In this study, we attempted to clarify whether puppy socialization and command training class, which was limited to puppies approximately 4 months of age, prevented behavior problems in dogs. We evaluated the behaviors of 142 dogs with 4 types of training experience by using a behavior test and the Canine Behavior Assessment and Research Questionnaire (C-BARQ). Dogs in the puppy class (PC) group (n=44) attended the class for 1 hr each week for 6 weeks, dogs in the puppy party (PP) group (n=39) attended a 1-hr “puppy party,” dogs in the adult class (AC) group (n=27) undertook basic obedience lessons for adolescent/adult dogs for 1 hr each week for 6 weeks, and dogs in the no class (NC) group (n=32) underwent no formal training. The behavior test evaluated each dog’s response to commands, owner’s recall, separation, a novel stimulus and strangers. The C-BARQ evaluated 15 canine behavioral factors. The behavior test results indicated that the PC and AC groups showed significantly higher response to commands than the PP or NC group. Thus, participation in puppy and adult classes improved the obedience behavior of dogs, regardless of age. Positive response to strangers in the PC group was significantly higher than that in the AC and NC groups and tended to be higher than that in the PP group. Therefore, PC may help prevent canine behavioral problems such as disobedience or fear of strangers.

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The aim of the undertaken research was to determine whether Foxp3⁺CD25⁺CD8⁺ cells exist in cattle and whether Foxp3 expression in CD8⁺ T cells is correlated with the intensity of CD25 expression. It has been found that 0.66 and 2.36% of CD8⁺ cells on average showed high and low expression of the CD25 molecule, respectively. On average, 11.61% of CD25^highCD8⁺ cells expressed Foxp3, while the mean percentage of Foxp3⁺ cells within CD25^lowCD8⁺ cells was 3.66%. The absolute count of Foxp3⁺CD25^highCD8⁺ cells was not significantly different from the absolute count of Foxp3⁺CD25^lowCD8⁺ cells. The obtained results indicate that CD8⁺ cells with the regulatory phenotype, i.e. Foxp3⁺CD25^highCD8⁺ and Foxp3⁺CD25^lowCD8⁺ cells, occur naturally in bovine peripheral blood, although both of these subpopulations are relatively small. There is a positive correlation between the intensity of CD25 expression and the expression of the transcription Foxp3 factor in bovine CD8⁺ cells.

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This study was to compare the degree of cell-mediated immunity induced by three commercial Mycoplasma hyopneumoniae bacterins using interferon-γ (IFN-γ) measurements, lymphocyte stimulation assays and delayed-type hypersensitivity tests. Serum IFN-γ levels were significantly elevated in all four vaccinated pig groups at 21 days post-vaccination (P<0.05). Lymphocytes isolated 21 days post-vaccination exhibited significantly more proliferation in response to M. hyopneumoniae than lymphocytes isolated 0 day pre-vaccination (P<0.05). Following intradermal injection of M. hyopneumoniae antigen at 14, 21 or 28 days post-vaccination, all pigs in the four vaccinated groups displayed skin reactions characterized by circumscribed, often erythematous nodules. Taken together, these demonstrate that all three commercial single-dose M. hyopneumoniae bacterins used in this study induce varying degrees of cell-mediated immunity.

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Monocyte chemoattractant protein-1 (MCP-1) is a member of the C-C family chemokines, which mobilizes monocytes from bone marrow to the site of inflammation. To evaluate the clinical utility of canine MCP-1 as a blood test item, we measured serum MCP-1 concentrations in normal and ill dogs. Reference interval of canine MCP-1 was established as 115.6–176.9 pg/ml. Serum MCP-1 concentrations increased in the dogs affected with neoplastic (518.0 ± 84.8 pg/ml), inflammatory (257.0 ± 42.5 pg/ml) or other diseases (360.3 ± 45.2 pg/ml). The results showed high sensitivity of MCP-1 to detect neoplasia and inflammation. Moreover, MCP-1 increased in some cases in which C-reactive protein didn’t increase. MCP-1 might be helpful as a screening blood test marker for detection of neoplasia and inflammation in dogs.

