To determine how to eliminate species difference in animal bone experiment, bone mineral content (BMC) was measured using dual energy X-ray absorptiometry (DXA) on the femurs of laboratory mice (Mus musculus) and rats (Rattus norvegicus), and common marmosets (Callithrix jacchus). Measures were taken on femurs in situ, detached from the body, skinned and defleshed, or dried completely. When the BMC of the bone measured in the intact limb attached to the trunk was set at 100%, the actual BMC of the dry bone was 58.7 ± 11.5% in mice and 103.2 ± 3.2% in rats. Similarly, the bone area (Area) and bone mineral density (BMD) of the dried femur was significantly lower in the mouse femurs than intact limb. Thus, soft limb tissue such as skin and muscle modified the BMC, Area, and BMD only in mouse but not in those from rats or marmosets. The bone mineral ratio (BMR; BMC divided by dry bone weight) was nearest to the human bone value in the rat femurs, whereas the mouse femur BMR was the most different. The BMR was proved to be a practical index in evaluating bone characteristics in laboratory animals, but the mouse femur might not be suitable as an animal model for research into the aging of human bone.

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The immunohistolocalization and gene expression of carbonic anhydrase (CA) isoenzymes CA-II and CA-VI in the canine lower airways and lung were examined using specific canine CA-II and CA-VI antisera and the RT-PCR method. Laryngeal, tracheal and bronchial epithelia, serous acinar and bronchiolar secretory cells and pulmonary great alveolar cells showed immunopositive reactions to anti-CA-II and anti-CA-VI antisera. However, all mucous cells showed immunonegative reactions. The physiological roles of CA-II and CA-VI in the lower airways and lung may involve the maintenance of pH balance and the protection of mucosal surfaces against the acidic milieu.

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The present study examined the courses of testicular efferent lymphatics in the rabbit, by injections of India ink directly into testicular parenchyma. In 58% of the testes examined, testicular efferent lymphatics were observed to course along the testicular artery to the lumbar trunk. In other testes, efferent lymphatics were found along the testicular artery as well as along either the ductus deferens (26%) or cremasteric muscle (8%). In the remaining 8% of the testes, efferent lymphatics were found along the testicular artery, ductus deferens, and cremasteric muscle. These findings demonstrate that testicular efferent lymphatics in the rabbit course not only along the testicular artery, but also along the ductus deferens and/or cremasteric artery in about 40% of the testes.

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We genetically characterized fowl adenoviruses (serotype 4 FAdV, FAdV-4) isolated from chickens with hydropericardium syndrome (HPS) in Japan by the polymerase chain reaction (PCR) method coupled with direct sequencing. Phylogenetic analysis based on the part of the hexon gene that included the L1 region revealed that all FAdV-4 isolates from chickens with HPS in Japan were identical and were distinguished completely from the cluster including FAdV strains from chickens with HPS in India and Pakistan. This suggested that FAdV-4 from the HPS chickens in India and Pakistan was derived from a common ancestor, but the origin of the FAdV-4 from the HPS chickens in Japan was completely different.

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We examined whooper swans naturally infected with avian influenza virus (H5N1) to evaluate the possible zoonotic risk of swan feathers. Viruses were isolated from feather calami. Immunohistochemical testing revealed that virus antigens were present in the feather epidermis and feather follicle wall epidermis of some feathers. RT-PCR and genetic sequencing using paraffin sections of swan feathers confirmed the presence of avian influenza virus (H5N1) in the feather tissue. These results indicate that the feathers could have the risk for zoonotic infection from infected wild swans.

