Phosphorylation of Janus kinase 1 and signal transducer and activator of transcription 3 and 6 in keratinocytes of canine atopic dermatitis.

BACKGROUND: Canine atopic dermatitis (cAD) is a disease associated with Type 2 helper T (Th2) immune responses in the acute phase of the disease. In humans, keratinocytes are activated by Th2 cytokines via the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway. However, the activation of keratinocytes by Th2 cytokines in cAD has not yet been demonstrated. HYPOTHESIS/OBJECTIVES: To evaluate keratinocyte activation based on the phosphorylation (p) of JAK1, STAT3 and STAT6. ANIMALS: Seven dogs with cAD and three healthy dogs. MATERIALS AND METHODS: Immunohistochemical analysis was performed to detect pJAK1, pSTAT3 and pSTAT6 in keratinocytes in normal canine skin, and the skin of atopic dogs. In the latter group samples were collected from both primary and secondary lesions, and nonaffected skin. RESULTS: The percentage of pJAK1-positive keratinocytes was significantly higher in primary cAD lesions than in healthy skin (p < 0.05). No significant differences were observed in pSTAT3-positive keratinocytes among the groups. The percentage of pSTAT6-positive keratinocytes was significantly higher in primary and secondary lesions than in healthy skin (p < 0.05, respectively). CONCLUSIONS AND CLINICAL RELEVANCE: The novel finding in this study was the activation of keratinocytes as demonstrated by the phosphorylation of JAK1/STATs in lesional and nonlesional cAD skin. These results suggest the potential of not only JAK1, but also of STAT6 as therapeutic targets for cAD.

Circadian rhythm affects the magnitude of contact hypersensitivity response in mice.

BACKGROUND: The circadian rhythm controls multiple biological processes, including immune responses; however, its impact on cutaneous adaptive immune response remains unclear. METHODS: We used a well-established cutaneous type IV allergy model, contact hypersensitivity (CHS). We induced CHS using dinitrofluorobenzene (DNFB). Mice were sensitized and elicited with DNFB in the daytime or at night. RESULTS: In mice, a nocturnally active animal, we found that ear swelling increased when mice were sensitized at night compared with in the daytime. In addition, cell proliferation and cytokine production in the draining lymph nodes (LNs) were promoted when sensitized at night. We hypothesized that these differences were due to the oscillation of leukocyte distribution in the body through the circadian production of adrenergic hormones. Administration of a ß2-adrenergic receptor (ß2AR) agonist salbutamol in the daytime decreased the number of immune cells in blood and increased the number of immune cells in LNs. In contrast, a ß2AR antagonist ICI18551 administration at night increased the number of immune cells in blood and decreased the number of immune cells in LNs. Accordingly, the severity of CHS response was exacerbated by salbutamol administration in the daytime and attenuated by ICI18551 administration at night. CONCLUSION: Our study demonstrated that the magnitude of adaptive CHS response depends on the circadian rhythm and this knowledge may improve the management of allergic contact dermatitis (ACD) in humans.

Guest edited collection serological study of SARS-CoV-2 antibodies in japanese cats using protein-A/G-based ELISA.

BACKGROUND: Little is known about the epidemic status of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in cats in Japan due to insufficiently reliable seroepidemiological analysis methods that are easy to use in cats. RESULTS: We developed a protein-A/G-based enzyme-linked immunosorbent assay (ELISA) to detect antibodies against SARS-CoV-2 in cats. The assay was standardized using positive rabbit antibodies against SARS-CoV-2. The ELISA results were consistent with those of a conventional anti-feline-immunoglobulin-G (IgG)-based ELISA. To test the protein-A/G-based ELISA, we collected blood samples from 1,969 cats that had been taken to veterinary clinics in Japan from June to July 2020 and determined the presence of anti-SARS-CoV-2 antibodies. Nine cats were found to have SARS-CoV-2 S1-specific IgG, of which 4 had recombinant receptor-binding domain-specific IgG. Of those 9 samples, one showed neutralizing activity. Based on these findings, we estimated that the prevalence of SARS-CoV-2 neutralizing antibodies in cats in Japan was 0.05% (1/1,969 samples). This prevalence was consistent with the prevalence of neutralizing antibodies against SARS-CoV-2 in humans in Japan according to research conducted at that time. CONCLUSIONS: Protein-A/G-based ELISA has the potential to be a standardized method for measuring anti-SARS-CoV-2 antibodies in cats. The infection status of SARS-CoV-2 in cats in Japan might be linked to that in humans.

