Transmission of Helicobacter pylori between a human and two dogs: A case report.

BACKGROUND: Whereas non-Helicobacter pylori helicobacters, which are frequently detected in the stomachs of dogs and cats as a source of zoonoses, have attracted considerable attention, the role of pets in H. pylori epidemiology is unclear. In our previous study, an H. pylori infection was detected in the stomach of a dog (Dog 1). Here, we investigated the H. pylori infection status in the female offspring of Dog 1 (Dog 2) and its owner within the same household. MATERIALS AND METHODS: Biopsy specimens were obtained from the dog's owner and tested for H. pylori. DNA from gastric biopsy samples of Dog 1, gastric fluid sediment of Dog 2, and bacteria from the stomach of the owner was obtained, and Helicobacter genus- and species-specific PCRs were performed. Then, sequence analyses of the partial region of the ureAB gene were conducted. RESULTS: Samples from both dogs and the owner reacted positively in the genus-specific PCR and negative in the Helicobacter felis-, Helicobacter bizzozeronii-, and Helicobacter heilmannii sensu stricto-specific PCRs. All three samples also reacted positively in the H. pylori-specific PCR. Sequences of the partial ureAB gene from all subjects were identical. CONCLUSIONS: The results suggested that the two dogs and their owner were infected with an identical H. pylori strain. This report is the first to demonstrate that H. pylori can be transmitted between humans and dogs. Further studies are required to investigate the risk factors for the transmission of H. pylori between humans and dogs from the perspective of preventive epidemiology.

Pneumatosis coli after partial ligation of congenital portosystemic shunt in a dog.

Pneumatosis coli is a rare intestinal disorder in dogs that is characterized by submucosal or subserosal emphysema of the colon. An 8-year-old castrated male Shih Tzu developed anorexia and hematochezia after undergoing surgery for a congenital splenophrenic shunt. Abdominal radiographic examination revealed linear radiolucency throughout the wall of the large intestine. Results of abdominal computed tomography revealed intramural gas tracking along the colon and rectum. Based on these findings, a diagnosis of pneumatosis coli was made. The dog was treated with antibiotics, but the general condition gradually deteriorated, and the dog died 6 days after the day of diagnosis. It was suspected that portal hypertension following partial ligation of congenital portosystemic shunt may have been associated with pneumatosis coli in this case.

Multiple acquired portosystemic shunts secondary to primary hypoplasia of the portal vein in a cat.

A 6-year 5-month-old spayed female Scottish Fold cat presented with a one-month history of gait abnormalities, increased salivation, and decreased activity. A blood test showed hyperammonemia and increased serum bile acids. Imaging tests revealed multiple shunt vessels indicating acquired portosystemic shunt. Histopathologic analysis of liver biopsy showed features consistent with liver hypoperfusion, such as a barely recognizable portal vein, increased numbers of small arterioles, and diffuse vacuolar degeneration of hepatocytes. These findings supported the diagnosis of primary hypoplasia of the portal vein/microvascular dysplasia, (PHPV/MVD). To our knowledge, this is the first case of feline PHPV/MVD that developed multiple acquired portosystemic shunts and presented with hepatic encephalopathy.

Analysis of fecal short chain fatty acid concentration in miniature dachshunds with inflammatory colorectal polyps.

Short chain fatty acids (SCFAs) play an important role in the maintenance of colonic homeostasis, and their depletion has been reported in various gastrointestinal disorders. Inflammatory colorectal polyps (ICRPs) are a recently recognized disease specific to miniature dachshunds (MDs), and fecal dysbiosis with a reduction of SCFA-producing bacteria has been reported with this disease. Therefore, this study was performed based on the hypothesis that a reduced SCFA concentration associates with the development of ICRPs. We recruited 11 ICRP-affected MDs and 25 control MDs. Their fecal SCFA concentrations and bacterial proportions were quantified using high performance liquid chromatography and quantitative real-time PCR, respectively. The feces of ICRP-affected MDs contained lower amounts of propionic acid and lower proportions of Bifidobacterium than the feces of control MDs. Furthermore, fecal proportions of Bifidobacterium, Firmicutes and Lactobacillus exhibited significant positive correlations with fecal concentrations of total SCFAs and/or propionic acid; fecal Escherichia coli proportions correlated negatively with fecal concentrations of total SCFAs, as well as acetic, propionic and butyric acid. This result indicates an association between fecal dysbiosis and fecal SCFA concentrations; these phenomena may contribute to ICRP pathogenesis in MDs. Potential therapeutic targeting of the reduced propionic acid concentration using probiotics, prebiotics or SCFA enemas merits further study.

