Tadashi Iwasaki

Suppression of Salmonella enterica serovar Enteritidis excretion by intraocular vaccination with fimbriae proteins incorporated in liposomes

Liposome-associated fimbriae antigens (SEF14 and SEF21) were prepared for intraocular immunization to seek protective efficacy for intestinal infection with Salmonella enterica serovar Enteritidis. Chickens were immunized intraocularly with the antigens at 8 and 10 weeks of age. Evidence of an IgA and IgG responses were found in the intestinal tract and in sera of these chickens. Antibody-secreting lymphocytes were detected in the Harderian gland of immunized chickens as determined by enzyme-linked immunospot assay. Two weeks after the booster immunization, the chickens were challenged orally with 1x10(7) live Salmonella Enteritidis, and fecal samples were examined for bacterial excretion from the intestinal tract. Significantly less fecal excretion of bacteria was observed in immunized chickens for 15 days after challenge. The numbers of bacteria in the intestinal contents (caecum and rectum) were also significantly lower in immunized chickens than in unimmunized controls. Detection of S. Enteritidis-specific DNA by the polymerase chain reaction was consistent with the bacterial observations. Intraocular immunization with liposome-associated SEF14 and SEF21 therefore elicits both systemic and mucosal antibody responses, so that bacterial colonization in the intestinal tract and excretion of S. Enteritidis in the feces are suppressed by immunization.

Metabolomic investigation of pathogenesis of myxosporean emaciation disease of tiger puffer fish Takifugu rubripes.

Serum biochemical analysis was undertaken to study the pathophysiological details of emaciation disease of the tiger puffer fish Takifugu rubripes (Temminck and Schlegel). Serum parameters were measured by biochemical analysis using automated dry chemistry and gas chromatography/mass spectrometry (GC/MS). Serum concentrations of albumin, amylase, calcium, creatinine, glucose and total protein were significantly lower in the emaciated fish when compared with those of normal fish. Regression analyses found close correlation between concentrations of total protein, albumin, amylase, glucose and progress of the disease. In contrast, serum alanine aminotransferase increased significantly in emaciated fish indicating liver function disorder. Further, GC/MS metabolic profiling of the puffer serum showed that the profile of the emaciated fish was distinct to that of non-infected control. The serum content of amino acids including glycine, 5-oxo-proline and proline, and ascorbic acid, fumaric acid and glycerol increased significantly in serum in moderately emaciated fish. The serum glucose, linolenic acid and tyrosine level decreased significantly in the late phase of the disease. Our results clearly show that prolonged intestinal damage caused by myxosporean infection impairs absorption of nutrients, resulting in extreme emaciation.

Production and characterization of a novel monoclonal antibody against Vibrio parahaemolyticus F0F1 ATP synthase's delta subunit and its application for rapid identification of the pathogen.

We raised monoclonal antibodies (MAbs) against Vibrio parahaemolyticus cell extracts. One of the MAbs, designated MAb-VP34, reacted in enzyme-linked immunosorbent assays (ELISAs) with 140 V. parahaemolyticus strains, regardless of serotype or origin. MAb-VP34 did not detectably react with 96 strains belonging to 27 other Vibrio species (except for Vibrio natriegens) or with 29 non-Vibrio species. These results show that MAb-VP34 is highly specific for V. parahaemolyticus. Western blotting and mass spectrometry analyses revealed that MAb-VP34 recognized V. parahaemolyticus F(0)F(1) ATP synthases delta subunit. Using MAb-VP34, a rapid and simple immunodot blotting assay (VP-Dot) was developed to determine whether bacterial colonies growing on selective agar, represented V. parahaemolyticus. To evaluate VP-Dot, 20 V. parahaemolyticus strains and 19 non-related strains were tested. The results indicated that VP-Dot is a reliable tool for identification of V. parahaemolyticus colonies. The simple VP-Dot procedure took 40min, indicating that the MAb-VP34 based immunological method will greatly reduce labor, time, and costs required to verify V. parahaemolyticus colonies as compared with the conventional biochemical test.

Development of a rapid and simple immunochromatographic assay to identify Vibrio parahaemolyticus.

To rapidly and simply determine whether or not bacterial colonies growing on agar were Vibrio parahaemolyticus, we developed an immunochromatographic assay (VP-ICA) using two different monoclonal antibodies (designated mAb-VP34 and mAb-VP109) against the delta subunit of V. parahaemolyticus-F0F1 ATP synthase. The epitopes recognized by mAb-VP34 and mAb-VP109 were mapped to sequences of eight ((47)LLTSSFSA(54)) and six amino acid residues ((16)FDFAVD(21)), respectively. An amino acid sequence similarity search of the NCBI database using BLASTP showed that both epitopic amino acid sequences were present together only in V. parahaemolyticus. When 124 V. parahaemolyticus strains and 94 strains of 27 other Vibrio species or 35 non-Vibrio species were tested using the VP-ICA, the VP-ICA identified V. parahaemolyticus with 100% accuracy. The VP-ICA rapidly and simply identified the pathogen directly from a single agar colony within 30 min, indicating that VP-ICA will greatly reduce labor and time required to identify V. parahaemolyticus compared with conventional biochemical tests.

Induction of antibody response in the oral cavity of dogs following intraocular (eye drop) immunization with Porphyromonas gingivalis cell lysate incorporated in pH-sensitive fusogenic polymer-modified liposomes.

Induction of mucosal immune responses against Porphyromonas gingivalis within the oral cavity of dogs was studied by immunizing with pH-sensitive fusogenic polymer (MGluPG)-modified liposome-associated cell lysate. Dogs immunized with P. gingivalis cell lysate-containing MGluPG-modified liposomes by intraocular (eye drop) route displayed significant levels of P. gingivalis cell lysate-specific serum IgG and IgA as well as mucosal IgA antibodies in saliva secretion. Serum and salivary antibodies generated by intraocularly immunized with MGluPG-modified liposome-associated P. gingivalis cell lysate revealed a significant aggregation activity against P. gingivalis, whereas serum and saliva from dogs receiving MGluPG-modified liposomes unentrapping P. gingivalis cell lysate did not show the aggregation activity against P. gingivalis. Furthermore, P. gingivalis-specific antibodies in saliva of immunized dogs inhibited the adherence of P. gingivalis to cultured HeLa cells. More importantly, salivary antibodies induced by intraocular immunization with P. gingivalis cell lysate-containing MGluPG-modified liposomes significantly inhibited the coaggregation of P. gingivalis with Actinomyces naeslundii and the cell damage activity of P. gingivalis against FaDu cells, an oral epithelial cell. These results suggest that intraocularly administered P. gingivalis cell lysate-containing MGluPG-modified liposomes should be an effective mucosal vaccine against P. gingivalis infection in dogs and may be an important tool for the prevention of periodontitis.