Competitive enzyme-linked immunosorbent assay for the detection of the antibodies specific to akabane virus.

A competitive enzyme-linked immunosorbent assay (C-ELISA) using neutralizing monoclonal antibodies (MAbs) against Akabane virus (AKAV) was developed to detect antibodies to AKAV in cattle sera. The performance of the test using 7 different competitor MAbs was evaluated in sequential serum samples and sera from cattle infected with various bovine arboviruses. The dynamics of the antibody response expressed by percentage of inhibition (PI) in C-ELISA coincided with those of neutralizing antibody titers in sequential serum samples from 2 cattle experimentally infected with AKAV. The value of PI in C-ELISA for convalescent sera from cattle infected with arboviruses correlated with the neutralizing antibody titer to AKAV but was unaffected by the antibodies to other arboviruses. In the validation experiment of C-ELISA using 286 bovine sera previously examined for the AKAV antibody by serum neutralization (SN) test, the relative specificity of C-ELISA was more than 98%, whereas the relative sensitivities of individual MAbs ranged from 49% to 82.2%. Overall agreement between C-ELISA and the SN test varied from 72% to 90% depending on the MAb. These results suggest that the C-ELISA is acceptable as a rapid and specific method for detecting antibodies to AKAV and is a potential alternative to the SN test.     

Relationship between transportation stress and polymorphonuclear cell functions of bottlenose dolphins, Tursiops truncatus

Dolphins in a captive environment are exposed to various kinds of stresses. Handling and transportation are stressful events for terrestrial mammals, and such stress may affect immune system function and increase susceptibility to infectious diseases. The same phenomenon could occur in dolphins, however, few studies have reported this in dolphins. The objective of this study was to evaluate the relationship between stress and polymorphonuclear (PMN) cell function of dolphins during transportation. Four bottlenose dolphins (Tursiops truncatus) were transported for 6 hr by truck. Serum cortisol levels, leukograms, phagocytosis, and superoxide production of PMN cells were evaluated during handling and transportation compared to resting values. The mean serum cortisol level was significantly increased during handling and transportation (p<0.05) when compared with the resting values. White blood cell (WBC) counts, eosinophil counts, phagocytosis, and superoxide production of PMN cells during handling and transportation stages decreased significantly in comparison with the resting stage (p<0.05). The concentration of serum cortisol was significantly correlated with the results of the WBC counts, eosinophil counts, superoxide production, and phagocytosis (p<0.01, p<0.05, p<0.05, and p<0.001, respectively). The present results indicate that handling and transportation are stressful events for dolphins and could affect their PMN cell functions, thereby leading to the impairment of the immune system.     

Colonic mast cell infiltration in rats with TNBS-induced visceral hypersensitivity

Colonic mucosal mast cells are implicated in the pathogenesis of visceral hypersensitivity associated with irritable bowel syndromes. This study was designed to investigate the roles of mucosal mast cells in development of an experimental visceral hypersensitivity induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS) in rats. TNBS, when injected into the proximal colon through laparotomy, produced a significant decrease in pain threshold of the distal colon to mechanical distention, indicating a visceral hypersensitivity. In the proximal colon that was directly insulted by TNBS, mucosal necrosis and extensive inflammatory cell infiltration were observed with concomitant increase in tissue myeloperoxide (MPO) activity. In the distal colon where distention stimuli were applied, the number of mucosal mast cells significantly increased following TNBS treatment, although neither mucosal injury nor increase in tissue MPO activity was observed. In an organ culture, spontaneous release of a mucosal mast cell-specific protease (RMCP-2) from the distal colon tissue of TNBS-treated rats was significantly larger than that of sham animals. Furthermore, TNBS-induced visceral hypersensitivity was significantly suppressed by subcutaneous pretreatment with a mast cell stabilizer doxantrazole in a dose-dependent manner. These findings suggest that prominent colonic mast cell infiltration associated with an enhanced spontaneous mediator release is responsible, at least partly, for development of visceral hypersensitivity induced by TNBS in rats.     

Comparison of dendritic cell-mediated immune responses among canine malignant cells

Dendritic cell (DC) vaccination is one of the most attractive immunotherapies for malignancies in dogs. To examine the differences in DC-mediated immune responses from different types of malignancies in dogs, we vaccinated dogs using autologous DCs pulsed with keyhole limpet hemocyanin (KLH) and cell lysate prepared from squamous cell carcinoma SCC2/88 (SCC-KLH-DC), histiocytic sarcoma CHS-5 (CHS-KLH-DC), or B cell leukemia GL-1 (GL-KLH-DC) in vitro. In vivo inductions of immune responses against these tumor cells were compared by the delayed-type hypersensitivity (DTH) skin test. The DTH response against SCC2/88 cells were observed in dogs vaccinated with autologous SCC-KLH-DC, while the response was undetectable against CHS-5 and GL-1 cells in dogs vaccinated with autologous CHS-KLH-DC and GL-KLH-DC. Skin biopsies taken from DTH challenge sites were then examined for immunohistochemistry, and recruitment of CD8 and CD4 T cells was detected at the site where SCC2/88 cells were inoculated in dogs vaccinated with SCC-KLH-DC. By contrast, neither CD8 nor CD4 T cell infiltration was found at the DTH challenge site in the dogs vaccinated with CHS-KLH-DC or GL-KLH-DC. These findings may reflect that the efficacy of immune induction by DC vaccination varies among tumor types and that immune responses could be inducible in squamous cell carcinoma. Our results encouraged further investigation of therapeutic vaccination for dogs with advanced squamous cell carcinoma in clinical trials.     

