Isolation of equine herpesvirus-1 lacking glycoprotein C from a dead neonatal foal in Japan.

We isolated a variant equine herpesvirus-1 (EHV-1), strain 5089, from the lung of a dead neonatal foal in Japan and characterized the biological nature of the virus. The virus spread in cultured cells mainly by cell-to-cell infection, unlike wild-type EHV-1, which spreads efficiently as a cell-free virus. The virus titer in cultured supernatant and the intracellular virus titer were low compared to those of wild-type EHV-1. Heparin treatment of the virus had no effect on viral infectivity in cell culture. Glycoprotein C (gC) was not detected by Western blotting and fluorescent antibody tests in 5089 virions and 5089-infected cells, respectively. RT-PCR analysis revealed that the expression level of 5089 gC mRNA was reduced considerably compared to that of wild-type EHV-1. Sequencing analysis of the 5089 gC coding region showed a point mutation in the promoter region of the gC open reading frame. However, the mutation did not affect the promoter activity. These results suggested that the lack of gC in 5089 virions might be one of the reasons for spread of the virus by cell-to-cell infection and that gC mRNA expression might not be activated efficiently due to factors other than the mutation in the gC promoter region.

Experimental vertical transmission of Borna disease virus in the mouse.

We demonstrated the experimental vertical transmission of Borna disease virus (BDV) in pregnant BALB/c mice. Giessen strain He/80 of BDV was used in the present study. Six six-week-old mice were inoculated intraperitoneally with 10(5) 50% tissue culture infective doses (TCID50), and were bred immediately. Four pregnant mice were sacrificed under anaesthesia on the 10th and 14th days after vaginal plug formation. Nine newborns from two maternal mice were sacrificed under anaesthesia on the 7th day after birth. Positive signals with RT-nested PCR techniques for BDV p24-RNAs were seen in the fetuses, placentas and brains of all newborn mice. No immunopositivities for BDV p40 were found in the fetuses or placentas at 10 days' gestation. BDV p40 immunopositivities were found in neurons of the fetal brains and in decidual cells of the placentas at 14 days' gestation. They were also found in neurons of the brains of newborn mice. At 10 days' gestation, no positive signals for BDV p40 sense or antisense riboprobes were seen in the fetal brains or placentas. Positive signals were found in neurons of the fetal brains and decidual cells of the placentas at 14 days' gestation. Positive signals for BDV p40 sense and antisense riboprobes were found in almost all neurons throughout the brains of nine newborn mice. These results suggest that persistent infection with BDV in newborn mice may be induced by vertical transmission during gestation.

Borna disease in a dog in Japan.

Borna disease (BD) was diagnosed in a 3-year-old male Welsh corgi suffering from a severe and acute progressive disorder of the central nervous system. Histopathologically, neuronal lesions were characterized by a non-suppurative encephalomyelitis dominated by large perivascular cuffs consisting of lymphocytes, macrophages and plasma cells; also present were inflammatory cell infiltrates in the neural parenchyma, neuronophagia and focal gliosis. Strong immunolabelling with BD virus (BDV) p40 antibody was diffusely distributed in the cytoplasm of small and large neurons in areas of the brain with and without inflammatory changes, and also in the spinal cord. Positive hybridization signals with BDV p40 sense and antisense riboprobes were seen in the nucleus and cytoplasm of the neurons throughout the whole brain and spinal cord. BDV p24 RNA in formalin-fixed brain tissue was detected by reverse transcriptase (RT)-nested polymerase chain reaction (PCR). BDV p24 RNA-positive signals were detected in the temporal lobe. This is the first report of spontaneous canine BD in Japan.

Interferon-gamma and tumor necrosis factor-alpha levels in sera and whey of cattle with naturally occurring coliform mastitis.

