Edema disease (ED) has become frequent in Japan, but no effective method for experimental infection has been developed. We report here the use of a capsule that resistant against gastric digestion to induce the ED in piglets. Four 21-day-old piglets were used. Shiga toxin 2e-producing Escherichia coli (STEC) cell pellet was encapsulated and administered orally. Two pigs received 1.0 × 10¹⁰ CFU for two days, and the others received 3.9 × 10⁸ CFU for three days. The high-dose group caused the typical clinical ED signs (palpebral edema or neurologic impairment). Eosinophil infiltration, swollen lymphoid follicles, and edema were observed in the ileum. The kidney had the thrombus in the glomerulus.

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Highly pathogenic avian influenza (HPAI) was diagnosed in broiler breeders, submitted to the National Veterinary Research and Quarantine Service in South Korea. Grossly, the dead breeders had lesions consistent with HPAI, including pancreatic mottling, splenomegaly, pulmonary edema and congestion, and hemorrhages in the mucosa of the proventriculus, gizzard and small intestine, and on the serosal surface. Microscopically, there were necrotized hepatitis and pancreatitis, lymphocytic meningoencephalitis, myocarditis, and interstitial pneumonia. Influenza viral antigen was demonstrated in areas closely associated with histopathologic lesions. The AI virus was isolated from cecal tonsils, feces, trachea, and kidney of the chickens. The isolated virus was identified as the highly pathogenic H5N1, with a hemagglutinin proteolytic cleavage site deduced amino acid sequences of QREKRKKR/GLFGAGLFGAIAG. In order to determine the pathogenicity of the isolate, eight 6-week-old specific pathogen free chickens were inoculated intravenously with the virus, and all the birds died within 24 hr after inoculation. This is the first report of an outbreak of HPAI in the chickens in South Korea.

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In this study, we cloned a cDNA that encodes a small heat shock protein, Hsp20 (αB crystallin-related protein), from a maturing rat testis by means of differential display. The full-length cDNA sequence was completely identical to that registered in the DNA databank. The expression of Hsp20 gene was detected strongly in the heart and slightly in the testis of a 9-week-old rat. The expression of Hsp20 increased gradually from three weeks to 9 weeks, and the strongest expression was observed in the testis at week fifteen. The expression was localized in spermatocytes and round spermatids. The gene expression was not affected by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) when it was administered into male rats during the nursling period.

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Frequency of the 4-bp deletion mutant in canine mdr1 gene was examined in 193 dogs of eight breeds in Japan. The mutant allele was found in Collies, Australian Shepherds, and Shetland Sheepdogs, where its respective frequencies were 58.3%, 33.3%, and 1.2%. The MDR1 protein was detected on peripheral blood mononuclear cells (PBMC) from a MDR1/MDR1 dog, but not on PBMC from a mdr1-1Δ/mdr1-1Δ Collie. Rhodamine 123 was extruded from MDR1/MDR1 lymphocytes. That excretion was inhibited by a MDR1 inhibitor, verapamil. On the other hand, Rh123 excretion was not observed from lymphocytes derived from a mdr1-1Δ/mdr1-1Δ Collie. These results indicated that the mutant mdr1 allele also existed in Collie-breed dogs in Japan at high rates and that mdr1-1Δ /mdr1-1Δ dogs have no functional MDR1.

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Intracerebral microdialysis combined with electroencephalographic recordings was performed on 4 dogs of a familial idiopathic epileptic Shetland sheepdog colony to identify the kinds of neurotransmitters responsible for seizure activity. Immunohistochemistry using glutamate (Glu), glutamate transporter (GLT-1 and GLAST), and glutamine synthetase (GS) antibodies was also carried out on the cerebrum of four familial dogs that died of status epilepticus (SE). High values for extracellular levels of Glu and aspartate (ASP) were detected in association with an increased number of spikes and sharp waves during hyperventilation in 3 of 4 the familial epileptic dogs. The values of other amino acids analyzed were not altered in any of the familial epileptic dogs. Immunohistochemically, Glu-positive granules were occasionally found in the perineuronal spaces of the cerebral cortex in 3 of the familial epileptic dogs that died of SE. Immunostains for GLT-1 antibody predominantly decreased in the cerebral cortex and lateral nucleus of the thalamus in all the dogs that died of SE, whereas there were no differences detected in immunolabellings for GLAST and GS antibodies between familial epileptic dogs and controls. These results suggest that an extracellular release of both Glu and Asp may play an important role in the occurrence of seizure activity in this epileptic colony, and that a decreased expression of astrocytic GLT-1 may be related to development of SE.

