Canine Gene Research Articles

Investigating the influence of the SIRT6 gene and alternative splicing on canine longevity: an in-depth bioinformatics analysis and experimental confirmation via NGS-based targeted sequencing.

Sirtuin 6 (SIRT6) has many functions, but its most notable contribution lies in the intricate regulation of cell senescence and lifespan. The effect of the SIRT6 gene on body size and longevity in dogs has not been extensively studied, particularly with regard to alternative splicing mechanisms. To address this gap, the present study used a comprehensive approach that integrated bioinformatics analysis, DNA sequence analysis, and next-generation sequencing-based targeted sequencing analyses. Our results show that, according to the reference genomes of different dog breeds, the canine SIRT6 gene exhibits different variants according to the dog breed. Except for the exonic variant g.55,146,051C > T (rs851065050) detected in the Boxer breed, all variants obtained from other genomes were determined to be intronic variants. The g.56,075,604 G > T (rs3343377774) intronic variant previously detected in the Labrador Retriever breed was only detected in the small breed group in our study. As a result of in silico analysis, the g.56,075,604 G > T variant has an exonic splicing enhancer (ESE) site; this variant has created the motif of the binding site for the splicing factor ESE_SRp55. The g.55.146,051C > T variant was associated with a change in the ratio of exonic splicing silencer/ESE binding motifs. This change indicates an increased probability of exon skipping for the mutant allele. Thus, as with the intronic variant g.56,075,604 G > T, the mis-splicing induced by the exonic variant g.55,146,051C > T could potentially be associated with an altered distribution of regulatory splicing factors of the canine SIRT6 gene.

Exonisation of an intronic L1 element in the dystrophin gene associated with X-linked muscular dystrophy in a Border Collie dog.

X-linked recessive dystrophinopathies are the most common muscular dystrophies (MDs) in humans and dogs. To date, 20 breed-specific MD-associated variants are described in the canine dystrophin gene (DMD), including one associated with dystrophin-deficient MD in the Border Collie mixed breed. Here, we report the diagnosis and follow-up of mild dystrophin-deficient MD in a 5-month-old male Border Collie, associated with a novel DMD variant. Diagnosis was based on neurological examination and laboratory evaluations including creatine kinase activity, electromyography and muscle biopsies with immunofluorescent staining. Inspection of the Sashimi plots of the RNA-seq data from the affected muscle biopsy led to the discovery of a 162-bp L1 pseudoexon in DMD intron 63, introducing a frameshift and a premature stop codon (NM_001003343.1: c.9271_9272insN[162] p.(Ala3091fs*21)). Reduced DMD mRNA levels were detected for both the non-pseudoexon (50× less) and pseudoexon (3× less) containing transcripts in the affected muscle, compared with the level of the non-pseudoexon containing transcript in a control muscle, resulting in very low dystrophin protein levels and the upregulation of utrophin. Because the variant was only found in the affected dog, not in the healthy mother and grandmother, or in 108 unrelated Border Collies from the Belgian population (46 males and 62 females), it was considered a de novo variant. Although the prognosis for dystrophinopathy is generally regarded as poor, the dog stabilised at the age of 6 months and is still clinically stable at the age of 2 years.

The markers of the predictive DNA test for canine hip dysplasia may have a stronger relationship with elbow dysplasia

Canine hip and elbow dysplasias, which are prevalent orthopedic conditions rooted in developmental and hereditary factors are yet to be comprehensively assessed. This study aimed to address this gap by exploring the prognostic significance of five markers linked to canine hip dysplasia using available genome-wide association studies (GWAS) data. The influence of these markers on both hip and elbow dysplasia was examined in dogs exposed to standardized environmental conditions. We made a groundbreaking discovery using custom primers, qPCR assays, and evaluation of fluorescent resonance energy transfer (FRET) probes. Three specific SNPs previously associated with the risk of canine hip dysplasia demonstrated a potentially stronger correlation with elbow dysplasia. Notably, the SNP at nucleotide position 22691322, located near the canine CHST3 gene, displayed significance as a marker in multivariable logistic regression analysis. Surprisingly, none of the initially targeted SNPs showed a direct association with hip dysplasia. The genomic positions of these SNPs reside within a region conserved across mammals. In silico analyses suggested that the relevant variant might be positioned in a region linked to bone and muscle structures. Our findings revealed a remarkable relationship between SNP2 genotypes and methylation patterns, shedding light on the underlying mechanism that partially explains the genotype-phenotype correlation in canine CHST3. These groundbreaking findings offer essential insights for future, more extensive investigations into canine orthopedic health. This research significantly contributes to our understanding of the molecular foundations of hip and elbow dysplasia in dogs by charting a course for advancements in veterinary medicine and the overall well-being of canine companions.

