Seiichi Higuchi

Development of a SYBR Green Real-Time Polymerase Chain Reaction Assay for Quantitative Detection of Babesia Gibsoni (Asian Genotype) DNA

A real-time fluorogenic polymerase chain reaction (PCR) assay based on SYBR green that allows for sensitive, reproducible, and accurate quantification of Babesia gibsoni (Asian genotype). DNA from peripheral blood of infected dogs was developed. Standard curves were created by plotting the input amount of a standard template, constructed with plasmid DNA containing 182 base pairs (bp) of the p18 gene, against threshold cycle numbers. The curves showed a wide dynamic range (1,000,000-fold input) and high correlation values (>0.99). The PCR amplification efficacy of the standard template was similar to that of intact genomic DNA obtained from peripheral blood with B. gibsoni infection. The detection limit of the assay was 9 parasites/μl of blood with B gibsoni infection. The intra-assay and interassay coefficients of variation of the threshold cycles ranged from 0.70% to 1.89% and from 1.18% to 1.92%, respectively. This assay system was found to be reproducible and accurate for the quantification of parasite DNA in experimentally infected dogs and far more sensitive than traditional microscopic examination.

Evaluation of NT-Pro BNP and CT-ANP as Markers of Concentric Hypertrophy in Dogs with a Model of Compensated Aortic Stenosis

BACKGROUND Serum C-terminal atrial natriuretic peptide (CT-ANP) and N-terminal pro B-type natriuretic peptide (NT-pro BNP) concentrations have not been measured serially in dogs with chronic pressure overload of the heart. HYPOTHESIS We investigated whether serial evaluation of CT-ANP and NT-pro BNP concentrations is a useful guide to the risk of cardiac remodeling in dogs with a model of aortic stenosis. ANIMALS Six male Beagles. METHODS After anesthesia, the aorta was constricted with a polyester band and mean left ventricular systolic pressure (LVPs) was 50 mmHg above baseline. Echocardiographic and intracardiac catheter examinations and blood sampling were performed before surgery and 3 and 6 months after surgery. RESULTS LVP and left ventricular end-diastolic pressure (LVEDP) were significantly higher at 6 months. Compared with baseline, end-diastolic intraventricular septum thickness (IVSd), left ventricular posterior wall thickness (LVPWd), and relative wall thickness (RWT) were significantly increased 3 and 6 months after aortic constriction. Serum CT-ANP concentrations were increased significantly at 3 months and serum NT-pro BNP concentrations were significantly higher 3 and 6 months after aortic constriction. Serum NT-pro BNP concentration was significantly correlated with LVEDP and IVSd whereas serum CT-ANP concentration was not correlated with any measurement. Stepwise regression analysis showed that LVEDP, IVSd, and RWT could predict serum NT-pro BNP. CONCLUSIONS AND CLINICAL IMPORTANCE This study indicated the differential regulation of NT-pro BNP and CT-ANP concentrations during pressure overload. NT-pro BNP assay may be used as an additional screening method to stratify early-stage ventricular remodeling because of aortic constriction.

Prevalence of Giardia intestinalis and other zoonotic intestinal parasites in private household dogs of the Hachinohe area in Aomori prefecture, Japan in 1997, 2002 and 2007.

An epidemiological study on canine intestinal parasites was undertaken to evaluate changes in the prevalence among private household dogs from the Hachinohe region of Aomori prefecture, Japan, in 1997, 2002 and 2007, using the formalin-ethyl acetate sedimentation technique. The risk of zoonotic transmission from household dogs to humans was also discussed. All intestinal parasites detected in the present study (Giardia intestinalis, Isospora spp., Toxocara canis, Ancylostoma caninum, Trichuris vulpis and Strongyloides stercoralis) showed no changes in prevalence over the past 10 years based on analysis considering canine epidemiological profiles. In particular, prevalence of Giardia intestinalis in dogs under 1 year old, derived from pet shops/breeding kennels and kept indoors was unchanged, remaining at a high level of >15.0% at each time point. Toxocara canis also showed no changes in the group of dogs under 1 year old, bred by private owners and kept outdoors, and the prevalence was >10.0% every year. The present results indicate that the prevalence of Giardia intestinalis and other intestinal parasites in private household dogs has not always decreased, and the potential for direct parasitic zoonotic transmission from dogs to humans may be relatively high level, than from the environment (indoors and outdoors). We recommend careful surveillance of intestinal parasites and aggressive use of anthelminthic in private household dogs under considering the epidemiological factors.