Antibiotic-resistant status and pathogenic clonal complex of canine Streptococcus canis-associated deep pyoderma.

BACKGROUND: Streptococcus canis causes deep pyoderma in canines, which raises concerns about the risk of isolates from lesions acquiring an antibiotic-resistant phenotype. It is necessary to identify effective antibiotics and the characteristics of the pathogenic cluster for S. canis-associated deep pyoderma. RESULTS: The signalment, molecular typing, and antibiotic-resistant status of S. canis isolated from deep pyoderma lesions (27 strains) and oral cavities (26 strains) were analyzed. Older dogs tended to have S. canis-associated deep pyoderma (15 of 27 dogs over 10 years old). Veterinarians chose quinolones for 10/16 cases (63%), even though the rate of quinolone-resistant strains of S. canis is 38-59%. Although 70% of the strains showed resistance to three or more antibiotic classes (37/53), 94% (50/53) strains showed sensitivity for penicillins. We also identified ß-lactamase activity among penicillin-resistant strains of S. canis. Clonal complex 13 (CC13) was detected only in lesions and formed independent clusters in the phylogenetic tree. One strain of CC13 was resistant to the anti-methicillin-resistant Staphylococcus aureus drugs, vancomycin and linezolid. CONCLUSION: Although antibiotic-resistant strains of S. canis are isolated at a high rate, they can currently be treated with ß-lactamase-inhibiting penicillins. CC13 may be a pathogenic cluster with high levels of antibiotics resistance.

Narrow-band ultraviolet B therapy attenuates cutaneous T-cell responses in hapten-induced, experimental contact dermatitis in beagles.

BACKGROUND: In human medicine, narrow-band ultraviolet B (NB-UVB) phototherapy has been used to treat various T-cell-mediated skin diseases. However, the effect of NB-UVB on inflamed canine skin remains uncertain. OBJECTIVES: To investigate the effect of NB-UVB phototherapy on the skin of dogs with hapten-induced contact dermatitis. ANIMALS: Seven healthy beagles without skin problems. METHODS AND MATERIALS: Dogs were irradiated with varying doses of NB-UVB to determine the minimal erythema dose (MED). After determining the MEDs of six dogs (excluding one of the seven whose skin did not show a visible reaction), we investigated the effect of NB-UVB on their inflamed skin by topically applying 2,4-dinitrochlorobenzene (DNCB), which causes type 1 helper T cell (Th1)- and cytotoxic T-cell (Tc)1-induced skin inflammation. We then irradiated the skin with NB-UVB. We analysed the treated skin samples via histopathological and immunohistochemical methods, and TdT-mediated dUTP nick-end labelling (TUNEL) to demonstrate apoptotic cells. We also analysed the cytokine gene transcription via real-time quantitative reverse transcription PCR. RESULTS: The NB-UVB MEDs caused mild inflammatory changes yet no severe epidermal exfoliations in the irradiated skin. In DNCB-treated skin irradiated by the NB-UVB MEDs, TUNEL-positive dermal apoptotic cells were increased significantly compared with those of DNCB-treated, nonirradiated skin. INF-γ and TNF-α transcription levels in DNCB-treated, irradiated skin were significantly lower than those in the DNCB-treated, nonirradiated skin. CONCLUSION AND CLINICAL RELEVANCE: Phototherapy using NB-UVB MEDs attenuated cutaneous Th1 and Tc1 cytokine responses with minimal skin damage in a canine model of hapten-induced contact dermatitis.

Outcomes of extensive hemilaminectomy with durotomy on dogs with presumptive progressive myelomalacia: a retrospective study on 34 cases.

