Virulence of Francisella tularensis Subspecies holarctica Biovar japonica and Phenotypic Change during Serial Passages on Artificial Media.

Francisella tularensis (F. tularensis) is the etiological agent of the zoonotic disease tularemia. F. tularensis subspecies holarctica biovar japonica has rarely been isolated in Japan and is considered to have moderate virulence, although the biological properties of fresh isolates have not been analyzed in detail. Here, we analyzed the virulence of two strains of F. tularensis subspecies holarctica biovar japonica (NVF1 and KU-1) and their phenotypic stability during serial passages in Eugon chocolate agar (ECA) and Chamberlain's chemically defined medium (CDM) based agar (CDMA). C57BL/6 mice intradermally inoculated with 101 colony-forming units of NVF1 or KU-1 died within 9 days, with a median time to death of 7.5 and 7 days, respectively. Both NVF1 and KU-1 strains passaged on ECA 10 times had comparable virulence prior to passaging, whereas strains passaged on ECA 20 times and on CDMA 50 times were attenuated. Attenuated strains had decreased viability in 0.01% H2O2 and lower intracellular growth rates, suggesting both properties are important for F. tularensis virulence. Additionally, passage on ECA of the KU-1 strains altered lipopolysaccharide antigenicity and bacterial susceptibility to ß-lactam antibiotics. Our data demonstrate F. tularensis strain virulence in Japan and contribute to understanding phenotypic differences between natural and laboratory environments.

Protective effects of the Francisella tularensis ΔpdpC mutant against its virulent parental strain SCHU P9 in Cynomolgus macaques.

Tularemia is a severe infectious zoonotic disease caused by Francisella tularensis. Although F. tularensis is considered to be a potential biological weapon due to its high infectivity and mortality rate, no vaccine has been currently licensed. Recently, we reported that F. tularensis SCHU P9 derived ΔpdpC strain lacking the pathogenicity determinant protein C gene conferred stable and good protection in a mouse lethal model. In this study, the protective effect of ΔpdpC was evaluated using a monkey lethal model. Two cynomolgus macaques (Macaca fascicularis) intratracheally challenged with the virulent strain SCHU P9 were euthanized on 7 and 11 days post-challenge after the development of severe clinical signs. The bacterial replication in alveolar macrophages and type II epithelial cells in the lungs would cause severe pneumonia accompanied by necrosis. Conversely, two animals subcutaneously immunized with ΔpdpC survived 3 weeks after SCHU P9 challenge. Though one of the two animals developed mild symptoms of tularemia, bacterial replication was limited in the respiratory organs, which may be due to a high level of humoral and cellular immune responses against F. tularensis. These results suggest that the ΔpdpC mutant would be a safe and promising candidate as a live attenuated tularemia vaccine.

Evaluating the contact rate between companion dogs during dog walking and the practices towards potential cases of rabies among dog owners in Japan.

This study aimed to examine the habits of dog walking in Japan using an internet survey of insured dog owners. About 96.4% of the respondents (n = 1,151) reported that they would take their dogs for a walk and they most frequently walk their dogs once or twice a day (75.9%) for 30 min to 1 hr (83.1%). The probability of a companion dog having contact with other dogs during dog walking was estimated to be 0.83 (95% confidence interval [CI]: 0.81-0.85), and the associated daily contact rate was estimated using log-normal distribution with a mean of 2.73 (95% CI: 2.42-3.11) and a standard deviation (SD) of 6.39 (95% CI: 5.18-7.84). Multiple linear regression revealed that the contact rate is mainly influenced by the social behaviour of the owner and to a lesser degree by his/her demographic characteristics including the area of residence, the breed size of dog and the age of the owner. In addition, ten Likert items measured on a 5-point scale were designed to assess the practices towards potential cases of rabies among dog owners. The respondents (n = 972) achieved a mean score of 2.99 (out of a full score of 4) with a SD of 0.90 in responding to situations related to dog bite incidents and injury from stray cat scratches during dog walking. They achieved a higher score in responding to situations related to sighting a stray or wild animal during dog walking and situations related to non-specific clinical signs of rabies and bite injuries from stray dogs or wild animals during dog walking with a mean of 3.70 (SD = 0.58) and 3.84 (SD = 0.34), respectively. The level of best practice was also proved to be significantly associated with the demographic characteristics of the dog owner.