Prevalence of tick-borne Rickettsia and Ehrlichia in Ixodes persulcatus and Ixodes ovatus in Tokachi district, Eastern Hokkaido, Japan

DNA from 111 ticks collected by flagging in Tokachi district, Eastern Hokkaido, Japan were examined for infection with Rickettsia and Ehrlichia, by PCR and sequencing methodology. For Rickettsia, analysis of the partial sequence of the citrate synthase gene was successfully performed on 11 DNA samples from I. persulcatus, and 7 of them showed 99.8% identical with Rickettsia helvetica while the other 4 showed 99.8% identical with ;Candidatus Rickettsia tarasevichiae'. For Ehrlichia, a partial sequence of the 16S rRNA gene detected from I. persulcatus was 100% identical with that from Ehrlichia muris, and another DNA sample from I. ovatus showed 99.8% identical with Ehrlichia species detected from I. ovatus. The results suggest that the pathogens detected here might be distributed in this area.     

Establishment of testicular and ovarian cell lines from Honmoroko (Gnathopogon caerulescens)

We succeeded to establish cell lines from endemic fish species Honmoroko Gnathopogon caerulescens, which inhabits Lake Biwa, the third oldest lake in the world. Two cell lines designated as RMT1 and RMO1 were established from testis and ovary of G. caerulescens, respectively. These cell lines were initially cultured in Leibovitz’s L-15 medium supplemented with fetal bovine serum (FBS), fish embryo extract, epidermal growth factor, and basic fibroblast growth factor. Further addition of forskolin and β-mercaptoethanol was required to establish and maintain these cell lines for more than 60 passages. RMT1 and RMO1 cells showed fibroblast- and epithelial-like morphology, respectively. From immunocytochemical staining and gene expression patterns, RMT1 cells showed a characteristic of testicular Sertoli cells and RMO1 cells did that of ovarian theca cells. Both RMT1 and RMO1 cells multiplied well in the medium supplemented with 10 % FBS at 28 °C and their minimum population doubling times were 24.4 and 28.8 h, respectively. At the 45th passage, most of the RMT1 and RMO1 cells had a hyperploid set of chromosomes (67.3 and 96.1 %, respectively). Cells with normal diploid chromosome set were not observed. RMT1 cells were transfected with an enhanced green fluorescent protein (EGFP) expression vector and human elongation factor 1 α promoter worked efficiently to express EGFP. In addition, EGFP-expressing cell lines were also established, suggesting that the cell lines could be utilized as an in vitro monitor system (biosensor) for the evaluation of endocrine disruptors which might affect gonadal function.     

Heparin inhibits plaque formation by cell-free Marek's disease viruses in vitro

The mechanisms of Marek's disease virus (MDV) entry to host cells have not yet been analyzed. Heparan sulfate (HS) on the cell surface serves as a receptor for several herpesviruses in mammalian species. In this study, we demonstrated that plaque formation by cell-free MDV is inhibited by the addition of soluble heparin to the cell culture. Moreover, pretreatment of susceptible cells, chicken embryo fibroblasts, with heparinase, partially reduced infectivity of the cell-free MDV. From these results, it was suggested that the MDV entry, at least in the case of cell-free MDV, is dependent on the presence of cell surface glycosaminoglycans, principally HS.     

Bovine leukaemia virus envelope peptides cause immunomodulation in BALB/c mice

Immunomodulatory activity of two bovine leukaemia virus envelope (BLVEnv) derived peptides were examined in BALB/c mice. One is peptide homologous to CKS-17 which is known as a 17-amino acid peptide derived from p15E of feline leukaemia virus (CKS-17/BLV), and the other is an 18-amino acid synthetic peptide of BLV Env 61-78 (pep61). Priming with CKS-17/BLV in vitro, as well as CKS-17, significantly suppressed the mitogen-induced proliferative responses of spleen cells in naive BALB/c mice. In addition, priming of spleen cells with pep61 in vitro and in vivo resulted in suppression of lipopolysaccaride-induced B-cell proliferative response. This suppression was partially due to the basic amino acid sequence in the peptide because if the pep61-derived peptide lacking Arg was used, this inhibitory activity was partially restored. In contrast, pep61 enhanced both concanavalin A-stimulated proliferative response and IL-2 production. These findings showed that pep61 may contribute to the modification of the host immune responses in the course of BLV infection.     

Development of a PCR test for the identification of Staphylococcus intermedius based on the 16S rDNA sequence

The development of a PCR assay based on the 16S ribosomal RNA gene (rDNA) sequence was carried out for the identification of Staphylococcus intermedius. Sixty-six strains of S. intermedius, 70 of Staphylococcus aureus and 2 of Staphylococcus hyicus were examined for the assay. The 16S rDNA, of which the PCR target fragment makes up 901 bp corresponding to the sequence data of the gene, was detected in all strains of S. intermedius, but it was not detected in any strains of either S. aureus or S. hyicus. These results suggest that the PCR allows a simple and precise identification of S. intermedius.