Yu Minagawa

Expression and one-step purification of bovine interleukin-21 (IL-21) in silkworms using a hybrid baculovirus expression system.

A hybrid baculovirus, a hybrid of the Autographa californica nuclear polyhedrosis virus and the Bombyx mori nuclear polyhedrosis virus, was used for the large-scale production of bovine interleukin-21 (IL-21) in silkworms. A recombinant hybrid baculovirus containing the full length of the cDNA of bovine interleukin-21 was constructed and used to infect silkworm larvae or silkmoth pupae. After the infection of the virus, bovine mature IL-21 was produced in the haemolymph or pupal cell lysates. A one-step purification of bovine mature IL-21 from haemolymph using a cation exchange column gave 0.5 mg. IL-21 from 30 ml haemolymph. The bovine IL-21 produced by silkworms strongly induced NK cell proliferation using a human NK cell-line, NK0, and enhanced the lymphokine activated killer (LAK) activity of bovine peripheral blood mononuclear cells.

Interleukin-18 expression in pig salivary glands and salivary content changes during acute immobilization stress.

Interleukin-18 (IL-18) has recently been considered a promising marker of stress responses. In this study, to evaluate IL-18 as a noninvasive stress marker in pigs, we investigated the expression of IL-18 in porcine salivary glands and its presence in saliva, and its dynamics during acute immobilization stress in pigs. IL-18 mRNA was detected robustly in the pig salivary glands by RT-PCR. Immunohistochemical staining of IL-18 protein expression revealed that the expression patterns differed among the three types of salivary glands (parotid, submandibular, and sublingual gland). IL-18 was also detected in pig saliva by ELISA, and a diurnal rhythm with a peak in the afternoon was observed. The IL-18 concentration in saliva was significantly increased during a 60-min acute immobilization stress in thirteen 5-month-old pigs. These results are the first evidence of a stress-related change of IL-18 in pig saliva. Salivary IL-18 may thus become a useful noninvasive marker for the evaluation of acute stress in pigs.

Immunostimulatory Effects of Recombinant Erysipelothrix rhusiopathiae Expressing Porcine Interleukin-18 in Mice and Pigs

ABSTRACT Interleukin-18 (IL-18), which was originally called gamma interferon (IFN-γ)-inducing factor, has been shown to play an important role in innate and acquired immune responses. In this study, attenuated Erysipelothrix rhusiopathiae strains were engineered to produce porcine IL-18 (poIL-18) and evaluated for their potential immunostimulatory effect in animals. Recombinant poIL-18 was successfully expressed in the recombinant E. rhusiopathiae strains YS-1/IL-18 and KO/IL-18. The culture supernatant of YS-1/IL-18 was confirmed to induce IFN-γ production in murine splenocytes in vitro, and this production was inhibited by incubation with anti-poIL-18 monoclonal antibodies. Furthermore, more IFN-γ production was induced upon stimulation of splenocytes with concanavalin A for splenocytes from mice that were intraperitoneally inoculated with YS-1/IL-18 than for splenocytes from control mice inoculated with the parent strain YS-1. Peritoneal macrophages from mice preinoculated with YS-1/IL-18 exhibited enhanced phagocytosis of Salmonella enterica subsp. enterica serovar Typhimurium compared with peritoneal macrophages from control mice preinoculated with YS-1. We also confirmed the immunostimulatory effect on humoral immune responses against antigens of E. rhusiopathiae and Mycoplasma hyopneumoniae in gnotobiotic pigs that were orally preinoculated with KO/IL-18. Thus, these results provide evidence that E. rhusiopathiae is a promising vector for the expression of host cytokines and suggest the potential utility of E. rhusiopathiae vector-encoded cytokines in the activation of host innate and acquired immune responses.

Allele-specific primer polymerase chain reaction for a single nucleotide polymorphism (C1205T) of swine toll-like receptor 5 and comparison of the allelic frequency among several pig breeds in Japan and the Czech Republic.

In the present study, an allele‐specific primer‐polymerase chain reaction (ASP‐PCR) for genotyping a single nucleotide polymorphism (SNP) of swine Toll‐like receptor 5 (TLR5) (C1205T; P402L) that is related to the impaired recognition of Salmonella enterica serovar Choleraesuis (SC) was developed. The allele frequencies in several pig breeds in Japan and the Czech Republic were also compared. The swine TLR5 C1205T mutation was successfully determined by ASP‐PCR using genomic DNA samples in Japan that had previously been genotyped by a sequencing method. Using the PCR condition determined, genomic DNA samples from blood obtained from 110 pigs from seven different breeds in the Czech Republic were genotyped by the ASP‐PCR. The genotyping results from the ASP‐PCR completely matched the results from the sequencing method. The allele frequency of the swine TLR5 C1205T mutation was 27.5% in the Landrace breed of the Czech Republic compared with 50.0% in Japanese Landrace. In Japan, the C1205T mutation was found only in the Landrace breed, whereas in the Czech Republic it was found in both the Landrace and Piétrain breeds. These results indicate the usefulness of ASP‐PCR for detecting a specific SNP for swine TLR5 affecting ligand recognition. They also suggest the possibility of genetically improving pigs to enhance their resistance against SC infection by eliminating or selecting this specific SNP of swine TLR5.

Evaluation of the Environmental Factors, Stress Markers and Economical Losses in a Swine Farm with High Mortality Rate

茨城県内の1養豚農家においては，呼吸器病や下痢による高い死亡率による損耗が大きな問題となっていた。当農家では，木造開放式の比較的環境の良い離乳期育成舎（以下育成舎A）と，糞尿処理能力が十分でないコンクリート製の離乳期育成舎（以下育成舎B）を有していた。今回当該農家の育成豚の追跡調査を行い，異なる育成舎の空気環境やそこで飼養される育成豚にかかるストレスや生産性について調査を行い，得られた結果より死亡事故による経済的損失の算出を試みた。