Concentrations of interferon-gamma (lFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) were determined in serum and whey samples from cattle with naturally occurring coliform mastitis for two weeks after onset using bovine INF-gamma and TNF-alpha-specific ELISA. In serum and whey samples from healthy cows. IFN-gamma was almost undetectable and TNF-alpha was detected at low levels. At the onset of illness, INF-gamma in sera and whey and TNF-alpha in whey from the mastitic cows were significantly higher than their respective values in healthy cows. Concentrations of IFN-gamma and TNF-alpha in whey from mastitic cattle decreased significantly as the cows recovered.

Effect of interleukin-1 receptor antagonist on concanavalin A response of peripheral blood mononuclear cells from newborn calves.

The coexistence of interleukin (IL)-1beta with IL-1 receptor antagonist (ra) in bovine colostrum and the possibility of simultaneous transfer of these cytokines to neonates via colostrum have been demonstrated. In the present study, we investigated the effect of IL-1ra on the mitogenic response of calf peripheral blood mononuclear cells (PBMC) stimulated by concanavalin A (ConA), which was mediated by IL-1. Pretreatment of PBMC with recombinant bovine (rb) IL-1ra alone significantly suppressed the proliferation of ConA-stimulated cells. However, in the presence of rbIL-1beta, the suppressive activity of rbIL-1ra was counteracted. These results suggest that coexistence of IL-1ra with IL-1 in colostrum may have no effect on the activation of the neonatal immune system by IL-1beta.

Oral administration of IL-1 beta enhanced the proliferation of lymphocytes and the O(2)(-) production of neutrophil in newborn calf.

Recently, we demonstrated the presence of IL-1 beta in the colostral whey from dairy cows. Here, authors examined oral transmission of colostral IL-1 beta and its immunological effects on the neonatal calves. Biotin-labeled recombinant bovine (rb) IL-1 beta was administered orally to newborn calves and monitored in the serum. The results disclosed the passive transfer of colostral cytokines via the oral route, and a potent increase in white blood cell (WBC) count was observed in all calves administered with rbIL-1 beta. Oral administration of IL-1 beta significantly increased the proliferation of peripheral blood mononuclear cells (PBMCs) stimulated with concanavalin A, and the O(2)(-) production of stimulates neutrophils in newborn calves. These results suggest that the oral administration of IL-1 beta has an immunostimulatory activity in the newborn calf.

Mucosal disease induced in cattle persistently infected with bovine viral diarrhea virus by antigenically different cytopathic virus.

Four cattle persistently infected with non-cytopathic (NCP) bovine viral diarrhea (BVD) virus were challenged with cytopathic (CP) BVD virus that was antigenically different from the persistent virus. Two of the animals were injected with dexamethasone (DM) and then challenged. They developed mucosal disease on days 21 and 33 post-challenge. CP-BVD viruses were isolated from their lymph nodes but not from the sera. The isolates were antigenically different from the persistent virus and the nucleotide sequence of a 787 base region in the E2 gene was markedly different. One of the isolates was indistinguishable from the challenge virus by virus neutralization tests and the nucleotide sequence showed high homology with that of the challenge CP-BVD virus. The other two cattle, challenged with the CP-BVD virus without DM treatment, developed mucosal disease at 30 and 264 days post-inoculation. CP-BVD virus was isolated from the sera as well as the lymph nodes of the cattle and was antigenically and genetically similar to the persistent virus and different from the challenge CP-BVD virus. The present results indicate that cattle persistently infected with NCP-BVD virus can develop mucosal disease induced by antigenically different CP-BVD viruses when their cellular immunity is suppressed, although they are not immunotolerant to the virus.

Concentrations of IL-6 in serum and whey from healthy and mastitic cows.

Inflammatory cytokines, such as interleukin (IL)-6, have been shown to reflect clinical signs in certain conditions in diseased animals. In this study, we quantified the IL-6 concentrations in the serum and milk whey from 94 dairy cows with acute clinical mastitis and 55 healthy lactating cows. The IL-6 concentrations in serum from mastitic cows were significantly higher on the first day of illness compared to those of normal cows. Higher concentrations of IL-6 were also detected in the whey from mastitic cows, whereas low concentrations of IL-6 were detected in both serum and whey samples from normal cows. IL-6 concentrations in the serum taken at the onset of illness from cows that later required euthanasia were significantly higher than those in samples from cows that later recovered. These results suggest that serum IL-6 concentrations may be of prognostic value in identifying cows with severe mastitis.