Evaluation of Plasma C-Terminal Atrial Natriuretic Peptide in Healthy Cats and Cats with Heart Disease

BACKGROUND The clinical implications of evaluating C-terminal atrial natriuretic peptide (ANP) concentration in cats are still controversial. HYPOTHESIS The objective of this study was to investigate the relationship between plasma C-terminal ANP concentration and left atrial pressure (LAP) in healthy cats with volume overload (study 1), and to compare plasma C-terminal ANP in normal cats and cats with cardiomyopathy (study 2). ANIMALS Five healthy adult cats were used in study 1, and clinically healthy cats (n=8) and cats with cardiomyopathy (n=14) were used in study 2. METHODS In study 1, cats were anesthetized and given acetated Ringers solution (100 mL/kg/h for 60 minute) via the cephalic vein. Hemodynamic measurements and blood samples, collected from the jugular vein, were performed at 10-min intervals. In study 2, blood samples from normal cats and cats with cardiomyopathy were collected from the cephalic vein. The plasma C-terminal ANP concentration was determined by radioimmunoassay for human alpha-ANP. RESULTS In study 1, volume overload significantly increased the C-terminal ANP concentration and LAP from baseline. The C-terminal ANP concentration was strongly correlated with the mean LAP. In study 2, age, E wave velocity, and the ratios of the left atrium to aorta were significantly higher in the cats with cardiomyopathy compared with the normal cats. The C-terminal ANP concentration was significantly higher in the cats with cardiomyopathy compared with the normal cats. CONCLUSIONS AND CLINICAL IMPORTANCE Our results suggest that the measurement of plasma C-terminal ANP in cats may provide additional information for the diagnosis of heart disease.

Matrix metalloproteinase-2 stimulates collagen-I expression through phosphorylation of focal adhesion kinase in rat cardiac fibroblasts

Collagen-I is thought to be the main component of the extracellular matrix in cardiac fibrosis, the accumulation of which occurs with excessive activation of matrix metalloproteinase-2 (MMP-2). MMP-2 degrades the extracellular matrix; however, the relative importance of MMP-2 to collagen-I synthesis in cardiac fibroblasts remains unclear. We investigated whether extracellular activation of MMP-2 regulates collagen-I synthesis and phosphorylation of focal adhesion kinase (FAK) in rat cardiac fibroblasts. Primary cultures of rat cardiac fibroblasts were incubated with purified active MMP-2 to determine whether extracellular MMP-2 affects collagen-I synthesis and FAK phosphorylation in cardiac fibroblasts. Exogenous MMP-2 significantly stimulated FAK (Tyr397) phosphorylation and induced collagen-I expression in a time-dependent manner. Simultaneous treatment with the FAK inhibitor PF573228 abolished exogenous MMP-2-enhanced FAK (Tyr397) phosphorylation and collagen-I expression. Cells were then stimulated with norepinephrine (NE) to investigate whether endogenous MMP-2 could also induce collagen-I expression through FAK (Tyr397) phosphorylation. NE-stimulated endogenous MMP-2 activation in conditioned medium was significantly attenuated by simultaneous treatment with the MMP inhibitor PD166793. Similarly, NE-induced FAK (Tyr397) phosphorylation and collagen-I expression were significantly inhibited by simultaneous treatment with PD166793 or PF573228. Furthermore, MMP-2 knockdown induced by small interfering RNA (siRNA) significantly abolished endogenous MMP-2 expression and activation. MMP-2 siRNA significantly abolished NE-induced FAK (Tyr397) phosphorylation and collagen-I expression. These findings suggest that the extracellular activation of MMP-2 accelerated collagen-I synthesis in rat cardiac fibroblasts and that FAK phosphorylation (Tyr397) plays a pivotal role in MMP-2-stimulated collagen-I synthesis.

Prevalence of intestinal parasites and genotyping of Giardia intestinalis in pet shop puppies in east Japan.

The current study examined the prevalence of intestinal parasites and genotypes of Giardia intestinalis in puppies from nine pet shops in east Japan. Fresh fecal samples from 1794 puppies (≦3 months old) were collected on one occasion. Giardia spp. was examined for specific coproantigen using ELISA kit (SNAP®Giardia, IDEXX Laboratories, Inc., USA). Other intestinal parasites were detected microscopically using the formalin-ethyl acetate sedimentation technique. Genotyping was determined for the random 29 stool samples identified as Giardia spp. positive using PCR and direct sequencing of the glutamate dehydrogenase (gdh) gene. Overall prevalence of protozoan Giardia spp. and Cystoisospora spp. revealed 23.4% and 11.3%, respectively. Prevalence of ascarids, Strongyloides spp. and hookworms were recorded 1.8%, 1.1% and 0.1%, respectively. Protozoan Giardia spp. and Cystoisospora spp., thus, represent important pathogens among pet shop puppies. All genotyped G. intestinalis isolates were belonged to assemblage C or D, identified as dog-specific genotypes. Zoonotic assemblage A and B were not demonstrated. The result suggests that the risk of zoonotic transmission of G. intestinalis from pet shops puppies to humans may be quite low in Japan.

Acute cardiac volume load-related changes in plasma atrial natriuretic peptide and N-terminal pro-B-type natriuretic peptide concentrations in healthy dogs

Plasma atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) have been used for the diagnosis of heart disease. The aim of the study was to investigate differences in secretory responses of plasma ANP and N-terminal proBNP (NT-proBNP) concentrations related to acute changes in preload. Six dogs were anaesthetised and infused intravenously with Ringers solution (90-100mL/kg/h) for 60 min. Thereafter, furosemide was administered and dogs were monitored for 60 min. Plasma ANP and NT-proBNP concentrations were determined by chemiluminescence enzyme immunoassay and enzyme immunoassay, respectively. Volume overload significantly increased plasma ANP and NT-proBNP concentrations (P<0.001); however, preload reduction significantly reduced plasma ANP concentrations (P<0.05) without concurrent changes in plasma NT-proBNP. Mean pulmonary capillary wedge pressures were strongly correlated with plasma ANP concentrations (r=0.53, P<0.001), but not plasma NT-proBNP. Thus, plasma ANP is a useful, non-invasive parameter for measuring rapid haemodynamic changes.

