Rikio Kirisawa

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 and identification of equine herpesvirus-1 and -4 by polymerase chain reaction

A rapid method for detection and identification of equine herpesvirus-1 and -4 (EHV-1 and EHV-4) was developed using polymerase chain reaction (PCR). Primers for PCR were designed from aligned nucleotide sequences of glycoprotein B genes of EHV-1 and EHV-4 to amplify specific regions for EHV-1 or EHV-4 or a common region of both viruses. By using type specific primer mixture, amplified fragments were identified as EHV-1 or EHV-4 in a one-step reaction. We have applied this technique on specimens from aborted fetuses. The samples contained only EHV-1 and there was complete accordance between the results of PCR and virus isolation. Our PCR system could differentiate the two virus types rapidly in a one-step reaction.

Similarities and dissimilarities in the structure and expression of viral genomes of various virus strains immunologically related to Marek's disease virus.

SummaryVarious strains immunologically related to Mareks disease virus (MDV) have been subdivided into three serotypes: serotype 1, pathogenic strains of MDV and attenuated or apathogenic variants derived from them; serotype 2, naturally occuring apathogenic strains of MDV; serotype 3, herpesvirus of turkey (HVT). The viral genome structures of these three serotypes were compared by a simple, practical method using total DNA extracted from virus-infected cells instead of viral DNA purified from virions. The restriction endonuclease-cleavage patterns of serotype 2 viral DNA were found to differ from those of either serotype 1 MDV or serotype 3. Under stringent conditions, no significant DNA homology was detected among the three serotype viruses, except in a restricted portion of these viral genomes. Northern blot hybridization experiments suggested that virus-specific polyadenylated RNA of about 2.4 kilobases was transcribed from a restricted portion showing close homology in these viruses. Southern blot hybridization under less stringent conditions revealed that regions with weak homology were distributed over most of the viral genomes of the three serotypes. Two types of virus-specific glycoproteins, gA and gB, were identified in the immunoprecipitates of the culture medium and cell lysates, respectively, of serotype 2-infected cultures with monoclonal antibodies or hyperimmune antisera cross reactive with serotype 1 MDV and serotype 3 HVT, and detected on the surface of serotype 2-infected cells by the membrane immunofluorescence test. These results indicate a close evolutionary relationship among these three viral serotypes.

Species-independent detection of RNA virus by representational difference analysis using non-ribosomal hexanucleotides for reverse transcription

A method for the isolation of genomic fragments of RNA virus based on cDNA representational difference analysis (cDNA RDA) was developed. cDNA RDA has been applied for the subtraction of poly(A)+ RNAs but not for poly(A)− RNAs, such as RNA virus genomes, owing to the vast quantity of ribosomal RNAs. We constructed primers for inefficient reverse transcription of ribosomal sequences based on the distribution analysis of hexanucleotide patterns in ribosomal RNA. The analysis revealed that distributions of hexanucleotide patterns in ribosomal RNA and virus genome were different. We constructed 96 hexanucleotides (non-ribosomal hexanucleotides) and used them as mixed primers for reverse transcription of cDNA RDA. A synchronous analysis of hexanucleotide patterns in known viral sequences showed that all the known genomic-size viral sequences include non-ribosomal hexanucleotides. In a model experiment, when non-ribosomal hexanucleotides were used as primers, in vitro transcribed plasmid RNA was efficiently reverse transcribed when compared with ribosomal RNA of rat cells. Using non-ribosomal primers, the cDNA fragments of severe acute respiratory syndrome coronavirus and bovine parainfluenza virus 3 were efficiently amplified by subtracting the cDNA amplicons derived from uninfected cells from those that were derived from virus-infected cells. The results suggest that cDNA RDA with non-ribosomal primers can be used for species-independent detection of viruses, including new viruses.

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.

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.

The genomic diversity among equine herpesvirus-1 strains isolated in Japan

SummaryThe DNAs from nine Japanese field isolates of equine herpesvirus-1 (EHV-1) were analyzed by digestion with the restriction endonuclease Bam HI and Southern hybridization. Comparing restriction profiles among the EHV-1 strains, there was no considerable difference between isolates before and after vaccine application, but some minor variations in the mobility ofBam HI fragments were observed. To identify these variable fragments, all genomic DNA sequences of the Japanese prototype of EHV-1 have been cloned asBam HI restriction fragments into the plasmid pUC-18. Physical maps of the virus DNA were constructed by a combination of Southern blot analysis and double enzyme digestion of the cloned fragments. By using these cloned fragments as probes in Southern blot analysis, the areas of heterogeneity observed among the field EHV-1 isolates were located in both terminals of UL, the center of UL, IR, US and TR regions of the genome.

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.

Borna disease in a heifer in Japan

(IUdR) and chloroform, as well as by electron microscopy, as described by Chang and others (2001). Results from the chloroform and IUdR treatment indicated the threadfin isolate to be a non-enveloped RNA virus. The grouper isolate was found to be an enveloped DNA virus. Electron microscopy of tissue culture fluid revealed that the threadfin isolate was an icosahedral virus approximately 70 to 80 nm in size, while the grouper isolate was an icosahedral virus approximately 180 nm in size. On the basis of size, structure and CPE, it was suggested that the threadfin isolate was a reo-like virus, while the grouper isolate was an irido-like virus. The pathology and clinical syndrome observed in the grouper corroborates with that described by Inouye and others (1992), Anderson (1993) and Chou and others (1998), in other iridovirus outbreaks in groupers. It is likely that the mortalities in the groupers were caused by this virus and it is possible that the virus might have been imported with the fish from Taiwan. Moraxella species and Streptococcus species have been associated with mortalities in farmed fish and were the most likely cause of the sudden mortalities in the threadfin. Although reoviruses have been implicated in previous outbreaks of fatal disease (Wolf 1988), the role of the virus in the pathogenesis of the current outbreak is uncertain. Further studies are underway to characterise these viruses and to study their contribution to disease outbreaks in farmed marine fish.