Detection and characterization of Clostridium species in soil of Zambia.

In the retrospective study of soil-borne diseases of cattle in Zambia, malignant edema and blackquarter were widespread. One hundred and sixty-five cases with malignant edema and 103 cases with blackquarter were reported between 1985 and 1997. It was found that specific soil-conditions associate the emergence of the soil-borne diseases. Soil samples from five areas in Zambia were examined for the presence of genus Clostridium. Direct immunofluorescent assay (IFA) examination showed that C. septicum, C. novyi and C. chauvoei were detected in the soil of specific areas in Zambia, respectively. Causal organisms such as C. perfringens were isolated from the soil samples. The information of area-specific distribution of Clositridium species may give an efficient program in protecting cattle and man.

Detection of cytokines in bovine colostrum.

Colostrum contains factors that are protective for the neonate and may be a source of immunomodulary molecules that positively influence the immune status of the neonate. To confirm that colostrum contains a variety of cytokines with immunomodulatory properties, we established a bovine cytokine specific ELISA and five cytokines (IL-1 beta, IL-6, TNF-alpha, INF-gamma or IL-1 receptor antagonist, IL-1ra) in the whey samples from cows at different stages of lactation were monitored. The expression of cytokine mRNAs (IL-1 beta, IL-6, TNF-alpha and INF-gamma) in the colostral cells was detected by RT-PCR. The concentrations of cytokines in colostrum were significantly higher concentrations than those in the mature milk. A positive correlation was observed between the concentrations of IL-1ra and IL-1 beta in the colostrum samples. In conclusion, colostrum contains high levels of cytokines that could be produced and secreted in the mammary gland and that may have an immunomodulatory activity and influence neonatal immunity.

ELISA for bovine interleukin-1 receptor antagonist and its application to mastitic sera and whey.

A sandwich ELISA for the bovine IL-1 receptor antagonist (bIL-1ra) was developed using recombinant (r) bIL-1ra produced by Escherichia coli, anti-rbIL-1ra rabbit IgG, its biotinylated one and avidin-peroxidase. This ELISA system enabled detection of rbIL-1ra at a concentration of more than 2 ng/ml. This ELISA was applied to quantitation of bIL-1ra in sera and whey of mastitic and healthy cows. The results indicate that although IL-1ra levels in healthy and mastitic sera and whey were comparable, serum IL-1ra/IL-1beta ratio of euthanized cows was significantly lower than that of the recovered.

Detection of Borna disease virus in a pregnant mare and her fetus.

A pregnant mare showing pyrexia, reduced appetite, ataxia and paresis was euthanized and examined for the presence of Borna disease virus (BDV). Her brain, showing multiple neuronal degeneration and necrosis with hemorrhage, and the histologically normal brain of the fetus were both positive for BDV RNA. The BDV nucleotide sequences were identical in the mare and fetus in the second open reading frame (ORF). This is the first report of the possible vertical transmission of BDV in a horse.

Effects of lipopolysaccharide on production of interleukin-1 and interleukin-6 by bovine mammary epithelial cells in vitro.

This investigation was performed to determine the effect of lipopolysaccharide (LPS) on production of interleukin (IL)-1 and IL-6 by bovine mammary epithelial cells in vitro. After confluence, the cells were stimulated with LPS (0.1, 1.0 or 10 micrograms/ml) for 4, 8, 24, and 48 hr. LPS increased production of both IL-1 and IL-6 production from mammary cells in a dose dependent manner. The expression of mRNA for IL-1 receptor antagonist (IL-1ra) was demonstrated by reverse transcription-polymerase chain reaction in bovine mammary epithelial cells.