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A 13-year-old, neutered male miniature dachshund was presented with a one-month history of bilateral symmetrical swelling in the pinnae and carpal, cubital and tarsal joints, and swelling in the tail. The lesions were histopathologically characterized by multiple dilated lymphatic vessels lined by a single attenuated layer of endothelial cells. The subcutis was predominantly involved. A number of spindle-shaped cells lining the irregular vessels were observed. Morphological atypia was not evident in these cells. Immunohistochemical analyses revealed that the proliferating endothelial cells were positive for factor VIII-related antigen and CD31. Based on the clinical presentation and histopathological features, the dog was diagnosed with lymphangiomatosis. Treatment with anti-inflammatory prednisolone improved the symptoms.

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Connexin 32 (Cx32) is a major gap junction protein in the liver. Neoplastic and non-neoplastic lesions were examined in Cx32-deficient (Cx32KO) mice maintained for 24-month, and compared with those in wild-type mice as a corresponding control. In neoplastic lesions, hepatocellular carcinoma increased significantly only in male Cx32KO mice, suggesting that Cx32 deficiency may be related to their pathogenesis. For females, the incidence of pituitary adenoma in the pars distalis of Cx32KO mice was lower than that of wild-type mice. No non-neoplastic lesions related to Cx32-deficiency were observed in the Cx32KO mice. In conclusion, these results demonstrate that the incidence of hepatocellular carcinoma increases only in male Cx32KO mice, presumably due to enhanced tumor promotion and progression signals associated with Cx32 deficiency.

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The aim of this study investigated whether nicotinamide affects parvalbumin expression in focal cerebral ischemic injury. Rats were treated with vehicle or nicotinamide (500 mg/kg) 2 hr after middle cerebral artery occlusion (MCAO), and cerebral cortex tissues were collected 24 hr after MCAO. Nicotinamide significantly decreases the volume of infarct areas in the cerebral cortex. A proteomic approach revealed that MCAO induces decreases of parvalbumin levels, while nicotinamide treatment prevents injury-induced decreases in parvalbumin. RT-PCR and Western blot analyses demonstrated that nicotinamide restores injury-induced decreases in parvalbumin. Moreover, immunohistochemical staining confirmed that the numbers of parvalbumin-positive cells were decreased in vehicle-treated animals with MCAO, and that nicotinamide averted this decrease. In cultured hippocampal cells, nicotinamide treatment prevents the glutamate exposure-induced increase in intracellular Ca²⁺ concentration and decrease in parvalbumin expression. These results suggest the fact that the maintenance of parvalbumin expression is mediated to the neuroprotective function of nicotinamide against ischemic brain injury.

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In this study, we attempted to apply new convenience gender sorting methods using sex-determining region Y (SRY) gene expression on Y spermatozoa to mice. Mouse spermatozoa labeled with Cy3-SRY antibody conjugate were used for intracytoplasmic sperm injection (ICSI). In addition, spermatozoa conjugated with SRY antibody were conjugated with magnetic beads (Mag) and were pulled to the bottom of the medium. The supernatant of the medium was used for in vitro fertilization (IVF). The rate of males reproduced by ICSI using the spermatozoa conjugated with Cy3-SRY antibody was 86.1%. The female proportion reproduced by IVF using the spermatozoa separated in the supernatant after Mag-SRY antibody conjugation was 67.3%. These gender sorting methods are effective for the reproduction of transgenic mice.

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Babesia ovata is a tick-transmitted hemoprotozoan parasite that infects cattle. In our study, bovine blood samples (n=2,034) were collected from 10 different countries (Brazil, China, Ghana, Japan, Mongolia, the Philippines, South Africa, Sri Lanka, Thailand and Vietnam) and DNA extracted. The DNA samples were screened using an established and specific polymerase chain reaction (PCR) assay targeting the Apical membrane antigen 1 (AMA-1) gene. Parasite DNA was detected among samples collected from Japan, Mongolia and Thailand. Sequence analyses confirmed that the PCR assay detected only B. ovata AMA-1, and that amplicons from different geographical locations were conserved. Our findings highlight the importance of designing adequate strategies to control B. ovata infection in Japan, Mongolia, and Thailand.