Adenocarcinoma of Barrett's esophagus in a dog.

An endoscopic examination revealed a mass in the distal esophagus of a 9-year-old intact male bulldog. Histopathologically, the mass was composed of cuboidal to columnar neoplastic epithelial cells and extended from the squamous epithelium of the esophageal mucosa, indicating that the tumor was derived from Barrett's esophagus. Moreover, highly atypical foci that exhibited a cribriform pattern and high mitotic indices were also observed. The epithelial cells on the surface of the lesion often produced mucus that was positive for Alcian blue and immunohistochemically positive for MUC5AC. The neoplastic epithelial cells were diffusely positive for cytokeratin 7 and p53, and occasionally positive for cytokeratin 20. Based on these findings, the tumor was diagnosed as an adenocarcinoma. This report describes the clinical and pathological features of a spontaneous case of adenocarcinoma of Barrett's esophagus in a dog.

Significance of clonal rearrangements of lymphocyte antigen receptor genes on the prognosis of chronic enteropathy in 22 Shiba dogs.

Shiba dogs are predisposed to chronic enteropathy (CE) and have poorer prognosis than other dog breeds. The objective of this study was to investigate the significance of polymerase chain reaction for antigen receptor rearrangement (PARR) results on clinical findings and prognosis of Shiba dogs with CE. We retrospectively collected data on 22 Shiba dogs diagnosed as having CE. Fifty-nine percent of the dogs had clonality-positive results on PARR analysis. Furthermore, on histopathology, epitheliotropic behavior of small lymphocytes of the intestinal mucosa was observed significantly more frequently in dogs with clonal rearrangement of antigen receptor genes (P=0.027). The median overall survival time of clonality-positive dogs was 48 days (range, 4-239 days), compared to 271 days (range, 45-1,316+ days) in clonality-negative dogs. The median overall survival time of epitheliotropism-positive dogs was 76 days (range, 30-349 days) compared to 239 days (range, 4-1,316+ days) for epitheliotropism-negative dogs. Statistical analysis revealed that the clonality-positive result was associated with significantly shorter survival time (P=0.036). In contrast, presence or absence of epitheliotropism had no statistically significant effect on survival time (P=0.223). These cases might appropriately be diagnosed as small T-cell intestinal lymphoma; there are some common clinical and pathogenic features with human enteropathy-associated T-cell lymphoma type 2. The pathogenesis and poor prognosis for Shiba dogs with CE seem to be associated with this type of lymphoma, although further investigation is warranted.

Epidemiological study of gastric Helicobacter spp. in dogs with gastrointestinal disease in Japan and diversity of Helicobacter heilmannii sensu stricto.

Epidemiological and pathological studies of Helicobacter spp. in canine stomachs in Japan were performed to investigate strain specific pathogenicity. Gastric biopsies from 144 dogs with gastrointestinal diseases were evaluated for the presence of Helicobacter spp. using genus and species specific PCRs for Helicobacter felis, Helicobacter bizzozeronii, Helicobacter heilmannii sensu stricto (s.s.) and Helicobacter pylori. PCR indicated that 50/144 (34.7%) dogs were infected with Helicobacter spp. Of the genus positive samples, 21/50 could not be amplified by any of the species specific PCRs. To investigate Helicobacter at the species level, partial ureAB gene sequences from 48/50 genus positive samples were determined; 47 strains were identified. Thirty-five strains from 45 cases were closely related to H. heilmannii s.s. (89-99% sequence similarity), seven strains from seven cases were closely related to H. bizzozeronii (95-99% sequence similarity), three strains from three cases were closely related to Helicobacter felis (86%, 98% and 99% sequence similarity), one strain from one case was closely related to Helicobacter salomonis (99% sequence similarity) and one strain from one case was closely related to H. pylori (99% sequence similarity). Dogs infected with Helicobacter spp. most similar to H. heilmannii s.s. had a higher frequency of moderate to severe gastritis than dogs negative for Helicobacter spp. (P=0.044). In conclusion, the predominant Helicobacter spp. detected in canine stomachs in our study were most closely related to H. heilmannii s.s. and displayed substantial genetic diversity. Infection with Helicobacter spp. may be associated with more severe gastritis in dogs.

