Role of DLA-DRB1 amino acids outside the shared epitope in dachshund susceptibility to immune-mediated polyarthritis.

Canine immune-mediated polyarthritis (IMPA) is an idiopathic disorder encompassing both erosive and non-erosive forms of rheumatoid arthritis (RA), with a clinical picture similar to human RA. Resemblance in major histocompatibility complex (MHC)-associated risk between the two was first noted within the specific amino acid motif known as the shared epitope (SE) on human leukocyte antigen DRB1. Following further identification of amino acids conferring risk for human RA outside the SE, this study was designed to examine amino acids both within and outside the classic SE in dachshunds, a breed with reported susceptibility to IMPA in Japan. Genome-wide association studies have linked positions 11, 13 and 71 with strong risk for human RA and important roles in antigen presentation to T cells. Sequence based genotyping of 16 case and 64 control dachshunds revealed strong associations comparable to human RA between IMPA risk and valine at position 11 (Val-11), phenylalanine at 13 (Phe-13), and arginine at 71 (Arg-71) on the dog leukocyte antigen (DLA)-DRB1 molecule (OR 2.89, 95%CI 1.3-6.4, p = 0.009), while association with the classic SE was significant only regarding homozygote frequency of the QRRAA haplotype-also carrying Val 11 and Phe 13 outside the SE (p = 0.04). Moreover, limited range in possible combinations of amino acids at positions 11, 13 and 71 starting with Val-11 among all DLA-DRB1 alleles registered with the GenBank and IPD-MHC canine databases, suggested potential of further single-breed analyses in dachshunds to clarify the disorder in terms of diagnosis, treatment, and epigenetic control, while clinical and immunopathogenetic similarities between human and dachshund RA also suggested the possibility of gaining insight into RA per se through study of canine IMPA as a spontaneous model of human RA.

Changes in renin-angiotensin-aldosterone system during cardiac remodeling after mitral valvuloplasty in dogs.

BACKGROUND: Information regarding changes in renin-angiotensin-aldosterone system (RAAS) during cardiac remodeling after mitral valvuloplasty (MVP) in dogs remains lacking. HYPOTHESIS/OBJECTIVES: To assess the longitudinal effects of MVP on circulating RAAS activity. ANIMALS: Eight client-owned dogs receiving MVP for myxomatous mitral valve disease (MMVD). METHODS: This is a cohort study. Plasma renin activity (PRA), angiotensin II (AT2), aldosterone (PAC), blood urea nitrogen (BUN), and creatinine concentrations, were measured in these dogs before (baseline) and at 3 consecutive monthly follow-ups (Post-1M, Post-2M, Post-3M). Echocardiography was concomitantly used to assess the process of cardiac recovery after MVP. RESULTS: The echocardiography revealed a significant decrease in LVIDDN, LA/Ao, FS, E velocity, E/A, E' sep, S' lat, E' lat, and A' lat after MVP compared with baseline (P < .05). There was a significant reduction in the PRA (2.45, 3.05, 2.74 vs 8.8 ng/mL/h; P = .002), AT2 (466, 315, 235 vs 1200 pg/mL; P = .009), and PAC (39.88, 47, 54.62 vs 179.5 pg/mL; P = .01), respectively at Post-1M, Post-2M, Post-3M compared to the baseline. Additionally, BUN and creatinine concentrations decreased from Post-1M. The RAAS variables showed significant, weak to moderate, relationship with selected echocardiographic variables. CONCLUSIONS AND CLINICAL IMPORTANCE: Mitral valvuloplasty contributes to decreased RAAS activity in MMVD dogs, which paralleled the process of cardiac reverse remodeling up to Post-3M. This information facilitates formulating strategies to optimize clinical outcomes for dogs after MVP.

Dog leukocyte antigen (DLA) class II genotypes associated with chronic enteropathy in French bulldogs and miniature dachshunds.

