Toshiyuki Masuzawa

Serologic and Molecular Studies of Leptospira and Leptospirosis among Rats in the Philippines

Rats are known to be the most important reservoirs and transmission sources of leptospirosis. However, the status of leptospirosis in the Philippines regarding reservoirs and transmission remains unknown. A survey was conducted in Metro Manila and Laguna that analyzed samples obtained from 106 rats. Using the microscopic agglutination test, we found that 92% of rat serum samples were positive for anti-Leptospira antibodies; the most common infecting serovars were Manilae, Hebdomadis, and Losbanos. On the basis of pulsed-field gel electrophoresis and gyrase B gene sequence analyses, four groups of rat kidney isolates were found: L. interrogans serovar Manilae, serovar Losbanos, and serogroup Grippotyphosa, and L. borgpetersenii serogroup Javanica. Most isolates were lethal after experimental infection of golden Syrian hamsters. Results showed that these four Leptospira serovars and serogroups are circulating among rats, and that these animals may be one of the possible transmission sources of leptospirosis in the Philippines.

Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum, Francisella tularensis and their co-infections in host-seeking Ixodes ricinus ticks collected in Serbia

To evaluate the prevalence rate of tick-borne bacterial pathogens, unfed adult Ixodes ricinus ticks were collected from vegetation in 2001, 2003, and 2004 at 18 localities throughout Serbia. A total of 287 ticks were examined by PCR technique for the presence of Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum, and Francisellatularensis. The highest prevalence rate was that for B. burgdorferi sensu lato (42.5%), followed by A. phagocytophilum (13.9%) and F. tularensis (3.8%). The presence of five B. burgdorferi sensu lato genospecies, namely, B. burgdorferi sensu stricto, B. afzelii, B. garinii, B. lusitaniae, and B. valaisiana was identified by restriction fragment length polymorphism (RFLP) analysis. The most frequent B. burgdorferi sensu lato genospecies was B. lusitaniae, followed by B. burgdorferi sensu stricto. Co-infection by B. burgdorferi sensu stricto and B. lusitaniae was frequently observed. Co-infection by B. burgdorferi sensu lato and A. phagocytophilum and co-infection by B. burgdorferi sensu lato and F. tularensis appeared in 24 ticks. Sequencing of p44/msp2 paralogs of Serbian A. phagocytophilum showed that they were unique and distinct from those of A. phagocytophilum in US and UK. This is the first report of B. garinii, B. lusitaniae, B. valaisiana, as well as A. phagocytophilum and F. tularensis infected ticks in Serbia. These findings indicate a public health threat in Serbia of tick-borne diseases caused by B. burgdorferi sensu lato, A. phagocytophilum and F. tularensis.

Prevalence of Anaplasma phagocytophilum and its coinfection with Borrelia afzelii in Ixodes ricinus and Ixodes persulcatus ticks inhabiting Tver Province (Russia) - a sympatric region for both tick species

Human granulocytic anaplasmosis (HGA) and Lyme borreliosis (LB) are tick-borne infectious diseases caused by Anaplasma phagocytophilum and Borrelia burgdorferi sensu lato species, respectively. In this study, p44/msp2 paralogues specific to A. phagocytophilum and 5S-23S rRNA gene-intergenic spacers specific to B. burgdorferi sensu lato species were detected by PCR in ticks collected in two regions, Tver (Kalinin) and Konakovo, of the Tver (Kalinin) Province located 150 km north-west of Moscow. The PCR amplicons obtained were further characterized by sequencing and RFLP analysis. In the total of 199 ticks collected, 8.8 % (7/80) and 33.8 % (27/80) of Ixodes ricinus, and 2.5 % (3/119) and 45.4 % (54/119) of Ixodes persulcatus, were found to be infected with A. phagocytophilum and B. burgdorferi sensu lato spp., respectively. Of those 199 ticks, 5 (2.5 %) were coinfected with A. phagocytophilum and Borrelia afzelii. Phylogenetic analysis revealed unique p44/msp2 paralogous genes in A. phagocytophilum-infected Russian ticks. The sequence similarities with those of A. phagocytophilum in the United States, UK and Japan ranged from 42 % to 80.4 %, and there were no sequences showing more than 90 % similarity with those sequences from the other countries. The results showed that the p44/msp2 sequence similarity groups may provide an index of adaptation of A. phagocytophilum strains to specific vector ticks or reservoir hosts in different countries and areas. These findings suggest that there is a public health threat from HGA and LB in Tver Province surrounding Moscow.

