Naoyoshi Suzuki

The role of CD4+ or CD8+ T cells in the protective immune response of BALB/c mice to Neospora caninum infection

The role of CD4(+) or CD8(+) T cells in the immune response of BALB/c mice against Neospora caninum infection was examined by using anti-CD4 and/or anti-CD8 monoclonal antibodies (mAbs). Mice were intraperitoneally inoculated with anti-CD4 and/or anti-CD8 mAbs before and after infection with N. caninum and observed for 30 days after infection. Most of the anti-CD4 mAb-treated mice and all of the anti-CD4 and anti-CD8 mAbs-treated mice died within 30 days post-infection (p.i.). In contrast, 100% of PBS-treated mice and 70% of anti-CD8 mAb-treated mice survived more than 30 days. When compared with phosphate-buffered saline (PBS)-treated mice, the weight of mice treated with mAbs tended to decrease. From these results CD4(+) T cells, but not CD8(+) T cells, have an important role for protection of mice against N. caninum infection. Serum antibody levels to N. caninum in infected-mice treated with anti-CD4 mAb or a mixture of anti-CD4 and anti-CD8 mAbs were lower than those in the infected mice treated with anti-CD8 mAb or PBS. The mice treated with anti-interferon-gamma (IFN-gamma) mAb produced high antibody levels to N. caninum, but all mice died within 18 days p.i. These results indicated that IFN-gamma is an important cytokine for protection against N. caninum infection at the early stage of infection. However, since CD4(+) T cells against N. caninum were essential to the production of specific antibody, these antibodies might have important roles in host protection at the later stage of infection.

Rapid Immunochromatographic Test Using Recombinant SAG2 for Detection of Antibodies against Toxoplasma gondii in Cats

ABSTRACT An immunochromatographic test using recombinant truncated surface antigen 2 for detection of antibodies against Toxoplasma gondii was developed. Evaluation of detection of the antibody in mice and cats suggests that this test is rapid, simple, accurate, relatively inexpensive, and suitable for use under field conditions.

Serodiagnosis of Toxoplasma gondii infection in cats by enzyme-linked immunosorbent assay using recombinant SAG1

The gene encoding surface antigen 1 (SAG1, P30) of Toxoplasma gondii (T. gondii) was cloned into the plasmid pGEX-4T-3 and subsequently expressed in Escherichia coli (E. coli) as a glutathione-S-transferase (GST) fusion protein. The recombinant SAG1 (rSAG1) was refolded using 8M urea solution followed by dialysis and thereafter evaluated in an enzyme-linked immunosorbent assay (ELISA) for serological diagnosis of toxoplasmosis. The test sera were adsorbed with GST to block non-specific reactivity to the GST-SAG1 fusion protein. The ELISA with rSAG1 was able to differentiate very clearly between sera from cats or mice experimentally infected with T. gondii and sera from normal cats or mice. The ELISA detected no cross-reactivity with sera from mice experimentally infected with the closely related parasite Neospora caninum (N. caninum). Some 193 cat sera were tested for antibodies to T. gondii, out of which 40 (20.7%) reacted positively by ELISA with the rSAG1 while another 79.3% cats reacted negative to the assay. Both positive and negative sera were confirmed by Western blot analysis. The results of ELISA were in agreement with those of a commercially available latex agglutination test (LAT) kit, although the former had higher titers than the latter.

Expression of Babesia equi Merozoite Antigen 1 in Insect Cells by Recombinant Baculovirus and Evaluation of Its Diagnostic Potential in an Enzyme-Linked Immunosorbent Assay

ABSTRACT The gene encoding the entire Babesia equi merozoite antigen 1 (EMA-1) was inserted into a baculovirus transfer vector, and a recombinant virus expressing EMA-1 was isolated. The expressed EMA-1 was transported to the surface of infected insect cells, as judged by an indirect fluorescent-antibody test (IFAT). The expressed EMA-1 was also secreted into the supernatant of a cell culture infected with recombinant baculovirus. Both intracellular and extracellular EMA-1 reacted with a specific antibody in Western blots. The expressed EMA-1 had an apparent molecular mass of 34 kDa that was identical to that of native EMA-1. The secreted EMA-1 was used as an antigen in an enzyme-linked immunosorbent assay (ELISA). The ELISA differentiatedB. equi-infected horse sera from Babesia caballi-infected horse sera or normal horse sera. The ELISA was more sensitive than the complement fixation test and IFAT. These results demonstrated that the recombinant EMA-1 expressed in insect cells might be a useful diagnostic reagent for detection of antibodies to B. equi.

Peyer’s Patches: Organized Lymphoid Structures for the Induction of Mucosal Immune Responses in the Intestine

Peyer’s patches (PP) comprise transmucosal clusters of lymphoid follicles overlaid with a specialized lympho-epithelium and consequently play a central role in the induction of mucosal immune responses in the gut. Despite considerable achievements in the last 3 decades, in our understanding of how PP are involved in the induction of immune responses, much remains to be learned about these major organized lymphoid organs. The history and current status of PP termed ‘the major inductive site of immune responses’ is reviewed. The present understanding of PP biology and function, taking into account their preferential and unique retention of immune competent cells at specific sites, is discussed.