Bile acid composition of gallbladder contents in dogs with gallbladder mucocele and biliary sludge.

OBJECTIVE To examine bile acid composition of gallbladder contents in dogs with gallbladder mucocele and biliary sludge. ANIMALS 18 dogs with gallbladder mucocele (GBM group), 8 dogs with immobile biliary sludge (i-BS group), 17 dogs with mobile biliary sludge (m-BS group), and 14 healthy dogs (control group). PROCEDURES Samples of gallbladder contents were obtained by use of percutaneous ultrasound-guided cholecystocentesis or during cholecystectomy or necropsy. Concentrations of 15 bile acids were determined by use of highperformance liquid chromatography, and a bile acid compositional ratio was calculated for each group. RESULTS Concentrations of most bile acids in the GBM group were significantly lower than those in the control and m-BS groups. Compositional ratio of taurodeoxycholic acid, which is 1 of 3 major bile acids in dogs, was significantly lower in the GBM and i-BS groups, compared with ratios for the control and m-BS groups. The compositional ratio of taurocholic acid was significantly higher and that of taurochenodeoxycholic acid significantly lower in the i-BS group than in the control group. CONCLUSIONS AND CLINICAL RELEVANCE In this study, concentrations and fractions of bile acids in gallbladder contents were significantly different in dogs with gallbladder mucocele or immobile biliary sludge, compared with results for healthy control dogs. Studies are needed to determine whether changes in bile acid composition are primary or secondary events of gallbladder abnormalities.

Intranuclear inclusions in a dog with B-cell leukemia.

A 7-year-old Shetland Sheepdog was presented with anorexia. A CBC indicated thrombocytopenia and neutropenia. Bone marrow cytology revealed that 67.7% of all nucleated cells (ANC) were anaplastic large mononuclear cells. These cells were confirmed to be of B-cell origin based on IgH rearrangement, immunohistochemical, and flow cytometric analysis. Microscopic examination revealed that the neoplastic cells had intranuclear inclusions resembling Dutcher bodies. Immunohistochemistry confirmed that the intranuclear inclusions were immunopositive for IgG antibodies. The periodic acid-Schiff reaction was negative for the presence of polysaccharides and related substances. Although the dog achieved complete remission with a multi-drug chemotherapy protocol, it ultimately died because of tumor progression and acute renal insufficiency on day 201. This is the first known case of canine acute B-cell leukemia with intranuclear inclusions resembling Dutcher bodies.

Fecal dysbiosis in miniature dachshunds with inflammatory colorectal polyps.

Chronic gastrointestinal disease is associated with the alteration of gastrointestinal microbiota. Inflammatory colorectal polyps (ICRPs) are commonly observed in miniature dachshunds (MDs) in Japan and are characterized by multiple polyps that are restricted in the colorectal mucosa with severe neutrophil infiltration. This study was aimed to compare the fecal microbiota of ICRP-affected MDs with that of healthy MDs. High-throughput sequencing of amplicons derived from the V3-V4 region of the 16S rRNA gene was applied using the Illumina MiSeq system. Principal coordinates analysis revealed that fecal microbiota of ICRP-affected MDs was significantly altered compared with that of healthy MDs. Proportions of Fusobacteriaceae, Helicobacteraceae, Porphyromonadaceae, and Turicibacteraceae were significantly more abundant in ICRP-affected MDs, while those of Lachnospiraceae were significantly less abundant in ICRP-affected MDs compared with healthy MDs. These results suggest that the dysbiosis is associated with ICRPs and is a potential therapeutic target, though further investigations are needed.

Detection of circulating tumor cells using GeneScan analysis for antigen receptor gene rearrangements in canine lymphoma patients.

The presence of circulating tumor cells (CTCs) serves as a prognostic marker and indicator of disease relapse, as well as a means of evaluating treatment efficacy in human and canine lymphoma patients. As an extension of our previous study for the construction of clinically useful GeneScan system, we utilized the GeneScan system for detecting CTCs in canine lymphoma patients. Samples from the primary lesion and peripheral blood mononuclear cells (PBMCs) were obtained from 32 dogs with lymphoma at initial diagnosis. All samples were subjected to polymerase chain reaction (PCR) for antigen receptor gene rearrangements (PARR) followed by GeneScan analysis. Common clonal rearrangements with identical amplified fragments were detected in both the primary lesion and PBMCs in 19 of the 32 dogs (59.4%). However, the detection rate of CTCs varied among the anatomical classification of lymphoma studied. GeneScan analysis following PARR would facilitate studies on determining the clinical significance of CTCs in canine lymphoma patients.