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Salmonella Enteritidis is the most common cause of salmonellosis in humans in South Korea. It has been recognized that the principal source of human infection with S. Enteritidis is chickens and their products such as meat and eggs. A total of 173 S. Enteritidis isolates from humans (65 isolates) and chickens or their products (108 isolates) were analyzed by antibiotic susceptibility assay, phage typing, and pulsed-field gel electrophoresis (PFGE). Drug resistance was found to streptomycin (32.3%), ampicillin (30.6%), nalidixic acid (30.1%), ticarcillin (30.1%), and tetracycline (28.3%). More than 70% of the isolates were found to be resistant to one or more antibiotics tested. The most frequent patterns of resistant isolates were resistance to nalidixic acid only (28.3%) and resistance to two antibiotics (four combinations; 20.2%). The most predominant phage type (PT) was PT1 (27.2%) followed by PT21 (20.8%) and PT4 (8.7%) in chicken and human isolates. Nineteen different PFGE patterns were found among the 173 isolates, and A1 was the most common PFGE pattern, followed by A6 (17.3%). Most S. Enteritis isolates (except two isolates with patterns B and C) showed similar PFGE patterns that differed by only a few bands. These results show that 2 or 3 subtypes of S. Enteritidis are shared to a large extent by humans and chickens. This implies the possibility of the spread of chicken S. Enteritidis to humans.

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It has been reported that administration of Candida albicans into mouse induces an antifungal activity in serum, which has been identified as transferrin. In the present study, we show that not only C. albicans, but also other fungus such as Cryptococcus neoformans or Aspergillus fumigatus similarly can induce an antifungal activity in mouse serum. This antifungal activity was inhibited by the addition of ferrous ion, indicating that the growth inhibition of C. albicans was due to deficiency of ferrous ion, which may be caused by transferrin. Indeed, addition of transferrin in an in vitro assay system using RPMI1640 culture medium inhibited the growth of C. albicans, C. neoformans or A. fumigatus. However, when C. albicans was grown in RPMI1640 medium with 10% fetal bovine serum (FBS), transferrin was unable to inhibit the growth of C. albicans, in sharp contrast, when C. albicans treated mouse serum was added instead of FBS, the growth of the organism was inhibited. Similar results were obtained when C. neoformans or A. fumigatus was used. Taken together, the results suggest that antifungal activity induced by C. albicans, C. neoformans or A. fumigatus was not due to transferrin but likely due to other unknown serum proteins, which may cut off the source of iron for the growth of these fungi.

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Pasteurella multocida is a Gram-negative bacterium known to infect a wide range of domestic and wild animals. The increasing incidence of P. multocida isolated from cases of fowl cholera and hemorrhagic septicemia has led to a renewed interest in this pathogen as well as in the development of vaccines. In this study, PCR primers were designed to amplify the fragment of plpB gene from P. multocida FC-Pakchong (A:1). The purified PCR product of plpB gene consisting of 831 base pairs was inserted into the pGEX-5X-1 plasmid, which expressed the GST protein, and then transformed into E. coli. The purified fusion GST-PlpB protein showed a major band of about 63 kDa on SDS-PAGE gel. After enzyme cleavage, the recombinant PlpB protein appeared at the estimated size of 36 kDa. The recombinant GST-PlpB protein was tested for the toxicity in vivo. The results showed no toxicity in mice at the highest tested concentration of the protein. Moreover, the immunoprotective property of the recombinant GST-PlpB protein was determined in mice after subcutaneous immunization and challenge with an approximate dose of 50-100 LD₅₀ of P. multocida serotype A:1 and A:3,4. It was demonstrated that this subunit vaccine provided 20-30% survival rate after subcutaneous immunization and challenge with an approximate dose of 50-100 LD₅₀ of P. multocida serotype A:1 and A:3,4 whereas all of the non-immunized mice died from P. multocida infection. In conclusion, our data indicated that the PlpB protein may not be an appropriate target as a candidate subunit vaccine for P. multocida infection.

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In order to investigate the origin of tetracycline resistance in Erysipelothrix rhusiopathiae, conjugative transpositions of Tn916 were tested. The frequency of transfer between strains of E. rhusiopathiae was about 10-fold higher than that between Enterococcus faecalis and E. rhusiopathiae. In addition, detection of a Tn916-like transposon was performed by PCR assay and DNA sequencing in E. rhusiopathiae field isolates. Of 49 tetracycline-resistant isolates, 38 (77.6 %) carried a Tn916-like transposon, while 11 (22.4 %) carried tet(M) only. These results suggested that Tn916-like transposon may be widely present in the E. rhsuiopathiae field isolates resistant to tetracycline.

