Fumitoshi Asai

Distribution of periodontopathic bacterial species in dogs and their owners.

OBJECTIVE Presently, a large number of individuals consider their companion animals as family members and have close contact with them in daily life. The purpose of the present study was to analyze the distribution of periodontopathic bacterial species in oral specimens taken from dogs and their owners. DESIGN Dental plaque specimens were collected from 66 dogs and 81 members of 64 families who came to an animal clinic or dog training school in Okayama, Japan, in 2011. Bacterial DNA was extracted from each specimen and PCR analyses using primers specific for 11 periodontopathic species, Porphyromonas gingivalis, Porphyromonas gulae, Treponema denticola, Tannerella forsythia, Capnocytophaga ochracea, Capnocytophaga sputigena, Prevotella intermedia, Prevotella nigrescens, Aggregatibacter actinomycetemcomitans, Campylobacter rectus, and Eikenella corrodens were performed. RESULTS P. gulae (71.2%), T. forsythia (77.3%), and C. rectus (66.7%) were frequently found in the dogs, whereas the detection rates of those species in humans were less frequent at 16.0%, 30.9%, and 21.0%, respectively. P. gulae was identified in 13 human subjects and each of their dogs was also positive for the species. Furthermore, E. corrodens and T. denticola in specimens obtained from dogs were correlated with their presence in specimens from owners who had close contact with them. CONCLUSIONS These results suggest that several periodontopathic species could be transmitted between humans and their companion dogs, though the distribution of periodontopathic species in both is generally different.

Molecular detection of human periodontal pathogens in oral swab specimens from dogs in Japan.

Periodontal diseases are known to be major diseases in humans, and are also common in dogs. The purpose of the present study was to analyze the distribution of periodontitis-related bacterial species using oral swab specimens collected from 26 pet dogs. The distribution of an animal gingival organism Porphyromonas gulae, in addition to 10 human periodontitis-related bacterial species, including Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, Capnocytophaga ochracea, Capnocytophaga sputigena, Prevotella intermedia, Prevotella nigrescens, Aggregatibacter actinomycetemcomitans, Campylobacter rectus, and Eikenella corrodens, were evaluated by polymerase chain reaction with species-specific sets of primers. Porphyromonas gulae, Tannerella forsythia and Campylobacter rectus were detected in almost all dogs analyzed, all of which should be regarded as common members of oral flora in dogs. Then, isolation and identification of the Porphyromonas species in swab specimens were performed. There were 35 strains isolated from 22 dogs, and broad-range polymerase chain reaction and sequencing methods revealed that approximately 70 % of them were Porphyromonas gulae. In contrast, the frequency of Porphyromonas gingivalis was extremely low. These findings indicate the presence of specific periodontitis-related pathogens in pet dogs, especially Porphyromonas gulae.

Role of insulin resistance in the pathogenesis and development of type 2 diabetes in WBN/Kob-Lepr(fa) rats.

ABSTRACT WBN/Kob-Leprfa (fa/fa) rats have been identified as a new animal model of type 2 diabetes (T2DM), as they are characterized by impaired pancreatic insulin secretion and severe insulin resistance. Our previous study demonstrated impaired insulin secretion and its involvement in hyperglycemia in fa/fa rats. The present study was aimed at elucidating the role of insulin resistance in the development and progression of diabetes in these animals. Troglitazone (TGZ) was used as an insulin sensitizer. Insulin resistance and insulin secretory capacity were measured by a homeostasis model assessment of insulin resistance and the area under the blood concentration–time curve for plasma insulin levels after intravenous glucose tolerance testing, respectively. The fa/fa rats exhibited marked insulin resistance between 5 and 11 weeks of age, compared with age-matched Wistar rats. The insulin secretory capacity of fa/fa rats was higher than that of Wistar rats at 5 weeks of age, but decreased by 50% between 9 and 11 weeks of age. The fa/fa rats were fed a standard diet, with or without 0.2% w/w TGZ, for 4 weeks. Treatment with TGZ significantly improved insulin resistance, hyperglycemia and hypertriglyceridemia in both prophylactic and therapeutic study groups. These results suggest that insulin resistance is markedly involved in the development and progression of T2DM in fa/fa rats.

Distribution and molecular characterization of Porphyromonas gulae carrying a new fimA genotype

Porphyromonas gulae is a gram-negative black-pigmented anaerobe which is known to be a pathogen for periodontitis in dogs. Approximately 41kDa filamentous appendages on the cell surface (FimA) encoded by the fimA gene are regarded as important factors associated with periodontitis. The fimA genotype was classified into two major types and strains in type B were shown to be more virulent than those in type A. In the present study, we characterized a strain with a novel fimA genotype and designated it as type C. The putative amino acid sequence was shown to be similar to the genotype IV fimA of Porphyromonas gingivalis, a major pathogen of human periodontitis. Analyses using an oral squamous cell carcinoma cell line derived from tongue primary lesions revealed that the type C strain inhibited proliferation and scratch closure more than genotype A and B strains. In addition, experiments using a mouse abscess model demonstrated that the type C strain could induce much higher systemic inflammation when compared with strains of the other genotypes. Furthermore, molecular analyses of oral swab specimens collected from dogs demonstrated that the detection frequencies of P. gulae and the genotype C in the periodontitis group were significantly higher than those in the periodontally healthy group. These results suggest that FimA of P. gulae is diverse with the virulence of genotype C strains the highest and that molecular identification of genotype C P. gulae could be a possible useful marker for identifying dogs at high risk of developing periodontitis.

