Masakazu Hattori

Analysis of mixed parasite populations of Theileria sergenti using cDNA probes encoding a major piroplasm surface protein

The gene for the 32 kDa surface protein (p32) of Theileria sergenti was cloned into lambda gt11 and its nucleotide sequence was determined. The gene encodes a protein of 283 amino acids as deduced from its nucleotide sequence with a 22 residue N-terminal signal peptide. Using this cDNA as a probe we have isolated another two clones from a cDNA library with a CDM8 vector system derived from the same parasite stock. Comparison with three cDNA clones revealed differential polyadenylation and differences in sequences of non-coding regions. Within the coding regions, there were nucleotide transitions which affected the Pst I-restriction site, and one of the transitions was also accompanied by an amino acid substitution (Ala to Gly). Southern blot analysis showed hybridization pattern changes among the parasites isolated from individual calves at different times after infection. From these results, we conclude that at least 3 genetically different parasite populations may coexist, and the transition to predominant parasite populations might occur during persistent infections in a host, possibly to evade the host immune responses.

Expression of a 32 kilodalton Theileria sergenti piroplasm surface protein by recombinant baculoviruses.

Previous studies detected a single amino acid substitution (Ala196 to Gly196) between cDNA clones encoding a 32 kDa antigen (p32) of Theileria sergenti (Chitose stock) obtained from a persistently infected calf. In this study, 2 different recombinant baculoviruses (pAc/p32-Ala196 and pAc/p32-Gly196) were constructed for the expression of p32. Molecular masses of the polypeptides produced in Spodoptera frugiperda cells infected with the recombinant baculoviruses were the same as that of authentic p32. pAc/p32-Ala196 produced additional polypeptides, with molecular masses higher than 32 kDa, which resulted from differential N-glycosylation as revealed by endo N-glycosidase treatment. The results indicate that a single amino acid substitution may lead to a conformational change in p32 which affected post-translational modification of recombinant products.

Characterization of extrathymic T cells of chickens

The function of CD4+ T cells in antibody production was examined by using T cell subset-depleted chickens. CD4- and CD8-depleted chickens, established by the combination of thymectomy and injection of T cell subset-specific monoclonal antibodies, were immunized with sheep red blood cells (SRBC). Titers of anti-SRBC antibody produced in CD4-depleted chickens were lower than those in control chickens, while no difference in the antibody production was observed between CD8-depleted and control chickens. In chickens depleted of both CD4+ and CD8+ T cells, the recovery of T cells in the periphery was demonstrated starting 3 weeks after T cell depletion. Those T cells recovered in the periphery predominantly expressed CD4 molecules. Although low titers of antibody against SRBC were detected in chickens depleted of both CD4+ and CD8+ T cells, an increase of anti-SRBC antibody production was coincidentally observed with the recovery of CD4+ T cells in the periphery. These results suggest that CD4+ T cells could differentiate in extrathymic environments in chickens, and have a helper function in antibody production similar to that of intrathymic T cells. These extrathymic T cells, however, showed a lower proliferative response to concanavalin A than intrathymic T cells, suggesting that these extrathymic T cells may have some properties distinct from intrathymic T cells.

Cell surface C-reactive protein of rainbow trout lymphocytes

C-reactive protein (CRP) expressed on rainbow trout (Oncorhynchus mykiss) lymphocytes was investigated to determine what role CRP might have on lymphocyte function. C-reactive protein was detected on the surface (S-CRP) of 25% of peripheral blood lymphocytes (PBL) and 4% of head kidney lymphocytes (HKL) by flow cytometry of cells labelled with biotinylated rabbit anti-rainbow trout CRP IgG and fluorescent-coupled avidin. Purified CRP, when added to cells in culture, bound to both PBL and HKL. Stimulation of PBL and HKL by concanavalin A, phytohemagglutinin, or lipopolysaccharide increased the percentage of S-CRP-positive cells, which suggests the production of CRP by lymphocytes. Treatment of lymphocytes with anti-rainbow trout CRP IgG and complement decreased the number of S-CRP-positive cells.

Expression of the A antigen (gp57-65) of Marek's disease virus by a recombinant baculovirus

A recombinant baculovirus expressing the A antigen (A Ag) of Mareks disease virus (MDV) was constructed. In Spodoptera fruiperda (Sf) cells infected with the recombinant virus, A Ag expression was localized to the cell surface. Only a small amount of recombinant A Ag was detected in the culture supernatant of infected Sf cells, but authentic A Ag is mainly secreted into the culture supernatant of MDV-infected chicken embryo fibroblasts. Cell surface-associated recombinant A Ag seemed to be slightly larger than authentic A Ag, whereas the secreted recombinant A Ag seemed to be smaller. The recombinant A Ag was shown to be reactive with the sera of MDV-infected chickens by immunodiffusion studies and ELISA. Sera of chickens immunized with recombinant A Ag formed a precipitin line with the culture supernatant of MDV-infected chicken embryo fibroblasts in an immunodiffusion test. These results indicate that the recombinant A Ag expressed by the recombinant baculovirus retains the antigenic and immunogenic properties of the authentic A Ag.