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In this study, the prevalence of Borrelia infections in Ixodes ticks from a site in Hokkaido, Japan, with confirmed cases of Lyme disease was determined by a PCR method capable of detecting and differentiating between strains of pathogenic Borrelia, with particular emphasis on Borrelia garinii (B. garinii) and Borrelia afzelli (B. afzelli), using tick-derived DNA extracts as template. A total of 338 ticks, inclusive of 284 Ixodes persulcatus (I. persulcatus), were collected by flagging vegetation in mid-spring. Ninety-eight (34.5%) of I. persulcatus tested positive for Borrelia species DNA, whereas the overall prevalence of Borrelia species in Ixodes ovatus and Haemaphysalis longicornis ticks was 19.5 and 7.7%, respectively. PCR-RFLP and sequence analysis of Borrelia rrf(5S)-rrl(23S) intergenic spacer DNA amplicons indicated that they originated from three different Borrelia species namely, B. garinii, B. afzelii and B. japonica. Among the I. persulcatus species, which is a known vector of human borreliosis, 86 were mono-infected with B. garinii, 2 ticks were mono-infected with B. afzelii and whereas 12 ticks had dual infections. Most significant, 11 of the I. persulcatus ticks were coinfected with Anaplasma phagocytophilum and B. garinii. The difference between the number of obtained and expected co-infections was significant (χ²=4.32, P=0.038).

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The association of renin and angiotensin II, which are potent components of the renin–angiotensin system, with the severity of chronic renal disease was investigated immunohistochemically in dogs and cats. Immunoreactivities of renin and angiotensin II were evaluated quantitatively, and their correlations with the degrees of glomerulosclerosis, glomerular hypertrophy, interstitial cell infiltration and interstitial fibrosis were statistically analyzed. Immunoreactivities for renin were detected in afferent arteries in both dogs and cats. The score of renin-positive signals showed no correlation with plasma creatinine concentration or any of the histopathological parameters, except for the diameter of glomeruli in dogs. Immunoreactivities for angiotensin II were detected in tubules (primarily proximal tubules) and interstitial mononuclear cells in both dogs and cats. The score of tubular angiotensin II correlated with glomerulosclerosis and cell infiltration in cats but not in dogs. The score of interstitial angiotensin II correlated with plasma creatinine concentration, glomerulosclerosis, cell infiltration and fibrosis in dogs and with glomerulosclerosis and cell infiltration in cats. In conclusion, the results of the study suggest that intrarenal renin-angiotensin system is correlated with the severity of kidney disease, with the underlying mechanism differing between dogs and cats.

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A 10-year-old male fossa (Crytoprocta ferox) exhibited clinical signs of diarrhea, anorexia and weight loss. Chemistry values and echographic results were suggestive of intestinal lymphoma. Postmortem examination revealed severe multifocal wall thickening of the small intestine with severe enlargement of the pancreatic lymph node. Microscopically, the small intestine was multifocally transmurally infiltrated by large neoplastic round cells also found in the pancreatic lymph node and the liver. On immunohistochemistry, the neoplastic cells stained intensely with CD3 and didn’t stain with CD79a. Based on these findings, a diagnosis of intestinal T-cell lymphoma with pancreatic lymph node and liver involvement was made. To the authors’ knowledge, this is the first report of a lymphoma with immunohistochemical phenotyping in a fossa.