A Comparative Review of Prevention of Rabies Incursion between Japan and Other Rabies-Free Countries or Regions.

Although rabies still kills many people, the global eradication of human rabies is considered to be feasible. Progress towards eradication may differ among regions with differing socio-economic statuses; therefore, states that successfully eradicate this disease must be vigilant for rabies re-emergence. Here, we discuss challenges that remain concerning current rabies prevention measures and risk assessment results concerning possible rabies introduction and spread in rabies-free Japan. We summarize the preventative measures undertaken by representative rabies-free countries and regions. Our risk assessment results show that the risk of rabies reintroduction under current circumstances is very low, and that subsequent spread of the disease would be minimal because of quite low value of basic reproduction number. Similar assessments conducted in other rabies-free areas also showed limited risks of introduction. The majority of rabies-free countries maintain their rabies-free status through strict import quarantine of carnivorous animals, efficient surveillance of animal rabies including wildlife, quick emergency responses, and raising public awareness of the disease. To maintain the current rabies-free status in Japan, we strongly recommend maintaining the current quarantine system and reinforcing stakeholder compliance for those involved in international movement of dogs. Moreover, sustainable surveillance systems targeting wildlife are indispensable.

Benefit-cost analysis of the policy of mandatory annual rabies vaccination of domestic dogs in rabies-free Japan.

Japan is one of the few rabies-free countries/territories which implement the policy of mandatory vaccination of domestic dogs. In order to assess the economic efficiency of such policy in reducing the economic burden of a future canine rabies outbreak in Japan, a benefit-cost analysis (BCA) was performed using probabilistic decision tree modelling. Input data derived from simulation results of published mathematical model, field investigation conducted by the authors at prefectural governments, literature review, international or Japanese database and empirical data of rabies outbreaks in other countries/territories. The current study revealed that the annual costs of implementing the current vaccination policy would be US$160,472,075 (90% prediction interval [PI]: $149,268,935-171,669,974). The economic burden of a potential single canine rabies outbreak in Japan were estimated to be US$1,682,707 (90% PI: $1,180,289-2,249,283) under the current vaccination policy, while it would be US$5,019,093 (90% PI: $3,986,882-6,133,687) under hypothetical abolition of vaccination policy, which is 3-fold higher. Under a damage-avoided approach, the annual benefits of implementing the current vaccination policy in expected value were estimated to be US$85.75 (90% PI: $55.73-116.89). The benefit-cost ratio (BCR) was estimated to be 5.35 X 10(-7) (90% PI: 3.46 X 10(-7)-7.37 X 10(-7)), indicating that the implementation of the current policy is very economically inefficient for the purpose of reducing the economic burden of a potential canine rabies outbreak. In worse-case scenario analysis, the BCR would become above 1 (indicating economic efficiency) if the risk of rabies introduction increased to 0.04 corresponding to a level of risk where rabies would enter Japan in 26 years while the economic burden of a rabies outbreak under the abolition of vaccination policy increased to $7.53 billion. Best-case analysis further revealed that under relatively extreme circumstances the economic efficiency of the current policy could be improved by decreasing the vaccination price charged to dog owners, relaxing the frequency of vaccination to every two to three years and implementing the policy on a smaller scale, e.g. only in targeted prefectures instead of the whole Japan.

Detection and phylogenetic analysis of phlebovirus, including severe fever with thrombocytopenia syndrome virus, in ticks collected from Tokyo, Japan.

