Takaji Iida

Degranulation of eosinophilic granular cells with possible involvement in neutrophil migration to site of inflammation in tilapia

Observations were made on stretched preparations from the swim bladder and peritoneum obtained from tilapia (Oreochromis niloticus) after injection of formalin-killed Escherichia coli, proteose peptone, compound 48/80 or HBSS into the swim bladder. The eosinophilic granular cells (EGCs) in the swim bladder degranulated rapidly after inoculation. However, the peritoneal EGCs did not degranulate, indicating that degranulation occurs only at the injected site. There was also a correlation between the ratio of the degranulated EGCs and number of the exudate neutrophils. Killed E. coli (1 mg/fish) produced the greatest degranulation response of EGCs and migration of neutrophils into the inflammatory site. Additionally, the rate of the degranulation and number of the neutrophils were lowest when HBSS was injected. The results of this study verify that degranulation of EGCs is involved in the neutrophil migration and suggest that fish EGCs are analogous to mammalian mast cells.

Fish granulocytes in the process of inflammation

Abstract Inflammation is a protective reaction of the host in response to injury, resulting in specific morphological and chemical changes in tissues and cells. In fishes as well, much basic research has been conducted on the process of inflammatory leucocyte migration, which is the most characteristic event of the acute phase. The first response of a host to injury is vasodilatation, followed by increased vascular permeability. These vascular reactions have significance in understanding the mechanism of leucocyte migration, which occurs through the injured blood vessels and in response to chemical mediators converted from certain plasma proteins. Neutrophils migrate more quickly than do monocytes and macrophages during acute inflammation, as has been observed in many fish species. These leucocytes are phagocytes which act to remove irritants, bacteria, or damaged cells and tissues. Rapid migration of basophils is also distinguishable in carp (Cyprinus carpio) and puffer (Takifugu niphobles), although the functions of the cells in inflammation have not been clarified. Leucocytic infiltration in inflammation can be explained by chemical mediators. Complement factors, leucotriene B4 and a lymphokine, have been identified as chemotactic and chemokinetic factors for fish neutrophils. Besides these host factors, bacterial formyl peptides are reported to be chemoattractive for plaice (Pleuronectes platessa) neutrophils. The process of leucocytic migration in various types of inflammation has specific features, which are controlled by a variety of such chemical mediators. However, our knowledge at present represents but a glimpse of the intricacies of fish inflammation.

The efficacy of five avirulent Edwardsiella tarda strains in a live vaccine against Edwardsiellosis in Japanese flounder, Paralichthys olivaceus.

We evaluated the tissue persistence and live vaccine efficacy of five avirulent Edwardsiella tarda strains (E22, SU100, SU117, SU138, and SU244) isolated from the Japanese eel (Anguilla japonica) and from the environment. The live vaccines, containing a single strain, were injected intraperitoneally into Japanese flounder (Paralichthys olivaceus). Viable bacteria from all the strains (excluding SU100) were recovered from trunk-kidney tissue 28 d post-injection. Japanese flounder inoculated with E22 had the highest relative percentage survival (RPS = 45%) in an artificial challenge with virulent E. tarda (NUF806). The serum of E22-vaccinated fish had a significantly higher agglutination titer against NUF806. In contrast, there was little or no increase in the agglutination titer of the fish that were inoculated with the remaining avirulent strains. Injection with avirulent E. tarda increased the expression of cytokine genes, including interleukin-1beta (IL-1beta), type 1 interferon (IFN), and IFN-gamma in head-kidney of the Japanese flounder.

Identification of Edwardsiella ictaluri and E. tarda by Species-Specific Polymerase Chain Reaction Targeted to the Upstream Region of the Fimbrial Gene

Phylogenetic analysis of nine strains of Edwardsiella ictaluri and eight strains of E. tarda (six typical motile strains and two atypical nonmotile strains) isolated from diseased fish was performed using the upstream region of the fimbrial gene cluster. Strains of E. ictaluri and E. tarda were significantly clustered into separate groups. Moreover, atypical E. tarda strains were clustered into a different group from the other strains. Three polymerase chain reaction (PCR) primer sets for differential detection of E. ictaluri as well as typical and atypical E. tarda were developed from the respective characteristic sequences. Strains of E. ictaluri, typical E. tarda, and atypical E. tarda were specifically detected by PCR using each primer set. No amplifications were observed after the use of these three primer sets with 25 other bacterial species, including fish pathogens. In addition, the three primer sets were able to detect the DNA of each target species from fish kidney and liver artificially infected with E. ictaluri or E. tarda.

Adaptive immune response to Edwardsiella tarda infection in ginbuna crucian carp, Carassius auratus langsdorfii

Edwardsiella tarda is an intracellular pathogen that causes edwardsiellosis in fish. Although cell-mediated immunity and innate immunity play a major role in protection against intracellular bacterial infection in mammals, their importance in protecting fish against E. tarda infection remain unclear. In this study, we examined cell-mediated and humoral immune responses in ginbuna crucian carp (Carassius auratus langsdorfii) after E. tarda infection. Innate immunity was observed to be the principal immune system for eliminating the majority of E. tarda, while a proportion of the bacteria might be resistant to its bactericidal activity. Bacterial clearance in kidney and spleen was also observed following higher cytotoxic activities of cytotoxic T lymphocytes (CTLs) and increased numbers of CD8α(+) cells, suggesting that CTLs might contribute to the elimination of E. tarda-infected cells with specific cytotoxicity. On the other hand, E. tarda-specific antibody titers did not increase until after bacterial clearance, indicating that induction of humoral immunity would be too late to provide protection against infection. Overall, these data suggest that both cell-mediated immunity and innate immunity may play important roles in the protection against intracellular bacterial infection, as they do in mammals. Our study would also contribute toward the understanding of immune responses that provide protection against other intracellular pathogens.