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Upregulation of hyaluronidase 2 (HYAL2), one of somatic hyaluronidase (HAase), was demonstrated in granulation tissue during the healing of equine superficial digital flexor tendon injuries. The activity of HAase was assessed by hyaluronan (HA)-containing gel zymography and in situ zymography using frozen sections obtained from normal and injured tendon tissues. Elevated HAase activity was identified in the extract from the tendinopathic tissues, with lower levels of the activity in normal tendons. In situ zymography using fluorescently-labeled HA demonstrated HAase activity in the granulation tissue formed in the injured region. In addition, in situ hybridization analysis indicated that fibroblastic cells in the granulation tissue of the injured tendon actively expressed HYAL2 but not HYAL1. Quantitative RT-PCR further confirmed a higher level of amplicons corresponding to HYAL2 in tendonitis-derived samples. These results suggest that elevated HYAL2 activity in the granulation tissue could participate in controlling the healing process in equine tendonitis.

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Methylglyoxal (MGO) is a metabolite of glucose and likely related to pathogenesis of diabetes-related vascular complications including hypertension. In this study, long-term effects of MGO on endothelial function were examined. Rat isolated mesenteric artery was treated for 3 days with MGO using an organ culture method. The contractility, morphology and protein expression of organ-cultured artery were examined. MGO (42 μM, 3 days) impaired acetylcholine (ACh: 1 nM–300 μM)-induced endothelium-dependent relaxation, while it had no effect on sodium nitroprusside (0.1 nM–10 μM)-induced endothelium-independent relaxation. MGO decreased ACh (3 μM)-induced nitric oxide (NO) production as measured by a fluorescence NO indicator, diaminofluorescein-2. Consistently, MGO inhibited ACh (3 μM)-induced phosphorylation of vasodilator stimulated phosphoprotein (an indicator of cyclic GMP production). MGO induced apoptosis in endothelium as detected by TdT-mediated dUTP-biotin nick-end labeling staining. MGO induced accumulation of superoxide in endothelium as detected by dihydroethidium staining. MGO decreased protein expression of endothelial NO synthase (eNOS). Gp91ds-tat (0.1 μM), an inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX), prevented the impairment of endothelium-dependent relaxation and the decrease in eNOS protein caused by MGO. The present results demonstrated that long-term MGO treatment impairs endothelium-dependent relaxation through NOX-derived increased superoxide-mediated endothelial apoptosis.

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Rickettsial infection in feral raccoons (Procyon lotor) in the western part of Japan (Shimane, Fukuoka, Saga and Nagasaki Prefectures) was surveyed by a nested polymerase chain reaction (PCR) assay detecting the rickettsial citrate synthase (gltA) gene. Four of one hundred and ninety-four feral raccoon spleens (2.1%) were positive for Rickettsia spp. One gltA gene sequence was identical to R. helvetica, whereas the other 3 sequences were identical and had the highest similarity (98.4%) to R. amblyommii. Simultaneously, we determined a partial sequence of the rickettsial 17-kilodalton (17K) genus-common antigen gene in the later 3 raccoon samples. Their sequences were identical and had the highest similarity (98.5%) to Rickettsia sp. Hj126. Based on the sequences of gltA and 17K antigen genes, these raccoons might be infected with spotted fever group (SFG) rickettsia most closely related to R. amblyommii and/or Rickettsia sp. Hj126. Feral raccoons may be a susceptible reservoir for SFG rickettsiae in Japan.

