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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., Fish Pathology, 53(1), pp.19-28; 2018

Lactococcus garvieae is recognized as a crucial bacterial pathogen of freshwater and marine fish species. It has been divided into two serological phenotypes, namely KG? and KG+. Difference of the two phenotypes is owing to the presence or absence of polysaccharide capsule, and a phenotypic change from KG? to KG+ occurs during stocking of isolates for a long period or by repeated subculturing. We found that the phenotypic change occurred more readily in L. garvieae isolates from cultured filefish, thread-sail filefish Stephanolepis cirrhifer and black scraper Thamnaconus modestus, than those from other fish species. Thus we studied the gene cluster for capsular polysaccharide biosynthesis (capsule gene cluster) of a filefish isolate, strain BSLG13015, and revealed that the strain possessed the same capsule gene cluster as those from other fish species, but that it was integrated in a newly identified plasmid. The plasmid, a size of 31,654 bp and circular, was named pBSLG13015. It was detected in all of KG? filefish isolates but not in KG+ filefish isolates or L. garvieae from other fish species. It is highly probable that the easier change from KG? to KG+ in L. garvieae filefish isolates is attributed to the loss of the plasmid., Fish Pathology, 52(2), pp.82-88; 2017

Streptococcus parauberis is a pathogen of streptococcosis in turbot Scophthalmus maximus and the Japanese flounder Paralichthys olivaceus. S. parauberis isolates from diseased Japanese flounder have been classified into several serological phenotypes. In this study we analyzed the DNA sequence of the genetic locus for capsular polysaccharide (CPS) biosynthesis of S. parauberis KRS02083 (subserotype Ia), NUF1003 (subserotype Ib), NUF1071 (subserotype Ic), NUF1032 (serotype II), 2007-1 (nontypeable/PFGE cluster I) and NUF1095 (nontypeable/PFGE cluster III) to elucidate the genetic basis for serological diversity. As a result, three kinds of cps locus were revealed among serotypes and subserotypes, namely the loci for subserotype Ia, subserotypes Ib/Ic and serotype II. The genetic structure of cps loci suggests that the capsules of S. parauberis are synthesized through the Wzy-dependent pathway. Subserotypes Ib and Ic possessed the same genetic structure, although single-base substitution at several regions or insertion of an IS (insertion sequence) element was found in subserotype Ic. The nontypeable strains, which agglutinated with both serotypes I and II antisera, possessed the same genetic structure as subserotype Ib/Ic or serotype II with single-base substitution at several regions., Fish Pathology, 50(4), pp.192-199; 2015

Tenacibaculum maritimum is the gliding bacterium that causes tenacibaculosis, an ulcerative disease in marine fish. In this study, we conducted a pathogenicity test to assess the effect of skin abrasion on the infectivity of gliding and non-gliding strains of T. maritimum, NUF1128 and NUF1129, respectively, and investigated the infection kinetics by enumeration and immunohistochemical observation of the adhered and proliferated T. maritimum on the abraded skin of Japanese flounder Paralichthys olivaceus. In the pathogenicity test, Japanese flounder whose dorsal skin was abraded with a cotton swab or blade or tip of dorsal fin was clipped with scissors were immersed for 30 min in seawater containing 106 CFU/mL of cultured NUF1128 or NUF1129 cells. As a result 100% mortality was achieved in the fish groups pretreated with blades or scissors followed by challenged with NUF1128. NUF1129 was unable to induce infection regardless of the treatments applied. The infection kinetic studies revealed that NUF1128 adhered more readily than NUF1129 to dermal connective tissues which were exposed by abrasion with blades and subsequently proliferated mainly in the dermal connective tissues and perimysium.

