Yukita Sato

Phylogenetic Analysis of Borrelia Species Based on Flagellin Gene Sequences and Its Application for Molecular Typing of Lyme Disease Borreliae

We determined almost complete flagellin gene sequences of various Borrelia species and aligned them with previously published sequences. A neighbor-joining phylogenetic analysis showed that the genus Borrelia was divided into the following three major clusters: New World relapsing fever borreliae (Borrelia turicatae, Borrelia parkeri, and Borrelia hermsii), Old World relapsing fever borreliae (Borrelia crocidurae, Borrelia duttonii, and Borrelia hispanica), and Lyme disease borreliae (Borrelia burgdorferi sensu stricto, Borrelia garinii, and Borrelia afzelii). Agents of animal spirochetosis (Borrelia coriaceae and Borrelia anserina) and species of unknown pathogenicity (Borrelia miyamotoi and Borrelia lonestari) were related to relapsing fever borreliae. Although the Lyme disease borreliae, two related species (Borrelia japonica and Borrelia andersonii), and some newly described genomic groups (groups PotiB2, VS116, DN127, Hk501, and Ya501) were closely related to each other, each taxon formed an independent branch on the phylogenetic tree. The data obtained in this study indicate that the flagellin genes are useful in Borrelia taxonomy. To distinguish the Lyme disease borreliae from related organisms easily, we designed an oligonucleotide primer set for the flagellin gene and performed a PCR-restriction fragment length polymorphism (PCR-RFLP) analysis. The primer set amplified an approximately 580-bp DNA fragment that included species-specific restriction sites, and HapII, HhaI, CelII, HincII, or DdeI digestion of the product resulted in distinctively different PCR-RFLP patterns. The PCR-RFLP typing method which we developed should facilitate rapid identification of Lyme disease borreliae and related organisms obtained from biological and clinical specimens.

Antibody production in Syphacia obvelata infected mice

Antibody response to Syphacia obvelata infection was observed in AKR/J mice by ELISA. Experimental infection with the pinworm eggs showed the presence of specific IgG against S. obvelata somatic antigens at 12 days postinfection, and that it increased steadily thereafter. Sera of S. obvelata-infected mice showed cross-reactivity with somatic antigens of other Syphacia species such as S. mesocriceti and S. muris, but not with Aspiculuris asiatica. Western blotting of S. obvelata antigen with sera of S. obvelata-infected mice showed a corresponding increase in the number of bands during the course of infection. Infected mice showed significantly higher antibody production to sheep red blood cells than the uninfected control mice. Thus, S. obvelata infection is shown to alter the humoral response to nonparasitic antigenic stimuli. These observations indicate that infection by helminths, which apparently do not produce clinical symptoms, might modulate the immune system of the host and, therefore, affect experimental results.

Genetic Diversity of Borrelia burgdorferi Sensu Lato Isolated in Far Eastern Russia

One‐hundred and fifty‐seven Borrelia isolated from adult ticks, Ixodes persulcatus, and wild rodents, Clethrionomys rufocanus and Apodemus peninsulae, in the far eastern part of Russia were characterized and identified by restriction fragment length polymorphism (RFLP) of the 5S‐23S rRNA intergenic spacer. Some isolates showed unique RFLP patterns and were determined as Borrelia garinii on the basis of a sequence analysis of the intergenic spacer amplicon and reactivity with species‐specific monoclonal antibodies (MAbs). 86.5 and 12.7% of the tick isolates, and 74.2 and 12.9% of the rodent isolates were determined as Borrelia garinii and Borrelia afzelii, respectively, but no Borrelia burgdorferi sensu stricto was detected. This finding is similar to the results obtained from Borrelia surveys of I. persulcatus and wild rodents in Hokkaido, Japan.

Competence of a migratory bird, red-bellied thrush (Turdus chrysolaus), as an avian reservoir for the Lyme disease spirochetes in Japan.

To evaluate the competence of migratory birds as reservoirs for the Lyme disease spirochetes, we examined two species of migrants, Red-bellied thrush (Turdus chrysolaus) and Black-faced bunting (Emberiza spodocephala) in Nemuro, the northern part of Japan. Spirochetes were found in four individual birds out of 11 T. chrysolaus, three isolates were detected from the skins and the other one was obtained from the liver. No spirochete was found to be infected in 20 E. spodocephala. As far as we know, this is the first record of direct detection of the spirochetes from migratory birds in Japan. The spirochetes were also isolated from immature ixodid ticks, Ixodes persulcatus, fed on those species of birds. The spirochetes were transmitted trans stadially to the next stage, when infected ticks molted. All of the isolates from birds and ticks were identified as Borrelia garinii by our ribotyping and flagellin gene sequence analyses. Our results strongly suggest that the migratory birds are reservoirs in the transmission of the Lyme disease spirochetes in Japan.