Canine chronic enteropathy (CE) is a group of immunogenetic disorders of unclear etiology characterized by chronic or recurrent gastrointestinal signs and inflammation. Diagnosis of CE subtypes by treatment response is a lengthy and challenging process, particularly in refractory cases of the disease. Given known association of dog leukocyte antigen (DLA) class II genotype and various immunogenetic disorders between and across breeds, this study was designed to examine the potential of determining susceptibility to refractory CE through identification of risk and protective genotypes in French bulldogs and miniature dachshunds-two popular dog breeds in Japan. Sequence-based genotyping of three DLA class II genes in 29 French bulldogs and 30 miniature dachshunds with refractory CE revealed a protective haplotype DLA-DRB1*002:01-DQA1*009:01-DQB1*001:01 against CE in French bulldogs (OR 0.09, 95 % CI 0.01-0.71, p = 0.0084). No statistical difference was noted between miniature dachshund cases and controls. These findings, largely disparate from a previous study on German shepherd dogs in the UK, were taken as possible indication of etiological differences in the refractory CE noted between and within breeds, and by extension, the potential of identifying such disease heterogeneity by DLA typing. The DLA-DQA1/DQB1 haplotype, protective against CE in our French bulldogs, has been reported as protective in various immune-mediated disorders such as Doberman hepatitis (Dyggve et al., 2011). Likewise, the DLA-DRB1*006:01 risk allele for Doberman hepatitis was noted in more French bulldogs with CE compared to controls, in line with reports on genotypes associated with both risk and protection being shared across various autoimmune diseases and breeds. These findings support an immunogenetic basis to the French bulldog-CE in our analysis, calling for further DLA studies working with larger samples and different breeds towards phenotypic clarification that may aid in early diagnosis, treatment, and prophylaxis through epigenetic approaches and breeding.

Effect of modified ultrafiltration on cytokines and hemoconcentration in dogs undergoing cardiopulmonary bypass.

Cardiac surgery using cardiopulmonary bypass (CPB) generates severe inflammatory reactions secondary to hemodilution and surgical stress. This study was conducted to evaluate whether modified ultrafiltration (MUF) could be performed safely and to clarify its effects during mitral valve repair in dogs in terms of hemodilution and the status of inflammatory cytokines. We retrospectively studied 38 dogs with mitral valve disease who underwent MUF immediately after mitral valve repair under CPB. To determine the effect of MUF, we measured the pre- and post-MUF blood dilution and blood cytokine levels. The levels of red blood cells, hematocrit (HCT), and albumin were significantly increased after MUF, whereas interleukin (IL)-6 levels were significantly increased from 24.3 (range 9.6-54.6) to 32.3 (15.9-65.1) pg/ml. The levels of IL-8 and IL-10 declined significantly after MUF, from 368.2 (246.1-669.4) and 45.4 (28.6-76.1) to 272.2 (174.1-414.4) and 28.8 (18.8-44.5) pg/ml, respectively. Our results demonstrated that MUF can be applied in dogs undergoing CPB and is effective in achieving hemoconcentration. Moreover, MUF may be useful for the removal of cytokines. Further studies are needed to validate these findings and clarify the effects of inflammatory cytokines after CPB.

Diagnostic utility of computed tomographic angiography in dogs with portal vein thrombosis.

Computed tomographic (CT) angiography, the gold standard for diagnosing portal vein thrombosis (PVT) in humans, is poorly documented in dogs. Therefore, we retrospectively reviewed dogs with PVT diagnosed by CT angiography. Medical records of 13 client-owned dogs diagnosed with PVT by CT angiography were reviewed. All dogs had chronic PVT, and the most frequent clinical sign was vomiting (5/13), with pancreatitis the most frequent concurrent disease (6/13). All dogs tested for plasma D-dimer concentration (12/12) revealed elevated levels. On CT angiography, a thrombus was detected as a non-contrast enhancement structure in the portal vessel of 13 dogs. There was no evidence of complete obstruction of the portal vein in any of the dogs. The median luminal filling of the portal vein was 60.4%. The thrombus extension was variable among dogs, with a median of 34.9 mm. CT angiography identified the thrombus in the main portal vein of 12/13 dogs and multiple thrombus formation other than the main portal vein in 9/13 dogs. CT angiography provided specific information such as detecting the presence, location, and number of PVT in dogs. Therefore, CT angiography might be useful for the diagnosis and follow-up evaluation of PVT in dogs.

Plasma renin activity and aldosterone concentration in dogs with acquired portosystemic collaterals.