Severe fever with thrombocytopenia syndrome (SFTS) was detected for the first time in China in 2011. Since then, human cases have been reported in endemic regions, including Japan. To investigate the presence of tick-borne pathogens in Tokyo, 551 ticks (266 samples) were collected from October 2015 to October 2016. Although the SFTS virus was not detected by RT-PCR, a novel phlebovirus was detected in one sample. In a phylogenetic analysis based on the partial nucleotide sequences of the L and S segments of the virus, the virus clustered with Lesvos virus (Greece), Yongjia tick virus, and Dabieshan tick virus (China). Further studies involving virus isolation are required to characterize this novel phlebovirus and to expand the epidemiological knowledge of related pathogens.

Seroepidemiology of non-primate hepacivirus (NPHV) in Japanese native horses.

Non-primate hepacivirus (NPHV) is recently identified as a closely related homologue of hepatitis C virus. The previous studies showed a high prevalence of NPHV infection among Japanese domestic horses originated from abroad. The historical distribution of NPHV among horses in Japan, therefore, is still unknown. In this study, seroepidemiological study of NPHV was conducted using 335 sera from five breeds of Japanese native horses. These horses are maintained as the pedigree and are reared apart from other horse breeds. The detection of antibodies against NPHV were conducted by western blot analysis using the recombinant protein of the NPHV core protein. The antibodies against NPHV were detected in all five breeds, 83 out of 335 (23.4%) horses. These results suggested that NPHV was circulating among Japanese native horses.

Evaluation of Francisella tularensis ΔpdpC as a candidate live attenuated vaccine against respiratory challenge by a virulent SCHU P9 strain of Francisella tularensis in a C57BL/6J mouse model.

Francisella tularensis, which causes tularemia, is an intracellular gram-negative bacterium. F. tularensis has received significant attention in recent decades because of its history as a biological weapon. Thus, development of novel vaccines against tularemia has been an important goal. The attenuated F. tularensis strain ΔpdpC, in which the pathogenicity determinant protein C gene (pdpC) has been disrupted by TargeTron mutagenesis, was investigated as a potential vaccine candidate for tularemia in the present study. C57BL/6J mice immunized s.c. with 1 × 106 CFUs of ΔpdpC were challenged intranasally with 100× the median lethal dose (LD50 ) of a virulent SCHU P9 strain 21 days post immunization. Protection against this challenge was achieved in 38% of immunized C57BL/6J mice administered 100 LD50 of this strain. Conversely, all unimmunized mice succumbed to death 6 days post challenge. Survival rates were significantly higher in vaccinated than in unimmunized mice. In addition, ΔpdpC was passaged serially in mice to confirm its stable attenuation. Low bacterial loads persisted in mouse spleens during the first to tenth passages. No statistically significant changes in the number of CFUs were observed during in vivo passage of ΔpdpC. The inserted intron sequences for disrupting pdpC were completely maintained even after the tenth passage in mice. Considering the stable attenuation and intron sequences, it is suggested that ΔpdpC is a promising tularemia vaccine candidate.

Evaluation of the efficacy of the Japanese rabies RC-HL strain vaccine in domestic dogs using past and present data: Prediction based on logistic regression and meta-analysis.

Japan is one of the few rabies-free countries or territories (under the OIE standard) which still implement the policy of mandatory vaccination of domestic dogs. Under the Rabies Prevention Law enacted since 1950, pet owners in Japan are obliged to vaccinate their dogs every year. However, the national vaccination rate is estimated to average 42% over the past decade. Given this low owner compliance, there is debate over whether or not the mandatory vaccination policy should be maintained and if it were to be maintained, whether the yearly booster requirement is necessary or not. Data on 144 companion dogs vaccinated with the Japanese rabies RC-HL strain vaccine were analysed using multiple logistic regression. An extensive literature review was conducted and five previous vaccination studies were selected for meta-analysis. Results of logistic regression indicate that the proportion of dogs having a satisfactory antibody level lasting for 12 months (P_protected12) with only one vaccination was 74.7% (95% prediction interval (PI): 51.4%-90.5%). By contrast, P_protected12 for dogs vaccinated 2-4 times and 5 times or more was estimated as 96.6% (95%PI: 83.1%-99.3%) and 98.7% (95%PI: 96.9%-99.6%), respectively. Moreover, P_protected for 36 months would drop to 33.4% (95%PI: 11.4%-71.6%) for dogs vaccinated only once, while it would be 83.0% (95% PI: 39.4%-97.1%) and 93.0% (95%PI: 59.7%-99.2%) for dogs vaccinated 2-4 times and 5 times or more, respectively. The pooled P_protected for at least 12 months from meta-analysis was estimated as 83.8% (95%CI: 66.1%-97.5%) for dogs vaccinated only once, while it was estimated as 94.7% (95%CI: 87.7%-99.1%) for dogs vaccinated at least twice. Therefore, the yearly booster requirement of the current mandatory vaccination policy in Japan is reasonable in terms of its frequency. However, there is potential for future policy amendment to one that requires less frequent boosters, i.e. a booster is required within one year after primary vaccination and then every two to three years.

