Yasutake Yanagihara

GENOMIC ANALYSIS OF BORRELIA JAPONICA SP NOV. ISOLATED FROM IXODES OVATUS IN JAPAN

Genetic characteristics of 12 Borrelia isolates from the tick, Ixodes ovatus, I. persulcatus, and the rodent, Apodemus speciosus ainu, in Japan were compared to members of the three genospecies of Borrelia burgdorferi sensu lato; B. burgdorferi sensu stricto, B. garinii and group VS461. The methods used in this study were the quantitative microplate DNA hybridization assay and restriction fragment length polymorphism (RFLP) analyses of the flagellin structural genes and the 16S rRNA genes. The six isolates from I. persulcatus and A. speciosus ainu were identified as genospecies B. garinii using RFLP analysis of the flagellin and 16S rRNA genes. In contrast, RFLP analysis of the six isolates from I. ovatus indicated that they were different from the three reported genospecies. DNA homology studies confirmed the RFLP results. The six isolates from I. ovatus had DNA homologies ranging from 85 to 99%, whereas DNA relatedness of the I. ovatus isolate with strains belonging to the three genospecies was 50 to 64%. These results suggest that the strains isolated from I. ovatus in Japan differ from the three genospecies and should be classified as a new genospecies of B. burgdorferi sensu lato. We propose that strains isolated from I. ovatus should be classified as B. japonica sp. nov.

Analysis of Borrelia species associated with Lyme disease by rRNA gene restriction fragment length polymorphism

We investigated the usefulness of rRNA gene restriction fragment length polymorphism (RFLP) for grouping of the Borrelia isolates associated with Lyme disease or from ixodid ticks. Genomic DNA was digested with a restriction enzyme, blotted and hybridized with an rrl (23S rRNA) gene probe. The sizes of the restriction bands showed a good correlation with the genotypes recently proposed, and Borrelia isolates of diverse geographic origin formed four distinct DNA groups. Group I included all of the USA isolates and some European isolates; group II contained European isolates and Asian isolates; group III comprised European and Asian isolates; group IV included Japanese isolates and an eastern Russian isolate. Groups I, II and III corresponded to Borrelia burgdorferi, B. garinii and group VS461, respectively. The RFLPs of Japanese isolates were rather divergent and some of the isolates were quite distinct from the USA and European isolates. RFLP analysis of the rRNA genes and flanking regions, using rrl gene probes as we reported here, may be useful in the taxonomic study of Borrelia.

Protective activity of glycolipid antigen against infection by Leptospira interrogans serovar canicola

A protective glycolipid antigen (PAg) was extracted from Leptospira interrogans serovar canicola with chloroform/methanol/water (1:2:0.8, by vol.) and partially purified by silica gel column chromatography. The PAg elicited a protective response in hamsters and in cyclophosphamide-treated mice subsequently challenged with homologous Leptospira. The PAg band was detected as a single smear-like band, corresponding to a protein of 23-30 kDa, by silver-staining in SDS-PAGE. In immunoblots, this band reacted with a monoclonal antibody, A5, which agglutinated serovar canicola and recognized a serovar-specific antigen. Furthermore, the PAg did not migrate on silica gel TLC, but was detected at the origin as a ninhydrin- and naphthol-positive spot. This suggests that PAg is a hydrophilic molecule with a carbohydrate chain that contains amino groups, possibly as amino sugars.

Biological Activities of Lipopolysaccharide-Like Substance (LLS) Extracted from Leptospira interrogans Serovar canicola Strain Moulton

The biological activities of lipopolysaccharide‐like substance (LLS) extracted from Leptospira interrogans serovar canicola strain Moulton by the hot phenol‐water method were studied in mice. The addition of 12.5 μg/ml or more of LLS fraction increased the incorporation of [3H] thymidine into in vitro cultured spleen cells of C57BL/6 mice, while the activity of the LLS fraction was about 20 times weaker than that of Salmonella typhimurium lipopolysaccharide (LPS). Pretreatment of murine spleen cells with rabbit anti‐mouse thymocyte antiserum did not diminish the mitogenic activity of leptospiral LLS, and the LLS could not increase the incorporation of [3H] thymidine into thymocytes, suggesting that LLS acts on a B‐lymphocyte population of lymphocytes. When sheep erythrocytes and LLS fraction were injected intraperitoneally into BALB/c mice, LLS exhibited an enhancing effect on antibody response in vivo. However, lethal toxicity of the LLS fraction was about 500 times lower than that of LPS in C57BL/6 mice loaded with galactosamine. No antitumor activity of leptospiral LLS (250–1,000 μg/mouse) against the ascites form of Ehrlich carcinoma in ddY mice was observed. The biological activities of the LLS fraction from the organism were weaker than those of gram‐negative bacterial LPS, suggesting that Leptospira possesses no typical LPS.

The 23S/5S ribosomal RNA genes (rrl/rrf) are separate from the 16S ribosomal RNA gene (rrs) in Borrelia burgdorferi, the aetiological agent of Lyme disease.

DNA fragments containing the rRNA genes for Borrelia burgdorferi strain B31 were cloned in bacteriophage lambda EMBL3. A restriction map of the fragments was constructed and the organization of the rRNA genes was determined by Southern hybridization. One genomic DNA fragment contained a single copy of the rrs sequence and another cloned fragment contained both rrl and rrf sequences. The results revealed that the rrs gene is located separately from the set of rrl/rrf genes, suggesting that these rRNA genes are expressed independently in B. burgdorferi.