BACKGROUND: The renin-angiotensin-aldosterone system (RAAS) is activated in humans with portal hypertension (PH) associated with liver disease. However, involvement of RAAS in dogs with intrahepatic PH is not clear. OBJECTIVE: To measure plasma renin activity (PRA) and plasma aldosterone concentration (PAC) in dogs with PH (chronic hepatitis [CH] and primary hypoplasia of the portal vein [PHPV]), dogs with extrahepatic congenital portosystemic shunt (EH-CPSS), and healthy dogs and to determine whether the RAAS is activated in dogs with PH. ANIMALS: Twenty-seven dogs with acquired portosystemic collaterals (APSCs; 15 dogs with CH, 12 dogs with PHPV), 9 dogs with EH-CPSS, and 10 healthy dogs. METHODS: Retrospective study. Plasma renin activity and PAC were measured by radioimmunoassay. RESULTS: Plasma renin activity was significantly higher in the CH group (median, 4.4 ng/mL/h) than in the EH-CPSS (median, 1.0 ng/mL/h; P < .01) and the healthy (median, 1.1 ng/mL/h; P < .01) groups. No significant differences were found between the PHPV group (median, 2.2 ng/mL/h) and other groups. Plasma aldosterone concentration was significantly higher in the CH (median, 111.0 pg/mL) and PHPV (median, 89.5 pg/mL) groups than in the EH-CPSS (median, 1.0 pg/mL; P < .001, P < .01, respectively) and healthy (median, 14.5 pg/mL; P < .001, P < .05, respectively) groups. CONCLUSIONS AND CLINICAL IMPORTANCE: Activation of the RAAS contributes to the pathophysiology of intrahepatic PH in dogs, suggesting that spironolactone may not only be effective for the treatment of ascites but also for the suppression of intrahepatic PH.

Comparison of conventional magnetic resonance imaging and nonenhanced three dimensional time-of-flight magnetic resonance angiography findings between dogs with meningioma and dogs with intracranial histiocytic sarcoma: 19 cases (2010-2014).

OBJECTIVE To compare conventional MRI and nonenhanced 3-D time-of-flight (TOF) magnetic resonance angiography (MRA) findings between dogs with meningioma and dogs with intracranial histiocytic sarcoma (IHS). DESIGN Retrospective case series. ANIMALS 14 dogs with meningioma and 5 dogs with IHS. PROCEDURES Medical records of dogs with meningioma or IHS that were examined at a tertiary veterinary hospital from 2010 through 2014 and underwent 3-D TOF MRA in conjunction with conventional MRI were reviewed. Findings for conventional MRI and 3-D TOF MRA were compared between the 2 groups of dogs to evaluate whether there were any characteristics that could be used to differentiate meningioma from IHS. RESULTS Tumor type was significantly associated with signal intensity on conventional T2-weighted and fluid-attenuated inversion recovery MRI images; most meningiomas were hyperintense, and most IHSs were isointense or hypointense on those images. Tumor type was not associated with signal uniformity, tumor location, tumor origin, or the presence of edema, midline shift, or brain herniation. On MRA, blood vessels adjacent to the tumor were identified and characterized for 9 of 14 dogs with meningioma and all 5 dogs with IHS. Vessels adjacent to meningiomas were displaced in 8 of 9 dogs, whereas vessels adjacent to IHSs were not displaced. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated nonenhanced 3-D TOF MRA findings provided additional information that can be assessed in conjunction with conventional MRI findings to help differentiate meningiomas from IHSs in dogs.

Methylation of free-floating deoxyribonucleic acid fragments in the bronchoalveolar lavage fluid of dogs with chronic bronchitis exposed to environmental tobacco smoke.

BACKGROUND: The etiology of canine chronic bronchitis (CB) is not completely understood, although exposure to environmental tobacco smoke (ETS) affects the airway inflammatory responses in some dogs with CB. The mechanism by which this occurs is unknown. FINDINGS: We investigated the concentrations and methylation rates of free-floating DNA fragments in bronchoalveolar lavage fluid (BALF) from dogs with chronic bronchitis. Based on serum cotinine levels, dogs with CB were divided into 2 groups: dogs that either had or had not been exposed to ETS. Our results demonstrated that the total nucleated cell and macrophage numbers increased in BALF of ETS-exposed dogs with CB. There were no significant differences in DNA concentrations and methylation rates in BALF between the 2 groups. However, 3 out of 8 dogs exposed to ETS had high DNA methylation rates in their BALF samples. CONCLUSION: Our results suggest that ETS exposure leads to epigenetic modifications of cellular components in BALF in dogs diagnosed with CB.