Multilocus Sequence Typing Implicates Rodents as the Main Reservoir Host of Human-Pathogenic Borrelia garinii in Japan

ABSTRACT Multilocus sequence typing of Borrelia garinii isolates from humans and comparison with rodent and tick isolates were performed. Fifty-nine isolates were divided into two phylogenetic groups, and an association was detected between clinical and rodent isolates, suggesting that, in Japan, human-pathogenic B. garinii comes from rodents via ticks.

PCR and Culture Identification of Pathogenic Leptospira spp. from Coastal Soil in Leyte, Philippines, after a Storm Surge during Super Typhoon Haiyan (Yolanda)

ABSTRACT Leptospirosis is a zoonosis caused by pathogenic Leptospira spp. Most of the outbreaks of leptospirosis occur after floods caused by heavy rain in countries where Leptospira spp. are endemic. It has been believed that the overflow of seawater rarely causes outbreaks of leptospirosis because the leptospires are killed by salt water. On 8 November 2013, a storm surge caused by Super Typhoon Haiyan (Yolanda) inundated the entire coastal areas of Tacloban and Palo in Leyte, Philippines. The present study was carried out in order to determine whether the environmental leptospires in soil were able to survive after the storm surge in the affected areas. We collected 23 wet soil samples along the coastal areas of Tacloban and Palo 2 months after the storm surge. The samples were suspended in HEPES buffer, and the supernatants were cultured in liquid or semisolid Korthofs medium supplemented with five antimicrobial agents to inhibit the growth of contaminants. Leptospires were isolated from primary cultures of 22 out of 23 samples. The DNA of pathogenic Leptospira species was detected in 11 samples (47.8%) by analysis of flaB by nested PCR. Eventually, two pathogenic Leptospira strains were isolated and showed the highest 16S rRNA gene sequence similarity to Leptospira kmetyi. When these isolates were experimentally mixed with soil, they were found to survive in seawater for 4 days. These results show the possibility that leptospires living in soil survived after the storm surge. Our findings may serve as a warning that when seawater inundates the land during a storm surge or a tsunami, an outbreak of leptospirosis could occur in the disaster-stricken area.

Structural analysis of a p44/msp2 expression site of Anaplasma phagocytophilum in naturally infected ticks in Japan.

Anaplasma phagocytophilum, an agent of human granulocytic anaplasmosis, infects neutrophils and causes an emerging tickborne febrile disease. The genome of this bacterium contains a large number of p44/msp2-related genes encoding 44 kDa major outer-membrane proteins, and it is known that a specific p44/msp2 gene is predominantly transcribed from a single expression locus. This study successfully characterized the genomic expression site for p44/msp2 (3.8 kb) in uncultured A. phagocytophilum from Ixodes persulcatus ticks inhabiting a northern part of Japan. Comparative analysis of the sequences revealed that the structures of the expression sites in Japanese A. phagocytophilum were similar to those of US strains from human patients and European strains from a dog and sheep, but omp-1N (upstream from p44/msp2) and a truncated recA (downstream from p44/msp2) in the p44/msp2 expression site seemed to share similarities with those of US and European strains. The central hypervariable region sequences of Japanese p44/msp2 were found to be quite diverse (24.4-100 % amino acid similarities) and distinct from their closest relatives from US human patients or animal host origins (56.3-97.6 % amino acid similarities) with some exceptions. Thus, this study provides significant information about the molecular characteristics of A. phagocytophilum in East Asia, as well as the global diversity of p44/msp2.

Brazilian propolis ameliorates trinitrobenzene sulfonic acid-induced colitis in mice by inhibiting Th1 differentiation

BACKGROUNDnPropolis is a resinous substance collected by honeybees from leaf buds and cracks in the bark of various plants. We previously demonstrated the inhibitory activity of Brazilian propolis ethanolic extract against the differentiation process of Th17 cells and pathogenesis of collagen-induced arthritis, which is an experimental animal model of rheumatoid arthritis. Th1 cells are also involved in several autoimmune and inflammatory diseases, such as inflammatory bowel disease. In the present study, we demonstrated for the first time that Brazilian propolis significantly inhibited the generation of Th1 cells. Furthermore, we hypothesized that the administration of propolis to a murine model of colitis would suppress Th1 cell differentiation and ultimately ameliorate colitis.nnnMATERIALS AND METHODSnCD4 T cells were stimulated under Th1-polarizing conditions (immobilized anti-CD3 and anti-CD28 antibodies with IL-12 plus an anti-IL-4 monoclonal antibody for 5days) with or without the propolis ethanolic extract. Cells were characterized for helper T cell subsets by flow cytometric analysis. Furthermore, we investigated the effects of propolis on 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis in a mouse model.nnnRESULTSnPropolis dose-dependently reduced the frequency of IFNγ-producing CD4 T cells under Th1-polarizing conditions. Furthermore, the inhibitory effect of propolis on Th1 differentiation was demonstrated in vivo. The severity of colitis in mice fed propolis was significantly lower than that of mice fed the control diet.nnnCONCLUSIONnThese findings suggest that the suppressive effect of Brazilian propolis on Th1 differentiation may be useful in controlling unbalanced cytokine networks in autoimmune diseases.

