Takuya Itou

Genetic diversity of bat rabies viruses in Brazil

SummaryThirty-three Brazilian bat rabies viruses (RVs) were studied by sequence analysis and were compared against sequences of bat-related RVs from other regions of the Americas. Phylogenetic analysis revealed that bat-related RVs formed several monophyletic lineages and that these were associated with bat species. Brazilian bat RVs were found to include nine major lineages, one of which grouped with RVs isolated from Lasiurus spp. from different regions of the Americas. These results suggest that there is considerable diversity among Brazilian bat RV variants and that some of these RV variants may be associated with bats from other countries.

Kinetics of oxygen metabolism during respiratory burst in Japanese eel neutrophils.

Two oxygen radicals, superoxide and hydrogen peroxide, were measured by ferricytochrome C reduction and scopoletin fluorescent assays to investigate oxygen metabolism during the respiratory burst in Japanese eel neutrophils. Maximal superoxide production was obtained using 0.1 microgram/mL of PMA. Oxygen consumption was almost equivalent to superoxide production, indicating that the consumed oxygen was almost fully converted to superoxide in eel neutrophils. Hydrogen peroxide production was approximately half that of oxygen consumption or superoxide production in neutrophils elicited by killed bacteria or casein. The amounts of superoxide and hydrogen peroxide production, by neutrophils elicited by irritants, were about 11.5 and 5.0 nM/10(7) cells/ min, respectively, which were significantly higher than those of unelicited neutrophils (2.4 and 0.3 nM/10(7) cells/min, respectively).

Discrimination between dog-related and vampire bat-related rabies viruses in Brazil by strain-specific reverse transcriptase-polymerase chain reaction and restriction fragment length polymorphism analysis

BACKGROUND There is a geographical overlap between the two main rabies epidemiological cycles maintained by dogs and vampire bats in Latin America. The geographical and temporal coincidence of rabies outbreaks of respective origins is not unusual in rural areas of Latin America. These circumstances make it difficult to discriminate the intraspecies and interspecies transmission pathways of rabies. OBJECTIVE This study was conducted to develop techniques to discriminate dog-related and vampire bat-related rabies virus isolates (DRRV and VRRV, respectively) in Brazil. STUDY DESIGN The 1396 nucleotides of the nucleoprotein gene of a total of 27 DRRV and VRRV were sequenced. Strain-specific (SS) primers were developed based on these sequences. Forty-nine rabies virus strains isolated from animals and humans in several parts of Brazil were examined by reverse transcriptase-polymerase chain reaction (RT-PCR) with SS primers. These rabies viruses were also amplified by RT-PCR with general rabies primers and the PCR products were cut by three restriction enzymes, Blp I, Bsu36 I and BspE I. RESULTS All the DRRV and VRRV were distinguished by RT-PCR with SS primers. The PCR products obtained from DRRV were cut at one site by Blp I, but not by Bsu36 I. The PCR products obtained from VRRV were cut at one or two sites by Bsu36 I, but not by Blp I. Blp I and Bsu36 I clearly discriminated DRRV and VRRV in restriction fragment length polymorphysim (RFLP) assays. The results of SS RT-PCR and RFLP were consistent. CONCLUSION SS RT-PCR and RFLP assays have been developed for determining the origins of rabies virus isolates in Brazil. These assays are simple and rapid, and will be useful for identifying the rabies virus reservoirs of field isolates in Brazil, especially when used together.