Characterization of triple-phase computed tomography in dogs with pancreatic insulinoma.

Little information is available regarding triple-phase computed tomography (CT) of canine pancreatic insulinoma. A few case reports with small numbers of cases have indicated that hyper-attenuation in the arterial phase was a common finding on multi-phasic CT in dogs with insulinoma. Our purpose was to clarify the characteristic findings of dogs with insulinoma on triple-phase CT. Nine dogs with insulinoma that underwent triple-phase CT were included in the present study. Attenuation patterns in the arterial phase indicated hypo-attenuation in 4 cases and hyper-attenuation in 2 cases. In the remaining 3 cases, 1 case showed hypo-attenuation and 1 case showed hyper-attenuation in the pancreatic phase, and 1 case presented hyper-attenuation in the later phase. Altogether, 5 cases showed hypo and 4 cases showed hyper-attenuation in at least one phase. The enhancement pattern was homogenous in 7 cases and heterogeneous in 2 cases. Tumor margins were well-defined in 5 cases and ill-defined in 4 cases. Capsule formation was present in 5 cases and absent in 4 cases. In conclusion, it is important to note that hypo-attenuation was as common as hyper-attenuation in dogs with insulinoma in triple-phase CT in at least one phase. Additionally, mass lesions were most conspicuous not only in the arterial phase but in the pancreatic and later phases in some cases. Therefore, it is important to perform triple-phase CT and notice about variable findings for the detection of canine pancreatic insulinoma.

Efficacy of leflunomide for treatment of refractory inflammatory colorectal polyps in 15 Miniature Dachshunds.

Inflammatory colorectal polyp (ICRP), common in miniature dachshunds, presents with hematochezia, tenesmus and mucoid feces. Although an 80% response rate has been reported when treated with prednisolone and cyclosporine, effective treatment is needed for the remaining 20% of ICRP dogs. Leflunomide is an immunosuppressive drug reported as effective in several immune-mediated diseases. In the present study, we retrospectively evaluated the efficacy and adverse effects of leflunomide in 15 ICRP dogs that were refractory to treatment with prednisolone and cyclosporine. Treatment efficacy was assessed by endoscopy, clinical symptoms and rectal palpation. Adverse effects were determined by clinical symptoms and blood testing during follow-up. The leflunomide treatment response rate was 93.3%. The median dosage of leflunomide and the median response time were 3 mg/kg (range: 1.7-4.0 mg/kg) and 35 days (range: 20-119 days), respectively. Adverse effects observed included lethargy (3 dogs), anorexia (1 dog), respiratory symptoms (1 dog), leukocytopenia (2 dogs), thrombocytopenia (1 dog), anemia (1 dog) and liver enzyme elevation (8 dogs). Most of the adverse effects improved with symptomatic treatment and leflunomide discontinuation or dosage reduction. In conclusion, leflunomide treatment is effective in ICRP dogs refractory to treatment with prednisolone and cyclosporine. Because several adverse effects were observed, close monitoring is needed during leflunomide treatment follow-up.

Association between lymphocyte antigen receptor gene rearrangements and histopathological evaluation in canine chronic enteropathy.

Although definitive diagnosis of chronic enteropathy (CE) and gastrointestinal (GI) lymphoma requires histopathological evaluation of the GI tract, these conditions are often still difficult to differentiate from each other. Polymerase chain reaction (PCR) for antigen receptor gene rearrangements (PARR) has been applied recently as an adjunctive for diagnosis of lymphoid tumors; however, its clinical value in canine CE and GI lymphoma remains unclear. The purpose of this study was to investigate the relationship between PARR and histopathological diagnosis, degree of enteritis or lymphoma, and long-term prognosis in dogs, in order to evaluate the clinical significance of PARR. Endoscopic biopsy specimens obtained from 96 dogs with chronic enteritis (mild, n=14; moderate, n=20; marked, n=62) and 21 dogs with GI lymphoma were used. Clonality was observed in 51% of the animals with chronic enteritis; interestingly, it was found in 29% of those with only mild enteritis. In dogs with marked enteritis, the rate of PARR was higher in those with lymphocyte epitheliotropism than in those without epitheliotropism. The sensitivity of PARR in animals with GI lymphoma was 76%. There was no significant prognostic difference between chronic enteritis with or without clonal rearrangements. In contrast, dogs histopathologically diagnosed with marked enteritis had a significantly shorter survival time than did those with mild or moderate enteritis. While the significance of PARR in the diagnosis of GI lymphoma remains uncertain, the pathological roles of clonally expanding lymphocytes in canine CE should be investigated further.