Borrelia yangtzensis sp. nov., a rodent-associated species in Asia, is related to Borrelia valaisiana

Twenty-nine isolates of Lyme borreliosis (LB) group spirochaetes collected from ticks and rodents in China and Japan were included in a multilocus sequence analysis (MLSA). Using a different typing system, three of these strains had previously been identified as being divergent from other LB spirochaete species and the name Borrelia yangtze sp. nov. was proposed. The data presented here confirm that the genetic distance, calculated using sequences of MLSA housekeeping genes, to other known LB group spirochaete species was <u200995u2009% and to Borrelia valaisiana was 96.67u2009% (which represents the most closely related species within the group of LB spirochaetes). This and the fact that these strains are ecologically distinct from B. valaisiana (rodent-transmitted vs bird-transmitted) provide strong support for the validation of the proposed species status. We suggest the name Borrelia yangtzensis sp. nov. The type strain is Okinawa-CW62T (u2009=u2009DSM 24625Tu2009=u2009JCM 17189T).Twenty nine isolates of Lyme borreliosis (LB) group spirochetes collected from ticks and rodents in China and Japan were included into multilocus sequence analysis (MLSA). Using a different typing system, three of these strains had previously been identified as being divergent from other LB spirochete species and the name Borrelia yangtze sp. nov. was proposed. The data presented here confirm that the genetic distance calculated using sequences of MLSA housekeeping genes to other known LB group spirochete species was <95 % and to B. valaisiana 96.67 % (which represents the closest related species within the group of LB spirochetes). This and the fact that these strains are ecologically distinct from B. valaisiana (rodent transmitted vs bird transmitted) provide strong support for the validation of the proposed species status. We suggest the name B. yangtzensis sp. nov.. The type strain is Okinawa CW62T (= DSM 24625 T = JCM 17189 T).

Brazilian propolis inhibits the differentiation of Th17 cells by inhibition of interleukin-6-induced phosphorylation of signal transducer and activator of transcription 3

Propolis is a resinous substance collected by honeybees from leaf buds and cracks in the bark of various plants. It has been reported to show immunomodulatory activity. Previously, we reported the protective effect of Brazilian propolis ethanolic extract against the pathogenesis of collagen-induced arthritis (CIA), an experimental animal model of rheumatoid arthritis (RA). Moreover, we found that the protective effect against CIA was involved in suppression of the production of interleukin-17 (IL-17) in CIA mice. In the present study, we demonstrated for the first time that propolis inhibited IL-6 plus transforming growth factor-β induced Th17 differentiation in vitro. Propolis also suppressed the IL-6-induced phosphorylation of signal transducer and activator of transcription 3 (STAT3), a cytokine-activated essential transcription factor in Th17 development, concomitantly with the enhanced suppressor of cytokine signaling 3 expression involved in the downregulation of STAT3 phosphorylation. These data suggest that the suppressive effect of propolis on Th17 differentiation might be useful for controlling unbalanced cytokine networks in autoimmune diseases.

Molecular detection of Anaplasma phagocytophilum and Borrelia burgdorferi in Ixodes ricinus ticks from Istanbul metropolitan area and rural Trakya (Thrace) region of north-western Turkey.

We demonstrated the presence of the agent of human granulocytic anaplasmosis (HGA), Anaplasma phagocytophilum, and the agent of Lyme borreliosis, Borrelia burgdorferi sensu lato, in north-western Turkey. A total of 241 questing Ixodes ricinus adult ticks were sampled by flagging from recreational parks of the Asiatic and European sides of the heavily populated Istanbul metropolitan area and rural forests of Kirklareli located in the Thrace region in 2008. Both tick-borne pathogens were detected and identified by PCR and DNA sequencing analysis. A. phagocytophilum infection rates were 2.7% in Istanbul and 17.5% in the Kirklareli area. B. burgdorferi sensu lato infection rates were 38.7% in Istanbul and 11.4% in the Kirklareli area. Only 3 of 241 ticks were coinfected with A. phagocytophilum and B. burgdorferi sensu lato.