Changes in Activation States of Murine Polymorphonuclear Leukocytes (PMN) during Inflammation: a Comparison of Bone Marrow and Peritoneal Exudate PMN

ABSTRACT To study different activation states in polymorphonuclear leukocytes (PMN) in mice, we compared the function of murine PMN obtained from the bone marrow (BMPMN) with those of PMN obtained by intraperitoneal induction with thioglycolate (TGPMN) or uric acid (UAPMN). When stimulated with chemotactic peptides, e.g., formyl-methionyl-leucyl-phenylalanine (fMLF), WKYMVM, or WKYMVm, the TGPMN and UAPMN showed greatly enhanced generation of reactive oxygen species (ROS) compared with BMPMN, which suggests that exudation to the peritoneum per se induces a primed state in the cells. The WKYMVm peptide was the most potent stimulant of ROS generation, and it desensitized for subsequent stimulation with fMLF or WKYMVM. This desensitization was broken by the addition of cytochalasin B. The TGPMN and UAPMN appeared to be fully primed, since no increase in response was induced by pretreatment with tumor necrosis factor α (TNF-α). In contrast, the BMPMN response was increased 2.5- to 3-fold. The differences in oxidative responses were supported by degranulation studies. Preincubation with TNF-α promoted CR3 expression on BMPMN, and this level of expression was also enhanced by WKYMVm. In contrast, CR3 expression on untreated TGPMN and UAPMN was already similar to that on TNF-α-primed BMPMN and could be only slightly enhanced by TNF-α treatment. Taken together, these results indicate that BMPMN are in a resting state and have the capacity to become primed, while peritoneal exudate PMN are already fully primed upon isolation. These results have major implications for murine neutrophil research and show the importance of defining which PMN subsets to use when investigating murine models.

Prevalence of calf diarrhea caused by bovine group A rotavirus carrying G serotype 8 specificity.

One hundred and seventeen rectal fecal specimens were collected in 1995 and 1996 from calves with diarrhea in Kagoshima Prefecture in Japan. The bovine group A rotavirus was detected by enzyme immunoassay in 43 of 117 specimens and isolated from 33 of the 43 specimens that were positive. G serotype, P serotype, and P genotype of 33 isolates were identified by reverse transcription-polymerase chain reaction, and 20 of 33 isolates (60.6%) were identified as G serotype 8. Thus, we discovered that calf diarrhea caused by bovine group A rotavirus carrying G serotype 8 specificity was prevalent in this research area during this research period. To our knowledge, this is the first report on the prevalence of calf diarrhea caused by the bovine group A rotavirus carrying G serotype 8 specificity.

Changes in the prevalence of rotavirus G and P types in diarrheic calves from the Kagoshima prefecture in Japan

G8 bovine group A rotavirus was the most predominant serotype in calf diarrheal fecal specimens examined between 1995 and 1996 in Japan [Vet. Microbiol. 66 (1999) 301]. To date, no evidence that G8 was the most predominant in the typeable specimens has been observed outside Japan. To investigate whether G8 continues to be as common as G6 and G10, the incidence of the main serotypes was determined in the same area (Kagoshima prefecture) between 1997 and 1998 by reverse transcription-polymerase chain reaction. From a total of 104 rotavirus-positive specimens, we successfully identified G type in 79 (76.0%) and P type in 71 (68.3%). The combination of G and P types varied periodically; in 1995, G10P[11] was most common. The most predominant types changed drastically in 1996, and G8 and mixed P types were the most predominant. A dramatic shift of the most predominant type occurred again in 1997 when G6P[5] was most common. This frequency of G6P[5] also continued in 1998. These results suggest that the serotypes prevailing in certain areas change periodically.

Flow cytometric analysis of the neutrophil respiratory burst of ayu, Plecoglossus altivelis: comparison with other fresh water fish.

Neutrophils of vertebrates undergo respiratory burst activity (RBA) as a defense mechanism against bacterial infections. We report here that ayu (Plecoglossus altivelis) have unusually high RBAs even when they are in a healthy condition. Kidney and blood leukocytes were obtained from ayu, rainbow trout (Oncorhynchus mykiss), carp (Cyprinus carpio), eel (Anguilla japonica), and pond smelt (Hypomesus nipponensis). Neutrophil RBA was measured by flow cytometry using dihydrorhodamine after stimulation with phorbol myristate acetate. The amount of RBA of neutrophils from both blood and kidney was significantly higher in ayu than in the other species (e.g. the fluorescence intensity of ayu blood neutrophils was about 3-7 times higher than that from trout and carp, and that of ayu kidney neutrophils was 2-19 times higher than that of rainbow trout, carp, eel, and pond smelt). This unique character of ayu neutrophils was invariable even at different ages, locations, and sex-maturation stages.

