A Novel Plasmid Carrying Capsule Gene Cluster Found in Lactococcus garvieae Isolated from Filefish

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

Streptococcus iniaeの免疫原性における莢膜の役割

Streptococcus iniae causes an acute systemic disease in Japanese flounder Paralichthys olivaceus, one of the important cultured fish in Japan. Although commercial S. iniae vaccines have been developed and used in farms since 2005, the immunoprotection mechanism in vaccinated fish has not been elucidated yet. To verify the involvement of capsular polysaccharides of S. iniae in immunoprotection, we compared the protective efficacy of formalin-killed cells (FKC) of S. iniae NUF631 (capsulated) with those of its isogenic capsular-deleted mutants in S. iniae infection of Japanese flounder. As a result, high protection was achieved by immunization with NUF631 FKC but not with mutant FKC. Viable count of intravenously inoculated NUF631 decreased in the kidney of flounder immunized with NUF631 FKC, and NUF631 cells opsonized with anti-NUF631 flounder serum elevated the phagocytic activity and reactive oxygen species production of flounder peritoneal macrophages. In electron microscopic examination, electronically dense materials were observed on the capsule of NUF631 cells pretreated with the antiserum, and in western blot analysis flounder antibody was detected in the antiserum components bound to NUF631 cells. These findings indicate that capsular polysaccharides are important protective antigens and anti-capsule antibody plays a protective role as an opsonin in flounder S. iniae infection

Streptococcus iniae 莢膜欠損変異株におけるビルレンスの低下

Streptococcus iniae is known as an important bacterial pathogen causing serious damage in fish culture industry worldwide. Although the capsule of this pathogen is suggested to play a role in the disease process, it has not been verified yet. In this study we created isogenic uncapsulated mutants of S. iniae NUF631 by Tn916 transposon mutagenesis and investigated the role of capsule in the virulence and resistance to phagocytic activities of peritoneal macrophages of Japanese flounder Paralichthys olivaceus. Mutants transformed from K+ phenotype to K- phenotype were all uncapsulated. They exhibited neither K+ type-specific precipitation line in an immunodiffusion test nor ladder-like banding pattern characteristic for bacterial acidic polysaccharides in polyacrylamide gel electrophoresis. Their virulence to Japanese flounder was highly attenuated. In contrast to the mutant strains, the parent capsulated strain resisted phagocytic and bactericidal activities of the macrophages and prevented chemiluminescence reaction of the macrophages upon phagocytosis. From these results, it is verified that the S. iniae capsule is an important factor for intramacrophage survival of the pathogen

Variation in the Integration Site for Capsule Gene Cluster in the Genome among Strains of Lactococcus garvieae

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

病魚由来 Streptococcus iniae の血清学的性状

ヒラメ由来株を主体とした日本の病魚由来Streptococcus iniae の血清学的性状を検討した。供試菌株は抗S. iniae ウサギ血清との凝集性により, 2つのタイプに分かれ, それは莢膜の有無に依存していた。両タイプ (K+およびK-タイプ) の菌体オートクレーブ抽出物には共通抗原が検出され, K+タイプにはさらに型特異抗原が確認された。K+タイプは莢膜を保有し, ヒラメに対して強い毒力を示した。一方, K-タイプには型抗原や莢膜が認められず, 毒力も弱かった。Streptococcus iniae is known as an important marine and freshwater fish pathogen worldwide. In this paper, we characterized two serological phenotypes in Japanese S. iniae isolates, mainly from Japanese flounder Paralichthys olivaceus. The two phenotypes, designated K+ and K-, were distinguished by the presence or absence of capsule. K- cells agglutinated both with anti-K- and K+ type sera, whereas K+ cells agglutinated only with anti-K+ type serum. These two types were indistinguishable by any ordinary biological or biochemical characterization tests. Immunodiffusion test demonstrated the common antigens and a K+ type-specific antigen in the autoclave-extracts of cells. Capsule was observed on the K+ cells, from which acidic polysaccharides were detected. Strains of K+ type were virulent for Japanese flounder, whereas those of K- type were avirulent. There is a direct relationship between the serological phenotypes and the existence of capsule in S. iniae with link to its virulence