Genomic characterization and distribution of bovine foamy virus in Japan.

Bovine foamy virus (BFV) is distributed through worldwide cattle herds. Although the biological features of BFV are not well understood, appearance of clinical manifestation by superinfection with other microorganisms is inferred. In Japan, reports of genomic characterizations and epidemiology of this virus are limited. In this study, we performed whole genomic sequencing of BFV strains Ibaraki and No.43, which were isolated in this country. Additionally, we investigated BFV in geographically distant four daily farms in Japan, to estimate the distribution of BFV and its correlation to bovine leukemia virus (BLV). BFV was distributed throughout Japan; the average positive rate was 12.7%. The nucleotide sequence identities of the isolates were 99.6% when compared with BFV strain isolated in the USA. The phylogenetic tree using env gene sequence showed strains Ibaraki, No.43 and Kagoshima were sorted in the same cluster including the USA and Chinese strains, while Hokkaido strain was in the other cluster including European strains. Although no clear correlation between BFV and BLV could be found, BFV and BLV infections were likely to increase with ages. Our data on epidemiology and characteristics of BFV will provide important information to reveal biological features of BFV.

[A Case of Ascending Colon Cancer with Essential Thrombocythemia].

Essential thrombocythemia(ET)is a rare myeloproliferative disorder characterized by thrombocytosis and a risk of thrombotic and hemorrhagic events. ET rarely occurs simultaneously with colorectal cancer. Including our case, only 5 cases of c o l orectal cancer with ET have been reported in Japan. Herein, we report a case of colon cancer in an ET patient who underwent laparoscopic right hemicolectomy. Our perioperative management avoided complications such as thrombosis or bleeding. An 81-year-old woman developed bloody stools. She was previously diagnosed with ET 9 years ago. Aspirin, cilostazol, and hydroxyurea(HU)were prescribed. Colonoscopy revealed a tumor at the ascending colon. Histopathological examination showed a well-differentiated tubular adenocarcinoma. Since the patient had anemia, aspirin and cilostazol were discontinued after diagnosis. HU was discontinued from the day before surgery to 2 days after surgery. Enoxaparin was subcutaneously administered for 1 to 3 days after surgery. Aspirin and cilostazol were resumed on the fourth day post-surgery. The patient could be discharged when her condition stabilizes with no thrombosis and bleeding after 8 days.

Production of lipid mediators in mastitic milk of cow.

Bovine mastitis is one of the most prevalent and costly diseases in the dairy industry. Lipid mediators are signaling molecules which coordinately and intricately modulate inflammation. They are produced from polyunsaturated fatty acids (PUFAs) in the cellular membrane via several enzymes including cyclooxygenase (COX) and lipoxygenase (LOX). In the present study, we performed comprehensive analysis of lipid production in milk obtained from clinical or subclinical mastitic cows using liquid chromatography/mass spectrometry. We detected 26, 24, and 40 kinds of lipid constantly in healthy, subclinical, and clinical mastitic milk, respectively. In clinical mastitic milk, the amount of a major n-6 PUFA, arachidonic acid (AA), tended to increase, whereas amounts of major n-3 PUFAs, eicosapentaenoic acid and docosahexaenoic acid, tended to decrease. The amounts of several AA-derived lipids including COX-catalyzed prostaglandin (PG) D2 and PGE2 , and LOX-catalyzed leukotriene (LT) B4 were increased in clinical mastitic milk. Although subclinical mastitic milk represented similar trend of lipid production to healthy milk, amounts of several lipids such as LTD4 , 14,15-dihydroxyeicosatrienoic acid, and 14-epoxyeicosatrienoic acid changed. These findings would be helpful for better understanding of mastitis pathology and give us some insights to develop a new diagnostic and therapeutic strategy.

Antigenic heterogeneity among phylogenetic clusters of influenza D viruses.

Influenza (flu) D virus, a possible causative agent of bovine respiratory disease, is genetically classified into three clusters: D/OK-, D/660-, and D/Japan-lineages. To evaluate antigenic heterogeneity among these clusters, we compared antibody titers to each lineage virus using bovine sera collected over time following virus infection. Antibody titers to D/Japan-lineage virus rose rapidly in the acute phase of infection, and were 4 times higher than those to the other clustered viruses. In the later phase of infection, titers to D/Japan-lineage virus were equivalent to those to D/OK-lineage virus, and still higher than those to D/660-lineage virus. These results suggest the existence of common and lineage-specific antigenic epitopes in the hemagglutinin-esterase-fusion protein of flu D viruses.