Transition of the intestinal microbiota of cats with age.

The transition of intestinal microbiota with age has been well described in humans. However, the age-related changes in intestinal microbiota of cats have not been well studied. In the present study, we investigated the composition of intestinal microbiota of cats in 5 different age groups (pre-weanling, weanling, young, aged, senile) with a culture-based method. For lactobacilli and bifidobacteria, we also quantified with molecular-based method, real-time PCR. The results suggested that the composition of the feline intestinal microbiota changes with age, while the changes were different from those of humans and dogs. Bifidobacteria which are predominant in human intestine or lactobacilli which are predominant in dog intestine, did not appear to be important in cat intestines. Enterococci, instead, seem to be major lactic acid producing bacteria in cats. We also identified lactobacilli and bifidobacteria at the species level based on 16S rRNA gene sequences and found that the species composition of Lactobacillus also changed with age.

Tolerance mechanisms of human-residential bifidobacteria against lysozyme.

We previously reported that lysozyme present in breast milk is a selection factor for bifidobacterial colonization in infant human intestines. This study is aimed at examining their underlying mechanisms. Human-residential bifidobacteria (HRB) generally exhibited higher tolerance than non-HRB to lysozymes, except B. bifidum subspecies. To assess the involvement of enzymatic activity of lysozyme, peptidoglycan (PG) was isolated and the degree of O-acetylation (O-Ac) in 19 strains, including both HRB and non-HRB, was determined. Variety in the degree of O-Ac was observed among each of the Bifidobacterium species; however, all purified PGs were found to be tolerant to lysozyme, independent of their O-Ac degree. In addition, De-O-Ac of PGs affected the sensitivity to lysozyme of only B. longum-derived PG. To examine the non-enzymatic antibacterial activity of lysozyme on bifidobacteria, lysozyme was heat-denatured. The HRB and non-HRB strains exhibited similar patterns of susceptibility to intact lysozyme as they did to heat-denatured lysozyme. In addition, strains of B. bifidum (30 strains), which showed various tolerance of lysozyme, also exhibited similar patterns of susceptibility to intact lysozyme as they did to heat-denatured lysozyme. These results suggest that bifidobacteria are resistant to the peptidoglycan-degrading property of lysozyme, and the tolerance to lysozyme among some HRB strains is due to resistance to the non-enzymatic antibacterial activity of lysozyme.

The genotypes of Orientia tsutsugamushi, identified in scrub typhus patients in northern Vietnam.

Background: There are an estimated one million patients with scrub typhus in the Asia-Pacific region. There are few reports describing the incidence of scrub typhus in Vietnam. Methods: Blood samples collected from 63 patients clinically diagnosed as having scrub typhus from July 2015 to September 2016 were subjected to genotyping of Orientia tsutsugamushi. Results and Conclusions: Of these patients, 42 (67%) tested positive for O. tsutsugamushi, and the most common genotype was identified to be Karp (55%). Other genotypes, TA763, Gilliam type in Japan variant, and Kato were also found in 17%, 17% and 12% of patients, respectively. To better understand the epidemiological landscape of scrub typhus in Vietnam, a countrywide study is needed. Accession numbers: LC110330-LC110333, LC110336-LC110351 and LC214804-LC214825.