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Anesthetic and cardiorespiratory effects of medetomidine, lidocaine, butorphanol and propofol total intravenous anesthesia (MLBP-TIVA) were evaluated in horses undergoing an experimental surgery. Ten horses were premedicated with an intravenous injection (IV) of medetomidine (5 μg/kg) and butorphanol (20 μg/kg). Anesthesia was induced by administration of 1% propofol (3 mg/kg, IV) at a rate of 1 mg/kg/min (n=5, group-1) or 2% propofol administered at a rate of 6 mg/kg/min (n=5, group-2) following administration of lidocaine (1 mg/kg, IV) and then maintained by infusions of propofol, medetomidine (3.5 μg/kg/hr), lidocaine (3 mg/kg/hr) and butorphanol (24 μg/kg/hr). The mean durations of anesthesia and propofol infusion rate required for maintaining surgical anesthesia were 130 ± 17 min and 0.10 ± 0.01 mg/kg/min in group 1 and 129 ± 14 min and 0.10 ± 0.02 mg/kg/min in group 2. Four horses in group 1 and 2 horses in group 2 paddled following recumbency during induction of anesthesia. The median quality scores for induction (0–4: poor-excellent) and recovery (0–5: unable to stand-excellent) were 3 and 4 for both groups, respectively. Transition to anesthesia (the first 20-min period after induction) was uneventful in group 2, while all horses showed a light plane of anesthesia in group 1. The quality score (0–3: poor-excellent) for the transition to anesthesia in group 2 was significantly higher than in group 1 (median 3 versus 1, P=0.009). Heart rate and arterial blood pressure were maintained within acceptable ranges, but hypercapnia occurred during anesthesia in both groups. In conclusion, MLBP-TIVA may provide clinically useful surgical anesthesia in horses. A rapid induction with propofol may improve the qualities of induction and transition to MLBP-TIVA.

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We report two feline cases of patent ductus arteriosus (PDA) with pulmonary hypertension (PH). The subjects were both intact domestic shorthair cats, a 4-month-old, 2.5 kg male (case 1) and an 8-month-old, 2.12 kg female (case 2). At the first presentation, left-sided congestive heart failure was diagnosed in case 1 and severe aortic stenosis (AS) in case 2. Following surgical ligation of the ductus arteriosus (DA), furosemide therapy was no longer required in case 1, and the severe AS improved to mild status in case 2 perhaps because of reduced volume overload. In case 2, severe hypoxemia was revealed after surgery; however, this normalized within 96 days after surgery.

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We report here the clinical presentation and successful surgical management of synovial hemangioma accompanied by cruciate ligament injury in a dog. Surgical correction of cruciate ligament injury was performed after removing torn cruciate ligaments, synovial mass and masses attached to the ligaments and distal femoral articular cartilage. At 10 months, the dog has showed no evidence of recurrence in the stifle joint. Synovial hemangioma, although a seemingly rare cause of stifle joint pathology, should be considered in the differential diagnosis for cruciate ligament injury when a circumscribed intra-articular soft tissue mass is evident radiographically with cranial or caudal drawer motion. This is the first case report in a dog to describe synovial hemangioma accompanied by cruciate ligament injury.

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A procedure developed for nonsurgical transvaginal ultrasound-guided oocyte aspiration in the mature sow was dependent upon development of a special chute. This chute was designed to immobilize and protect the sow. The floor of the chute possessed a metal bar that was elevated via a hydraulic lift until the weight of the sow rested on the bar, while her hooves were still in contact with the floor. This exact positioning of the female prevented her from resisting procedure and allowed for a comfortable setting for technician to manipulate the ovaries (per rectum) and perform transvaginal ultrasound guided oocyte aspiration (TUGA). Oocyte collection was performed in six sows, and none experienced detrimental health effects from the procedure.

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True hermaphrodites are animals of equivocal sex in which both male and female gonads develop simultaneously in the same individual. The frequency of true hermaphroditism is relatively higher in pigs than in other domestic animals. Two Korean pigs were diagnosed with true hermaphroditism showing ovotestes, epididymides, a penis and uteri. The testicular tissues consisted of Sertoli cells that were devoid of spermatogenic cells and showed proliferation of interstitial cells. However, uteri looked normal and had well-developed endometrial glands. Samples showed the interpretation of 38, XX female karyotype and sex-determining region Y (SRY) gene expression was negative. These findings could be helpful to understand true porcine hermaphroditism for animal research as well as for the industry of Korean domestic animals.