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A cDNA library was constructed from salivary glands of partially-fed adult female Haemaphysalis longicornis (hard tick). Randomly selected clones were sequenced and a total of 633 sequences were analyzed by bioinformatic programs. The sequences were grouped into 213 clusters, with each cluster being considered to be composed of mRNAs derived from the same gene or closely related genes. About 36% of the mRNA sequences showed significant similarity to known proteins in the non-redundant protein database by the NCBI blastx program and appeared to be coding for functional predicted proteins, whereas the remaining 64% had no similar sequences. Two thirds of the predicted proteins were annotated as basic cellular proteins (housekeeping proteins). Among the functional predicted protein sequences, other than the housekeeping proteins, several protease inhibitors including anticoagulants, two metalloproteases and a potential immunosuppressive protein could be identified. These proteins may play important roles during tick feeding and could be novel anti-tick vaccine candidates.

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Endocrine patterns were compared in 2 strains of Japanese black cattle with growth retardation; MHO- and HSK-paternal strains (MHO and HSK cattle, respectively). MHO cattle (n=8) displayed lower serum concentrations of insulin-like growth factor-1 (IGF-1), triiodothyronine (T3), thyroxine (T4), and cortisol (31.1 ± 20.7 ng/ml, 73.9 ± 51.9 ng/dl, and 2.9 ± 2.9 μg/dl, 1.3 ± 0.7 μg/dl, respectively) than those in both HSK cattle (n=5) (64.9 ± 47.6 ng/ml, 97.8 ± 40.7 ng/dl, 4.1 ± 2.1 μg/dl and 1.8 ± 1.1 μg/dl, respectively), and the controls (n=6) (314.7 ± 197.2 ng/ml, 140.2 ± 21.3 ng/dl, 5.8 ± 1.7 μg/dl, and 3.0 ± 1.4 μg/dl, respectively). The area under the concentration curve of growth hormone (GH-AUC 0-600 min) in MHO cattle (22210 ± 18951 ng·min/ml) tended to be greater than those in HSK (7887 ± 6340 ng·min/ml) and the controls (2811 ± 1275 ng·min/ml). MHO cattle showed a high GH-AUC_0-600 min in contrast to a low serum IGF-1 concentration, as well as lower serum T3, T4, and cortisol concentrations. HSK cattle exhibited the same secretory patterns, but much more moderately. Growth retardation in Japanese black cattle exhibits some variations based on pedigree.

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We explored the involvement of endothelin-1 (ET-1) in the pathophysiology of dog dirofilariasis (heartworm disease caused by Dirofilaria immitis) by analyzing mRNA levels of preproendothelin-1 (PPET-1), the precursor form of ET-1, in cardiopulmonary organs as well as ET-1 peptide levels in plasma. To determine the cDNA sequence and primary protein structure of dog PPET-1, we performed molecular cloning of the full-length cDNA. Based on the determined sequence information, comparative expression analysis of PPET-1 mRNA was carried out by real-time polymerase chain reaction on cardiopulmonary organs from healthy (n=5) and filarial (n=5) dogs. Filarial dogs showed a significantly (p<0.05) higher mRNA expression level in the heart (about one hundred times) and lung (about ten times) than healthy dogs. Analysis of plasma ET-1 levels in healthy (n=10) and filarial (n=10) dogs showed that filarial dogs (6.9 ± 2.7 pg/ml) have significantly (p<0.01) increased plasma ET-1 levels compared with healthy dogs (1.4 ± 0.3 pg/ml). To assess the pathophysiological significance of ET-1 in dirofilariasis relative to other cardiopulmonary disorders, plasma ET-1 levels determined in dogs diagnosed with mitral regurgitation (n=10), tricuspid regurgitation (n=5), ventricular septal defect (n=5), and patent ductus arteriosus (n=5) were compared to plasma ET-1 levels in filarial dogs. Filarial dogs, which commonly develop serious pulmonary hypertension, exhibited by far the highest ET-1 levels of the disease states examined. Based on the fact that ET-1 is a potent bioactive mediator that induces vasoconstriction and promotes vascular remodeling, these findings suggest that ET-1 plays an important role in the pathophysiology of dog dirofilariasis as an aggravating factor by inducing pulmonary hypertension.

