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.     

Cyclopedic protein expression analysis of cultured canine mammary gland adenocarcinoma cells from six tumours

We characterised cultured canine mammary gland adenocarcinoma cells by exhaustive step protein expression analysis to identify factors associated with tumour progression or metastasis of canine mammary gland tumour. Cultured adenocarcinoma cells derived from a total of 3 primary and 3 metastatic lesions from 3 dogs (CHMp/m, CIPp/m and CNMp/m, where CHM, CIP, and CNM indicate the 3 animals) were used in this study. The expression of 24 proteins reported to be related to tumourigenesis or malignancy of human breast cancers were examined by Western blot analysis using 24 antibodies. The expression of sialyl Lewis X [sLe(x)] was only observed in CHMm cells, which were derived from pleural effusion. This expression was further confirmed by immunohistochemistry. The levels of some factors, such as 14-3-3sigma, cyclinD1 and Rb, differed among cells or between the primary and metastatic cells in the pair. Though the difference in their expression was not consistent within the cells from primary and metastatic origin, this characterisation should provide useful information for further molecular analysis of these cultured cells. Since some of the factors, such as sLe(x), 14-3-3sigma, cyclinD1 and Rb, showed different levels of expression in the pair, these cultured cells might be meaningful tools for clarification of distant metastasis in canine mammary gland tumours.     

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.     

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.     

Utilization of biochar impregnated with anaerobically digested slurry as slow‐release fertilizer

We examined the possibility of an environment‐friendly slow‐release fertilizer (SRF) made of biochar impregnated by anaerobically digested slurry. The biochar materials were produced from three types of feedstocks (orange peel, residual wood, water‐treatment sludge) at different temperatures of 300°C, 500°C, and 700°C via pyrolysis. The release behaviors of the water‐soluble K⁺, Ca²⁺, and Mg²⁺ were similar for all impregnated biochars and the commercial SRF used. The water‐retention capacity was greatly improved by mixing the biochar‐SRF with the soil. The yield of lettuce was lower for the biochar‐SRF applications of 3.7 to 34.2 t ha^–1 than for the commercial SRF application of 51.4 t ha^–1. This might be due to excessive increase of soil pH for the biochar‐SRF application. Based on these results, the authors concluded that the biochar impregnated with nutrients could become an effective slow‐release K⁺ fertilizer.     

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.     

Cell cycle analysis of bovine cultured somatic cells by flow cytometry

This study was undertaken to examine the cell cycle characteristics of bovine fetal and adult somatic cells (fetal fibroblasts, adult skin and muscle cells, and cumulus cells) after culture under a variety of conditions; 1) growth to 60-70% confluency (cycling), 2) serum starvation, 3) culture to confluency. Cell -cycle phases were determined by flow cytometry with propidium iodide staining enabling the calculation of percentages of cells in G0 /G1, S and G2 /M. The majority was in G0/G1 regardless of cell type and treatment. Serum-starved or confluent cultures contained higher percentages of cells in G0/G1 (89.5-95.4%; P < 0.05). Percentages of cells in G0/G1 increased as cell size decreased regardless of the cell type and treatment. In the serum-starved and confluent cultures, about 98% of small cells were in G0/G1 . Serum-starved cultures contained higher percentages of small cells (38.5-66.9%) than cycling and confluent cultures regardless of cell type (P < 0.05) . After trypsinization of fetal fibroblasts and adult skin cells that were serum-starved and cultured to confluency, the percentages of cells in G0/G1 increased (P < 0.05) on incubation for 1.5 (95.7-99.5%) or 3 hr (95.9-98.6%). These results verify that serum starvation and culture to confluency are efficient means of synchronizing bovine somatic cells in G0/G1, and indicate that a more efficient synchronization of the cells in G0/G1 can be established by incubation for a limited time period after trypsinization of serum-starved or confluent cells.     

Infection of bovine immunodeficiency virus and bovine leukemia virus in water buffalo and cattle populations in Pakistan

A survey of antibodies to bovine immunodeficiency virus (BIV) known as bovine lentivirus and bovine leukemia virus (BLV) was conducted with samples from water buffalo and cattle populations in Pakistan. A total of 370 water buffaloes and 76 cattle were tested, and 10.3% and 15.8%, respectively, were found positive for anti-BIV p26 antibodies determined by Western blotting, while 0.8% of water buffaloes and no cattle were positive for anti-BLV antibodies determined by immunodiffusion test. BIV-seropositive water buffaloes and cattle were found to have BIV proviral DNA in the peripheral blood mononuclear cells determined by nested polymerase chain reaction. This is the first report of BIV infections in water buffaloes.