Alopecia X is a hair cycle arrest disorder in Pomeranians. Histologically, kenogen and telogen hair follicles predominate, whereas anagen follicles are sparse. The induction of anagen relies on the activation of hair follicle stem cells and their subsequent proliferation and differentiation. Stem cell function depends on finely tuned interactions of signaling molecules and transcription factors, which are not well defined in dogs. We performed transcriptome profiling on skin biopsies to analyze altered molecular pathways in alopecia X. Biopsies from five affected and four non-affected Pomeranians were investigated. Differential gene expression revealed a downregulation of key regulator genes of the Wnt (CTNNB1, LEF1, TCF3, WNT10B) and Shh (SHH, GLI1, SMO, PTCH2) pathways. In mice it has been shown that Wnt and Shh signaling results in stem cell activation and differentiation Thus our findings are in line with the lack of anagen hair follicles in dogs with Alopecia X. We also observed a significant downregulation of the stem cell markers SOX9, LHX2, LGR5, TCF7L1 and GLI1 whereas NFATc1, a quiescence marker, was upregulated in alopecia X. Moreover, genes coding for enzymes directly involved in the sex hormone metabolism (CYP1A1, CYP1B1, HSD17B14) were differentially regulated in alopecia X. These findings are in agreement with the so far proposed but not yet proven deregulation of the sex hormone metabolism in this disease.

The transcriptome profile and differential gene expression in telogen and late anagen microdissected hair follicles and the interfollicular epidermis of healthy dogs was investigated by using RNAseq. The genes with the highest expression levels in each group were identified and genes known from studies in other species to be associated with structure and function of hair follicles and epidermis were evaluated. Transcriptome profiling revealed that late anagen follicles expressed mainly keratins and telogen follicles expressed GSN and KRT15. The interfollicular epidermis expressed predominately genes encoding for proteins associated with differentiation. All sample groups express genes encoding for proteins involved in cellular growth and signal transduction. The expression pattern of skin-associated genes in dogs is similar to humans. Differences in expression compared to mice and humans include BMP2 expression mainly in telogen and high KRT17 expression in the interfollicular epidermis of dogs. Our data provide the basis for the investigation of the structure and function of canine skin or skin disease and support the use of dogs as a model for human cutaneous disease by assigning gene expression to specific tissue states.

Bald thigh syndrome is a common hair loss disorder in sighthounds. Numerous possible causes, including environmental conditions, trauma, stress, endocrinopathies and genetic components have been proposed, but only endocrinopathies have been ruled out scientifically. The overall goal of our study was to identify the cause of bald thigh syndrome and the pathological changes associated with it. We approached this aim by comparing skin biopsies and hair shafts of affected and control dogs microscopically as well as by applying high-throughput technologies such as genomics, transcriptomics and proteomics. While the histology is rather unspecific in most cases, trichogram analysis and scanning electron microscopy revealed severe structural abnormalities in hair shafts of affected dogs. This finding is supported by the results of the transcriptomic and proteomic profiling where genes and proteins important for differentiation of the inner root sheath and the assembly of a proper hair shaft were downregulated. Transcriptome profiling revealed a downregulation of genes encoding 23 hair shaft keratins and 51 keratin associated proteins, as well as desmosomal cadherins and several actors of the BMP signaling pathway which is important for hair shaft differentiation. The lower expression of keratin 71 and desmocollin 2 on the mRNA level in skin biopsies corresponded with a decreased protein expression in the hair shafts of affected dogs. The genetic analysis revealed a missense variant in the IGFBP5 gene homozygous in all available Greyhounds and other sighthounds. Further research is required to clarify whether the IGFBP5 variant represents a predisposing genetic risk factor. We conclude from our results that structural defects in the hair shafts are the cause for this well-known disease and these defects are associated with a downregulation of genes and proteins essential for hair shaft formation. Our data add important knowledge to further understand the molecular mechanisms of HF morphogenesis and alopecia in dogs.

We characterized 72 isolates with reduced susceptibility to carbapenems (50 Acinetobacter spp., 13 Proteus mirabilis, five Escherichia coli, one Morganella morganii, one Enterobacter cloacae, one Providencia rettgeri, and one Pseudomonas aeruginosa) from a hospital in Sofia, Bulgaria. Different β-lactamase genes were identified by polymerase chain reaction and sequencing. Bacterial strain typing was performed by enzymatic macrorestriction and pulsed-field gel electrophoresis (PFGE) typing as well as multilocus sequence typing for selected isolates. The majority of Acinetobacter baumannii (46/50) and one Acinetobacter pittii isolate harbored carbapenemase genes bla or bla; two A. baumannii contained both genes. PFGE typing of all A. baumannii showed the presence of nine different clones belonging to eight sequence types ST350, ST208, ST436, ST437, ST449, ST231, ST502, and ST579. Molecular characterization of the remaining isolates confirmed the presence of one NDM-1-producing E. coli-ST101 clone (five isolates) and one P. mirabilis clone (13 isolates) with VIM-1 and CMY-99. Furthermore, NDM-1 was identified in P. rettgeri and M. morganii and VIM-2 in the P. aeruginosa isolate. The permanent introduction of OXA-23/72 carbapenemase-producing A. baumannii clones into the hospital and the repeated occurrence of one VIM-1-producing P. mirabilis and one NDM-1-producing E. coli-ST101 clone over a period of more than 1 year is of concern and requires intensified investigations.