Transition of the intestinal microbiota of dogs with age.

Although it is established that the composition of the human intestinal microbiota changes with age, transition of the intestinal microbiota of animals with age has not been well studied. In the present study, we collected fresh fecal samples from dogs of 5 different age groups (pre-weanling, weanling, young, aged, senile) and analyzed the compositions of their intestinal microbiota with a culture-based method. The results suggested that the composition of the canine intestinal microbiota also changes with age. Among intestinal bacteria predominant in dog intestines, lactobacilli appeared to change with age. Both the number and the prevalence of lactobacilli tended to decrease when dogs became older. Bifidobacteria, on the other hand, was not predominant in the intestine of the dogs. We also identified lactobacilli at the species level based on 16S rRNA gene sequences and found that the species composition of Lactobacillus also changed with age. It was further suggested that bacteria species beneficial to host animals may differ depending on the host species.

The rise and fall of rabies in Japan: A quantitative history of rabies epidemics in Osaka Prefecture, 1914-1933.

Japan has been free from rabies since the 1950s. However, during the early 1900s several large-scale epidemics spread throughout the country. Here we investigate the dynamics of these epidemics between 1914 and 1933 in Osaka Prefecture, using archival data including newspapers. The association between dog rabies cases and human population density was investigated using Mixed-effects models and epidemiological parameters such as the basic reproduction number (R0), the incubation and infectious period and the serial interval were estimated. A total of 4,632 animal rabies cases were reported, mainly in dogs (99.0%, 4,584 cases) during two epidemics from 1914 to 1921, and 1922 to 1933 respectively. The second epidemic was larger (3,705 cases) than the first (879 cases), but had a lower R0 (1.50 versus 2.42). The first epidemic was controlled through capture of stray dogs and tethering of pet dogs. Dog mass vaccination began in 1923, with campaigns to capture stray dogs. Rabies in Osaka Prefecture was finally eliminated in 1933. A total of 3,805 rabid dog-bite injuries, and 75 human deaths were reported. The relatively low incidence of human rabies, high ratio of post-exposure vaccines (PEP) and bite injuries by rabid dogs (minimum 6.2 to maximum 73.6, between 1924 and 1928), and a decline in the proportion of bite victims that developed hydrophobia over time (slope = -0.29, se = 3, p < 0.001), indicated that increased awareness and use of PEP might have prevented disease. Although significantly more dog rabies cases were detected at higher human population densities (slope = 0.66, se = 0.03, p < 0.01), there were fewer dog rabies cases detected per capita (slope = -0.34, se = 0.03, p < 0.01). We suggest that the combination of mass vaccination and restriction of dog movement enabled by strong legislation was key to eliminate rabies. Moreover, the prominent role of the media in both reporting rabies cases and efforts to control the disease likely contributed to promoting the successful participation required to achieve rabies elimination.

Administration of bovine casein-derived peptide prevents cognitive decline in Alzheimer disease model mice.

