Miamiensis avidus is the causative agent of scuticociliatosis in various marine fish species. The virulence factors of the parasite have not been identified, so far. In this study, we examined M. avidus extracellular proteinases (ECPs) as potential virulence factors, using culture supernatants as an ECPs source. We investigated the substrate specificity of ECPs using artificial peptides, and the cytotoxicity of the ECPs was examined using CHSE-214 cells. To elucidate the role of ECPs in ciliate growth, M. avidus was cultured on CHSE-214 cells in the presence of proteinase inhibitors. We detected proteinase activities from the supernatant of M. avidus. Viable CHSE-214 cells decreased significantly in number, when incubated in a medium supplemented with the culture supernatant of M. avidus. The growth of ciliates on CHSE-214 cells was delayed in the presence of PMSF (serine proteinase inhibitor) and E-64 (cysteine proteinase inhibitor). These results suggested that the culture supernatant contained ECPs showing cytotoxicity, and the proteinases facilitated nutrient uptake by the ciliates. Thus, ECPs may be responsible for virulence factors of M. avidus.
Determination of numbers of T cell subsets in tissues is important to assess the functional maturity of the immune system. The distribution of T cells in lymphoid tissues during ontogenic development has been examined in several fish species using pan-T cell markers. However, information on the age-related changes in the number of T cell subsets in tissues is limited due to the lack of antibodies against CD4 and CD8 in fish. In the present study we examined the percentage dynamics of CD4-1+ and CD8α+ cells in various tissues accompanied with age of ginbuna crucian carp using flow cytometry. We found rapid increases in both CD4-1+ and CD8α+ T cells up until 6 months post-hatch (mph). CD4 and CD8 double-positive cells (DP cells) are present only in the thymus and DP cells increased, while double negative cells decreased, in fish up to 3-6 mph. The percentages of CD4-1+ T cells were always higher than those of CD8α+ T cells in the various tissues examined, except for the thymus and intestine. In contrast, significantly higher percentages of CD8α+ T cells than CD4-1+ T cells were found in the intestine during the period between 6 mph and 2 years post-hatch.
Lactococcus garvieae is known as a pathogen of freshwater and marine fish species worldwide. L. garvieae isolates of serotype I have been divided into two serological phenotypes, namely KG- and KG+, which are differentiated by the presence or absence of polysaccharide capsule, and a phenotypic change from KG- to KG+ occurs during repeated subculturing. When we subcultured laboratory collections of L. garvieae KG- strains repeatedly, they were divided into two groups. One group consisted of the strains which changed phenotypically after a relatively small number of times of subculturing, and the other group consisted of those which hardly changed. Genetic analyses revealed that the capsule gene cluster of the strains in the former group was integrated in a plasmid (CPS plasmid) and that in the latter group it was integrated in the chromosome or in both CPS plasmid and chromosome simultaneously. In the present study, we found three novel CPS plasmids. These plasmids were similar in structure to the CPS plasmid pBSLG13015 which was previously found in L. garvieae filefish isolates. In addition, we found a novel integration site of chromosomal capsule gene cluster. The majority of strains isolated before 1991 were those whose capsule gene cluster was integrated in the CPS plasmids.
Outbreaks of disease caused by the infection of Edwardsiella ictaluri in ayu Plecoglossus altivelis have been reported in rivers in Japan since 2007. In this study, we performed comparative proteomic analysis between the virulent strain FPC1091 and the less virulent strain FPC1214 to identify virulence factors of E. ictaluri to ayu. In the experimental infection, cumulative mortalities of the ayu intraperitoneally injected with FPC1091 (1.2 × 107 CFU/fish) and FPC1214 (2.2 × 107 CFU/fish) were 100% and 26.7%, respectively. The genetic fingerprint profile of FPC1214 was identical to that of FPC1091 in the amplified-fragment length polymorphism (AFLP) analysis. The analysis of two-dimensional polyacrylamide gel electrophoresis and mass spectrometry between FPC1091 and FPC 1214 revealed 23 proteins that were either specific to or produced in greater amounts in the virulent strain FPC1091. These proteins included components of the type III and type VI secretion systems, and the proteins that seem to be involved in the resistance against oxidative stress by host phagocytic cells, suggesting that these proteins are the virulence factors of E. ictaluri to ayu.
To develop an accurate diagnosis of “new ulcer disease” in koi carp, we produced four monoclonal antibodies (mAbs) against a strain of atypical Aeromonas salmonicida isolated from koi carp. These mAbs did not cross-react with an isolate of atypical A. salmonicida from Japanese flounder and other pathogenic bacteria. Re-isolation from artificially infected koi carp was achieved by selecting the blue colonies on agar medium containing Coomassie brilliant blue, and some of the colonies were detected by immunofluorescent staining using the mAbs. These results suggested that the mAbs can distinguish atypical A. salmonicida from koi carp from resident aeromonads.
Mass mortalities due to bacterial kidney disease (BKD) occurred after 10-15 months rearing in two groups of chum salmon Oncorhynchus keta which had been raised from asymptomatically infected eggs and fry transferred from private hatcheries. Mortality reached 56.1% and 19.0% in the two groups, respectively, 146 days after the first death. A Renibacterium salmoninarum-specific gene was detected from various organs of the subclinical and dead fish. In the dead fish, the number of gene copies was higher than those in the subclinical fish in all the eight organs examined. These results suggested that the bacterium distributed in the bodies of asymptomatic fish proliferated systemically, resulting in an outbreak of BKD.
Mass mortalities of goldfish Carassius auratus occurred at some farms and wholesalers in Japan, where fish were held at around 33°C to control herpesviral hematopoietic necrosis. All dead fish showed light to moderate Clinostomum infection. Cysts containing metacercariae were accompanied by hemorrhage and congestion, and metacercariae were activated and excysted during the high temperature water treatment. When infected fish were experimentally treated at 20°C, 31°C, 33°C and 35°C, mortality rates reached to 0%, 5%, 40% and 100%, respectively. The experimental results indicated that the elevation of water temperature against the virus infection caused the mass mortalities in Clinostomum-infected goldfish.