Molecular and geographic analyses of vampire bat-transmitted cattle rabies in central Brazil

BackgroundVampire bats are important rabies virus vectors, causing critical problems in both the livestock industry and public health sector in Latin America. In order to assess the epidemiological characteristics of vampire bat-transmitted rabies, the authors conducted phylogenetic and geographical analyses using sequence data of a large number of cattle rabies isolates collected from a wide geographical area in Brazil.MethodsPartial nucleoprotein genes of rabies viruses isolated from 666 cattle and 18 vampire bats between 1987 and 2006 were sequenced and used for phylogenetic analysis. The genetic variants were plotted on topographical maps of Brazil.ResultsIn this study, 593 samples consisting of 24 genetic variants were analyzed. Regional localization of variants was observed, with the distribution of several variants found to be delimited by mountain ranges which served as geographic boundaries. The geographical distributions of vampire-bat and cattle isolates that were classified as the identical phylogenetic group were found to overlap with high certainty. Most of the samples analyzed in this study were isolated from adjacent areas linked by rivers.ConclusionThis study revealed the existence of several dozen regional variants associated with vampire bats in Brazil, with the distribution patterns of these variants found to be affected by mountain ranges and rivers. These results suggest that epidemiological characteristics of vampire bat-related rabies appear to be associated with the topographical and geographical characteristics of areas where cattle are maintained, and the factors affecting vampire bat ecology.

Molecular epidemiology of rabies from Maranhão and surrounding states in the northeastern region of Brazil.

Summary.Although many outbreaks of rabies have been reported in northern Brazil, few epidemiological studies of these outbreaks have been undertaken. In this study, molecular epidemiological analyses were performed using 41 rabies virus samples isolated in the Maranhão (MA), Pará (PA), and Tocantins (TO) states of northeastern Brazil. A 599-bp region of the glycoprotein (G) gene was first amplified from each sample by RT-PCR, then sequenced and subjected to phylogenetic analysis. A phylogenetic tree divided the 41 isolates into two clades: Clade I was associated with terrestrial carnivores and Clade II was associated with vampire bats. The Clade I isolates were further sub-divided into two groups. The first group was closer to carnivore isolates that predominate in central Brazil, whereas the second group more closely resembled wild fox isolates from the northeastern coastal state of Paraíba (PB). MA isolates of Clade II formed an entirely separate group. These results demonstrate that bat- and dog-transmitted rabies occur in northwestern Brazil.

A method for simultaneous detection and identification of Brazilian dog- and vampire bat-related rabies virus by reverse transcription loop-mediated isothermal amplification assay.

At present, the sporadic occurrence of human rabies in Brazil can be attributed primarily to dog- and vampire bat-related rabies viruses. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) was employed as a simultaneous detection method for both rabies field variants within 60 min. Vampire bat-related rabies viruses could be distinguished from dog variants by digesting amplicons of the RT-LAMP reaction using the restriction enzyme AlwI. Amplification and digestion could both be completed within 120 min after RNA extraction. In addition, the RT-LAMP assay also detected rabies virus in isolates from Brazilian frugivorous bats and Ugandan dog, bovine and goat samples. In contrast, there were false negative results from several Brazilian insectivorous bats and all of Chinese dog, pig, and bovine samples using the RT-LAMP assay. This study showed that the RT-LAMP assay is effective for the rapid detection of rabies virus isolates from the primary reservoir in Brazil. Further improvements are necessary so that the RT-LAMP assay can be employed for the universal detection of genetic variants of rabies virus in the field.