BACKGROUND: Progressive myelomalacia (PMM) is a fatal complication of progressive ascending and descending necrosis of the spinal cord after acute spinal cord injury. A recent study suggested that extensive hemilaminectomy with durotomy (EHLD) at the intramedullary T2-hyperintense region which performed immediately after magnetic resonance imaging (MRI) improved the survival rate in dogs with presumptive PMM. The objective of this retrospective study was to evaluate the effects of EHLD on halting the progression of PMM in dogs presumptively diagnosed with PMM which had the interval between MRI and surgery. RESULTS: Thirty-four dogs with presumptive PMM which had undergone EHLD with the delay following MRI examination (range, 0 to 3 days) were included. The cranial side of EHLD was set depending on the delay time after MRI, MRI findings, neurological examination and intraoperative macroscopic appearance. Two weeks after surgery, the perioperative survival rate was 97% (33/34). During follow-up with a median time period of 82.5 weeks (range, 0-290 weeks), the postoperative survival rate was 91% (31/34). At the end of the follow-up period, 31 out of 34 dogs were alive without severe postoperative complications while the remaining 2 dogs died from causes not directly attributable to the surgery. There was no improvement in the pelvic limb function of all dogs. CONCLUSIONS: EHLD appears to be effective in halting the progression of presumptive PMM and preventing morbidity even in dogs which had the interval between MRI and EHLD. Our algorithm of determining the range of EHLD may enable to set the appropriate ranges of EHLD in the cases which develop signs consistent with PMM after MRI examination.

Serum canine thymus and activation-regulated chemokine (TARC/CCL17) concentrations correlate with disease severity and therapeutic responses in dogs with atopic dermatitis.

BACKGROUND: Thymus and activation-regulated chemokine (TARC/CCL17) has been implicated in the pathogenesis of canine atopic dermatitis (cAD). Serum TARC concentrations are a reliable biomarker for human atopic dermatitis; however, their potential as a biomarker for cAD has not been investigated. HYPOTHESIS/OBJECTIVES: To investigate whether serum TARC concentrations correlate with disease severity and therapeutic responses for cAD. ANIMALS: Thirty-nine dogs with cAD and 42 healthy dogs were recruited. METHODS AND MATERIALS: Serum TARC concentrations in dogs with cAD and healthy dogs were measured by sandwich ELISA with anti-canine TARC antibodies. The clinical severity of cAD was scored using the validated Canine Atopic Dermatitis Extent and Severity Index, 4th iteration (CADESI-04). Serum TARC concentrations were compared between dogs with cAD and healthy controls, and their relationship with CADESI-04 was examined. Serum TARC concentrations also were measured in 20 dogs with cAD treated with prednisolone or oclacitinib for four weeks. RESULTS: Serum TARC concentrations were significantly higher in dogs with cAD than in healthy dogs (P < 0.001). In dogs with cAD, serum TARC concentrations correlated with CADESI-04 scores (ρ = 0.457, P < 0.01). Furthermore, serum TARC concentrations significantly decreased in treated dogs with the attenuation of clinical signs (P < 0.001). Changes in serum TARC concentrations before and after treatment correlated with those in CADESI-04 scores (ρ = 0.746, P < 0.001). CONCLUSIONS AND CLINICAL RELEVANCE: Serum TARC concentrations have potential as a clinical and research tool for the objective evaluation of disease severity and therapeutic responses for cAD.

Expression of IL-33 in chronic lesional skin of canine atopic dermatitis.

BACKGROUND: In humans, interleukin (IL)-33 plays a critical role in the enhancement of allergic skin inflammation. However, it currently remains unclear whether IL-33 is involved in the pathogenesis of canine atopic dermatitis (cAD). OBJECTIVES: To examine the expression of IL-33 in chronic lesional skin of cAD. ANIMALS: Eight dogs with spontaneous cAD and five healthy dogs were used. METHODS: The transcription of il-33 in chronic lesional skin of cAD was quantified by quantitative reverse transcription PCR. The expression of IL-33 was evaluated immunohistochemically using an anti-human IL-33 monoclonal antibody with cross-reactivity to canine IL-33. RESULTS: The transcription levels of il-33 in chronic lesional skin of cAD were significantly higher than those in normal skin of healthy dogs. Keratinocytes were a major cellular source of IL-33 production in chronic lesional skin of cAD. CONCLUSIONS AND CLINICAL IMPORTANCE: The results indicate that IL-33 is involved in chronic lesional skin of cAD.

Expression of ZO-1 and claudin-1 in a 3D epidermal equivalent using canine progenitor epidermal keratinocytes.