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Humic substances are formed during the decomposition of organic matter in humus, and are found in many natural environments in which organic materials and microorganisms have been present. In the present study, oral administration of humus extract to ayu fish (Plecoglossus altivelis) induced effective protection against experimental Flavobacterium psychrophilum infection (cold water disease). Mortality of fish and development of skin lesions, such as erosion and hemorrhages on the skin, gill cover or mouth, were significantly suppressed in fish treated with 10%, 5% or 1% humus extract adsorbed on dry pellets. Although F. psychrophilum was not re-isolated from gills and erosion lesions of the skin of dead fish, bacterial gyrB DNA could be amplified in these specimens from dead fish and surviving control fish using the polymerase chain reaction. The protective effect of the extract was not the results of direct killing of bacteria or antibiotic activity of the extract since no obvious reduction in the bacterial number was observed at 5 times to 5,000 times dilution of the humus extract having pH 5.45 to 7.40. These results clearly show that treating fish with humus extract is effective in preventing cold water disease.

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Use of porcine tissues has been suggested as a promising solution for severe shortage of transplantable human organs. The immediate hurdle for xenotransplantation is acute immune/inflammatory vascular rejection of the transplant. Because endothelial cells play a key role in the initiation and the amplification of inflammation, alteration of gene expression in human endothelial cells, by various inflammatory stimulators has been studied extensively. However, transcriptional changes induced by human and other inflammatory stimulators in porcine endothelial cells have thus far not been studied. In this study, we treated porcine endothelial cells with human tumor necrosis factor (TNF)-α, porcine interferon (IFN)-γ, H₂O₂ and lypopolysaccharide (LPS) and profiled transcriptional change at 1 hr, 6 hr and 24 hr, using pig oligonucleotide 13K microarray. We found that mRNA species such as chemokine (C-X-C motif) ligand 6 (CXCL6) and Cathepsin S were significantly induced in porcine endothelial cells, as was previously reported with human endothelial cell. We also found that mRNA species including secreted frizzled-related protein 2 (SFRP2), radical S-adenosyl methionine domain containing 2 (RSAD2), structure specific recognition protein 1 (SSRP1) also were highly overexpressed in porcine endothelial cells. This result shows clues to understand underlying mechanisms of xenotransplantation rejection and the highly responsive porcine genes may serve as novel targets to be regulated for improving the function of grafted porcine donor organs.

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This study investigated the feasibility of using the values of tissue Doppler imaging (TDI)-derived myocardial velocity during isovolumic relaxation (V_IR) and myocardial acceleration during isovolumic relaxation (ACC) obtained from the left ventricular (LV) free wall to evaluate LV relaxation in normal dogs. Seven dogs were anesthetized, and dobutamine or esmolol was infused at a rate of 5.0 and 10.0 μg/kg/min or 100 and 500 μg/kg/min, respectively, via a cephalic vein. The order of drug administration (dobutamine or esmolol) was assigned to each dog. Simultaneous pulsed-Doppler (PD) echocardiography, TDI and hemodynamic measurements were performed. Compared with the baseline values, dobutamine significantly increased dP/dt min, but significantly shortened tau (τ). Similarly, esmolol significantly decreased dP/dt min, but significantly prolonged τ. Compared with the baseline values, dobutamine significantly increased V_IR and ACC, and esmolol significantly decreased V_IR and ACC. Both dP/dt min and τ were significantly correlated with TDI-derived IVRT (r=-0.43 and 0.74), V_IR (r=0.85 and -0.49) and ACC (r=0.84 and -0.52). These results indicate that the TDI-derived V_IR and ACC values obtained from the LV free wall can potentially be used to assess LV relaxation in dogs.