Increased proportions of CCR4(+) cells among peripheral blood CD4(+) cells and serum levels of allergen-specific IgE antibody in canine chronic rhinitis and bronchitis.

Canine chronic rhinitis (CR) and bronchitis (CB) are suspected to be allergic diseases. The present study tested whether dogs diagnosed with CR or CB present an atopic predisposition based on the ratio of CC chemokine receptor 4 (CCR4)-positive cells among peripheral blood CD4-positive cells (CCR4/CD4) and the serum levels of allergen-specific IgE antibodies. We found that most dogs with CR and CB have a possibility of atopic predisposition, and macrolide therapy constitutes an alternative to corticosteroid therapy in controlling the clinical signs.

Expression of selectins and P-selectin glycoprotein ligand-1 in dogs with lymphocytic-plasmacytic enteritis.

Lymphocytic-plasmacytic enteritis (LPE) is the most common form of inflammatory bowel disease (IBD) affecting the canine small intestine; however, the molecular pathogenesis of this disease remains unclear. Although selectins and their ligands play a critical role as cell adhesion molecules during inflammation, there is very little information about their involvement in canine LPE. The aim of this study was to evaluate transcript expression of selectins (E-, L-, and P- selectin) and P-selectin glycoprotein ligand-1 (PSGL-1) in the duodenal mucosa of 21 dogs with LPE and 10 healthy laboratory beagles. Duodenal expression of E-selectin, L-selectin, P-selectin, and PSGL-1 was quantified by real-time reverse-transcription PCR. Correlations between clinical severity, histopathological grade, selectins, and PSGL-1 were analyzed by Spearman's rank test. Transcript expression of duodenal E- and P-selectins and PSGL-1 was higher in dogs with LPE than in healthy laboratory beagles; however, there was no difference in L-selectin expression. Positive correlations between E- and L-selectin and between L- and P-selectin were observed in the duodenum of LPE dogs. The selectins and ligand may recruit circulating inflammatory cells into the lesion. These findings improve our understanding of the inflammatory cascade of canine LPE.

Evaluation of a disintegrin-like and metalloprotease with thrombospondin type 1 repeat motifs 13 (ADAMTS13) activity enzyme-linked immunosorbent assay for measuring plasma ADAMTS13 activity in dogs.

A disintegrin-like and metalloprotease with thrombospondin type 1 repeat motifs 13 (ADAMTS13) is a von Willebrand factor (vWF)-cleaving protease. Deficiencies in ADAMTS13 activity are known to cause thrombotic diseases in human beings. The present study evaluated whether the human ADAMTS13 activity enzyme-linked immunosorbent assay (ELISA) kit containing human vWF73 (a minimal substrate) and anti-N10 antibody (which specifically recognizes the decapeptide of the C-terminal edge of cleaved vWF by human ADAMTS13) is applicable to the measurement of canine plasma ADAMTS13 activity. Human vWF73 fused with a GST-tag and a His-tag (GST-hvWF73-His) was reacted with recombinant canine (rc)ADAMTS13, canine plasma, and human plasma, and then used in Western blotting using anti-N10 antibody. Linearity and intra- and interassay reproducibility of the human ADAMTS13 activity ELISA kit in canine plasma were further evaluated. Finally, plasma ADAMTS13 activity was measured in 13 healthy dogs and 6 dogs with bacteremia using the human ADAMTS13 activity ELISA kit. Cleaved products with a 28-kDa GST-hvWF73-His were detected specifically in rcADAMTS13 as well as in human ADAMTS13, and also in canine plasma by anti-N10 antibody, showing excellent linearity. Intra-assay coefficient of variation (CV) was 3.0-12.4%, and interassay CV was 11.5-12.5%. The ADAMTS13 activity was significantly lower in dogs with bacteremia than in healthy dogs (P = 0.0025). The current study revealed that the human ADAMTS13 activity ELISA kit is applicable for measurement of canine plasma ADAMTS13 activity to elucidate the pathology of thrombotic diseases in dogs.

Case report: First confirmed case of canine peritoneal larval cestodiasis caused by Mesocestoides vogae (syn. M. corti) in Japan.