Canine hyper-sociability structural variants associated with altered three-dimensional chromatin state

Strong selection on complex traits can lead to skewed trait means and reduced trait variability in populations. An example of this phenomenon can be evidenced in allele frequency changes and skewed trait distributions driven by persistent human-directed selective pressures in domesticated species. Dog domestication is linked to several genomic variants; however, the functional impacts of these variants may not always be straightforward when found in non-coding regions of the genome. Four polymorphic transposable elements (TE) found within non-coding sites along a 5 Mb region on canine CFA6 have evolved due to directional selection associated with heightened human-directed hyper-sociability in domesticated dogs. We found that the polymorphic TE in intron 17 of the canine GTF2I gene, which was previously reported to be negatively correlated with canid human-directed hyper-sociability, is associated with altered chromatin looping and hence distinct cis-regulatory landscapes. We reported supporting evidence of an E2F1-DNA binding peak concordant with the altered loop and higher expression of GTF2I exon 18, indicative of alternative splicing. Globally, we discovered differences in pathways regulating the extra-cellular matrix with respect to TE copy number. Overall, we reported evidence suggesting an intriguing molecular convergence between the emergence of hypersocial behaviors in dogs and the same genes that, when hemizygous, produce human Williams Beuren Syndrome characterized by cranio-facial defects and heightened social behaviors. Our results additionally emphasize the often-overlooked potential role of chromatin architecture in social evolution.

Preclinical evaluation and first-in-dog clinical trials of PBMC-expanded natural killer cells for adoptive immunotherapy in dogs with cancer

BackgroundNatural killer (NK) cells are cytotoxic cells capable of recognizing heterogeneous cancer targets without prior sensitization, making them promising prospects for use in cellular immunotherapy. Companion dogs develop spontaneous cancers...

Genetic analysis of PALB2 gene WD40 domain in canine mammary tumour patients.

DNA repair mechanisms are essential for tumorigenesis and disruption of HR mechanism is an important predisposing factor of human breast cancers (BC). PALB2 is an important part of the HR. There are similarities between canine mammary tumours (CMT) and BCs. As its human counterpart, PALB2 mutations could be a predisposing factor of CMT. In this study, we aimed to investigate the impacts of PALB2 variants on tumorigenesis and canine mammary tumor (CMT) malignancy. We performed Sanger sequencing to detect germline mutations in the WD40 domain of the canine PALB2 gene in CMT patients. We conducted in silico analysis to investigate the variants, and compared the germline PALB2 mutations in humans that cause breast cancer (BC) with the variants detected in dogs with CMT. We identified an intronic (c.3096+8C>G) variant, two exonic (p.A1050V and p.R1354R) variants, and a 3' UTR variant (c.4071T>C). Of these, p.R1354R and c.4071T>C novel variants were identified for the first time in this study. We found that the p.A1050V mutation had a significant effect. However, we could not determine sufficient similarity due to the differences in nucleotide/amino acid sequences between two species. Nonetheless, possible variants of human sequences in the exact location as their dog counterparts are associated with several cancer types, implying that the variants could be crucial for tumorigenesis in dogs. Our results did not show any effect of the variants on tumor malignancy. The current project is the first study investigating the relationship between the PALB2 gene WD40 domain and CMTs. Our findings will contribute to a better understanding of the pathogenic mechanism of the PALB2 gene in CMTs. In humans, variant positions in canines have been linked to cancer-related phenotypes such as familial BC, endometrial tumor, and hereditary cancer predisposition syndrome. The results of bioinformatics analyses should be investigated through functional tests or case-control studies.

