Katsuro Hagiwara

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.

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.

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.

Long-term shedding of hepatitis E virus in the feces of pigs infected naturally, born to sows with and without maternal antibodies.

Pigs are presumed reservoirs for hepatitis E virus (HEV) transmission to humans. To examine infection kinetics, two litters of domestic pigs (A and B, each containing 10 piglets) infected naturally with HEV were studied until pigs were 6 months old. Maternal IgG and IgA antibodies were detected in litter A piglets, but not in litter B ones. All pigs shed HEV in feces when they were 30–110 days old, and 17 developed viremia at 40–100 days of age. Phylogenetic analysis revealed a highly close sequence of HEV genotype 3 in all pigs. The serum levels of specific IgG and IgA were similar in all pigs, although IgA was not detected in the feces. Interestingly, the onset of both viremia and seroconversion was delayed significantly in litter A pigs. The kinetics of fecal virus shedding was similar in both litters; shedding was not detected after the pigs were 120 days old. The differences in the infection kinetics between litters A and B suggested that maternal antibodies delayed the onset of viremia and seroconversion. Quantitative real‐time reverse transcriptase‐polymerase chain reaction revealed that HEV RNA in feces peaked 10 days after initial shedding of approximately 106.0 copies/g. The viral load was much lower in the serum than in the feces. At 200 days of age, HEV RNA was found in the internal organs of 3 out of 13 pigs. These study findings improve the understanding of the dynamics of natural HEV transmission in pigs, which could help in controlling virus transmission from pigs to humans. J. Med. Virol. 82:69–76, 2010.

Seroprevalence of Borna disease virus in domestic animals in Xinjiang, China

To investigate the animals infected with Borna disease virus (BDV) in Xinjiang, China, we examined for BDV antibodies in the sera from groups of 20 horses, sheep and cattle, and from 165 wild rodents (18 species) by ELISA and immunoblot. The serological study disclosed the presence of antibodies to both BDV-p24 and -p40 in the horses (20%) and sheep (25%), whereas no apparent positive reaction was detected either in cattle or rodents. The results suggested that BDV is prevalent in horses and sheep in the district investigated.

Proinflammatory Cytokines in Bovine Colostrum Potentiate the Mitogenic Response of Peripheral Blood Mononuclear Cells from Newborn Calves through IL-2 and CD25 Expression

Bovine colostrum contains high concentrations of cytokines, and colostral cytokines are considered to be an important factor in stimulation of maturation of the immune system in newborns. In this study, 5 proinflammatory cytokines (IL‐1β, IL‐6, TNF‐α, IFN‐γ and IL‐1 receptor antagonist, IL‐1ra) present in colostrum were tested for their potential to enhance mitogenic response and to elicit expression of IL‐2 mRNA and CD25 in peripheral blood mononuclear cells (PBMC) from newborn calves before being fed colostrum. PBMC were pretreated with each recombinant bovine cytokine for 2 hr before stimulation with concanavalin A (ConA). Pretreatment of PBMC from newborn calves with IL‐1β, TNF‐α or IFN‐γ significantly enhanced the ConA response, whereas IL‐1ra inhibited the response. The degree of enhancement or inhibition of mitogenic response by these cytokines was more pronounced in PBMC from newborn calves than in those from adult cows. Although IL‐2 mRNA expression in ConA‐stimulated PBMC from newborn calves was weaker than that in those from adult cows of ConA‐stimulated controls, the expression levels became comparable after pretreatment with IL‐1β, TNF‐α or IFN‐γ. The CD25 expression in PBMC from newborn calves was also enhanced by pretreatment with IL‐1β, TNF‐α and IFN‐γ. These results suggest that pretreatment of neonatal PBMC with IL‐1β, TNF‐α or IFN‐γ promotes mitogenic response to ConA through up‐regulating the production of IL‐2 and the expression of the mature IL‐2 receptor.

Theileria sergenti proliferates in SCID mice with bovine erythrocyte transfusion

The unavailability of in vitro or in vivo experimental systems has been the major factor hampering the progress of research studies on Theileria sergenti, causative agent of theileriosis, a major disease of cattle in Japan. We report the first successful propagation of T. sergenti in SCID mice into which uninfected bovine erythrocytes (Bo-RBC) were supplied periodically. The infectivity of T. sergenti proliferated in an SCID mouse was ascertained by successful transfer of infection into another SCID mouse into which uninfected Bo-RBC were supplied periodically.

Hepatitis E virus in Norway rats (Rattus norvegicus) captured around a pig farm

BackgroundHepatitis E virus (HEV) transmitted via the oral route through the consumption of contaminated water or uncooked or undercooked contaminated meat has been implicated in major outbreaks. Rats may play a critical role in HEV outbreaks, considering their negative effects on environmental hygiene and food sanitation. Although the serological evidence of HEV infection in wild rodents has been reported worldwide, the infectivity and propagation of HEV in wild rats remain unknown. To investigate if rats are a possible carrier of HEV, we studied wild Norway rats (Rattus norvegicus) that were caught near a pig farm, where HEV was prevalent among the pigs.MethodsWe examined 56 Norway rats for HEV. RNA from internal organs was examined for RT-PCR and positive samples were sequenced. Positive tissue samples were incubated with A549 cell line to isolate HEV. Anti-HEV antibodies were detected by ELISA.ResultsSixteen rats were seropositive, and the HEV RNA was detected in 10 of the 56 rats. Sequencing of the partial ORF1 gene from 7 samples resulted in partially sequenced HEV, belonging to genotype 3, which was genetically identical to the HEV prevalent in the swine from the source farm. The infectious HEVs were isolated from the Norway rats by using the human A549 cell line.ConclusionsThere was a relatively high prevalence (17.9%) of the HEV genome in wild Norway rats. The virus was mainly detected in the liver and spleen. The results indicate that these animals might be possible carrier of swine HEV in endemic regions. The HEV contamination risk due to rats needs to be examined in human habitats.

Extent of hepatitis E virus elimination is affected by stabilizers present in plasma products and pore size of nanofilters

Background and Objective  To investigate the physico‐chemical properties of hepatitis E virus (HEV) with regard to inactivation/removal, we have studied four isolates with respect to sensitivity to heat during liquid/dry‐heating as well as removal by nanofiltration.

Experimental vertical transmission of Borna disease virus in the mouse.

Summary. 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 105 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.