Transmission of the Lyme disease spirochete, Borrelia garinii, between infected and uninfected immature Ixodes persulcatus during cofeeding on mice

The ixodid tick, Ixodes persulcatus, serves as a vector for the Lyme disease spirochete, Borrelia garinii, in Japan. Transmission of the spirochete from infected nymphs to uninfected larvae during their cofeeding on mice was studied under laboratory conditions. Using feeding chambers, infected nymphs and uninfected larvae were allowed to cofeed on heads of normal BALB/c mice. In another group of mice, we separately exposed nymphs to the head and larvae to the back. The resultant engorged larvae were reared and the molted nymphs were examined for spirochetes. Spirochetal infections were found only in ticks that had fed together with infected ticks. The data strongly suggest that spirochetes can migrate from infected feeding ticks to their uninfected neighbors by way of host skin. Moreover, nymphs that had become infected through the cofeeding process could transmit spirochetes to mice.

Rapid diagnosis of lyme disease: Flagellin gene-based nested polymerase chain reaction for identification of causative Borrelia species

Abstract Objective: Each of Borrelia burgdorferi sensu stricto, Borrelia garinii , and Borrelia afzelii has characteristic restriction sites in its flagellin gene. The authors focused on this gene and developed a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis for rapid diagnosis of Lyme disease. Methods: External and internal primer sets were designed for nested PCR to amplify an approximately 580 by fragment of the flagellin gene that includes species-specific restriction sites. DNA extracted from tissue samples of mice and humans were used as templates for PCR. The amplicons obtained were digested with Hap II, Hha I, Cel II Hinc II, or Dde l endonuclease. Results: In mice experimentally infected with each of B. burgdorferi sensu stricto, B. garinii , and B. afzelii , borrelial DNA was detected irrespective of differences in the causative species. However, RFLP of the amplicons was able to identify the species. Skin biopsy samples from 11 Japanese patients with erythema migrans were subjected to both PCR and culture tests. Borrelial infections were detected in seven cases (64%) by PCR and eight cases (73%) by culture. All PCR-positive samples were also positive by culture. The causative species in human infections was easily identified as B. garinii by RFLP analysis of the amplicons. Conclusion: The nested PCR-RFLP system appears to be an easy and reliable diagnostic tool for the detection and species identification of borreliae in human cutaneous biopsies.

Refeeding activity of immature ticks of Ixodes persulcatus and transmission of lyme disease spirochete by partially fed larvae

The refeeding activity of immature ticks of Ixodes persulcatus was studied under laboratory conditions. BALB/c mice were used as blood sources. Feeding larvae and nymphs spontaneously detached themselves from parasitized mice when the mice were killed 1 or 2 days after tick exposure. Partially fed ticks were reexposed on other mice. Both larvae and nymphs were able to refeed on mice. The resultant engorged ticks normally molted to the next developmental stages. We also examined the transmission of the Lyme disease spirochete, Borrelia garinii, by bites of partially fed ticks. Larvae that had fed on infected mice for 1 or 2 days transmitted the spirochete to mice by refeeding.

Cystic metacestodes of a rat-adapted Taenia taeniaeformis established in the peritoneal cavity of scid and nude mice

In vitro-hatched (but not activated) oncospheres of a rat-adapted strain of Taenia taeniaeformis intraperitoneally inoculated into severe combined immunodeficiency (scid), congenitally athymic (nude) and immunocompetent (normal) female BALB/c mice developed into cystic metacestodes in the peritoneal cavity (but not in the liver) of scid and nude mice exclusively. This suggests that cystic metacestodes of this parasite, usually harboured in the liver only, can establish in scid and nude mice provided that the oncospheres are inoculated into the peritoneal cavity. Immunodeficient mice, especially scid mice, may be a good experimental animal model for the intermediate host of any taeniid species, of human, domestic- or wild-animal origin.

Experimental infection of larval Echinococcus multilocularis in the rodent brain as a model for cerebral alveolar echinococcosis

Experimental infection of larval Echinococcus multilocularis in the rodent brain was attempted to establish a murine model for cerebral alveolar echinococcosis. Balb/c mice and jirds were injected intracranially with 10% of a homogenated hydatid cyst mass. Small cystic larvae were observed macroscopically in the cranial cavity 1, 2 and 5 months post-infection in both mice and jirds. Some larval cysts from both rodents contained mature or immature protoscoleces. In mice, the laminated layer was found in the lateral ventricle 2 months post-infection but without protoscoleces. At five months post-infection, larger larval cysts were found in the cranial cavity of a mouse, which also demonstrated partial palsy of the legs. A laminated layer with mature protoscoleces was observed in the third ventricle and the mouse also harboured, in the left lung, a larval cyst containing protoscoleces surrounded by lymphocytes. Jirds were also found to be infected with metacestodes in the cranial cavity, but neither unusual behaviour nor establishment of cysts inside the brain was observed in jirds during the course of infection.