Abstract 1772: A naturally occurring canine model of peripheral T-cell lymphoma, not otherwise specified

Abstract Despite being the most common subtype of human peripheral T-cell lymphoma (PTCL), PTCL-not otherwise specified (PTCL-NOS) remains a poorly understood diagnosis of exclusion with poor survival times and treatment responses. Canine PTCL clinically and immunophenotypically resembles human PTCL-NOS, and PTCL is more common in dogs than humans, leading to their interest as a naturally occurring preclinical model. Canine models of cancer offer several unique advantages: in contrast to traditional rodent models, these are spontaneous tumors in genetically diverse and immunocompetent animals living in a shared environment with humans, but relative to their human counterparts, they offer accelerated lifespans with shorter clinical courses of disease. Here, we analyzed the gene expression profile of 96 canine CD4+ PTCLs to determine similarities to human PTCL-NOS and investigate a possible cell of origin. Bulk RNA-seq was performed on lymph node aspirates of 96 cases of canine CD4+ PTCL diagnosed by flow cytometry and sorted CD4+ nodal lymphocytes and CD4+ thymocytes from healthy control dogs. Raw reads were aligned to the Ensembl canine reference genome (ROS_Cfam_1.0) and tabulated. Data normalization and differential expression analysis was conducted with DESeq2, and differentially expressed genes were compared to human PTCL-NOS and various stages of T-cell development via gene set enrichment analyses (GSEA). Global gene expression in canine CD4+ PTCL was analogous to that of human PTCL-NOS (NES = 2.3, p < 0.001). Additionally, canine CD4+ PTCL had increased expression of GATA3 (log2fc = 1.9, padj = 6.5e-07) and was enriched for gene signatures associated with downregulation of PTEN (NES = 1.7, p = 0.0001) and upregulation of PI3K/AKT/mTOR signaling (NES = 1.5, padj = 0.0007), resembling the more aggressive human GATA3-PTCL subtype. Canine CD4+ PTCL was also enriched for human and murine gene signatures associated with early thymocyte progenitor cells and had increased expression of genes of immaturity, including CD34 (log2fc = 7.8, padj = 2.6e-14), KIT (log2fc = 3.3, padj = 7.7e-05), and CCR9 (log2fc = 4.8, padj = 3.9e-11), although surface CD34 expression is not detected by flow cytometry. These findings were confirmed when we compared the canine PTCL gene expression programs to a canine single cell transcriptomic atlas of hematopoietic precursors and T cells across canine bone marrow, thymic, and lymph node tissues. Canine CD4+ PTCL cells were significantly (padj < 0.05) enriched for gene signatures associated with early thymic and bone marrow precursors (NES 1.3-2.1), and negatively enriched for gene signatures associated with naïve nodal CD4+ T cells (NES = -3.2, padj = 0.006). In conclusion, the gene expression profile of canine CD4+ PTCL resembles human PTCL-NOS, supporting its potential as a naturally occurring preclinical model, and a subset of these neoplasms may arise from a thymic precursor cell of origin. Citation Format: Eileen Owens, Adam Harris, Anne Avery. A naturally occurring canine model of peripheral T-cell lymphoma, not otherwise specified [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1772.

A New Benzo[6,7]oxepino[3,2-b] Pyridine Derivative Induces Apoptosis in Canine Mammary Cancer Cell Lines

CMC is the most frequently diagnosed cancer and one of the leading causes of death in non-spayed female dogs. Exploring novel therapeutic agents is necessary to increase the survival rate of dogs with CMC. MPOBA is a BZOP derivative that has a significant anticancer effect in a human cell line. The main goal of this study was to investigate the anticancer properties of MPOBA against two CMC cell lines (REM134 and CMGT071020) using a 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, a wound healing assay, a transwell migration assay, an Annexin V-FITC apoptosis assay with a flow cytometry analysis, a mRNA expression analysis using quantitative real-time PCR (qRT-PCR), and an immunohistochemistry (IHC). According to the accumulated studies, MPOBA caused significant concentration- and time-dependent reductions in cell proliferation and cell migration and induced apoptosis in both CMC cell lines. In gene expression analysis, nine canine genes, including TP53, BCL-2, BAX, epidermal growth factor receptor (EGFR), snail transcription factor (SNAIL), snail-related zinc-finger transcription factor (SLUG), TWIST, E-cadherin, and N-cadherin, were investigated. The mRNA expression results revealed that MPOBA induced upregulation of TP53 and overexpression of the pro-apoptotic gene BAX, together with an inhibition of BCL-2. Moreover, MPOBA also suppressed the mRNA expression levels of SNAIL, EGFR, and N-cadherin and induced upregulation of E-cadherin, crucial genes related to the epithelial-to-mesenchymal transition (EMT). However, there was no significant difference in the IHC results of the expression patterns of vimentin (VT) and cytokeratin (CK) between MPOBA-treated and control CMC cells. In conclusion, the results of the present study suggested that MPOBA exhibited significant anticancer activity by inducing apoptosis in both CMCs via upregulation of TP53 and BAX and downregulation of BCL-2 relative mRNA expression. MPOBA may prove to be a potential candidate drug to be further investigated as a therapeutic agent for CMC.

