Kazutaka Kanai

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.

Inhibitory Effects of Oral Disulfiram on Endotoxin-Induced Uveitis in Rats

Purpose: Disulfiram (DSF) exhibits a wide variety of biological activities, including an anti-inflammatory action, on which we focused our attention. The aim of the present study was to investigate the effect of oral DSF on endotoxin-induced uveitis (EIU) in rats. Methods: We investigated its effect upon cellular infiltration and protein leakage, as well as on the concentration of tumor necrosis factor-α (TNF-α), nitric oxide (NO), and prostaglandin E2 (PGE2) in the anterior chamber. Some eyes were enucleated for histologic examination and immunohistochemical analysis. EIU was induced in male Lewis rats by a footpad injection of lipopolysaccharide (LPS). One hour before the LPS injection, either 250, 500, or 750 mg/kg DSF was administered orally. Twenty-four hours later, the aqueous humor was collected from both eyes, and the number of infiltrating cells and protein concentration in the aqueous humor were determined. Levels of TNF-α, NO, and PGE2 were determined by enzyme-linked immunosorbent assay. Immunohistochemical analysis in the iris ciliary body (ICB) cells was perfomed to determine the expression of activated nuclear factor kappa B (NF-κB), inducible-nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Results: The oral administration with DSF suppressed, in a dose-dependent manner, the number of inflammatory cells, the protein concentration, and the levels of TNF-α, NO, and PGE2 in the aqueous humor and improved the histiologic status of the ocular tissue. The expression of activated NF-κB-positive cells in the ICB was significantly inhibited by oral administrated with DSF 3 hr after the LPS injection. The LPS-induced increased expressions of iNOS and COX-2 proteins in the ICB were also inhibited by oral DSF 24 hr after LPS injection. Conclusions: The present results indicate that oral DSF suppresses the inflammation in EIU by inhibiting the NF-κB-dependent pathway and the subsequent production of pro-inflammatory mediators.

Circadian variations in salivary chromogranin a concentrations during a 24-hour period in dogs

The purpose of this study was to determine if salivary chromogranin a secretion in dogs exhibits a circadian rhythm. Saliva sampling was performed during three different sessions occurring in three nonconsecutive 24-h periods. Sixteen healthy adult beagle dogs (8 males and 8 females) were moved to a sampling room and housed individually in cages. Saliva samples were obtained every 4 h from 12:00 p.m. to 12:00 p.m. the following day. In the interest of habituation, saliva was obtained hourly from each dog 3 h before the experiment was started. Salivary chromogranin A concentrations were measured using an enzyme-linked immunosorbent assay. No circadian rhythm was detected for salivary chromogranin A secretion, and no differences in salivary chromogranin A concentrations measured every 4 h were demonstrated during the 24-h cycle in dogs.

Prevalence of intestinal parasites in private-household cats in Japan

The present study is the first national investigation of intestinal parasites in private-household cats in Japan. A total of 942 faecal samples were collected from private-household cats. Giardia species was assessed using an enzyme-linked immunosorbent assay kit and other intestinal parasites were identified microscopically. The overall prevalence of intestinal parasites was 10.1%; two protozoan parasites (Giardia species and Cystoisospora species) and five helminths (Toxocara cati, Toxascaris leonina, Ancylostoma tubaeforme, Taenia species and Spirometra erinacei) were detected. The total prevalence of intestinal parasite infection was significantly higher in cats aged ≤6 months old than in cats older than 6 months because of a significantly higher prevalence of Cystoisospora species and T cati. The total infection prevalence was higher among outdoor cats as a result of the significantly higher prevalence of T cati and S erinacei. Sex and faecal condition were not related to intestinal parasite infections. Regional differences were observed in Cystoisospora species and A tubaeforme.