Streptococcus parauberis is the etiologic agent of streptococcosis in Japanese flounder Paralichthys olivaceus. Two serotypes, termed serotypes I and II, are known among the Japanese S. parauberis isolates. In the course of serodiagnosis, we found several strains that did not agglutinate with rabbit anti-serotype I or II sera. In this study, we investigated the serological and genetic relationships among the stocked S. parauberis strains including the non-agglutinating ones using a newly prepared rabbit antiserum against a non-agglutinating strain (NUF1071) as well as previously prepared antiserotype I and II sera, and pulsed-field gel electrophoresis (PFGE). An antiserum cross-absorption test and microtiter agglutination test revealed that the serotype I was divided into three subserotypes, tentatively designated Ia, Ib and Ic. The non-agglutinating strains belonged to the subserotype Ic. Of the 104 serotype I strains, 6, 91 and 7 strains belonged to subserotypes Ia, Ib and Ic, respectively. Formalin-killed cells of subserotype Ia and Ic strains were agglutinated with the anti-subserotype Ia serum (so far being used as an anti-serotype I serum) and Ic serum, respectively. Subserotype Ib strains were agglutinated with both sera. In PFGE analysis, the stocked 188 S. parauberis strains were divided into three clusters corresponding to subserotypes Ib/Ic, Ia and serotype II., 魚病研究, 50(2), pp.75-80; 2015

Tenacibaculum maritimum is the gliding bacterium that causes tenacibaculosis, an ulcerative disease in marine fish. In this study, we conducted a pathogenicity test to assess the effect of skin abrasion on the infectivity of gliding and non-gliding strains of T. maritimum, NUF1128 and NUF1129, respectively, and investigated the infection kinetics by enumeration and immunohistochemical observation of the adhered and proliferated T. maritimum on the abraded skin of Japanese flounder Paralichthys olivaceus. In the pathogenicity test, Japanese flounder whose dorsal skin was abraded with a cotton swab or blade or tip of dorsal fin was clipped with scissors were immersed for 30 min in seawater containing 106 CFU/mL of cultured NUF1128 or NUF1129 cells. As a result 100% mortality was achieved in the fish groups pretreated with blades or scissors followed by challenged with NUF1128. NUF1129 was unable to induce infection regardless of the treatments applied. The infection kinetic studies revealed that NUF1128 adhered more readily than NUF1129 to dermal connective tissues which were exposed by abrasion with blades and subsequently proliferated mainly in the dermal connective tissues and perimysium., 魚病研究, 50(2), pp.44-52; 2015

A multiplex PCR assay for differentiation of Streptococcus parauberis serotypes was developed. The three primer pairs for subserotypes Ia and Ib/Ic and serotype II were designed from the serotype-specific sequences of the wzy gene in the loci for capsular polysaccharide biosynthesis. All of 188 S. parauberis isolates from Japanese flounder showed positive reaction with the expected size of PCR product for each serotype, which was consistent with the results of agglutination test using rabbit antisera. The nontypeable isolates, which are not differentiated by agglutination test, could be identified as subzero type Ib/Ic or serotype II. Other streptococci including S. parauberis derived from cow and major bacterial fish pathogens showed negative reaction., Fish Pathology, 50(4), pp.213-215; 2015

We determined the nucleotide sequence of a 8.6 kb DNA region containing etfA encoding a putative fimbrial major subunit from chromosomal DNA of Edwardsiella tarda KG8401 which expresses mannose-resistant hemagglutination (MRHA). This region contained three novel genes, etfBCD, at the downstream region of etfA. The deduced amino acid sequences of EtfABCD contained conserved fimbrial domains; fimbrial protein, fimbrial chaperone, fimbrial usher and fimbrial protein, respectively. Escherichia coli transformed with the cloned etf operon expressed MRHA and fimbriae that reacted with rabbit antiserum against the fimbrial major subunit of E. tarda, showing the implication of the fimbriae in the hemagglutination of E. tarda., 魚病研究 = Fish pathology 39(2) pp.87-93; 2004

長崎大学水産学部研究報告 v.84 p.53-63 , 2003

Presence of fimbrial genes among fish pathogenic and non-pathogenic strains of Edwardsiella tarda was investigated by polymerase chain reaction (PCR). Four primer sets for PCR were designed to detect the four genes (etfA, etfB, etfC, etfD) of the type 1 fimbrial gene cluster of E. tarda. All the four genes were successfully amplified with the four primer sets in all the pathogenic strains. On the other hand, any of etfA, etfB and etfC were not detected in 13 of 14 non-pathogenic strains. The results suggest that fimbriae play a role in the pathogenicity of E. tarda and that detection of etfA, etfB and etfC by PCR is useful to distinguish between pathogenic and non-pathogenic strains of E. tarda., 魚病研究 = Fish Pathology 42(2) pp.115-117; 2007