A kinase-dead natural polymorphism in the canine Tnni3k gene.

Most mammalian cardiomyocytes become polyploid in the neonatal period, concurrent with their loss of proliferative capacity. In mice, natural or engineered mutation of the cardiomyocyte-specific kinase gene Tnni3k causes a higher level of diploid CMs and a higher capacity to support proliferation after adult injury. Here, we identified a polymorphism in the canine Tnni3k gene that is particularly common in the West Highland White Terrier breed, and show that this variant eliminates Tnni3k kinase activity. Thus, in several species, natural Tnni3k polymorphisms exist that are predicted to contribute to variation in diploid CM level and heart regenerative ability.

Generation of canine induced pluripotent stem cells under feeder-free conditions using Sendai virus vector encoding six canine reprogramming factors

Although it is in its early stages, canine induced pluripotent stem cells (ciPSCs) hold great potential for innovative translational research in regenerative medicine, developmental biology, drug screening, and disease modeling. However, almost all ciPSCs were generated from fibroblasts, and available canine cell sources for reprogramming are still limited. Furthermore, no report is available to generate ciPSCs under feeder-free conditions because of their low reprogramming efficiency. Here, we reanalyzed canine pluripotency-associated genes and designed canine LIN28A, NANOG, OCT3/4, SOX2, KLF4, and C-MYC encoding Sendai virus vector, called 159cf. and 162cf. We demonstrated that not only canine fibroblasts but also canine urine-derived cells, which can be isolated using a noninvasive and straightforward method, were successfully reprogrammed with or without feeder cells. ciPSCs existed in undifferentiated states, differentiating into the three germ layers invitro and invivo. We successfully generated ciPSCs under feeder-free conditions, which can promote studies in veterinary and consequently human regenerative medicines.

Development of a next-generation sequencing protocol for the canine T cell receptor beta chain repertoire

Profiling the T cell receptor (TCR) repertoire using next-generation sequencing has become common in both human and translational research. Companion dogs with spontaneous tumors, including canine melanoma, share several features, e.g., natural occurrence, shared environmental exposures, natural outbred population, and immunocompetence. T cells play an important role in the adaptive immune system by recognizing specific antigens via a surface TCR. As such, understanding the canine T cell response to vaccines, cancer, immunotherapies, and infectious diseases is critically important for both dog and human health. Off-the-shelf commercial reagents, kits and services are readily available for human, non-human primate, and mouse in this context. However, these resources are limited for the canine. In this study, we present a cost-effective protocol for analysis of canine TCR beta chain genes. Workflow can be accomplished in 1–2 days starting with total RNA and resulting in libraries ready for sequencing on Illumina platforms.

Genomic analysis across 53 canine cancer types reveals novel mutations and high clinical actionability potential.

A genomic understanding of the oncogenic processes and individual variability of human cancer has steadily fueled improvement in patient outcomes over the past 20 years. Mutations within tumour tissues are routinely assessed through clinical genomic diagnostic assays by academic and commercial laboratories to facilitate diagnosis, prognosis and effective treatment stratification. The application of genomics has unveiled a wealth of mutation-based biomarkers in canine cancers, suggesting that the transformative principles that have revolutionized human cancer medicine can be brought to bear in veterinary oncology. To advance clinical genomics and genomics-guided medicine in canine oncology, we have developed and validated a canine cancer next-generation sequencing gene panel for the identification of multiple mutation types in clinical specimens. With this panel, we examined the genomic landscapes of 828 tumours from 813 dogs, spanning 53 cancer types. We identified 7856 alterations, encompassing copy number variants, single nucleotide variants, indels and internal tandem duplications. Additionally, we evaluated the clinical utility of these alterations by incorporating a biomarker framework from comprehensive curation of primary canine literature and inferences from human cancer genomic biomarker literature and clinical diagnostics. Remarkably, nearly 90% of the cases exhibited mutations with diagnostic, prognostic or therapeutic implications. Our work represents a thorough assessment of genomic landscapes in a large cohort of canine cancers, the first of its kind for its comprehensive inclusion of multiple mutation types and structured annotation of biomarkers, demonstrating the clinical potential of leveraging mutation-based biomarkers in veterinary oncology.

