and may serve as a contributor to opportunistic infections. Due to the lack of current literature, more insights and improved knowledge about Schaalia spp. and their pathogenicity will be useful to choose appropriate therapy regimens and improve the treatment success rate and outcome in exotic and endangered species.

Phenotypic and Genomic Analysis of Cystic Hygroma in Pigs.

Cystic hygroma is a malformation of the lymphatic and vascular system and is recognized as a benign congenital tumor that affects humans and animals in the perinatal period. This congeni-tal disorder is rarely described in animals, and until today, cystic hygroma in pigs has not been described in the literature. In a purebred Piètrain litter with twelve live-born piglets, cystic hy-groma was noticed on the rump of two male pigs within the first week of life. In addition, a third case of a crossbred weaner (Large White × Landrace) was detected during a herd examina-tion. To rule out common differential diagnoses, e.g., abscess or hematoma, further clinical and pathological investigations were conducted. During clinical examination, a painless and soft mass, which was compressible, was detected on the rump of all affected animals. The ultra-sonographic examination revealed a fluid-filled and cavernous subcutaneous structure. In addi-tion, a puncture of the cyst was conducted, revealing a serosanguinous fluid with negative bacte-riological culture. In all cases, a necropsy was performed, showing that the animals had fluid-filled cysts lined by well-differentiated lymphatic endothelium. Based on the clinicopathological examination, cystic hygroma was diagnosed. Furthermore, SNP array genotyping and whole-genome sequencing was performed and provided no evidence for a chromosomal disorder. In the Piètrain family, several genome regions were homozygous in both affected piglets. None-theless, a dominant acting de novo germline variant could not be ruled out, and therefore differ-ent filtering strategies were used to find pathogenic variants. The herein presented lists of pri-vate variants after filtering against hundreds of control genomes provide no plausible candidate and no shared variants among the two sequenced cases. Therefore, further studies are needed to evaluate possible genetic etiology. In general, systematic surveillance is needed to identify ge-netic defects as early as possible and to avoid the occurrence of losses in the pig population.

An Evolutionary Cancer Epigenetic Approach Revealed DNA Hypermethylation of Ultra-Conserved Non-Coding Elements in Squamous Cell Carcinoma of Different Mammalian Species.

BACKGROUND: Ultra-conserved non-coding elements (UCNEs) are genomic sequences that exhibit > 95% sequence identity between humans, mammals, birds, reptiles, and fish. Recent findings reported their functional role in cancer. The aim of this study was to evaluate the DNA methylation modifications of UNCEs in squamous cell carcinoma (SCC) from different mammal species. METHODS: Fifty SCCs from 26 humans, 17 cats, 3 dogs, 1 horse, 1 bovine, 1 badger, and 1 porcupine were investigated. Fourteen feline stomatitis and normal samples from 36 healthy human donors, 7 cats, 5 dogs, 5 horses, 2 bovines and 1 badger were collected as normal controls. Bisulfite next generation sequencing evaluated the DNA methylation level from seven UCNEs (uc.160, uc.283, uc.416, uc.339, uc.270, uc.299, and uc.328). RESULTS: 57/59 CpGs were significantly different according to the Kruskal-Wallis test (p < 0.05) comparing normal samples with SCC. A common DNA hypermethylation pattern was observed in SCCs from all the species evaluated in this study, with an increasing trend of hypermethylation starting from normal mucosa, through stomatitis to SCC. CONCLUSIONS: Our findings indicate that UCNEs are hypermethylated in human SCC, and this behavior is also conserved among different species of mammals.

Dystrophic Mineralization of Costal Cartilage in Hartley Guinea Pigs.

Hartley guinea pigs are widely used animal models of disease, particularly in studies of osteoarthritis. The purpose of this study was to investigate lesions in the costal cartilage from 16 male, 5- to 6-month-old Hartley guinea pigs. Routine histological sections from the costal cartilage and costochondral junction (longitudinal and cross sections) and sternum (for evaluation of bone marrow) were examined. All 16 (100%) animals had histological lesions involving the costal cartilage that included matrix degeneration and mineralization, reduced cellularity, and evidence of chondrocyte necrosis. Of the 16, 4 (25%) of the lesions contained blood vessels and 3 (19%) contained central osseous metaplasia. The cartilage lesions were accompanied by degeneration (sometimes with regeneration and/or fibrosis) in adjacent skeletal muscle in 15 of the 16 (94%) animals. The lesions in the costal cartilage were interpreted as dystrophic mineralization of unknown cause and appear to be incidental findings, although they bear some resemblance to lesions occurring in Tietze's disease in humans. The significance of the lesions in skeletal muscle is unclear. Histological lesions of cartilage matrix degeneration and mineralization in these sites have not, to our knowledge, been reported previously.

The role of the dynein light intermediate chain in retrograde IFT and flagellar function in Chlamydomonas.

The assembly of cilia and flagella depends on the activity of two microtubule motor complexes, kinesin-2 and dynein-2/1b, but the specific functions of the different subunits are poorly defined. Here we analyze Chlamydomonas strains expressing different amounts of the dynein 1b light intermediate chain (D1bLIC). Disruption of D1bLIC alters the stability of the dynein 1b complex and reduces both the frequency and velocity of retrograde intraflagellar transport (IFT), but it does not eliminate retrograde IFT. Flagellar assembly, motility, gliding, and mating are altered in a dose-dependent manner. iTRAQ-based proteomics identifies a small subset of proteins that are significantly reduced or elevated in d1blic flagella. Transformation with D1bLIC-GFP rescues the mutant phenotypes, and D1bLIC-GFP assembles into the dynein 1b complex at wild-type levels. D1bLIC-GFP is transported with anterograde IFT particles to the flagellar tip, dissociates into smaller particles, and begins processive retrograde IFT in <2 s. These studies demonstrate the role of D1bLIC in facilitating the recycling of IFT subunits and other proteins, identify new components potentially involved in the regulation of IFT, flagellar assembly, and flagellar signaling, and provide insight into the role of D1bLIC and retrograde IFT in other organisms.

A differential cargo-loading model of ciliary length regulation by IFT.

BACKGROUND: During the assembly and maintenance of cilia, precursor proteins need to be transported from the cell body into the organelle. Intraflagellar transport (IFT) is assumed to be the predominant protein transport pathway in cilia, but it remains largely unknown how ciliary proteins use IFT to reach their destination sites in the cilium and whether the amount of cargo transported by IFT is regulated. RESULTS: Single-particle imaging showed that DRC4, a structural protein of the axoneme, moves in association with IFT particles inside Chlamydomonas reinhardtii cilia. IFT is required for DRC4 transport both into and within the cilium. DRC4 cargoes dissociate from IFT trains at the tip as well as at various sites along the length of the cilium. Unloaded DRC4 diffuses before docking at its axonemal assembly site. In growing cilia, DRC4 transport by IFT was strongly increased over the steady-state level, and the frequency decreased linearly with the increasing ciliary length. The frequency of DRC4 transport was similarly elevated in short growth-arrested cilia and remained high even when the amount of DRC4 available in the cell body was reduced. CONCLUSIONS: DRC4 is a bona fide cargo of IFT. Incompletely assembled cilia trigger an increase in the amount of DRC4 cargo transported by IFT particles, and DRC4 transport is downregulated as cilia approach their steady-state length. We propose a model in which ciliary length is controlled by regulating the amount of cargo transported by IFT particles.