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A 7-year-old female Boston terrier was referred to Hokkaido University Veterinary Teaching Hospital with a history of hemoglobinuria and anemia for several days. Abdominal radiographs showed splenomegaly, and ultrasonography revealed a hypoechoic splenic parenchyma with interspersed linear echoes consistent with the ultrasonographic appearance of splenic torsion. Ultrasonography and computed tomography (CT) indicated a C-shaped spleen. Exploratory laparotomy confirmed the diagnosis of splenic torsion. A splenectomy was performed, and the dog recovered well without complications. This is the first report of splenic torsion in Boston terriers, and the usefulness of ultrasonographic and CT findings of the splenic torsion was also confirmed.

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A 6-year-old, intact female Maltese dog was presented with generalized seizures. Based on the neurological and physical examinations, intracranial lesion was suspected. Magnetic resonance imaging (MRI) of the brain was performed at three different magnetic field strengths (0.2, 1.5 and 7.0 T). Diffuse hypo- and hyperintense lesions involving the left caudate nucleus and internal capsule to the cranial diencephalon were identified on T2-weighted images. The detailed anatomical locations, the inflammatory and hemorrhagic changes of the lesions could be detected more apparently at 7.0 T. Histopathologically, granulomatous meningoencephalitis (GME) was diagnosed. This is the first case describing histopathologically confirmed GME lesions using 0.2, 1.5 and 7.0 T clinical MR scanner.

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We have reported that treatment with trientine, Cu-chelating agent, inhibits tumor growth in a murine transplantation model using fibrosarcoma and induces apoptosis in tumor cells in vivo and in vitro. When fibrosarcoma cells were treated with 10 mM trientine, the activities of p38 MAPK in treated cells were approximately 3-4 times higher than those in untreated cells. Proportions of cells in which apoptosis was induced by trientine increased in an incubation time-dependent manner from days 2 to 6. The proportions of apoptotic cells in the cells treated with trientine and SB203580, an inhibitor of p38 MAPK, were approximately 50% in those of cells treated with trientine alone. The present results showed that the p38 MAPK pathway may play an important role in induction of apoptosis in fibrosarcoma cells by trientine.

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A 5-year-old, male Bichon-Frise dog presented with a cutaneous mass in the basal region of the auricle. Histologically, the cutaneous neoplasm was comprised of lobules with solid cellular proliferation separated by thin fibrous septa. Neoplastic cells varied in size, with moderate to abundant amounts of PAS-positive cytoplasm, large nuclei and prominent nucleoli. Immunohistochemical examinations showed that tumor cells were positive for pan-cytokeratin (CK) (AE1/AE3 and CAM5.2), CK8 and CK18, but negative for pan-CK (KL1), CK7, CK14, CK16 and CK20. Double-labeled immunofluorescence testing indicated that neoplastic cells frequently co-expressed CK and vimentin, suggesting divergent differentiation of tumor cells. Based on these findings, the tumor was diagnosed as canine clear cell adnexal carcinoma.

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The effects of various selective phosphodiesterase (PDE) inhibitors on muscle contractility and cyclic nucleotide contents in porcine iris sphincter were investigated. Forskolin and sodium nitroprusside inhibited carbachol (CCh)-induced contraction in a concentration-dependent manner. Various selective PDE inhibitors, vinpocetine (type 1), erythro -9-(2-hydroxy-3-nonyl)adenine (EHNA, type 2), milrinone (type 3), Ro20-1724 (type 4) and zaprinast (type 5), also inhibited CCh-induced contraction in a concentration-dependent manner. The rank order of potency of IC₅₀ was zaprinast > Ro20-1724 > EHNA ≥ milrinone > vinpocetine. In the presence of CCh (0.3 μM), vinpocetine, milrinone and Ro20-1724 increased cAMP, but not cGMP, contents. In contrast, zaprinast and EHNA both increased cGMP, but not cAMP, contents. This indicates that vinpocetine-, milrinone- and Ro20-1724-induced relaxation is correlated with cAMP, while EHNA- and zaprinast- induced relaxation is correlated with cGMP in porcine iris sphincter.