Identification of novel genetic risk factors of dilated cardiomyopathy: from canine to human

BackgroundDilated cardiomyopathy (DCM) is a life-threatening heart disease and a common cause of heart failure due to systolic dysfunction and subsequent left or biventricular dilatation. A significant number of cases have a genetic etiology; however, as a complex disease, the exact genetic risk factors are largely unknown, and many patients remain without a molecular diagnosis.MethodsWe performed GWAS followed by whole-genome, transcriptome, and immunohistochemical analyses in a spontaneously occurring canine model of DCM. Canine gene discovery was followed up in three human DCM cohorts.ResultsOur results revealed two independent additive loci associated with the typical DCM phenotype comprising left ventricular systolic dysfunction and dilatation. We highlight two novel candidate genes, RNF207 and PRKAA2, known for their involvement in cardiac action potentials, energy homeostasis, and morphology. We further illustrate the distinct genetic etiologies underlying the typical DCM phenotype and ventricular premature contractions. Finally, we followed up on the canine discoveries in human DCM patients and discovered candidate variants in our two novel genes.ConclusionsCollectively, our study yields insight into the molecular pathophysiology of DCM and provides a large animal model for preclinical studies.

Olfaction evaluation in dogs with sudden acquired retinal degeneration syndrome.

To evaluate olfaction in dogs with sudden acquired retinal degeneration syndrome (SARDS) compared with sighted dogs and blind dogs without SARDS as control groups. Forty client-owned dogs. Olfactory threshold testing was performed on three groups: SARDS, sighted, and blind/non-SARDS using eugenol as the test odorant. The olfactory threshold was determined when subjects indicated the detection of a specific eugenol concentration with behavioral responses. Olfactory threshold, age, body weight, and environmental room factors were evaluated. Sixteen dogs with SARDS, 12 sighted dogs, and 12 blind/non-SARDS dogs demonstrated mean olfactory threshold pen numbers of 2.8 (SD = 1.4), 13.8 (SD = 1.4), and 13.4 (SD = 1.1), respectively, which correspond to actual mean concentrations of 0.017 g/mL, 1.7 × 10-13 g/mL and 4.26 × 10-13 g/mL, respectively. Dogs with SARDS had significantly poorer olfactory threshold scores compared with the two control groups (p < .001), with no difference between the control groups (p = .5). Age, weight, and room environment did not differ between the three groups. Dogs with SARDS have severely decreased olfaction capabilities compared with sighted dogs and blind/non-SARDS dogs. This finding supports the suspicion that SARDS is a systemic disease causing blindness, endocrinopathy, and hyposmia. Since the molecular pathways are similar in photoreceptors, olfactory receptors, and steroidogenesis with all using G-protein coupled receptors in the cell membrane, the cause of SARDS may exist at the G-protein associated interactions with intracellular cyclic nucleotides. Further investigations into G-protein coupled receptors pathway and canine olfactory receptor genes in SARDS patients may be valuable in revealing the cause of SARDS.

Transcriptomic analysis reveals immune infiltration status and potential biomarkers of canine colorectal cancer

Colorectal cancer (CRC) in dogs has been shown to have similar molecular characteristics to human colorectal cancer. Although researchers have explored the pathogenesis and immune status of human CRC, the canine CRC has been far less studied. As a result, we analyzed canine colorectal tumors and normal canine intestinal samples by Gene Set Enrichment Analysis (GSEA) and found significant enrichment of immune-related pathways, including the TNF-α signaling pathway and IL6-STAT3 signaling pathway. In addition, the differential infiltration of naive B cells and regulatory T cells suggested that canine CRC was in a state of immunosuppression. Weighted gene co-expression network analysis (WGCNA) revealed the gene modules that contribute to differences in regulatory T cell inetfiltration, Further cross-validation of canine and human CRC differential genes obtained three core genes that are both species-conserved and differentially expressed, CD44, NAT10, and ETV4, of which NAT10 and ETV4 have been little studied in the immune status of colorectal cancer. Our findings may have implications for the pathogenesis and progression of CRC in dogs and could be a new potential therapeutic target for CMT and provide a bioinformatics foundation for later clinical experiment validation.