Canine peritoneal larval cestodiasis (CPLC) is an unusual parasitic disease in dogs that is caused by asexual proliferation of larval Mesocestoides. A 12 year-old spayed Shetland sheepdog with abdominal distension was referred to the Animal Medical Center at Nihon University, Japan. The presence of ascites was confirmed by abdominal ultrasonography and X-ray imaging. In addition, a number of parasites were observed in the ascitic fluid collected by abdominal paracentesis. Each of the whitish colored parasites was less than 1mm in size. The parasites were morphologically identified as Mesocestoides sp. tetrathyridia. The parasites had four suckers and calcareous corpuscles, but no hooks or rostellum. Mitochondrial (mt) 12S rDNA and mt cytochrome c oxidase subunit 1 DNA amplified from the tetrathyridia were used for molecular identification to species level. DNA sequence analysis showed that the tetrathyridia shared more than 99% identity with M. vogae (syn. M. corti) for each gene. The patient was treated with a standard dose (5mg/kg) of praziquantel, which was administered subcutaneously twice at an interval of 14 days. This resulted in successful deworming. This is the first case that CPLC was diagnosed in a dog that had never been taken outside of Japan, indicating that M. vogae is distributed in this country.

Regional variations and age-related changes detected with magnetic resonance spectroscopy in the brain of healthy dogs.

OBJECTIVE: To investigate age-related and regional differences in estimated metabolite concentrations in the brain of healthy dogs by means of magnetic resonance spectroscopy (MRS). ANIMALS: 15 healthy Beagles. PROCEDURES: Dogs were grouped according to age as young (n = 5; all dogs were 2 months old), adult (5; mean age, 4.5 years), or geriatric (5; all dogs were 12 years old). Imaging was performed by use of a 1.5-T MRI system with T1- and T2-weighted spin-echo and fluid-attenuated inversion recovery sequences. Signal intensity measurements for N-acetyl aspartate, creatine, choline, and lactate-alanine (the spectroscopic peaks associated with alanine and lactate could not be reliably differentiated) were determined with MRS, and areas under the spectroscopic peaks (representing concentration estimates) were calculated. Ratios of these metabolite values were compared among age groups and among brain regions with regression analysis. RESULTS: The choline-to-creatine ratio was significantly higher in young dogs, compared with other age groups. The N-acetyl aspartate-to-choline ratio was significantly lower in young dogs and geriatric dogs than in adult dogs. When all age groups were considered, the choline-to-creatine ratio was significantly higher and N-acetyl aspartate-to-choline ratio was significantly lower in the frontal lobe than in all other regions. The N-acetyl aspartate-to-creatine ratio was significantly lower in the cerebellum than in other regions. CONCLUSIONS AND CLINICAL RELEVANCE: Metabolite ratios varied significantly among age groups and brain regions in healthy dogs. Future studies should evaluate absolute concentration differences in a larger number of dogs and assess clinical applications in dogs with neurologic diseases.

Gene expression of growth factors and growth factor receptors for potential targeted therapy of canine hepatocellular carcinoma.

The purpose of this study was to evaluate the gene expression of growth factors and growth factor receptors of primary hepatic masses, including hepatocellular carcinoma (HCC) and nodular hyperplasia (NH), in dogs. Quantitative real-time reverse transcriptase-polymerase chain reaction was performed to measure the expression of 18 genes in 18 HCCs, 10 NHs, 11 surrounding non-cancerous liver tissues and 4 healthy control liver tissues. Platelet-derived growth factor-B (PDGF-B), transforming growth factor-α, epidermal growth factor receptor, epidermal growth factor and hepatocyte growth factor were found to be differentially expressed in HCC compared with NH and the surrounding non-cancerous and healthy control liver tissues. PDGF-B is suggested to have the potential to become a valuable ancillary target for the treatment of canine HCC.

Bronchoalveolar lavage fluid and serum canine surfactant protein A concentrations in dogs with chronic cough by bronchial and interstitial lung diseases.

We measured bronchoalveolar lavage fluid (BALF) and serum canine surfactant protein (cSP)-A concentrations in dogs with chronic cough. There were no significant differences between bronchial and interstitial lung diseases in BALF cSP-A concentrations. However, serum cSP-A concentrations in dogs with the interstitial lung disease as diffuse panbronchiolitis and idiopathic pulmonary fibrosis were significantly higher than those in dogs with the bronchial disease as chronic bronchitis. These results suggest that serum cSP-A concentrations may be a useful and noninvasive biomarker to understand the existence of interstitial lung damage in dogs with chronic cough.