Enzymatic amplification and expression of bovine interleukin-1 receptor antagonist cDNA.

cDNA generated from lipopolysaccharide-stimulated bovine peripheral blood mononuclear cells was used to amplify and clone the bovine interleukin-1 receptor antagonist (IL-1ra) using primers derived from semi-conserved regions between human and mouse IL-1ra sequences. 5' and 3' terminal sequences of bovine IL-1ra were amplified by 5' and 3' rapid amplification of cDNA ends. The deduced amino acid sequence of bovine IL-1ra demonstrated 80%, 78%, 78%, 77% and 76% homology with human, mouse, rat, rabbit and equine sequences, respectively. Recombinant bovine IL-1ra produced in Escherichia coli suppressed the growth inhibitory activity of bovine IL-1beta on A375 cells in a dose-dependent manner, indicating that the present bovine IL-1ra cDNA encodes biologically active proteins.

Protective effect of passive immunization against TNF-alpha in mice infected with Sendai virus.

TNF-alpha has been reported to be induced in mice infected with Sendai virus. We evaluated the role of TNF-alpha in the virus infection. TNF-alpha was induced locally in proportion to virus titers in the lung. The activity was correlated with suppression of body weight gain. Passive immunization against TNF-alpha improved body weight gain and ameliorated pneumonic lesions in infected mice, and prevented them from lethal infection, but lung virus induced emaciation, pneumonic lesions and death were mediated by TNF-alpha.

Antigenic and genetic diversities of Babesia ovata in persistently infected cattle.

Exploring the antigenic and genetic diversities of Babesia ovata, we obtained several field isolates from grazing cattle in the Okushiri island, Japan. Parasite isolation was greatly facilitated by using bovine red blood cell-substituted SCID mice (Bo-RBC-SCID mice), into which the blood samples of the cattle were inoculated. Isolates from different individuals within a herd of cattle were compared in immunoblot analysis with an anti-B. ovata serum and also in Southern blot analysis with a probe for the small subunit ribosomal RNA gene. In both analyses, the isolates exhibited banding patterns that were significantly different from each other. We were also able to obtain a series of parasite isolates from a single cow in different seasons of a nine months period, including winter when active vector ticks were not in the field environment. Different seasonal isolates showed different banding patterns in both immunoblot and Southern blot analyses. By contrast, these analyses detected little difference among the parasites that had been passed various times in Bo-RBC-SCID mice, where no specific immune responses should be generated. These results indicate that individual animals within a herd of cattle were infected with antigenically and genetically diversified populations of B. ovata, and that the parasites could persistently infect a single animal with dynamic change in their predominant subpopulations.

Abnormal G1 arrest in the cell lines from LEC strain rats after X-irradiation.

The effect of X-irradiation of cell lines from LEC and WKAH strain rats on a progression of cell cycle was investigated. When WKAH rat cells were exposed to 5 Gy of X-rays and their cell cycle distribution was determined by a flow cytometer, the proportion of S-phase cells decreased and that of G2/M-phase cells increased at 8 hr post-irradiation. At 18 and 24 hr post-irradiation, approximately 80% of the cells appeared in the G1 phase. On the contrary, the proportion of S-phase cells increased and that G1-phase cells decreased in LEC rats during 8-24 hr post-irradiation, compared with that at 0 hr post-irradiation. Thus, radiation-induced delay in the progression from the G1 phase to S phase (G1 arrest) was observed in WKAH rat cells but not in LEC rat cells. In the case of WKAH rat cells, the intensities of the bands of p53 protein increased at 1 and 2 hr after X-irradiation at 5 Gy, compared with those of unirradiated cells and at 0 hr post-irradiation. In contrast, the intensities of the bands were faint and did not significantly increase in LEC rat cells during 0-6 hr incubation after X-irradiation. Present results suggested that the radioresistant DNA synthesis in LEC rat cells is thought to be due to the abnormal G1 arrest following X-irradiation.