Protective Effect of HER2 Gene Polymorphism rs24537331 in the Outcome of Canine Mammary Tumors.

The role of HER2 in canine mammary tumors is not completely elucidated, and the contradictory results published so far may be, in part, explained by the genetic variability recognized in the canine HER2 gene. Single nucleotide polymorphism (SNPs) in HER2 were recently associated with less aggressive canine mammary tumor histotypes. This study assesses the relationship between SNPs rs24537329 and rs24537331 in canine HER2 gene and clinicopathological characteristics and outcome of mammary tumors in a group of 206 female dogs. Allelic variants were observed in 69.8% and 52.7% of the dogs for SNP rs24537329 and rs24537331, respectively. Our results demonstrated that SNP rs24537331 was associated with decreased tumoral necrosis (HR: 3.09; p = 0.012) and with longer disease-specific overall survival (HR: 2.59; p = 0.013). However, no statistically significant associations were found between SNP rs24537329 and the tumors' clinicopathological characteristics or survival. Our data suggest that SNP rs24537331 may have a protective effect in canine mammary tumors, allowing the identification of a subgroup of animals prone to develop less aggressive forms of the disease. This study emphasizes the importance of the genetic tests associated with clinical images and histological examinations when assessing CMT outcomes.

Establishment of a BRAF V595E-mutant canine prostate cancer cell line and the antitumor effects of MEK inhibitors against canine prostate cancer.

Canine prostate cancer (cPCa) is a malignant neoplasm with no effective therapy. The BRAF V595E mutation, corresponding to the human BRAF V600E mutation, is found frequently in cPCa. Activating BRAF mutations are recognized as oncogenic drivers, and blockade of MAPK/ERK phosphorylation may be an effective therapeutic target against BRAF-mutated tumours. The aim of this study was to establish a novel cPCa cell line and to clarify the antitumor effects of MEK inhibitors on cPCa in vitro and in vivo. We established the novel CHP-2 cPCa cell line that was derived from the prostatic tissue of a cPCa patient. Sequencing of the canine BRAF gene in two cPCa cell lines revealed the presence of the BRAF V595E mutation. MEK inhibitors (trametinib, cobimetinib and mirdametinib) strongly suppressed cell proliferation in vitro, and trametinib showed the highest efficacy against cPCa cells with minimal cytotoxicity to non-cancer COPK cells. Furthermore, we orally administered 0.3 or 1.0mg/kg trametinib to CHP-2 xenografted mice and examined its antitumor effects in vivo. Trametinib reduced tumour volume, decreased phosphorylated ERK levels, and lowered Ki-67 expression in xenografts in a dose-dependent manner. Although no clear adverse events were observed with administration, trametinib-treated xenografts showed osteogenesis that was independent of dosage. Our results indicate that trametinib induces cell cycle arrest by inhibiting ERK activation, resulting in cPCa tumour regression in a dose-dependent manner. MEK inhibitors, in addition to BRAF inhibitors, may be a targeted agent option for cPCa with the BRAF V595E mutation.

An Improved Duplex Real-Time Quantitative RT-PCR Assay with a Canine Endogenous Internal Positive Control for More Sensitive and Reliable Detection of Canine Parainfluenza Virus 5.