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The usefulness of computed tomography (CT) for the diagnosis of feline hepatic lipidosis (FHL) was evaluated. Liver CT number was 54.7 ± 5.6 HU (mean ± SD) in 26 healthy cats. We fast 6 healthy cats for 72 hr to induced FHL experimentally and the cats were assessed by CT and serum biochemical analysis. Liver CT number of the six cats was 53.8 ± 3.0 HU before fasting, 46.8 ± 2.4 HU after fasting, and 50.2 ± 3.6 HU two weeks after restarted feeding. The decreased CT number was associated with the elevation of serum non-esterified fatty acid (NEFA) and β-hydroxybutyrate levels. These results indicate that measurement of CT number of the liver is an effective procedure for the diagnosis of FHL.

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Nucleotide sequences of ribosomal internal transcribed spacer (ITS1) and mitochondrial NADH dehydrogenase I (NDI) gene were analyzed to genetically characterize aspermic Fasciola forms in Korea. From the difference in ITS1 sequences, Korean flukes were divided into 3 haplotypes represented by Kor1, Kor2 and Kor1/2, which had nucleotides identical to F. hepatica, F. gigantica and those overlapped between the two species, respectively. NDI sequences also showed that Korean flukes could be classified into 3 distinct haplotypes (Kor1: F. hepatica-type, Kor2a and Kor2b: F. gigantica-type). The sequences of Kor1 and Kor2a were 100% identical to those of the haplotypes Fsp1and Fsp2, respectively, which are major Fasciola forms in Japan. These findings strongly suggest that aspermic Fasciola forms in Korea and Japan originated from same ancestors and have recently spread throughout both countries.

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A retrospective cohort study was conducted to clarify the association between seropositive reactions to Neospora caninum and subsequent reproductive disorders among dairy cattle in Japan. A statistically significant association between Neospora seropositive reactions and abortions was observed (P=0.016), and seropositive cattle were 6.1 times more likely to abort compared to seronegative cows. No significant differences were observed between seropositive reactions and other reproductive disorders such as conception failure, perinatal death and calf mortality. As indicated by estimation of the attributable fraction, 83.6% of abortions in Neospora seropositive animals may be attributed to N. caninum. Considering seroprevalence of N. caninum in the cattle which aborted in Japan, 21.8% of abortions were estimated to be caused by neosporosis in Japan.

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In this study, we performed experiments to evaluate the extend of the process of apoptotic cell death by foot-and-mouth disease virus (FMDV). Apoptosis can also occur in some virus-infected cells, and ability of viruses to either inhibit or promote apoptosis may influence the pathologic outcome of infection. In this study, to determine if apoptosis plays a role in the outcome of FMDV infection in swine, we evaluated apoptosis in diseased tissues collected from pigs inoculated with two different stains of FMDV (O1 Campos and O Taiwan). And host cell DNA fragmentation in diseased tissue from animals which were infected with either virus was evaluated by occurrence of a laddering pattern characteristic of apoptosis. Infection of cultured keratinocytes from swine tongue failed to demonstrate apoptosis in the first few hours of infection, suggesting that cell-to-cell correlation between viral antigen and apoptotic changes, e.g. cytokine secretions by immune system cells, could be critical to initiating apoptosis. Consistent with this finding, we were able to detect the pro-inflammatory cytokine TNF-α in diseased tissues. A clear difference in the pathogenicity of the two different FMDV isolates to pigs was not demonstrated in our study.

