Sequence comparison of the ORF 7 region of transmissible gastroenteritis viruses isolated in Japan

The 3' end region nucleotide sequence, including ORF7, of nine Japanese and two U.S.A. isolates of transmissible gastroenteritis virus (TGEV) were determined and compared. Nine Japanese TGEV strains have been isolated over the past 40 years (1956-1997). From the comparison of determined nucleotide sequences, we could divide the TGEV Japanese isolates into two groups and distinguish them from TGEV U.S.A. isolates.     

Efficiency of fecal steroid hormone measurement for assessing reproductive function in the Hokkaido brown bear (Ursus arctos yesoensis)

The present study aimed to establish simple systems for measuring fecal steroid hormones in order to monitor the reproductive profiles of captive Hokkaido brown bears. The efficiency of fecal sample processing at the steps of dehydration and extraction and the correlation between steroid concentrations in matched fecal and blood samples were studied. Then, monthly changes in fecal estradiol-17 beta and progesterone in female bears, and testosterone in male bears were examined. The procedure was finalized as follows. Fecal samples were dried at 100 degrees C for 3 hr and extracted with diethyl ether. The diethyl ether in the extracts was evaporated and residues were reconstituted in ethanol for the assays. Hormone concentrations were quantified using enzyme immunoassays. Concentrations of progesterone and testosterone in fecal and plasma samples were correlated in the systems. The changes in fecal progesterone and testosterone concentrations were similar to those in serum concentrations of bears as reported previously. In contrast, fecal estradiol concentrations did not correlate with plasma levels probably because of the time lag in excretion. However, the changes in estradiol-17 beta concentrations in feces in the present study were similar to those reported in serum. In conclusion, fecal progesterone and testosterone assay systems appear practical for monitoring ovarian and testicular activities without immobilization, though methodological improvements and further validation may be required. For the fecal estradiol-17 beta assay, there is a need to solve the problem of excretion time lag before the system can be used in the study of reproductive physiology.     

Immunological characterization of peripheral blood leukocytes using vaccine for mycoplasmal pneumonia of swine (MPS) in swine line selected for resistance to MPS

This study was conducted to evaluate immunological changes in peripheral blood leukocytes in pigs that were genetically selected for their improved resistance to mycoplasmal pneumonia of swine (MPS), using MPS vaccine as an antigen. Twelve castrated MPS‐selected Landrace pigs were compared with the same number of pigs from a nonselected line by using a time‐course analysis at the hematological level. After the second sensitization with MPS vaccine, the percentages of B cells, CD4⁺ T cells, and natural killer (NK) cells in total leukocytes were lower in the selected line than in the nonselected line, whereas the percentage of granulocytes in total leukocytes increased in the MPS‐selected line. We also assessed the proliferative ability of peripheral blood mononuclear cells (PBMCs) stimulated with Mycoplasma hyopneumoniae, lipopolysaccharide or concanavalin A, and found that although the proliferative ability of the PBMC was not different between the two lines at a steady state, the nonselected line showed a significantly higher proliferative ability after sensitization with MPS vaccine than the selected line regardless of antigens used. These results thus indicate that the selection of pigs on the basis of MPS resistance changes their immunophenotype, and would give us beneficial information for the prevention of MPS infection.     

Methicillin‐resistant Staphylococcus aureus keratitis in a dog

The purpose of this study is to report a case of methicillin‐resistant Staphylococcus aureus (MRSA) keratitis in a dog. A 7‐year‐old intact male American cocker spaniel that had undergone removal of a nictitating gland was referred for severe ulcerative keratitis. Slit‐lamp examination showed swelling of the eyelid, mucopurulent discharge, conjunctival injection and chemosis, diffuse corneal edema and opacity, and a deep ulcer in central cornea. Gram staining of discharge from the eye demonstrated Gram‐positive cocci. Despite topical ofloxacin, oxytetracycline and polymyxin B ophthalmic solution and intravenous cefazolin, there was no improvement. Cultures revealed MRSA that was sensitive only to chloramphenicol, vancomycin, lincomycin, and clindamycin. The antibiotic regimen was changed to topical and systemic chloramphenicol. After 9 days of treatment, although inflammation started to be resolved, the dog developed nonregenerative anemia. The antimicrobial regimen was changed again to topical and systemic vancomycin. Inflammation continued to improve over the next week. MRSA should be considered a potential organism in infectious keratitis, especially when general antibiotics are not effective. Although topical and systemic chloramphenicol and/or vancomycin are effective for treating MRSA keratitis, vancomycin should only be used when culture and susceptibility results indicate it is appropriate and no other options are available. To our knowledge, this is the first detailed case report of MRSA keratitis in a dog.     