Effects of high-fat diet and fructose-rich diet on obesity, dyslipidemia and hyperglycemia in the WBN/Kob-Leprfa rat, a new model of type 2 diabetes mellitus

Obesity and type 2 diabetes mellitus (T2DM) are occurring at epidemic-like rates, and these epidemics appear to have emerged largely from changes in daily diet. In the present study, we compared effects of high-fat diet (HFD) and fructose-rich diet (FRD) in WBN/Kob-Leprfa (WBKDF) rats that spontaneously develop obesity, dyslipidemia and T2DM. After a 4-week feeding of each diet, WBKDF-HFD and WBKDF-FRD rats exhibited aggravated obesity and dyslipidemia compared with WBKDF rats fed standard diet (STD). In contrast, hyperglycemia developed in WBKDF-STD rats was significantly inhibited in WBKDF-FRD rats, but not in WBKDF-HFD rats. The present study demonstrated that the 4-week feeding of HFD and FRD caused diet-induced obesity with a distinct phenotype in the glucose metabolism in WBKDF rats.

Plasma 2-hydroxyglutarate and hexanoylcarnitine levels are potential biomarkers for skeletal muscle toxicity in male Fischer 344 rats

To identify new candidate biomarkers for skeletal muscle toxicity, an unbiased metabolomic analysis was performed in rats treated with two distinct myotoxicants, cerivastatin (CER) and tetramethyl-p-phenylenediamine (TMPD). Skeletal muscle toxicity was induced in male Fischer 344 rats by administering CER or TMPD and monitored using established endpoints, such as increased plasma creatine kinase (CK) activity and histopathology, and a metabolomic analysis of skeletal muscle and plasma samples. Plasma CK levels in CER-treated rats were markedly elevated at Day 11; however, those in TMPD-treated rats showed a statistically significant decrease at 24 hr after dosing. Light microscopy revealed that vacuolated or necrotic fibers were evident in all CER-treated rats on Day 11, and slightly vacuolated fibers were observed in TMPD-treated rats at 6 and 24 hr after dosing. Metabolomic analysis of the rectus femoris indicated increases in 2-hydroxyglutarate (2HG) in CER-treated rats and hexanoylcarnitine in CER- and TMPD-treated rats. There were also increases in plasma 2HG in CER-treated rats on Days 8 and 11 and in TMPD-treated rats at 24 hr after dosing and increases in plasma hexanoylcarnitine in CER-treated rats on Day 11 and in TMPD-treated rats at 6 and 24 hr after dosing. These experiments demonstrated the potential of plasma 2HG and hexanoylcarnitine as specific and easily detectable biomarkers for skeletal muscle toxicity in rats and demonstrated the value of metabolomics for biomarker detection and identification in toxicological studies.

The Effects of High‐Sodium Intake on Systemic Blood Pressure and Vascular Responses in Spontaneously Diabetic WBN/Kob‐Leprfa/fa Rats

The prevalence of type 2 diabetes mellitus (T2DM) and hypertension has markedly increased worldwide. The purpose of the present study was to examine the effects of a high‐salt intake on the systolic blood pressure (SBP) and vascular responses in WBN/Kob‐Leprfa/fa (WBKDF) rats, a new spontaneous animal model of T2DM. Male WBKDF rats and age‐matched Wistar rats at 6 weeks of age were each divided into two groups and fed either a normal‐sodium (NS, 0.26%) diet or high‐sodium (HS, 8%) diet for 14 weeks: (i) Wistar rats on NS diet (Wistar‐NS); (ii) Wistar rats on HS diet (Wistar‐HS); (iii) WBKDF rats on NS diet (WBKDF‐NS); (iv) WBKDF rats on HS diets (WBKDF‐HS). Neither WBKDF‐NS nor Wistar‐NS rats showed significant changes in SBP throughout the experiment, but both WBKDF‐HS and Wistar‐HS exhibited significant elevation of SBP, which was more prominent (P<.01) in WBKDF‐HS than in Wistar‐HS. Phenylephrine‐induced contractions of isolated thoracic aortic rings were significantly (P<.01) enhanced in WBKDF‐HS and Wistar‐HS compared with the respective strain of rats on the NS diet. In contrast, acetylcholine‐ and nitroprusside‐induced relaxation were significantly (P<.01) diminished in both WBKDF‐HS and Wistar‐HS, and these HS diet‐induced changes were more profound (P<.01) in WBKDF rats than in Wistar rats. Significantly (P<.05) higher plasma concentrations of 8‐iso‐prostaglandin F2α and sodium ions were observed in WBKDF‐HS than in Wistar‐HS. The current study demonstrated that WBKDF‐HS rats developed salt‐sensitive hypertension associated with vascular dysfunction. The WBKDF rat may be a useful model for investigating the etiology of hypertension with T2DM.