Detection of Babesia caballi infection by enzyme-linked immunosorbent assay using recombinant 48-kDa merozoite rhoptry protein

The 48-kDa Babesia caballi merozoite rhoptry protein was expressed using a pGEX4T expression vector in Escherichia coli as glutathione S-transferase fusion protein (GST-BC48), and the expressed GST-BC48 was used in an ELISA to detect specific antibodies in serum samples. No cross-reaction was observed with sera from horses experimentally infected with Babesia equi. GST-BC48 ELISA was a highly sensitive and specific test when compared with the CFT. A total of 209 horse sera obtained from Central Mongolia were examined with the GST-BC48 ELISA and 46.4% (97/209) were found to be seropositive for B. caballi, suggesting that the GST-BC48 ELISA can be successfully used for both quarantine and epidemiological studies.

Growth-inhibitory effects of interferon-γ on Neospora caninum in murine macrophages by a nitric oxide mechanism

Abstract To clarify the effector pathway of Neospora caninum growth-inhibitory activity induced by interferon-γ (IFN-γ) in murine macrophages we examined the relationship between IFN-γ and nitric oxide (NO). Production of NO was enhanced in cultures of macrophages supplemented with IFN-γ, and dose-dependent growth inhibition was observed. These findings suggest that the inhibitory activity induced in macrophages by IFN-γ is mediated NO molecules. A competitive inhibitor of the L-arginine-dependent effector pathway, NG-monomethyl-L-arginine, virtually abolished the inhibitory effects induced by IFN-γ. From this finding it appears that the inhibitory effects induced by IFN-γ in macrophages may be mediated by an L-arginine-dependent effector pathway that involves NO production. In vivo, mice with a targeted disruption of the inducible NO synthase gene (iNOS−/−) were more susceptible than wild-type mice to N. caninum. Therefore, the production of NO in macrophages induced by IFN-γ is an important mechanism for the killing of intracellular N. caninum.

Effects of immune lymphocyte products and serum antibody on the multiplication of Toxoplasma in murine peritoneal macrophages.

SummaryIn vitro studies on cell-mediated immunity against Toxoplasma infection were carried out by estimating the ability of antigenically stimulated lymphocytes. Splenic lymphocytes from normal mice and from hyper-immunized mice were cultured in the presence or absence of Toxoplasma lysate antigen. The cell-free supernatant fluids from these lymphocyte cultures were assessed for their ability to alter the functional capacities of normal macrophages. When glycogen-induced peritoneal macrophages were incubated with the culture supernatant from immune lymphocytes reacting with specific antigen, the intracellular multiplication of the organisms was inhibited remarkably.In contrast, the addition of antitoxoplasma antibody to culture medium had no effect on the enhancement of phagocytic activity of cultured macrophages. However, when extracellular organisms were exposed to fresh or heat-inactivated immune serum just before infection of the monolayers, the intracellular multiplication of Toxoplasmas was inhibited significantly by either activated or normal macrophages.

Cloning of a Truncated Babesia equi Gene Encoding an 82-Kilodalton Protein and Its Potential Use in an Enzyme-Linked Immunosorbent Assay

ABSTRACT To isolate Babesia equi genes encoding immunodominant proteins, a cDNA expression library prepared from B. equi mRNA was immunoscreened with B. equi-infected horse serum. Eighteen positive cDNA clones were obtained, and the clone that showed the strongest immunoreactivity, designated Be82, was further characterized. The Be82 gene consisted of 1,953 bp and contained a partial open reading frame lacking the 5′-terminal sequence. As shown by Western blot analyses, immune sera from mice intraperitoneally injected with the Be82 gene product recognized the 82- and 52-kDa proteins of B. equi but not those of Babesia caballi. The glutathione S-transferase fusion protein expressed in Escherichia coli that was purified and used as the antigen in the enzyme-linked immunosorbent assay reacted specifically with B. equi-infected horse sera. These results suggest that the Be82 gene product is a potential diagnostic antigen candidate in the detection of B. equi infection in horses that will be useful both in the performance of epidemiological studies and in the granting of quarantine passes.

Transfer of antibodies to kittens from mother cats chronically infected with Toxoplasma gondii

By indirect immunofluorescence assay, anti-Toxoplasma gondii antibody levels were examined in fetuses and kittens born from chronically infected cats. Titer of anti-T. gondii IgG in sera of kittens born from infected cats was significantly high on the seventh day post-birth, and decreased to a serologically non-detectable level at 8-12 weeks post-birth under continuous suckling of maternal milk. Littermates nursed by a non-infected cat showed a faster rate of IgG antibody depletion. In sera of fetuses obtained from infected cats, anti-T. gondii IgG titer was lower than that of offspring born from infected cats. Anti-T. gondii IgM titer was non-detectable in sera of all kittens and fetuses. Kittens born from infected cats inoculated with T. gondii oocysts on Day 35 after birth shed oocysts and showed a transient increase of anti-T. gondii IgM titer. Findings in this study suggest that anti-T. gondii antibody IgG in kittens is transferred mainly via colostrum and the kittens that receive maternal anti-T. gondii antibodies develop inadequate resistance to T. gondii infection.