Summary Major characteristics of coat variation in dogs can be explained by variants in only a few genes. Until now, only one missense variant in the KRT71 gene, p.Arg151Trp, has been reported to cause curly hair in dogs. However, this variant does not explain the curly coat in all breeds as the mutant 151Trp allele, for example, is absent in Curly Coated Retrievers. We sequenced the genome of a Curly Coated Retriever at 22× coverage and searched for variants in the KRT71 gene. Only one protein‐changing variant was present in a homozygous state in the Curly Coated Retriever and absent or present in a heterozygous state in 221 control dogs from different dog breeds. This variant, NM_001197029.1:c.1266_1273delinsACA, was an indel variant in exon 7 that caused a frameshift and an altered and probably extended C‐terminus of the KRT71 protein NP_001183958.1:p.(Ser422ArgfsTer?). Using Sanger sequencing, we found that the variant was fixed in a cohort of 125 Curly Coated Retrievers and segregating in five of 14 additionally tested breeds with a curly or wavy coat. KRT71 variants cause curly hair in humans, mice, rats, cats and dogs. Specific KRT71 variants were further shown to cause alopecia. Based on this knowledge from other species and the predicted molecular consequence of the newly identified canine KRT71 variant, it is a compelling candidate causing a second curly hair allele in dogs. It might cause a slightly different coat phenotype than the previously published p.Arg151Trp variant and could potentially be associated with follicular dysplasia in dogs.

Hereditary nasal parakeratosis (HNPK), described in the Labrador Retriever breed, is a monogenic autosomal recessive disorder that causes crusts and fissures on the nasal planum of otherwise healthy dogs. Our group previously showed that this genodermatosis may be caused by a missense variant located in the SUV39H2 gene encoding a histone 3 lysine 9 methyltransferase, a chromatin modifying enzyme with a potential role in keratinocyte differentiation. In the present study, we investigated a litter of Greyhounds in which six out of eight puppies were affected with parakeratotic lesions restricted to the nasal planum. Clinically and histologically, the lesions were comparable to HNPK in Labrador Retrievers. Whole genome sequencing of one affected Greyhound revealed a 4-bp deletion at the 5'-end of intron 4 of the SUV39H2 gene that was absent in 188 control dog and three wolf genomes. The variant was predicted to disrupt the 5'-splice site with subsequent loss of SUV39H2 function. The six affected puppies were homozygous for the variant, whereas the two non-affected littermates were heterozygous. Genotyping of a larger cohort of Greyhounds revealed that the variant is segregating in the breed and that this breed might benefit from genetic testing to avoid carrier × carrier matings.

Background Keratinocyte organoids can be used as a tool to evaluate epidermal structure, function and dysfunction. Objectives To optimize the canine keratinocyte organoid system and produce organoids that are structurally equivalent to in vivo canine epidermis, in order to enable studies that focus on epidermal diseases and diseases resulting from an impaired epidermal barrier. Animals Skin biopsies were obtained from five recently euthanized dogs of different breeds with no skin abnormalities. Methods and materials Cells derived from microdissected interfollicular epidermis were seeded in basement membrane extract and epidermal organoids were grown under different media conditions. Organoids were characterized to assess cell morphology and architecture in haematoxylin and eosin‐stained slides and expression of selected epidermal markers (keratin 5, keratin 10, loricrin and filaggrin) by immunohistochemical analysis and quantitative reverse transcription PCR. Results The selected epidermal markers were expressed in the same epidermal layers in the organoids cultured in expansion medium and differentiation medium as in normal interfollicular epidermis, yet restriction to the distinct layers was best achieved with expansion medium. Comparison of the mRNA expression levels of these markers revealed that relative expression is similar in organoids cultured in expansion medium and normal canine epidermis, while it differs in organoids cultured in differentiation medium. Conclusion and clinical importance Organoids cultured in expansion medium have an equivalent structure to the interfollicular epidermis and express key marker proteins in similar proportions. Epidermal organoids are therefore a promising in vitro model to study epidermal structure, function and dysfunction.

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.