Characterization of Th1 activation by Bartonella henselae stimulation in BALB/c mice: Inhibitory activities of interleukin-10 for the production of interferon-gamma in spleen cells

This study was conducted to analyze cytokine production mechanisms in mice after Bartonella henselae stimulation. BALB/c mice were inoculated intraperitoneally with 3 x 10(6) colony forming units of B. henselae (Houston-1 strain) twice at 10-day interval. Spleen cells were harvested from the mice and stimulated with the organisms. Following the stimulation, interferon-gamma (IFN-gamma) and interleukin-4 (IL-4), IL-10, IL-12 and tumor necrosis factor-alpha (TNF-alpha) were measured in the culture supernatants of the spleen cells by ELISA. The spleen cells specifically secreted IFN-gamma, but not IL-4, indicating that T helper 1 (Th1) cells were activated following B. henselae stimulation. In addition, IL-10 and TNF-alpha productions were also detected in the culture supernatants of spleen cells. Neutralization of IL-10 in the culture supernatants significantly enhanced the production of IFN-gamma from the spleen cells stimulated with B. henselae. These results indicate that B. henselae predominantly stimulated Th1 cells and resulted in secreting IFN-gamma, however the production was partially inhibited by IL-10, which was produced simultaneously.     

The Enhancing Effects of Hyperbaric Oxygen on Mouse Skin Carcinogenesis

The effects of hyperbaric oxygen (HBO) on mouse skin two-stage chemical carcinogenesis were examined. Six-week-old inbred CD-1 female mice were divided into the following five groups: group 1, normoxia and application of 25 nmol 7,12-dimethylbenz[a]anthracene (DMBA) and 8.5 nmol 12-O-tetradecanoylphorbol-13-acetate (TPA) (n=19); group 2, HBO and DMBA/TPA (n=21); group 3, HBO and DMBA/acetone (n=3); group 4, normoxia and acetone (n=3); and group 5, non-treatment group (n=5). HBO was started at the same time as DMBA. Mice were euthanized at 23 weeks after the start of the experiment. Mice in group 2 showed the occurrence of tumors at 8 weeks after the beginning of the experiment, while the occurrence of tumors in mice in group 1 was observed beginning at 9 weeks. There was a difference in occurrence among low-grade papillomas, high-grade papillomas and SCCs in both groups 1 and 2 by the χ²-test at end of the experiment (p<0.05). The Ki-67 labeling indices of tumors revealed that the percentages of positive cells in low-grade papillomas in groups 1 and 2 were 15.27 ± 2.54% and 29.67 ± 2.82%, respectively (p<0.01). The results suggested that the tumors in group 2, which was treated with HBO, were more progressive than those in group 1, which was not treated with HBO. In this study, HBO accelerated tumor cell proliferation and advanced tumor progression in skin carcinogenesis by DMBA/TPA.     

Autophagy-dependent viral recognition by plasmacytoid dendritic cells

Plasmacytoid dendritic cells (pDCs) detect viruses in the acidified endosomes by means of Toll-like receptors (TLRs). Yet, pDC responses to certain single-stranded RNA (ssRNA) viruses occur only after live viral infection. We present evidence here that the recognition of such viruses by TLR7 requires transport of cytosolic viral replication intermediates into the lysosome by the process of autophagy. In addition, autophagy was found to be required for the production of interferon-alpha by pDCs. These results support a key role for autophagy in mediating ssRNA virus detection and interferon-alpha secretion by pDCs and suggest that cytosolic replication intermediates of viruses serve as pathogen signatures recognized by TLR7.