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Pantothenic acid (PaA) is a water-soluble vitamin required to sustain various physiological functions in animals. The physiological roles of PaA on testicular function, in particular, testicular endocrinology and sperm mortility, were investigated in rats. Male rats at 3 weeks of age were fed a PaA-free diet or a 0.0016% PaA diet (control) for 7 weeks. Total body weight, as well as the weights of the liver, kidney, pituitary, testis, epididymis, seminal vesicle and prostate; sperm motility; and the plasma concentrations of luteinizing hormone (LH), follicle-stimulating hormone (FSH), testosterone and corticosterone were measured in rats at 10 weeks of age. Body weight gain decreased from 5 weeks of age in rats fed the PaA-free diet compared with the control. The relative weights of the testes were significantly higher in the PaA-deficient group compared with the control group. Several parameters of sperm motility were significantly reduced in the PaA-deficient group compared with the control group. In addition, the plasma concentrations of testosterone and corticosterone were significantly lower in the PaA-deficient group compared with the control group, whereas the plasma concentrations of FSH and LH showed no change. These results clearly demonstrate that PaA is an essential factor in testicular endocrinology and sperm motility in male rats.

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In this study, we examined the effects of four chemical reagents, 5 azacytidine (5azaC), all-trans-retinoic acid (ATRA), Phorbol 12-myristate 13-acetate (TPA) and trichostatin A (TSA), on an MCM-B2 canine mammary tumor cell line. Growth of the MCM-B2 cells was inhibited by addition of any of these four chemical reagents in a dose-dependent manner. TPA-treated cells became blast-cell-like and had high expressions of cyclin D, cyclin B and PCNA. Both ATRA-treated and 5azaC-treated cells showed decreased expression of cell cycle related molecules and increased expressions of the mammary epithelial marker cytokeratin 18 and underwent morphological changes. ATRA-treated cells were converted from the originally spindle-shaped cells into cubic-shaped cells, and 5azaC-treated cells became fibroblast-like. In the TSA-treated cells, the cytoplasm was enlarged, increasing the cytoplasm/nucleus ratio, and the expressions of cell cycle-promoting molecules (cyclin D and PCNA), as well as cell cycle-inhibiting factors (p21^WAF1 and p27^kip1), were increased. These results demonstrated the growth-inhibiting, differentiation-inducing and antitumor effects of each of these four chemicals on the unique phenotype of canine mammary tumors.

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The bispectral index (BIS) was evaluated as an indicator of central nervous system (CNS) depression in horses anesthetized with propofol. Five non-premedicated horses were anesthetized with 7 mg/kg, IV propofol and the minimum infusion rate (MIR) of propofol required to maintain anesthesia was determined during intermittent positive pressure ventilation in each horse. The BIS was determined 20 min later and after stabilization at 2.0 MIR, 1.5 MIR, and 1.0 MIR. The BIS was also recorded after the cessation of propofol infusion when the horses regained spontaneous breathing and swallowing reflex. The MIR and plasma concentration (Cp) of propofol were 0.20 ± 0.03 mg/kg/min and 17.5 ± 4.0 μg/ml, respectively. The BIS value and Cp were 59 ± 13 and 26.7 ± 8.6 μg/ml at 2.0 MIR, 63 ± 9 and 22.9 ± 9.7 μg/ml at 1.5 MIR, 64 ± 13 and 20.1 ± 5.9 μg/ml at 1.0 MIR, 64 ± 24 and 13.0 ± 2.8 μg/ml at return of spontaneous breathing, and 91 ± 4 and 11.0 ± 3.4 μg/ml when the swallowing reflex returned, respectively. The BIS value was significantly less in anesthetized horses compared to horses once swallowing returned (p=0.025). The BIS value was significantly correlated with the propofol Cp (r=-0.625, p=0.001). There was not a significant difference in the BIS values during the MIR multiples of propofol. The BIS was a useful indicator of awakening but did not indicate the degree of CNS depression during propofol-anesthesia in horses.