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To compare the roles of chondromodulin-I (ChM-I) and bone morphogenetic protein-6 (BMP-6) in ectopic mesenchymal tissue formation in canine mammary gland tumors, 33 tumors and 2 normal mammary glands were examined. Immunohistochemical analysis revealed co-expression of ChM-I and BMP-6 in canine mammary tumors. In mixed tumors, newly formed woven bone with ossified cartilage matrix was observed in 4/9 cases. The osteoblasts lining the woven bone showed moderate immunoreactivity to ChM-I and BMP-6. Almost all chondrocytes and proliferative myoepithelial cells within the basement membrane showed intense immunoreactivity to both, and the myoepithelial cells adjacent to the mature cartilage showed the most intense immunoreactivity. The immunoreactivity to ChM-I and BMP-6 of the interstitial myoepithelial cells in the myxomatous stroma varied in each focus of mixed tumors. Similar findings were found in complex adenomas. In simple adenomas, hyperplasic myoepithelial cells within the basement membrane showed moderate immunoreactivity to both markers. Western blot analysis detected a 25 kDa band of ChM-I in fresh tissue samples from three mixed tumors. Our results support the hypothesis that proliferating myoepithelial cells with ChM-I and BMP-6 expression play important roles in mesenchymal metaplasia in canine mammary tumors.

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The effectiveness of Doxil as a new chemotherapeutic agent against canine transmissible venereal tumor was evaluated, using NOD/ SCID and CD1-nu xenograft mouse models and the response between the two mouse strains was compared. Samples of xenografted venereal tumor were inoculated SC into 20 six week-old NOD/SCID mice and 20 six week-old CD1-nu mice. Seven weeks later, tumor-bearing mice were divided into treatment and control groups. Treatment group was injected with Doxil (6 mg/kg, IP, as a single injection). Control group was injected with buffered saline (0.75cc, IP). Tumor size was determined by caliper measurements and tumor response was assessed according to standard criteria. In both strains there was a significant decrease in tumor size in response to Doxil treatment (P<0.0001). In CD1-nu eight out of nine tumors (88%) responded to the treatment, and in 2 cases complete remission was observed. In NOD/SCID group response to the treatment was seen in eight out of ten tumors (80%) but none regressed fully. Response to the treatment was statistically equal in both strains even though the apoptotic rate, confirmed by TUNEL staining, was higher in NOD/SCID than in CD-1-nu (8.65% and 0.7%, respectively) and tumor infiltrating cells were different: eosinophils in NOD/SCID and CD45R-positive B lymphocytes, and plasma cells in CD-1-nu. In untreated CD1-nu mice, tumor progress was slower than in NOD/SCID. Our results indicate that Doxil is effective against CTVT in mouse xenograft models.

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A tumor sized in 2.0 × 2.0 × 2.5 cm developed in the cerebellum of a female Beagle was pathologically investigated. Histopathologically, the tumor grew by compression and partially by infiltration into the adjacent cerebellar parenchyma. There were a large number of necrotic lesions and proliferation of collagen fibers. The tumor cells had oval nucleus showing cellular atypia and a high mitotic index. The tumor cells were reacted with vimentin antibody on immunostain. Electron microscopic examination revealed the tumor cells interdigitated with cytoplasmic processus where the desmosomes developed on cell junction. This tumor was diagnosed as anaplastic meningioma, which is rarely observed in dogs.

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Previously, we demonstrated that plasma cortisol (Cor) levels were increased by road transportation in castrated male goats, but the extent of the increase was significantly reduced by 5α-dihydrotestosterone (DHT) implantation. This study aims to clarify whether the reduction of Cor secretion by androgen during transportation results from reduced plasma adrenocorticotropic hormone (ACTH). Castrated goats were implanted separately with cholesterol (Cho), testosterone (T) or DHT, followed by transportation. Plasma Cor levels increased during transportation regardless of hormone treatment, but the levels in T and DHT treated animals were lower than those in animals treated with Cho. Plasma ACTH levels also increased during transportation, and those in T treated animals were significantly lower than in those treated with Cho. However, plasma ACTH levels in DHT treated animals varied among the animals and did not differ from those in Cho treated animals. Significant and highly positive correlations between the logarithm of plasma ACTH levels and plasma Cor levels were found in every treatment group. The areas under the regression curves between plasma ACTH levels and plasma Cor levels associated with T and DHT treatments were significantly lower than those with Cho treatment. In conclusion, T was shown to reduce ACTH secretion in response to transportation in castrated goats. However, this suppression of the increase in Cor secretion during transportation by androgen is suggested to be mainly a result of suppression of the responsiveness of the adrenal cortex to ACTH.