Successful resolution of esophageal granulomas in a dog infected with Spirocerca lupi.

A 13-year-old female Labrador Retriever presented with chronic regurgitation. Radiography and computed tomography (CT) revealed nodules in the caudal esophagus. Upper gastrointestinal endoscopy followed by histopathological examination revealed esophageal granulomas caused by Spirocerca lupi. The infection was treated with milbemycin oxime. The therapy was successful, and a remarkable reduction in granulomas was observed. This case report describes the diagnostic imaging findings and treatment outcome of a dog with S. lupi infection in Japan.

Activation of nuclear factor-kappa B and cell adhesion molecule mRNA expression in duodenal mucosa of dogs with lymphocytic-plasmacytic enteritis.

Lymphocytic-plasmacytic enteritis (LPE) is the most common form of inflammatory bowel disease (IBD) affecting the canine small intestine; however, the molecular basis of the pathogenesis remains unclear. It has recently been hypothesized that the primary defect is impaired innate immune function, as is the case for human IBD. Nuclear factor-kappa B (NFkappaB) plays a central role in innate immunity, and is a major transcriptional regulator of several proinflammatory cytokines, pattern recognition receptors (PRRs) such as toll-like receptors (TLRs), nucleotide-binding oligomerization domain-like receptors and cell adhesion molecules (CAMs). The purpose of this study was to evaluate, in the duodenal mucosa of 21 dogs with LPE and 8 control dogs, the degree of NFkappaB activity and the mRNA expression of two selected cytokines, nucleotide oligomerization domain two (NOD2) receptor and three selected CAMs, all of which are regulated by NFkappaB, using the electrophoretic mobility shift assay and real-time reverse transcription PCR. NFkappaB binding activity was significantly higher in the inflamed duodenal mucosa of the LPE dogs as compared to healthy controls. Furthermore, expression of mRNA for intercellular cell adhesion molecule 1 (ICAM-1) and mucosal addressin cell adhesion molecule 1 (MAdCAM-1) was significantly higher and vascular cell adhesion molecule 1 (VCAM-1) mRNA significantly lower in LPE dogs than in healthy controls. However, there was no significant difference in the mRNA levels for TNFα, IL1ß and NOD2 between the two groups. These results suggest that NFkappaB and CAMs may play important roles in the pathogenesis of canine LPE.

The characteristics of short- and long-term surviving Shiba dogs with chronic enteropathies and the risk factors for poor outcome.

BACKGROUND: The objectives of this study were to investigate the differences in the characteristics of short- and long-term surviving dogs, and the factors that predict poor outcome in Shiba dogs with chronic enteropathies (CE). METHODS: A total of 25 Shiba dogs were included in this study, and classified as either short-term (≤6 months) survivors (Ss; n=16) or long-term (>6 months) survivors (Ls; n=9). The clinical and clinicopathological variables, histopathology, response to therapy, and outcomes were investigated between groups. Furthermore, these factors were tested for their ability to predict poor outcome. RESULTS: All CE dogs were diagnosed as having inflammatory bowel disease (IBD) with lymphocytic-plasmacytic enteritis (LPE). Age and canine inflammatory bowel disease activity index (CIBDAI) were significantly higher in the Ss group than in the Ls group (age: p = 0.035, CIBDAI: p = 0.018), as determined via univariate logistic regression analysis. According to receiver operator characteristic (ROC) curve analysis, the best predictors of poor outcome were age and CIBDAI, with the cutoffs determined as 7 years and 9 points, respectively. The majority of the cases (84%) responded to initial treatment; in particular, 75% of dogs in Ss group responded to therapy. The time to response (days) to the initial treatment in the Ss group (median 42.5 days, range: 20-91 days) was significantly shorter than that of the Ls group (median 285 days, range: 196-1026 days). Approximately half (55.5%) of the dogs in the Ls group died due to relapse of CE. CONCLUSIONS: This study suggested that there is a high risk of early mortality in Shiba dogs with CE, particularly if the dogs are older (>7 years) and have a high CIBDAI score (>9 points). There appears to be a possibility of early mortality even if the initial treatment was efficacious. Furthermore, Shiba dogs with CE that become less responsive to initial therapy in the short-term (approximately 3 months) are more likely to have an early mortality. Thus, it is necessary to follow-up Shiba dogs with CE in the long-term, as approximately half of the long-term survivors eventually died due to a relapse of the signs.