Comparison of OspA Serotypes for Borrelia burgdorferi Sensu Lato from Japan, Europe and North America

Sixty‐one Borrelia burgdorferi sensu lato strains from various sources (ticks, human, and wild animals) in Japan and two strains from ticks in Far Eastern Russia were classified on the basis of reactivity with 16 monoclonal antibodies (mAb) to outer surface protein A (OspA) and by DNA‐DNA hybridization assay. Eleven OspA serotypes (J1 to J11) were recognized among the Japanese and the Far East Russian isolates (serotypes J1 to J9 were identified as B. garinii, serotype J10 was identified as B. afzelii, and serotype J11 corresponded to B. japonica), whereas 7 OspA serotypes for North American and European isolates previously reported (Bettina Wilske et al, J. Clin. Microbiol. 31:340‐350, 1993) were not observed except for OspA serotype 2 which showed identical reactivity with OspA serotype J10. This finding provides helpful information for understanding the geographical distribution of Lyme disease borrelia and the development of vaccine and diagnostic tests. In conclusion: 1. B. burgdorferi sensu stricto has not been observed in Japan, 2. Japanese B. afzelii isolates are closely related to those from Europe, 3. B. garinii isolates from Japan are highly heterogeneous and apparently different from European B. garinii isolates.

Vibrio trachuri sp. nov., a new species isolated from diseased Japanese horse mackerel.

A new species, Vibrio trachuri sp. nov., was isolated from the cultured Japanese horse mackerel (Trachurus japonicus). These Vibrio were Gram negative, motile rods and formed yellow colonies on BTB teepol and TCBS plate, turned TSI medium to yellow and was sensitive to 150 μM O/129 (2,4‐diamino‐6,7‐diisopropyl pteridine phosphate) like Listonella anguillarum which has been described as Vibrio anguillarum. However, the results of VP test and decarboxylation of lysine or dihydrolation of arginine suggested that these Vibrio are rather closely related to V. parahaemolyticus. DNA similarity determined by the microplate hybridization technique revealed that these Vibrio are genetically quite distant from Listonella anguillarum or V. parahaemolyticus and rather close to V. harveyi, although there was no Vibrio species which had more than 70% similarity value. From these results we propose to nominate Vibrio trachuri sp. nov. for this new Vibrio species.

Polymerase Chain Reaction Analysis of Borrelia Species Isolated in Japan

Primer reactivities of 25 Borrelia burgdorferi sensu lato isolates from the ticks, Ixodes persulcatus and I. ovatus, in Japan and 10 isolates in Europe and North America were investigated. The methods used in this study were the polymerase chain reaction (PCR) on the flagellin structural gene (fla), the outer surface protein A gene (osp A) and the outer surface protein B gene (osp B), and the restriction fragment length polymorphism (RFLP) analysis of PCR products from osp A and osp B, The flagellin PCR primer set reacted with all the Borrelia strains tested. Four genospecies, B. burgdorferi sensu stricto, B. garinii, B. afzelii and B. japonica, were differentiated by PCR using osp A and osp B primers combined with RFLP analysis. Some Japanese isolates from I. persulcatus were identified as B. garinii or B. afzelii. The other isolates from I. persulcatus did not fit in any of the 4 genospecies.

Anti-Cephalexin Monoclonal Antibodies and Their Cross-Reactivities to Cephems and Penams

Three cell lines producing monoclonal antibodies (MAbs), Cep1-2, 2-2 and 6, against cephalexin were established and the immunoglobulin class of the MAbs was IgM. The cross-reactions of the MAbs with penams and cephems were examined by enzyme-linked immunosorbent assay (ELISA). The cross-reactivities of Cep1-2 and 2-2 were scarcely influenced by the structures of acyl side chains of cephems and penams. The cross-reactivities of Cep1-2 were affected by the presence or absence of dihydrothiazolidine ring of cephem nucleus in hapten-protein conjugates which were prepared by alkaline method, MBS method and activated ester method but the cross-reactivities of Cep2-2 were not. The findings suggest that Cep1-2 recognize the degradate product(s) of cephem nucleus and Cep2-2 recognize a new antigenic determinant (NAD), which is formed by the conjugation of beta-lactam and carrier protein. On the other hand, the cross-reactivities of Cep6 were influenced by the structure of amino acyl side chain. It seems that Cep6 recognize specifically the acyl side chain at the C-7 of cephem. In ELISA inhibition test, three MAbs showed different inhibition pattern. The reaction of Cep1-2 with cephalexin-HSA was inhibited by cephalexin lysate. Cep2-2 and Cep6 were weakly inhibited by the binding to cephalexin-HSA by cephalexin lysate. Furthermore, the reactions of all MAbs were remarkably inhibited by penicillamine. The above results indicate that the MAbs can recognize at least three epitopes of the degradate product(s) of cephem nucleus, NAD and acyl side chain in cephalexin-protein conjugate.

Chemical compositions of cell walls and polysaccharide fractions of spirochetes.

Cellular polysaccharide fractions of various representative members of genera of the family Spirochaetaceae were obtained by the ammonium hydroxide extraction method. The sugar composition of the polysaccharide preparations was complex and many kinds of sugars such as rhamnose, fucose, ribose, xylose, mannose, galactose, and glucose were detected in all of the spirochetes tested. Of particular interest was the presence of 4‐O‐methylmannose as a constituent polysaccharide in members of the genus Leptospira. This sugar was not detected in the polysaccharides of Spirochaeta, Borrelia, and Treponema.