Evidence for the existence of cytochrome b558 in fish neutrophils by polyclonal anti-peptide antibody

When western blotting analysis of Japanese eel neutrophil lysate was performed using antibody against the synthetic peptide corresponding to the carboxyl terminal region of human cytochrome b558 large subunit, a broad band was specifically detected at approximately 90 kDa. The antibody recognized the epitope present in eel neutrophils only after the cells were permeabilized with detergent. These results indicate that the large subunit of cytochrome b exists in fish neutrophils and the epitope is exposed to the cytoplasmic side of membrane, and suggest that cytochrome b is conserved across species.

Identification of Major Antigenic Proteins of Edwardsiella Tarda Recognized by Japanese Flounder Antibody

Edwardsiella tarda is a fish pathogen that causes systemic infections in fresh water and marine fish. Determining the antigenic proteins is important for the development of an immunodiagnostic tests and a vaccine for effective infection control in fish. In the current study, antigens were detected by immunoblotting and affinity column chromatography using a Japanese flounder (Paralichthys olivaceus) antibody produced by experimental infection with E. tarda. GroEL, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), outer membrane protein A, filament protein, 30S ribosomal protein S6, 50S ribosomal protein L9, cold shock protein, and carbon storage protein were identified as antigens of E. tarda through biochemical analyses of the molecular weights, isoelectric points, and N-terminal amino-acid sequences. These proteins can be easily detected in flounder infected with E. tarda and are potential diagnostic markers.

Identification of novel putative virulence factors, adhesin AIDA and type VI secretion system, in atypical strains of fish pathogenic Edwardsiella tarda by genomic subtractive hybridization.

Edwardsiella tarda, which is known to be the causative agent of edwardsiellosis in freshwater and marine fish, has two motility phenotypes. Typical strains exhibiting motility are isolated mainly from freshwater fish and Japanese flounder. Atypical strains exhibiting non‐motility are isolated mainly from marine fish, with the exception of Japanese flounder. Subtractive hybridization was performed to identify genomic differences between these two phenotypes. Two fragments which showed homology to potential virulence factors were isolated from atypical strains: the autotransporter adhesin AIDA and a component of T6SS. We analysed DNA sequences of about 5 kbp containing these fragments and identified two partial ORF, and ORF encoding for other components of T6SS. The predicted amino acid sequences showed remarkably low homology to components of T6SS reported in the typical E. tarda strain PPD130/91. Furthermore, the organization of these ORF was different from the gene cluster of the typical E. tarda strain. AIDA and T6SS may therefore be associated with different pathogenicity in typical and atypical E. tarda hosts.

ANALYSIS OF LOCALIZATION AND FUNCTION OF THE COOH-TERMINAL CORRESPONDING SITE OF CYTOCHROME B558 IN FISH NEUTROPHILS

Using an antibody against the synthetic peptide corresponding to the COOH-terminal region of human cytochrome b558 large subunit, a broad band was specifically detected in neutrophil lysates from 6 marine fish and 2 freshwater fish by western blotting. Immunofluorescence assay showed that the antibody recognized the epitopes in eel and tilapia neutrophils permeabilized with detergent. These results suggest that the cytochrome b large subunit universally exists in fish neutrophils and that the epitopes are exposed to the cytoplasmic side of fish neutrophils as well as human neutrophils. Furthermore, a synthetic peptide corresponding to the COOH-terminus of the large subunit apparently blocked superoxide production in a specific and dose-dependent fashion in eel and tilapia neutrophils, indicating that the region equivalent to the COOH-terminus of cytochrome b large subunit is responsible for superoxide generation in fish neutrophils.

Molecular cloning and identification of bottle-nosed dolphin flavocytochrome b gp91phox and p22phox subunits

The bottle-nosed dolphin (Tursiops truncatus) gp91(phox) and p22(phox) cDNA were cloned from mitogen stimulated leukocytes RNA utilizing the reverse transcription-polymerase chain reaction. The sequences of these cDNAs showed that dolphin gp91(phox) and p22(phox) clones contained open reading frames encoding 569 and 192 amino acids, respectively. Analysis of the gp91(phox) amino acids sequence showed three potential N-linked glycosylation sites. Comparison of the deduced amino acid showed that dolphin gp91(phox) sequence shared 95.4, 93.8, 91.4 and 89.5% similarity with the bovine, porcine, human and mouse gp91(phox) sequences, respectively. Similarly, the amino acid sequence showed that dolphin p22(phox) shared 89.7, 84.6, 84.1, 83.6 and 83.6% similarity with the bovine, mouse, porcine, human and rattus p22(phox) sequences, respectively. Western blotting analysis with anti-peptide antibodies supported the molecular weights of the dolphin gp91(phox) and p22(phox) homologous proteins predicted from the cDNAs and amino acids sequence data.