Do Cattle Choose Preferred Plant Species Without Visual Sense? (Advanced Studies on Sustainable Animal Production: Interrelationships among Human, Animal and Environment, 8th International Symposium of Integrated Field Science)

Poster Sessio

Quantification of Bite Size, Bite Rate and Intake Rate in Cattle Foraging Japanese Native Grasses (Advanced Studies on Sustainable Animal Production: Interrelationships among Human, Animal and Environment, 8th International Symposium of Integrated Field Science)

Poster Sessio

Characterization of an epimastigote-stage-specific hemoglobin receptor of Trypanosoma congolense

Background: Since Trypanosorna spp. lack a complete heme synthesis pathway, the parasites are totally dependent on their host for heme throughout all of the stages of their life -cycle. We herein report the identification and characterization of a T. congolense epimastigote form (EMF)-specific hemoglobin (Hb) receptor. The gene was initially reported to encode a T. congolense haptoglobin (Hp)-Hb complex receptor (TcHpHbR) based on its similarity to a gene encoding a T brucei Hp-Hb complex receptor (TbHpHbR). Methods: Trypanosorna congolense IL3000 was used in this study. A TcHpHbR gene was PCR amplified from the parasite genome. The recombinant protein was used as an immunogen to raise antibodies for immunofluorescence assay and immunoblotting. Hemoglobin uptake by the parasite was examined by using Alexa 488 labelled Hb and visualized by confocal laser scanning microscopy. The qualitative and quantitative interaction between TcHpHbR and its ligand were measured using a surface plasmon resonance assay. Results: We found that, unlike TbHpHbR, TcHpHbR was exclusively expressed in the EMF stage at RNA and protein levels. The recombinant TcHpHbR (rTcHpHbR) was co-precipitated with free-Hb in a GST-pull down assay. Surface plasmon resonance revealed that rTcHpHbR binds free-Hb with high affinity (dissociation constant (K,A) =2.1x10(-8) M) but free-Hp with low affinity (Kd = 2.2x10(-7) M). Furthermore, Alexa 488-labelled-Hb was only taken up by the EMF and co-localized with tomato lectin, which is a marker of endocytic compartments (flagellar pocket and lysosome). Conclusion: We conclude that the T. congolense EMF takes up free-Hb via TcHpHbR, a receptor which is specific to this developmental stage. We therefore propose renaming TcHpHbR as T congolense EMF-specific Hb receptor (TcEpHbR)

腸管出血性大腸菌経口感染マウスに誘導される腸管内特異免疫応答

腸管粘膜感染菌である腸管出血性大腸菌(enterohemorrhagic Escherichia coli、EHEC)O157:H7の経口感染マウスを用い、粘膜免疫応答の誘導機構の解析を行った。特異抗体は、EHEC O157:H7菌体(Whole cell)を抗原とした酵素免疫測定法(Filtration ELISA)を用い、菌体表面に対する抗体価として評価した。EHEC野生株をICRマウスに経胃接種した場合、本菌は盲腸上皮細胞に接着して4週間以上定着し、接種4週後の糞便中にEHEC菌体に対するIgA抗体の著明な上昇がみられた。一方、EHECの上皮細胞への接着に関わる因子の遺伝子を変異させた菌株はマウス腸管への定着性を示さず、糞便中IgA抗体の誘導もみられなかった。変異株は繰り返し接種しても血中抗体は誘導されるものの糞便中IgA抗体は誘導されなかった。さらに、マウスを抗生物質処置することによって変異株を腸管内に長期定着させた場合も、糞便中IgA抗体は誘導されなかった。以上のように、EHECの上皮細胞への接着は全身性免疫応答の誘導には必ずしも必要ではないが、粘膜免疫応答の誘導には必須であることが示された。The induction mechanism of mucosal immune responses was examined in mice infected orally with an intestinal pathogen, enterohemorrhagic Escherichia coli (EHEC) O157:H7. Antibodies were evaluated as those specific to EHEC O157:H7 cell surface by enzyme linked-immunosorbent assay using EHEC O157:H7 whole cells as an antigen (Filtration ELISA). When the EHEC wild type was inoculated intragastlically into ICR mice, the bacteria adhered on the cecal epithelia and colonized over four weeks, and fecal IgA antibody to the EHEC whole cells markedly increased at four weeks after the inoculation. On the other hand, mutants deleted adherent factors of the EHEC did not colonize in the intestine and did not induce fecal IgA antibody. When the mutant was inoculated repeatedly, fecal IgA antibody was not induced although the serum antibodies were induced. Furthermore, the fecal IgA antibody was not induced even when the mutants resided persistently in the intestine of mice by treatment with an antibiotic. Thus, the adhesion of EHEC to epithelial cells was indispensable to induce the mucosal immune responses but not systemic immune responses

Gliding Motility of Babesia bovis Merozoites Visualized by Time-Lapse Video Microscopy

BACKGROUND: Babesia bovis is an apicomplexan intraerythrocytic protozoan parasite that induces babesiosis in cattle after transmission by ticks. During specific stages of the apicomplexan parasite lifecycle, such as the sporozoites of Plasmodium falciparum and tachyzoites of Toxoplasma gondii, host cells are targeted for invasion using a unique, active process termed "gliding motility". However, it is not thoroughly understood how the merozoites of B. bovis target and invade host red blood cells (RBCs), and gliding motility has so far not been observed in the parasite. METHODOLOGY/PRINCIPAL FINDINGS: Gliding motility of B. bovis merozoites was revealed by time-lapse video microscopy. The recorded images revealed that the process included egress of the merozoites from the infected RBC, gliding motility, and subsequent invasion into new RBCs. The gliding motility of B. bovis merozoites was similar to the helical gliding of Toxoplasma tachyzoites. The trails left by the merozoites were detected by indirect immunofluorescence assay using antiserum against B. bovis merozoite surface antigen 1. Inhibition of gliding motility by actin filament polymerization or depolymerization indicated that the gliding motility was driven by actomyosin dependent process. In addition, we revealed the timing of breakdown of the parasitophorous vacuole. Time-lapse image analysis of membrane-stained bovine RBCs showed formation and breakdown of the parasitophorous vacuole within ten minutes of invasion. CONCLUSIONS/SIGNIFICANCE: This is the first report of the gliding motility of B. bovis. Since merozoites of Plasmodium parasites do not glide on a substrate, the gliding motility of B. bovis merozoites is a notable finding