[A Case of Breast Cancer with Metastatic Nodules in Additionally Resected Specimens].

A 68-year-old woman who had leftbreastcancer (cT2N0M0, cStage ⅡA)underwentbreast -conserving therapy and sentinel lymph node biopsy. Pathological diagnosis of the resected specimen revealed a 60mm cancer lesion including a 50 mm invasive ductal carcinoma with surrounding ductal carcinoma in situ, although the pre-operative MRI suggested a 30mm invasive cancer. The surgical margin was positive with the exposure of ductal carcinoma in situ. Additional resection was performed with a resection margin of 20mm from the head-side stump of the previous surgery. Pathological diagnosis of the additionally resected specimen revealed a 6mm invasive carcinoma with its exposure on the surface of the specimen around the new surgical stump distant from the initial surgical margin, where no remnant cancer was noted. She underwent left mastectomy. Pathological diagnosis further revealed 7mm and 2mm invasive carcinomas in the remnant breast. The preoperative imaging was reviewed retrospectively, and it was found that identifying the nodules in the remnant breast was quite difficult based on the images, including MRI. We report a case of breast cancer with metastatic nodules in additionally resected specimens.

[Clinical Experience of a Primary Accessory Breast Cancer Patient].

A 59-year-old woman attended a previous hospital complaining of a nodule of the right axilla. Although ultrasonography had shown no evidenceof malignancy, a growth of thenodulewas found on follow-up. Excisional biopsy revealed a primary accessory breast cancer. Because the resected margins were involved, she was referred to our hospital for additional treatment. Based on imaging, both bilateral mammary glands and axillary lymph nodes were reported normal, and distant metastasis was not observed. We performed additional resection of the right axillary tissue around the biopsy site and the right axillary lymph nodedisse ction. Histo-pathological examination revealed the residual invasive ductal carcinoma in the resected specimen. Both the new surgical margins and the lymph nodes were free of disease. Accessory breast cancer is relatively rare, with the incidence being less than 1% of all breast cancers. It is most frequent in the axillary region. Local extensive resection with sufficient surgical margin and axillary lymph node dissection are generally required. This case report presents our clinical experience of accessory breast cancer with some discussion of the literature.

[Study of Breast Cancer Patients Diagnosed Preoperatively with Ductal Carcinoma In Situ].

Although ductal carcinoma in situ(DCIS)is generally cured by surgical resection, it has been suggested that resection is over-treatment for some patients with DCIS. The aim of this study was to reconsider operative indications for patients with DCIS by examining clinicopathological features of 23 patients who underwent surgical resection for DCIS in our institute over a single year. Postoperative histological examination revealed that there were Luminal and HER2-positive subtypes, but no triple negative cancers. We found coincidental invasive ductal carcinoma(IDC)in 5 patients, and in all 5 the tumor size exceeded 60 mm. There was no coincidence of IDC in patients with a Ki-67 index ≤5%. Positive surgical margins were observed in 7 patients, all of which were histologically diagnosed as DCIS. Only 1 of the 7 patients underwent additional surgical resection; the 6 remaining patients, including 2 patients who received no treatment, did not undergo additional resection. All patients including those with positive surgical margins have had a 5-year relapse-free survival. Our findings imply that the subgroup of DCIS patients without IDC could be followed up without surgery.

Effect of Treadmill Exercise and Hydrogen-rich Water Intake on Serum Oxidative and Anti-oxidative Metabolites in Serum of Thoroughbred Horses.