There is a growing interest in identifying natural food ingredients that may serve to prevent dementia such as that due to Alzheimer disease (AD). Peptides derived from food proteins have been demonstrated to have various physiological activities such as a hypotensive action. Recent findings have indicated possible associations of hypertension with AD progression, and suggest that angiotensin converting enzyme (ACE) inhibitors with potential to pass through the blood brain barrier (BBB) may reduce the risk of AD. In this study, we investigated the effect of milk peptide (CH-3) on cognitive function in AD model mice. CH-3 contains a tripeptide (methionine-lysine-proline, MKP) that has been found to have a strong ACE inhibitory effect and the potential to pass through the BBB. Adult male ddY mice were used in this study, and an animal model of AD was induced by intracerebroventricular (ICV) injection of Aß1-42. CH-3 (250 mg/kg/day) or MKP (0.5 mg/kg/day) was orally administered every day starting 2 days before ICV injection. At 3 weeks after ICV injection, cognitive function was evaluated by the Morris water maze test. Brain samples were obtained after behavioral testing, and expression of inflammatory cytokines and NADPH oxidase subunits was measured by real-time quantitative RT-PCR. ICV injection of Aß1-42 significantly impaired cognitive function compared with that in PBS-injected mice. Daily administration of CH-3 markedly attenuated this Aß1-42-induced cognitive decline. Aß1-42 injection significantly enhanced the expression of tumor necrosis factor-α (TNF-α), inducible nitric oxide synthase (iNOS) and p22phox in the mouse hippocampus compared with PBS injection, and showed a tendency to increase the expression of monocyte chemoattractant protein-1 (MCP-1), p47phox and gp91phox, whereas CH-3 treatment markedly reduced Aß1-42-induced TNF-α, MCP-1, iNOS, p47phox and gp91phox expression. Finally, administration of MKP also attenuated Aß1-42-induced cognitive impairment with an increase in cerebral blood flow. The present study demonstrated that repeated oral administration of CH-3 to AD model mice not only improved cognitive function but also suppressed the expression of inflammatory cytokines and production of oxidative stress, and suggests its therapeutic potential for preventing cognitive impairment in AD.

Elephant's breast milk contains large amounts of glucosamine.

Hand-reared elephant calves that are nursed with milk substitutes sometimes suffer bone fractures, probably due to problems associated with nutrition, exercise, sunshine levels and/or genetic factors. As we were expecting the birth of an Asian elephant (Elephas maximus), we analyzed elephant's breast milk to improve the milk substitutes for elephant calves. Although there were few nutritional differences between conventional substitutes and elephant's breast milk, we found a large unknown peak in the breast milk during high-performance liquid chromatography-based amino acid analysis and determined that it was glucosamine (GlcN) using liquid chromatography/mass spectrometry. We detected the following GlcN concentrations [mean ± SD] (mg/100 g) in milk hydrolysates produced by treating samples with 6M HCl for 24 hr at 110°C: four elephant's breast milk samples: 516 ± 42, three cow's milk mixtures: 4.0 ± 2.2, three mare's milk samples: 12 ± 1.2 and two human milk samples: 38. The GlcN content of the elephant's milk was 128, 43 and 14 times greater than those of the cow's, mare's and human milk, respectively. Then, we examined the degradation of GlcN during 0-24 hr hydrolyzation with HCl. We estimated that elephant's milk contains >880 mg/100 g GlcN, which is similar to the levels of major amino acids in elephant's milk. We concluded that a novel GlcN-containing milk substitute should be developed for elephant calves. The efficacy of GlcN supplements is disputed, and free GlcN is rare in bodily fluids; thus, the optimal molecular form of GlcN requires a further study.

Quantitative risk assessment of the introduction of rabies into Japan through the illegal landing of dogs from Russian fishing boats in the ports of Hokkaido, Japan.

Japan has been free from rabies since 1958 and various preventive measures are in place protecting the country from the introduction of the disease. Historical reviews indicate that the illegal landing of dogs from Russian fishing boats in the ports of Hokkaido occurred frequently especially in the early 2000s and this could potentially be a source of introduction of rabies into Japan. The method of scenario tree modelling was used and the following entry and exposure pathway was considered the most likely route of rabies entry: a rabies-infected dog arriving on a Russian fishing boat lands in a port of Hokkaido in Japan, it becomes infectious, contacts and infects a susceptible domestic animal (companion dog, stray dog or wildlife). Input parameter values were based on surveys of Russian fishermen, expert opinion and scientific data from the literature. At present (2006-2015), the probability of the introduction of rabies as a result of one Russian fishing boat arriving at a port of Hokkaido is 8.33×10(-10) (90% Prediction Interval (PI): 7.15×10(-11)-5.34×10(-9)), while this probability would have been 7.70×10(-9) (90% PI: 6.40×10(-10)-4.81×10(-8)) in the past (1998-2005). Under the current situation (average annual number of boat arrivals is 1106), rabies would enter Japan every 1,084,849 (90% PI: 169,215-20,188,348) years, while the disease would have been introduced every 18,309 (90% PI: 2929-220,048) years in the past (average annual number of boat arrivals is 7092). The risk of rabies introduction has decreased 59 fold due to both the effective control of the issue of illegal landing of dogs and the decline in the number of Russian boat arrivals. Control efforts include education of Russian fishermen, establishment of warning signs, daily patrols and regular port surveillance of potential dog landing activity. Furthermore, scenario analysis revealed that the policy of mandatory domestic dog vaccination does not contribute effectively to Japan's rabies prevention system under rabies-free situation. Although the current risk of rabies introduction is minimal, control measures against the illegal landing of dogs must be maintained. Further risk management measures, such as the removal of wildlife from the port area and regular monitoring of the rabies situation in Russia (particularly the easternmost regions), can be established to strengthen the current rabies prevention system in Hokkaido.