Polymorphisms of nucleotide-binding oligomerization domain 2 (NOD2) gene in miniature dachshunds with inflammatory colorectal polyps.

Inflammatory colorectal polyps (ICRPs) frequently occur in miniature dachshunds (MDs) in Japan, typically form multiple polyps with severe neutrophil infiltration. ICRPs are speculated as a novel, breed-specific canine inflammatory bowel disease. Pattern recognition receptors (PRRs) play an important role in the differentiation of pathogens from commensal bacteria and food antigens, and polymorphisms of various PRRs have been shown to be associated with human and canine IBD. We recently reported that the reactivity of nucleotide-binding oligomerization domain 2 (NOD2), toll-like receptor (TLR) 1/2, TLR2, and TLR2/6 are greater in ICRP-affected MDs than that in controls. Therefore, this study was aimed to investigate single nucleotide polymorphisms (SNPs) of PRRs associated with ICRPs in MDs. Mutational analysis of canine NOD2, TLR1, TLR2, and TLR6 genes was performed with six ICRP-affected MDs, five control MDs, and five healthy beagles. The mutational analysis identified 13 non-synonymous SNPs in NOD2, TLR1, TLR2, and TLR6 genes, of which six SNPs in NOD2 exon 3 were further analyzed in an association study using 63 ICRP-affected MDs, 82 control MDs, and 237 control dogs of various breeds. Four of the SNPs (A1532G, T1573C, C1688G, and G1880A of the NOD2 gene) were in Hardy-Weinberg equilibrium and in complete linkage disequilibrium in MDs, and their minor allele frequencies were significantly lower in ICRP-affected MDs than in control MDs (0.016 vs. 0.140, P=0.0002). The calculated inheritance model was an additive model (odds ratio=0.10, 95% confidence interval=0.02-0.45, P=0.0001), which indicates that the haplotype with minor alleles in these SNPs (A, T, C, and G in A1532G, T1573C, C1688G, and G1880A) possess a protective effect regarding the development of ICRPs. However, these SNPs were not specific for MDs, although the minor allele frequencies of these SNPs in control MDs were significantly lower than in other breed dogs. These results suggest that the identified four SNPs (A1532G, T1573C, C1688G, and G1880A in the NOD2 gene) may play a role in the pathogenesis of ICRPs in MDs. Because the majority of MDs and other breed dogs do not have the protective alleles, their absence may not be a specific cause of ICRPs in MDs but rather contribute to the development of inflammation.

Functional analysis of pattern recognition receptors in miniature dachshunds with inflammatory colorectal polyps.

Inflammatory colorectal polyps (ICRPs) frequently occur in miniature dachshunds (MDs) in Japan. MDs with ICRPs develop multiple polyps with severe neutrophil infiltration that respond to immunosuppressive therapy. Therefore, ICRPs are thought to constitute a novel, breed-specific form of canine inflammatory bowel disease (IBD). Pattern recognition receptors (PRRs) play a key role in the distinction of pathogens from commensal bacteria and food antigens. Dysfunction resulting from genetic disorders of PRRs have been linked to human and canine IBD. Therefore, we analyzed the reactivity of PRRs in MDs with ICRPs. Twenty-six MDs with ICRPs and 16 control MDs were recruited. Peripheral blood-derived monocytes were obtained from each dog and then stimulated with PRR ligands for 6 and 24 hr; subsequently, messenger RNA (mRNA) expression levels and protein secretion of IL-1ß were quantified using quantitative real-time PCR and ELISA, respectively. The levels of IL-1ß mRNA and protein secretion after stimulation with a nucleotide-binding oligomerization domain 2 (NOD2) ligand were significantly greater in monocytes from ICRP-affected MDs than in those from control MDs. In addition, IL-1ß protein secretion induced by toll-like receptor (TLR) 1/2, TLR2 and TLR2/6 stimulation was also significantly greater in ICRP-affected MDs. These results suggest that reactivity against NOD2, TLR1/2, TLR2 and TLR2/6 signals is enhanced in ICRP-affected MDs and may play a role in the pathogenesis of ICRPs in MDs. Additional studies of the genetic background of these PRRs should be performed.