The present study aimed to clarify changes of oxidative stress and antioxidative functions in treadmill-exercised Thoroughbred horses (n=5, 3 to 7 years old), using recently developed techniques for measurement of serum d-ROMs for oxidative stress, and BAP for antioxidative markers. Also, the effect of nasogastric administration of hydrogen-rich water (HW) or placebo water preceding the treadmill exercise on these parameters was examined. Each horse was subjected to a maximum level of treadmill exercise in which the horses were exhausted at an average speed of 13.2 ± 0.84 m/sec. Blood samples were taken 4 times, immediately before the intake of HW or placebo water at 30 min preceding the treadmill exercise, immediately before the exercise (pre-exercise), immediately after the exercise (post-exercise) and at 30 min following the exercise. In all horses, both d-ROMs and BAP values significantly increased at post-exercise. The increase in d-ROMs tended to be lower in the HW trial, as compared to the placebo trial at pre-exercise. The increase in BAP was considerable at approximately 150% of the pre-exercise values in both the HW and placebo treatment trials. The BAP/d-ROMs ratio was significantly elevated at post-exercise in both treatment trials, while a significant elevation was also observed at pre-exercise in the HW trial. BAP, d-ROM, and the BAP/d-ROM ratio tended to decline at 30 min after the exercise, except BAP and BAP/d-ROMs in the placebo trial. These results demonstrate that the marked elevation of oxidative stress and anitioxidative functions occurred simultaneously in the intensively exercised horses, and suggest a possibility that HW has some antioxidative efficacy.

Breed Differences in Dopamine Receptor D4 Gene (DRD4) in Horses.

Genetic polymorphisms in genes related to neurotransmitters or hormones affect personality or behavioral traits in many animal species including humans. In domestic animals, the allele frequency of such genes has been reported to be different among breeds and it may account for breed differences in behavior. In this study, we investigated breed differences in horses in the dopamine receptor D4 gene (DRD4), which has been reported to affect horse personality. We collected samples from seven horse breeds including those native to Japan and Korea, and compared the sequence of the DRD4 exon3 region among these breeds. We found that there were two types of polymorphisms (VNTR and SNPs) in the exon3 region, and some of them seemed to be breed-specific. In addition, we found that the allele frequency of G292A, reported to be associated with horse personality, differed greatly between native Japanese horses and Thoroughbred horses. The frequency of the A allele which is associated with low curiosity and high vigilance, was much lower in native Japanese horses (Hokkaido, 0.03; Taishu, 0.08) than in Thoroughbreds (0.62). This difference may account for breed differences in personality or behavioral traits. Further studies of the function of these polymorphisms and their effect on behavior are indicated.

Epizootology and experimental infection of Yokose virus in bats.

To reveal whether bats serve as an amplifying host for Yokose virus (YOKV), we conducted a serological survey and experimentally infected fruit bats with YOKV isolated from microbats in Japan. YOKV belongs to the Entebbe bat virus group of vector unknown group within the genus Flavivirus and family Flaviviridae. To detect antibodies against YOKV, we developed an enzyme-linked immunosorbent assay (ELISA) using biotinylated anti-bat IgG rabbit sera. Serological surveillance was conducted with samples collected in the Philippines and the sera supplied from Malaysia. One of the 36 samples from the Philippines (2.7%) and 5 of the 26 samples from Malaysia (19%) had detectable ELISA antibodies. In the experimental infections, no clinical signs of disease were observed. Moreover, no significant viral genome amplification was detected. These findings revealed that YOKV replicates poorly in the fruit bat, suggesting that fruit bats do not seem to serve as an amplifying host for YOKV.

Incorporation of beta-amyloid protein through the bovine ileal epithelium before and after weaning: model for orally transmitted amyloidoses.

To determine the mechanism of bovine intestinal incorporation of the pathogen, and the pathogenesis of prion protein in the early stage, cows suckling and weaning were orally given a fusion protein of Abeta-EGFP. Abeta-EGFP was incorporated through the villous columnar epithelial cells and accumulated in crypt patches in the ileum of suckling cows. The sites of the uptake and accumulation of Abeta-EGFP are very close to the peripheral nervous system; however, such uptake of Abeta-EGFP was not observed in 6-month-old post-weaning cows. The present study, therefore, suggests that the weaning period is very important for the risk of transmission.

Production of a transgenic pig expressing human albumin and enhanced green fluorescent protein.

We introduced a fusion gene of human albumin and enhanced green fluorescent protein (EGFP) into porcine oocytes using the sperm vector method, and produced a piglet that showed clear expression of GFP in the hooves and skin. PCR and Southern blotting analysis of genomic DNA extracted from the piglet's tissues, including the liver, showed that the tissues carried the transgene. RT-PCR analysis demonstrated that both the human albumin and EGFP genes were expressed in the tissues. The fact that human albumin gene was integrated and expressed in the liver of the transgenic pig opened a way for us to achieve our goal, which was the use of transgenic pigs for the bioartificial liver support system.