BACKGROUND: Previous studies indicate that tight junctions are involved in the pathogenesis of canine atopic dermatitis (cAD). An in vitro skin model is needed to elucidate the specific role of tight junctions in cAD. A 3D epidermal equivalent model using canine progenitor epidermal keratinocytes (CPEK) has been established; the expression of tight junctions within this model is uncharacterized. HYPOTHESIS/OBJECTIVES: To investigate the expression of tight junctions in the 3D epidermal equivalent. ANIMALS: Two normal laboratory beagle dogs served as donors of full-thickness skin biopsy samples for comparison to the in vitro model. METHODS: Immunohistochemical techniques were employed to investigate the expression of tight junctions including zonula occludens (ZO)-1 and claudin-1 in normal canine skin, and in the CPEK 3D epidermal equivalent. RESULTS: Results demonstrated the expression of ZO-1 and claudin-1 in the CPEK 3D epidermal equivalent, with staining patterns that were similar to those in normal canine skin. CONCLUSIONS AND CLINICAL IMPORTANCE: The CPEK 3D epidermal equivalent has the potential to be a suitable in vitro research tool for clarifying the specific role of tight junctions in cAD.

Transcriptional analysis of the IL-33 receptor suppression of tumourigenicity 2 and its effects on canine Type 2 T helper cells: a preliminary study.

BACKGROUND: Interleukin (IL)-33 has been implicated in the pathogenesis of canine atopic dermatitis, a Type 2 T helper cell (Th2)-associated disease. In humans, IL-33 mediates its biological effects through the receptor suppression of tumourigenicity 2 (ST2), which is preferentially expressed on Th2 cells. The effects of IL-33 on canine Th2 cells are unclear. HYPOTHESIS/OBJECTIVES: ST2 may be preferentially expressed on canine Th2 cells; IL-33 may induce the transcription of Th2 cytokines from these cells. ANIMALS: Three healthy dogs were used. METHODS: The transcription level of st2 was quantified in helper T cells, cytotoxic T cells and Th2 cells isolated from healthy dogs. The transcription levels of Th2 cytokines including il-4, il-5, il-13 and il-31 were quantified in Th2 cells stimulated with recombinant canine (rc) IL-33 and/or recombinant human (rh) IL-2. RESULTS: Transcription of st2 was the strongest in Th2 cells. Th2 cells also transcribed the genes for il-5 and il-13 after being stimulated with rcIL-33 and rhIL-2. CONCLUSIONS AND CLINICAL IMPORTANCE: These results indicate that canine Th2 cells activated by IL-33 enhance Th2-mediated inflammation through the production of IL-5 and IL-13.

Phenotypic analysis of mice xenografted with canine epitheliotropic cutaneous T-cell lymphoma cells.

BACKGROUND: In canine epitheliotropic cutaneous T-cell lymphoma (ECTCL), neoplastic cells cause skin lesions and potentially metastasize to lymph nodes, blood and other organs. Murine models are potentially valuable for elucidating the molecular mechanisms responsible for regulation of ECTCL cell migration. HYPOTHESIS/OBJECTIVES: To describe a phenotype of mice xenografted with canine ECTCL cells (EO-1 cells). ANIMALS: Four NOD.CB17-Prkdcscid /J (NOD SCID) mice were used. METHODS AND MATERIALS: EO-1 cells were subcutaneously xenografted into NOD SCID mice. After four weeks, the development of tumour lesions in skin and other organs was investigated. RESULTS: Mice developed skin lesions with metastasis to the lymph nodes, spleen, lung, blood and liver. CONCLUSIONS AND CLINICAL IMPORTANCE: Mice xenografted with EO-1 cells may be useful for studying the pathogenesis of canine ECTCL.

A review of the roles of keratinocyte-derived cytokines and chemokines in the pathogenesis of atopic dermatitis in humans and dogs.

BACKGROUND: Dysfunction of the physical and chemical barriers of the skin may play roles in the pathogenesis of atopic dermatitis (AD) by facilitating penetration of antigens through the skin and consequently evoking aberrant immune reactions. It is now emerging that keratinocytes are actively involved in cutaneous immune reactions by producing various soluble factors initiated by inflammatory stimuli, including mechanical injury or activation of Toll-like receptors and protease-activated receptors. Among the soluble factors, keratinocyte-derived cytokines and chemokines skew Type 2 helper T (Th2) cell-dominant immune reactions, with the recruitment of Th2 cells. OBJECTIVE: To review the roles of keratinocyte-derived cytokines and chemokines in the pathogenesis of AD in humans and dogs. CONCLUSION AND CLINICAL IMPORTANCE: Keratinocyte-derived cytokines such as thymus and activation-regulated chemokine, granulocyte-macrophage colony stimulating factor, thymic stromal lymphopoietin and interleukin-33 are involved in the pathogenesis of human AD and possibly in canine AD. These cytokines and chemokines may possibly be used as subjective clinical markers and therapeutic targets for both human and canine AD.