Characteristics of WBN/Kob diabetic fatty rats supplemented with a fructose-rich diet as a metabolic syndrome model: response to a GLP-1 receptor agonist

The incidence of metabolic syndrome is rapidly increasing worldwide, and adequate animal models are crucial for studies on its pathogenesis and therapy. In the search of an adequate experimental model to simulate human metabolic syndrome, the present study was performed to examine the pharmacological response of WBN/Kob-Leprfa (WBKDF) rats supplemented with a fructose-rich diet (FRD) to liraglutide, a GLP-1 receptor agonist. Male WBKDF rats fed FRD at 7 weeks of age were divided into 3 groups, and administered liraglutide (75, 300 µg/kg subcutaneously) or saline (control group), once daily for 4 weeks. All rats in the control group became overweight, and developed hyperglycemia, hypertension and dyslipidemia as they aged. The rats given liraglutide exhibited a dose-dependent reduction in body weight, visceral fat content and food intake compared with control rats. In addition, liraglutide suppressed the development of hyperglycemia, hypertension and dyslipidemia. An intravenous glucose tolerance test revealed that liraglutide improved glucose tolerance, insulin secretion and insulin resistance. On histological examination, decreased hepatic fatty degeneration was observed in the liraglutide groups. The present study demonstrated that liraglutide protected against obesity, hyperglycemia, hypertension, dyslipidemia, and hepatic steatosis in WBKDF rats fed FRD, suggesting that WBKDF rats fed FRD may be a useful model to investigate the etiology of human metabolic syndrome.

Plasma 2-hydroxyglutarate, a promising prognostic biomarker candidate for skeletal muscle injury in Fischer 344 rats

Previously, we have demonstrated the potential of plasma 2-hydroxyglutarate (2HG) as an easily detectable biomarker for skeletal muscle injury in rats. Here, we examined whether plasma 2HG was superior to conventional skeletal muscle damage biomarkers, including aspartate aminotransferase (AST), creatine kinase (CK), and skeletal muscle-type CK isoenzyme (CK-MM) levels, in rats. Skeletal muscle injury was induced in 4- or 9-week-old male Fischer 344 rats by cerivastatin (CER) or tetramethyl-p-phenylenediamine (TMPD) administration. Plasma 2HG levels were measured on days 4, 8, and 11 (CER group) and at 6 and 24 hr post-administration (TMPD group). Plasma AST, CK, and CK-MM activities and histopathological changes in the rectus femoris muscle were evaluated at the study endpoints. In the CER group, AST, CK, and CK-MM increased in 4- and 9-week-old rats, whereas increases in CK (4- and 9-week-old rats) and CK-MM (4-week-old rats) were not obvious in the TMPD group. In both 4- and 9-week-old rats, plasma 2HG increased on day 8 and at 24 hr post-administration in the CER and TMPD groups, respectively. Histopathological analysis revealed myofiber vacuolation and necrosis in both groups. The histopathological damage to the rectus femoris muscle was more severe in the CER than in the TMPD group. Increased plasma 2HG was associated with CER- and TMPD-induced skeletal muscle injuries in rats and was not affected by age differences or repeated blood collection. The results suggest that plasma 2HG is superior to CK and CK-MM as a biomarker for mild skeletal muscle injury.

High-Salt Intake Ameliorates Hyperglycemia and Insulin Resistance in WBN/Kob-Leprfa/fa Rats: A New Model of Type 2 Diabetes Mellitus

High-salt intake is a major risk factor for developing hypertension in type 2 diabetes mellitus, but its effects on glucose homeostasis are controversial. We previously found that high-salt intake induces severe hypertension in WBN/Kob diabetic fatty (WBKDF) rats. In the present study, we examined the effects of a high-salt intake on glucose homeostasis in WBKDF rats. Male WBKDF rats and age-matched Wistar rats at 6 weeks of age were each divided into two groups and fed either a normal-sodium (NS, 0.26%) diet or high-sodium (HS, 8%) diet for 7 weeks. Systolic blood pressure and urine volume were increased in WBKDF-HS and Wistar-HS. Body weight gain and food consumption were comparable between NS and HS in both strains. Plasma and urine glucose levels were significantly increased in WBKDF-NS but not in WBKDF-HS. HOMA-IR in WBKDF-HS was significantly lower compared with that in WBKDF-NS. The high plasma adiponectin level in WBKDF-NS compared with that in Wistar-NS was further enhanced in WBKDF-HS. Glycogen deposits and fat droplets in the livers of WBKDF-HS were reduced compared with those of WBKDF-NS. The present study demonstrated that HS intake ameliorated hyperglycemia and insulin resistance in WBKDF rats, which may be due to increased plasma levels of adiponectin.