Effects of instillation of eyedrops containing disulfiram and hydroxypropyl-β-cyclodextrin inclusion complex on endotoxin-induced uveitis in rats.

Objective: To investigate the anti-inflammatory effects of the instillation of disulfirum (DSF) eyedrops that enhance solubility using 2-hydroxypropyl-β-cyclodextrin (HPβCD) and hydroxypropylmethylcellulose (HPMC) on endotoxin-induced uveitis (EIU) in rats and mechanisms related to ocular inflammation. Methods: EIU was induced in male Lewis rats by subcutaneous injection of 200 μg lipopolysaccharide (LPS). DSF (0.125%, 0.25% and 0.5%) or commercially available 0.05% dexamethasone (Dexa) was topically applied to both eyes of rats 1 hour before, immediately after, and 1 and 2 hours after injection of LPS. The aqueous humor (AqH) was collected 24 hours after LPS injection, and the number of infiltrating cells, protein concentration, and levels of tumor necrosis factor-α (TNF-α), nitric oxide (NO) and prostaglandin E2 (PGE2) were determined. Immunohistochemical analysis of the iris ciliary body (ICB) cells was performed to determine the expression of activated nuclear factor κB (NF-κB), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Results: The topical administration with DSF suppressed, in a dose-dependent manner, the number of inflammatory cells, the protein concentration, and the levels of NO, TNF-α and PGE2 in the AqH and improved the histologic status of the ocular tissue. The anti-inflammatory potency of 0.5% DSF treatment was as strong as that of 0.05% Dexa. Topical DSF treatment also suppressed the activated NF-κB 3 hours after LPS injection, and iNOS and COX-2 expression in the ICB 24 hours after LPS injection. Conclusions: The present results demonstrate that the topical instillation of DSF eyedrops suppresses the inflammation in EIU, suggesting a possible novel approach for the treatment of ocular inflammation.

Clinical implications of measurement of plasma atrial natriuretic peptide concentration in dogs with spontaneous heart disease.

OBJECTIVE To determine whether measurement of plasma atrial natriuretic peptide (ANP) concentration could be used to identify heart disease in dogs and to assess disease severity in affected dogs. DESIGN Cross-sectional study. ANIMALS 37 healthy dogs and 78 dogs with heart disease. PROCEDURES Dogs were divided into 5 groups on the basis of plasma ANP concentration: healthy, ANP-1 (< 50 pg/mL; n = 19), ANP-2 (50 to 100 pg/mL; 24), ANP-3 (101 to 200 pg/mL; 20), and ANP-4 (> 200 pg/mL; 15). All dogs underwent physical examination, echocardiography, thoracic radiography, and blood sampling before treatment. RESULTS Compared with healthy dogs, dogs with increased plasma ANP concentration had significant concomitant increases in heart rate, cardiothoracic ratio, vertebral heart score, fractional shortening, ratio of left atrial-to-aortic root diameter, and mitral early diastolic flow (E wave) velocity and a significant decrease in relative wall thickness. Use of plasma ANP concentration > 25 pg/mL to identify dogs with heart disease (International Small Animal Cardiac Health Council class > I) had a sensitivity of 91.0% and specificity of 94.7%. Use of plasma ANP concentration > 100 pg/mL to identify dogs with International Small Animal Cardiac Health Council class IIIb heart disease had a sensitivity of 81.0% and specificity of 81.1 %. CONCLUSIONS AND CLINICAL RELEVANCE Results may provide reference values for plasma ANP concentration in dogs and suggest that plasma ANP concentration may help to distinguish dogs with cardiac disease from clinically normal dogs. Measurement of plasma ANP concentration may be a useful marker for predicting the severity of heart disease in dogs.

Protective immunity and mast cell and eosinophil responses in mice infested with larval Haemaphysalis longicornis ticks

WBB6F1 ‐+‐ /+ mice were infested with larval Haemaphysalis longicornis ticks twice at an interval of 14 days: apparent resistance against ticks was expressed in the second infestation. The first infestation induced degranula‐tion of a small number of mast cells at the feeding sites within 6 days, and resulted in two‐fold increases of mast cell numbers on day 14 with a significant elevation of total immunoglobulin E (IgE) levels in sera and high proportion of IgE‐bound mast cells. The second infestation resulted in the intensive degranulation of the increased mast cells at the feeding sites. Eosinophils infiltrated into the feeding sites of ticks: the second infestation led to a greater maximal level of the infiltrating eosinophils. These data suggest that the resistance against larval H. longicornis ticks in mice may be expressed as a result of immediate hypersensitivity and many eosinophils infiltrating from the blood to the feeding sites might contribute to the tick rejection.