Transcription of thymic stromal lymphopoietin via Toll-like receptor 2 in canine keratinocytes: a possible association of Staphylococcus spp. in the deterioration of allergic inflammation in canine atopic dermatitis.

BACKGROUND: Colonization, overgrowth and subsequent infection by Staphylococcus spp. is frequently observed in canine atopic dermatitis (CAD), where it contributes to the intensity of cutaneous inflammation. The mechanisms by which staphylococci contribute to the pathogenesis of CAD are unclear. Studies suggest that thymic stromal lymphopoietin (TSLP), a cytokine induced by a cell wall component of Staphylococcus spp., may play a critical role in Th2 responses including the pathogenesis of CAD. HYPOTHESIS/OBJECTIVE: To determine if synthetic triacylated lipopeptide (TLR1/2 ligand), a cell wall component of Staphylococcus spp., induces the transcription of TSLP via TLR2 in canine keratinocytes. METHODS: Transcription of TSLP was quantified in a canine keratinocyte cell line after stimulation with synthetic triacylated lipopeptide, and again after inhibition of TLR2 by a targeted small interfering RNA. RESULTS: The transcription of TSLP was enhanced 6 h after stimulation with the synthetic triacylated lipopeptide; it was completely suppressed by knockdown of TLR2. CONCLUSIONS AND CLINICAL IMPORTANCE: The results demonstrated that a synthetic cell wall component of Staphylococcus spp. induced transcription of TSLP via TLR2 in canine keratinocytes. Additional studies will be required to investigate whether Staphylococcus spp. contributes to Th2 responses in CAD through TLR2-mediated TSLP production.

Gene transcription of pro-inflammatory cytokines and chemokines induced by IL-17A in canine keratinocytes.

BACKGROUND: Pro-inflammatory cytokines and chemokines produced by activated keratinocytes play an important role in the pathogenesis of canine atopic dermatitis (AD) as well as human AD. Recent studies suggest that keratinocytes activated by IL-17A are involved in the pathogenesis of human AD. However, the role of IL-17A in canine keratinocytes is poorly understood. HYPOTHESIS/OBJECTIVES: Interleukin-17A would induce the transcription of pro-inflammatory cytokines and chemokines in canine keratinocytes. METHODS: The transcription levels of pro-inflammatory cytokines and chemokines were quantified in a canine keratinocyte cell line stimulated with recombinant canine (rc) IL-17A. RESULTS: The transcription of GM-CSF, S100A8, IL-8 and IL-19 in cultured keratinocytes was significantly enhanced at 24 h after stimulation with rcIL-17A. CONCLUSIONS AND CLINICAL IMPORTANCE: Keratinocytes activated by IL-17A have the ability to produce various pro-inflammatory cytokines and chemokines, suggesting that IL-17A may play a central role of the development of Th2-associated inflammation in canine AD.

Up-regulated inflammatory signatures of the spinal cord in canine degenerative myelopathy.

Canine degenerative myelopathy (DM) is an adult-onset fatal disease characterized by progressive degeneration of the spinal cord. Affected dogs have homozygous mutations in superoxide dismutase 1, and thus DM is a potential spontaneous animal model of human familial amyotrophic lateral sclerosis (ALS). Neuroinflammation is the pathological hallmark of ALS, whereby proinflammatory cytokines and chemokines are overproduced by activated glial cells such as astrocytes and microglia. However, the detailed pathogenesis of spinal cord degeneration in DM remains unknown. To further characterize the pathological mechanism of DM, we analyzed the caudal cervical cords of ten Pembroke Welsh Corgis pathologically diagnosed with DM by quantitative real-time reverse transcription polymerase chain reaction, immunohistochemistry (IHC), and double immunofluorescence. Compared to control spinal cord tissues, we found significantly enhanced transcriptions of interleukin-1ß, tumor necrosis factor-α, CC motif chemokine ligand (CCL) 2 and vascular cell adhesion molecule -1 mRNA in the spinal cords of DM dogs. Moreover, IHC for the class II major histocompatibility complex molecules HLA-DR and CCL2 indicated that the immunopositive areas of activated macrophages/microglia and CCL2 protein were significantly increased in DM, and CCL2 protein was mainly overproduced by astrocytes. Our results suggest a proinflammatory state of the microenvironment in the DM spinal cord in which activated microglia and astrocytes play important roles by secreting a set of cytokines, chemokines, and expressing adhesion molecules.