Virulence of representative Japanese Francisella tularensis and immunologic consequences of infection in mice.

Francisella tularensis, which causes tularemia, is widely distributed in the Northern hemisphere. F. tularensis strains isolated in Japan are genetically unique from non-Japanese strains; however, their phenotypic properties have not been well studied. Thus, mice were infected with representative Japanese strains of F. tularensis and their virulence and mouse immune responses to them assessed. Of four representative Japanese strains, the Ebina, Jap and Tsuchiya strains were susceptible to H2 O2 and did not grow well intracellularly. Only Yama strain grew intracellularly and was lethal to mice. Infection with Yama strain resulted in drastic increases in IFN-γ, CD4 and CD8 double-positive T cells and Th1 cells (CD3, CD4 and Tim3-positive cells), and a decrease in the ratio of CD8-positive CD4-negative T cells in mice. C57BL/6J mice that survived infection produced IgM antibodies to LPS and IgG2c antibodies to 43, 19 and 17 kDa proteinase K-sensitive components. These data are valuable for understanding the phenotypic properties of F. tularensis in Japan.

Prevalence and Phylogenetic Analysis of Orientia tsutsugamushi in Small Mammals in Hanoi, Vietnam.

Rodents are important reservoirs of many human pathogens transmitted via arthropod vectors. Arthropod-borne bacteria belonging to the family Rickettsiaceae cause acute febrile diseases in humans worldwide, but the real burdens of rickettsial diseases appear to be underestimated in Hanoi, Vietnam, because differential diagnosis on the basis of clinical signs and symptoms is confounded by the presence of other tropical infectious diseases with similar signs and symptoms. To know the prevalence of bacteria of the family Rickettsiaceae among small mammals in Hanoi, 519 animals thriving in the public places were captured and examined for the presence of bacterial sequences using duplex PCR. Nucleotide sequences specific for Orientia tsutsugamushi were detected in seven samples (1.3%). Out of seven animals, two were captured in a market, whereas five were in hospitals. None of the captured small mammals tested positive for the genus Rickettsia. The nucleotide sequence analysis of the genes encoding the 47-kDa high-temperature requirement A (47-kDa HtrA) and 56-kDa type-specific antigen (TSA) showed that these seven isolates were indistinguishable from each other. O. tsutsugamushi isolated in this study was closely related phylogenetically to the Gilliam strain, which was originally isolated at the border of Assam and Burma, rather than to those isolated in the central to southern part of Vietnam. It should be emphasized that Vietnamese hospitals were heavily infested by small rodents and some of them harbored O. tsutsugamushi. Strict hygienic control should be implemented to mitigate the potential risk posed by O. tsutsugamushi in hospital settings.

Survey of Francisella tularensis in Wild Animals in Japan in Areas Where Tularemia is Endemic.

Samples taken from 428 wild animals and 126 ticks, collected from a tularemia-endemic area in Japan between 2005 and 2013, were analyzed for the presence of Francisella tularensis. F. tularensis was isolated from a Japanese hare carcass whereas the samples from live animals and ticks were negative for F. tularensis by real-time PCR. Our results suggest that F. tularensis is still present in Japan although its prevalence is considerably low even in areas where tularemia is endemic.