A duplex real-time quantitative reverse transcription-polymerase chain reaction (dqRT-PCR) assay was successfully developed to simultaneously detect canine parainfluenza virus 5 (CPIV5) and a canine endogenous internal positive control (EIPC) in canine clinical samples. Two sets of primers and probes for the CPIV5 L and canine 16S rRNA genes were included in the dqRT-PCR assay to detect CPIV and monitor invalid results throughout the qRT-PCR process. The developed dqRT-PCR assay specifically detected CPIV5 but no other canine pathogens. Furthermore, 16S rRNA was stably amplified by dqRT-PCR assay in all samples containing canine cellular materials. The assay's sensitivity was determined as below ten RNA copies per reaction, with CPIV5 L gene standard RNA and 1 TCID50/mL with the CPIV5 D008 vaccine strain, which was 10-fold higher than that of the previous HN gene-specific qRT-PCR (HN-qRT-PCR) assays and was equivalent to that of the previous N gene-specific qRT-PCR (N-qRT-PCR) assays, respectively. Moreover, the Ct values of the CPIV5-positive samples obtained using the dqRT-PCR assay were lower than those obtained using the previous HN- and N-qRT-PCR assays, indicating that the diagnostic performance of the dqRT-PCR assay was superior to those of previous HN- and N-qRT-PCR assays. The calculated Cohen's kappa coefficient values (95% confidence interval) between dqRT-PCR and the HN- or N-specific qRT-PCR assays were 0.97 (0.90-1.03) or 1.00 (1.00-1.00), respectively. In conclusion, the newly developed dqRT-PCR assay with high sensitivity, specificity, and reliability will be a promising diagnostic tool for the detection of CPIV5 in clinical samples and useful for etiological and epidemiological studies of CPIV5 infection in dogs.

Tailored Multiplex Real-Time RT-PCR with Species-Specific Internal Positive Controls for Detecting SARS-CoV-2 in Canine and Feline Clinical Samples

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections have been frequently reported in companion dogs and cats worldwide during the ongoing coronavirus disease. However, RT-qPCR methods developed for humans have been used for the diagnosis of SARS-CoV-2 infections in suspected companion dogs and cats owing to the lack of the companion animal-tailored methods. Therefore, we developed a multiplex RT-qPCR (mRT-qPCR) using newly designed primers and probes targeting RdRp and N genes of all currently circulating SARS-CoV-2 variants as well as the canine or feline 16S rRNA gene as an endogenous internal positive control (EIPC) for reliable diagnosis of SARS-CoV-2 infection from suspected dogs and cats. The developed mRT-qPCR assay specifically detected the target genes of SARS-CoV-2 but no other canine or feline pathogens. Furthermore, canine and feline EIPCs were stably amplified by mRT-qPCR in samples containing canine- or feline-origin cellular materials. This assay has high repeatability and reproducibility, with an optimal limit of detection (<10 RNA copies per reaction) and coefficients of variation (<1.0%). The detection rate of SARS-CoV-2 of the developed mRT-qPCR was 6.6% for canine and feline nasopharyngeal samples, which was consistent with that of a commercial mRT-qPCR kit for humans. Collectively, the newly developed mRT-qPCR with canine and feline EIPC can efficiently diagnose and evaluate the viral load in field specimens and will be a valuable tool for etiological diagnosis, epidemiological study, and controlling SARS-CoV-2 infections in canine and feline populations.

Hyperkeratotic erythema multiforme variant in 17 dogs.

A new canine subgroup defined as 'old-dog' or 'hyperkeratotic' erythema multiforme (HKEM) with marked hyperkeratosis and parakeratosis has been proposed without any detailed description of larger case series. We report herein the signalment, clinical signs, treatment outcome, and histopathological and immunological findings in 17 dogs with HKEM. Inclusion criteria were the presence of (i) scaly skin lesions with or without crusting; and (ii) microscopic lesions typical of EM (i.e. a panepidermal cytotoxic lymphocytic dermatitis with or without basal keratinocyte apoptosis); and (iii) microscopic ortho- and/or parakeratotic hyperkeratosis affecting the interfollicular epidermis. Clinical questionnaires and skin biopsies were reviewed. Polymerase chain reactions for epidermotropic viruses and direct immunofluorescence were performed. Various breeds were affected with an over-representation of males in their mid-to-late adulthood (median age 9 years). Generalised skin lesions included multifocal-to-coalescing, linear and annular macules and plaques with erythema and adherent firm crusting. Microscopic lesions were specific for EM and featured prominent superficial epidermal apoptosis with lymphocytic satellitosis and parakeratosis. No drug triggers were identified. Polymerase chain reactions for canine herpesvirus polymerase gene, canine parvovirus and canine distemper virus were negative in all HKEM and canine erosive EM (15 dogs) biopsies. Lesions failed to respond to oral and/or topical antimicrobials. Complete remission of signs was achieved in 9 of 17 dogs (53%) using immunosuppressive regimens. Hyperkeratotic erythema multiforme (HKEM) is a chronic, persistent and clinically distinctive erythema multiforme (EM) variant that differs from 'classic' vesiculobullous erosive-to-ulcerative EM in dogs.