Expression of microRNAs in plasma and in extracellular vesicles derived from plasma for dogs with glioma and dogs with other brain diseases.

OBJECTIVE: To measure expression of microRNAs (miRNAs) in plasma and in extracellular vesicles (EVs) derived from plasma for dogs with glioma and dogs with other brain diseases. SAMPLE: Plasma samples from 11 dogs with glioma and 19 control dogs with various other brain diseases. PROCEDURES: EVs were isolated from plasma samples by means of ultracentrifugation. Expression of 4 candidate reference miRNAs (let-7a, miR-16, miR-26a, and miR-103) and 4 candidate target miRNAs (miR-15b, miR-21, miR-155, and miR-342-3p) was quantified with reverse transcription PCR assays. Three software programs were used to select the most suitable reference miRNAs from among the 4 candidate reference miRNAs. Expression of the 4 target miRNAs was then calculated relative to expression of the reference genes in plasma and EVs, and relative expression was compared between dogs with glioma and control dogs with other brain diseases. RESULTS: The most suitable reference miRNAs were miR-16 for plasma and let-7a for EVs. Relative expression of miR-15b in plasma and in EVs was significantly higher in dogs with glioma than in control dogs. Relative expression of miR-342-3p in EVs was significantly higher in dogs with glioma than in control dogs. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that miR-15b and miR-342-3p have potential as noninvasive biomarkers for differentiating glioma from other intracranial diseases in dogs. However, more extensive analysis of expression in specific glioma subtypes and grades, compared with expression in more defined control populations, will be necessary to assess their clinical relevance.

Th17 cells increase during maturation in peripheral blood of healthy dogs.

Recent studies have indicated that T helper 17 (Th17) cells are involved in the pathogenesis of various inflammatory diseases in dogs. However, age-related changes in canine Th17 cells have not yet been investigated. In the present study, the proportion of Th17 cells was examined in the peripheral blood mononuclear cells (PBMCs) of healthy dogs at various ages: Group 1 (n = 16; less than 1 year of age), Group 2 (n = 25; 1-5 years), and Group 3 (n = 19; 6-9 years), using flow cytometry and an anti-human interleukin (IL)-17A monoclonal antibody that reacts with canine IL-17A. The proportion of circulating Th17 cells positively correlated with age. The age-related differences were observed in the proportion of Th17 cells among Group 1 (mean ± SD: 1.52 ± 1.18%), Group 2 (mean ± SD: 3.81 ± 1.94%) and Group 3 (mean ± SD: 7.49 ± 2.54%). Our results suggest that age-related changes in Th17 cells need to be considered in future research on Th17-related diseases in dogs.

Identification of reference genes for microRNAs of extracellular vesicles isolated from plasma samples of healthy dogs by ultracentrifugation, precipitation, and membrane affinity chromatography methods.

OBJECTIVE: To compare ultracentrifugation, precipitation, and membrane affinity chromatography methods for isolation of extracellular vesicles (EVs) from canine plasma samples and to identify suitable reference genes for incorporation into a quantitative reverse transcription PCR assay of microRNA expression in plasma EVs of healthy dogs. ANIMALS: 6 healthy Beagles. PROCEDURES: Plasma samples were obtained from each dog, and EVs were isolated from 0.3 mL of these samples via ultracentrifugation, precipitation, and membrane-affinity chromatographic methods. Nanoparticle tracking analysis was performed to determine the concentration and size distribution of EVs isolated by the ultracentrifugation method. Expression levels (cycle threshold values) of 4 microRNAs (let-7a, miR-16, miR-26a, and miR-103) were then compared by means of quantitative reverse transcription PCR assay. Three statistical programs were used to identify the microRNAs most suitable for use as reference genes. RESULTS: Results indicated that ultracentrifugation was the most stable of all 3 methods for isolating microRNAs from 0.3 mL of plasma. Nanoparticle tracking revealed that EV samples obtained by the ultracentrifugation method contained a mean ± SD of approximately 1.59 × 1010 vesicles/mL ± 4.2 × 108 vesicles/mL. Of the 4 microRNAs in plasma EVs isolated by ultracentrifugation, miR-103 was the most stable. CONCLUSIONS AND CLINICAL RELEVANCE: The ultracentrifugation method has potential as a stable method for isolating EVs from canine plasma samples with a high recovery rate, and miR-103 may provide the most stable reference gene for normalizing microRNA expression data pertaining to plasma EVs isolated by ultracentrifugation.