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Warfarin is a rodenticide commonly used worldwide. It inhibits coagulation of blood by inhibiting vitamin K 2,3-epoxide reductase (VKOR) activity. An inadequate supply of vitamin K blocks the production of prothrombin and causes hemorrhage. Recently, warfarin-resistant brown rats (Rattus norvegicus) were found around the Aomori area of Japan. There is no significant difference in the metabolic activity of warfarin in sensitive and resistant brown rats. To clarify the mechanism underlying warfarin resistance, we cloned the VKORC1 gene from rats and identified a novel substitution of arginine to proline at position 33 of the VKORC1 amino acid sequence. Then, we determined the differences in kinetics of VKOR activity between warfarin-resistant and sensitive rats. Hepatic microsomal VKOR-dependent activity was measured over a range of vitamin K epoxide concentrations from 6.25 to 150 μM. The V_max values of resistant rats (0.0029 ± 0.020 nmol/min/mg) were about one tenth of those of sensitive rats (0.29 ± 0.12 nmol/min/mg). The K_m values of resistant rats (47 ± 32 μM) were similar to those of sensitive rats (59 ± 18 μM). Warfarin-sensitive rats exhibited enzyme efficiencies (V_max/K_m) which were ten-fold greater than those observed in resistant rats. It may mean that VKOR activity of warfarin-resistant Aomori rats is almost lost, because their enzymatic efficiencies are very low even without warfarin. Further studies are needed to clarify how these rats can survive with a markedly reduced VKOR activity and how they simultaneously exhibit warfarin resistance.

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Although cisplatin (cis-diamminedichloroplatinum II) is one of the most effective chemotherapeutic agents, its clinical use is limited because of its strong side effects on the kidneys. The aim of this study was to investigate the renoprotective effect of bovine lactoferrin (bLf) in cisplatin-induced renal failure in rats. To assess the renoprotective effect of bLf, oral bLf (300 mg/kg) was administered from the day before to the fifth day after cisplatin (7 mg/kg, i.p.) injection. Daily administration of bLf histologically reduced renal tubular injury induced by cisplatin and suppressed the deterioration of renal function. Accumulated platinum content in the kidney was significantly decreased by the daily administration of bLf. Moreover, the administration of intravenous bLf caused a significant increase in urine volume in a dose-dependent manner. These results suggest that pretreatment with bLf produces a protective effect against cisplatin-induced nephrotoxicity. This protective effect of bLf involves the reduction of accumulated cisplatin in the kidney.

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Bovine papillomavirus type 12 (BPV-12) was recently identified in epithelial papillomas on the cattle tongue in our previous study. Along with the full-length genome, one deleted circular genome, named BPV-12-del, was detected in the same papilloma lesion. BPV-12-del is 3363 base pairs in length, and a total of 3,384 base pairs, including E1, E2 and E4 genes and partial E7 and L2 ORFs, were deleted compared with the complete genome. Real-time PCR results showed that the percentage of BPV-12-del was 42% of the total genomes in the sample. Southern Blot analysis also confirmed the presence of the deleted genome. This is the first report describing a circular genome deletion detected in a naturally BPV-infected sample.

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The values of CFU were described incorrectly.
Error Correction
100 CFU/g ⇒ 10 CFU/g
400 CFU/g ⇒ 40 CFU/g
>100 CFU/g ⇒ >10 CFU/g
Error : The levels of contamination in 3 salami samples and 1 raw ham sample that tested positive were below 100 CFU/g, which is an acceptable level at which this bacterium does not grow, according to food safety criteria for RTE foods by Codex [2] (Table 3). However, bacteria at a level of 100 CFU/g were isolated from 1 salami sample and at a level of 400 CFU/g from a raw ham sample; these levels exceed the limits specified by the criteria established by Codex.
Correction : The levels of contamination in 3 salami samples and 1 raw ham sample that tested positive were below 10 CFU/g (Table 3). Bacteria at a level of 10 CFU/g were isolated from 1 salami sample and at a level of 40 CFU/g from a raw ham sample. Therefore, all positive samples showed the CFU below an acceptable level (<100 CFU/g) at which this bacterium does not grow, according to food safety criteria for RTE foods by Codex [2].

The authors would like to offer our sincere apologies for these mistakes.

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