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It is well known that the minimum alveolar concentration (MAC) of inhalation anesthetic decreases with increasing age. However, there is limited information regarding the effect of age on MAC in dogs. This study was designed to determine the effect of age on sevoflurane MAC in dogs. MAC was determined in 6 young (2 years old) and 6 old beagle dogs (8 to 10 years old) under artificial ventilation. Anesthesia was induced via mask induction and maintained with sevoflurane in oxygen, and MAC was determined by using a tail clamp method. The sevoflurane MAC for the older dogs was significantly less than that for the younger dogs (1.86 ± 0.29% vs 2.25 ± 0.15%, P=0.007). The MAC of sevoflurane is profoundly affected by age in dogs.

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In order to examine reproductive characteristics of feral raccoons in Kamakura, 335 raccoons were collected from March 2005 to March 2007. Raccoons were classified into five age classes: Class I, less than 5 months old; Class II, 5-11 months; Class III, 12-17 months; Class IV, 18-23 months; and Class V, over 23 months old. Females were examined for their age specific pregnancy rate and litter size. To determine when raccoon births occur in the region, birth months of fetuses were estimated by the fetal growth rate, and birth months of Class I individuals were examined by tooth eruption. From fetuses of 18 pregnant females and 47 Class I individuals, it was found that the raccoon births occur from February to October. Of 163 females examined, all of Class I-II females were nulliparous. Pregnancy rate was 47.6% in Class III females, which was significantly lower than 75.0% in Class IV and 78.1% in Class V. The litter size of fetuses ranged from 2 to 5, and 3.9 on average; and that of placental scars ranged from 1 to 7, 3.8 on average. Our findings suggest that parturition of raccoons is a bimodal distribution and age at first parturition may occur between 12 and 17 months old. In order to reduce the raccoon population successfully, females of all ages should be captured throughout the year.

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An equine herpesvirus type 1 (EHV-1) mutant, ΔgE, defective in glycoprotein E (gE) was evaluated as a modified live virus (MLV) vaccine. Colostrum-deprived Thoroughbred foals inoculated intranasally (i.n.) or intramuscularly (i.m.) with ΔgE did not exhibit any clinical signs of respiratory disease except for a mild nasal discharge in 1 i.n. inoculated foal on Days 1 and 3 post-infection. In contrast, the intranasal inoculation of foals with the revertant of ΔgE resulted in biphasic pyrexia, mucopurulent nasal discharge and swelling of submandibular lymph nodes. These results indicated that gE plays an important role as regards EHV-1 virulence in horses. The ability of ΔgE to protect against wild type EHV-1 challenge infection was assessed using i.m. vaccinated foals. Foals inoculated twice i.m. with 10⁵ or 10⁶ plaque-forming units (pfu) of ΔgE at an interval of 3 weeks exhibited no clinical evidence of local inflammation, respiratory disease or deleterious systemic responses. Remarkable increases in SN antibody titer to EHV-1 were observed in all vaccinated foals after the 2nd inoculation with ΔgE. Following a wild type EHV-1 challenge infection, vaccinated foals showed milder clinical symptoms than foals vaccinated with a placebo. Specifically, 1 of 3 foals vaccinated with 10⁶ pfu of ΔgE exhibited no clinical symptoms other than a mild nasal discharge for 1 day. Additionally, the virus load of nasal shedding and viremia were reduced by vaccination. These results suggest that ΔgE would be a good candidate as an MLV vaccine.

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Nine isolates of rabbit hemorrhagic disease virus (RHDV) were used for the genetic characterization of RHDV strains collected from rabbits in Korea between 2006 and 2008. A phylogenetic analysis of the complete VP60 region was performed and the sequences were divided mainly into two groups. The one group consisted of original RHDV and the other contained antigenic variant strain known as RHDVa strains. Most of the Korean isolates clustered with Chinese RHDV strains and belonged to the RHDVa subtype. A comparison of the amino acid sequences among RHDVa strains and original RHDV strains revealed significant substitutions of two amino acids in the A region, two in the B region, two in the F region, and nine amino acids in the E region. Taken together, the recent RHDVa strains have gradually replaced the original RHDV and are the predominant strains in Korea.

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