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Although the release of growth hormone (GH) is known to be regulated mainly by GH-releasing hormone (GHRH) and somatostatin (SRIF) secreted from the hypothalamus, ghrelin also may be involved in GH release during juvenile period. We have examined plasma concentrations of acylated ghrelin, desacyl ghrelin, and GH in juvenile beagle dogs. Plasma acylated and desacyl ghrelin levels changed through aging; however, there was no closely correlation between ghrelin, body weight and circulating GH levels during juvenile period. The increase in body weight was essentially linear until 8 months of age, whereas plasma GH concentrations exhibited bimodal peaks for the meanwhile. The results suggest that ghrelin may not play internal cueing in GH secretion in juvenile beagle dogs.

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Diseases of companion animals are shifting from infectious diseases to neoplasms (cancer), and since radiation therapy is one of the effective choices available for cancer treatment, the application of radiotherapy in veterinary medicine is likely to increase. However tumor tissues have different radiosensitivities, and therefore it is important to determine the intrinsic radiosensitivity of tumors in individual patients in advance of radiotherapy. We have studied the relationship between the surviving cell fraction measured by a clonogenic assay and DNA double strand breaks detected by a comet assay under neutral conditions in three canine tumor cell lines, after γ-ray and carbon ion irradiation. In all the cell lines, cell death assessed by the clonogenic assay was much higher following irradiation with carbon ions than with γ-rays. The initial and residual (4 hr) DNA damage due to γ-ray and carbon ion irradiation were higher in a radiosensitive cell line than in a radioresistant cell line. The surviving cell fraction at 2 Gy (SF2) showed a tendency for correlation with both the initial and residual DNA damage. In particular, the residual damage per Gy was significantly correlated with SF2, regardless of the type of radiation. This indicates that cellular radiosensitivity can be predicted by detection of radiation-induced residual DNA damage.

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Canine epididymides were excised and immediately stored at 4°C for 48 hr, and the qualities of caudal epididymal sperm after recovery and cryopreservation were evaluated. To confirm the fertility of the cryopreserved caudal epididymal sperm, artificial intrauterine insemination was performed. The sperm motility (61.0%) immediately after recovery from caudal epididymis stored at 4°C for 48 hr was significantly lower than those of sperm stored for 0 and 24 hr (88.6 and 80.7%, respectively), but there was no significant difference after freeze-thawing (0-, 24-, and 48-hr storage groups: 27.9, 24.3, and 28.3%, respectively). The incidence of abnormal sperm immediately after recovery was significantly higher in the 24-hr and 48-hr storage groups (19.3 and 27.7%, respectively) than in the 0-hr storage group (5.6%), and a significant difference was also observed after freeze-thawing. The incidence of immature sperm with cytoplasmic droplets was significantly higher in the 48-hr storage group (18.4%) than in the 0-hr storage group (4.7%), but there was no difference after freeze-thawing. By unilateral intrauterine insemination (2 × 10⁸ sperm), 4 of 5 bitches (80%) conceived. The above findings demonstrated that sperm motility was good even enough the incidence of abnormal sperm was high in canine epididymal sperm that were recovered from the epididymis stored at 4°C for 48 hr and cryopreseved, and that artificial intrauterine insemination resulted in a high conception rate.

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Herpesviral DNA was detected in 24/261 DNA samples that were extracted from whole blood of 13 wild animal species in South Africa. Herpesviral DNA was shown in 22 impalas (Aepyceros melampus) and 2 springboks (Antidorcas marsupialis). All of DNA sequences detected in impalas were identical, whereas two DNA sequences detected in springboks were different each other. These three sequences showed 44 to 72% homology to the corresponding gene of the Gammaherpesvirinae, indicating that these three viruses should be unrecognized novel gammaherpesviruses. The putative novel herpesviruses were tentatively designated as Aepyceros melampus (impala) herpesvirus 1 (ImHV-1), Antidorcas marsupialis (springbok) herpesviruses 1 (SpHV-1) and 2 (SpHV-2). ImHV-1 seems to be a relatively independent virus. SpHV-1 belongs to a group of ruminant lymphotropic herpesviruses and SpHV-2 is closer to a malignant catarrhal fever virus group. Potential threat of these herpesviruses to domestic animals should be considered.

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