Inflammatory colorectal polyps in miniature Dachshunds frequently develop ventrally in the colorectal mucosa.

This study explored the hypothesis that inflammatory colorectal polyps (ICRPs) in miniature Dachshunds are more likely to occur ventrally in the colorectum. Angle-fixed colonoscopic images were collected from 11 miniature Dachshunds with ICRPs and randomly rotated. Macroscopic severity at 12 divided angles was scored by four veterinarians blinded to the rotation angle. Mean prevalence and severity scores of ICRPs were significantly higher ventrally than dorsally (P < 0.01).

Expression profiling of pattern recognition receptors and selected cytokines in miniature dachshunds with inflammatory colorectal polyps.

Inflammatory colorectal polyps (ICRPs) are commonly seen in miniature dachshund (MD) dogs; typically, multiple polyps form with severe neutrophil infiltration. ICRP is thought to be a novel form of inflammatory bowel disease (IBD), but its etiology has not been investigated. The innate immune system is implicated in the pathogenesis of both human and canine IBD. Therefore, the aim of the current study was to evaluate the messenger RNA (mRNA) expression profiles of pattern recognition receptors (PRRs) and cytokines in ICRPs. Polyp tissues were collected by colonoscopic biopsies from 24 MDs with ICRPs. Non-polypoid colonic mucosa was collected from all MDs with ICRPs and 21 clinically healthy beagles (as the controls). The expression levels of the mRNAs encoding toll-like receptors (TLRs) 1-10; nucleotide-binding oligomerization domain (NOD)-like receptors NOD1 and NOD2; and cytokines IL-1ß, IL-6, IL-8/CXCL8, and TNF-α were evaluated by quantitative real-time RT-PCR. Three of the 10 well-known candidate reference genes were selected as housekeeper genes based on analyses from the GeNorm, NormFinder, and BestKeeper programs. Levels of TLR1, TLR2, TLR4, TLR6, TLR7, TLR8, TLR9, TLR10, NOD2, and all cytokines were significantly upregulated in the polyps relative to those in the controls. There was significant decrease in the expression levels of TLR3 and NOD1 in the polyp tissues compared to the non-polypoid colonic mucosa obtained from MDs with ICRPs. All upregulated PRR mRNAs were positively correlated with all proinflammatory cytokine mRNAs. This study demonstrated the dysregulation of PRRs and proinflammatory cytokines in ICRPs of MDs, which may play an important role in the pathogenesis of this disease.

Prognostic analyses on anatomical and morphological classification of feline lymphoma.

The present study was carried out to analyze the prognosis of 163 cats with lymphoma classified anatomically and cytomorphologically. Anatomically, alimentary lymphoma was the most common form and showed significantly shorter survival than mediastinal and nasal lymphomas in cats. Cytomorphologically, there was no predominant subtype in feline lymphomas. Immunoblastic type (18%), centroblastic type (16%), globule leukocyte type (15%), lymphocytic type (12%), lymphoblastic type (12%), pleomorphic medium and large cell type (10%) and anaplastic large cell type (7%) were relatively common subtypes. Most of the cats with globule leukocyte lymphoma had the alimentary form. Comparing median survival time among classifications, cats with globule leukocyte lymphoma showed significantly shorter survival than those with high-grade and other low-grade lymphomas. Furthermore, cats with high-grade lymphomas showed significantly shorter survival than cats with other low-grade lymphomas. The present study indicated the clinical significance of anatomical and cytomorphological evaluation in feline lymphomas.

Phase I dose-escalation study of nimustine in tumor-bearing dogs.

Nimustine (ACNU) is an alkylating agent of the nitrosourea and can be an antineoplastic agent in dogs. But, there has been no report on its dose-limiting toxicity (DLT) in dogs. This study was a phase I dose-escalation clinical trial to determine the maximum tolerated dose (MTD) and DLT of ACNU in tumor-bearing dogs. The starting dosage was 25 mg/m(2), and subsequent dosages were administered in increments of 5 mg/m(2) in cohort of 3 dogs. Eight dogs were included, the MTD was determined to be 25 mg/m(2), DLT was neutropenia, and the optimal interval was considered to be 21 days. The data herein provide a basis for the subsequent phase II trial of ACNU in dogs.