Diagnostic system for the detection of severe fever with thrombocytopenia syndrome virus RNA from suspected infected animals.

BACKGROUND: Severe fever with thrombocytopenia syndrome virus (SFTSV) causes severe hemorrhagic fever in humans and cats. Clinical symptoms of SFTS-infected cats resemble those of SFTS patients, whereas SFTS-contracted cats have high levels of viral RNA loads in the serum and body fluids. Due to the risk of direct infection from SFTS-infected cats to human, it is important to diagnose SFTS-suspected animals. In this study, a reverse transcription polymerase chain reaction (RT-PCR) was newly developed to diagnose SFTS-suspected animals without non-specific reactions. METHODOLOGY/PRINCIPLE FINDINGS: Four primer sets were newly designed from consensus sequences constructed from 108 strains of SFTSV. A RT-PCR with these four primer sets successfully and specifically detected four clades of SFTSV. Their limits of detection are 1-10 copies/reaction. Using this RT-PCR, 5 cat cases among 56 SFTS-suspected animal cases were diagnosed as SFTS. From these cats, IgM or IgG against SFTSV were detected by enzyme-linked immunosorbent assay (ELISA), but not neutralizing antibodies by plaque reduction neutralization titer (PRNT) test. This phenomenon is similar to those of fatal SFTS patients. CONCLUSION/SIGNIFICANCE: This newly developed RT-PCR could detect SFTSV RNA of several clades and from SFTS-suspected animals. In addition to ELISA and PRNT test, the useful laboratory diagnosis systems of SFTS-suspected animals has been made in this study.

A retrospective survey of the seroprevalence of severe fever with thrombocytopenia syndrome virus in wild animals in Japan.

Severe fever with thrombocytopenia syndrome (SFTS) has a high fatality rate and is caused by SFTS virus (SFTSV). Currently, SFTS is endemic to some areas in western Japan, and wild animals are considered to play important roles in the circulation of SFTSV in the environment. Previous retrospective surveys using samples mainly obtained between 2006 and 2015 revealed serological evidence of SFTSV infection in wild animals; however, seroprevalence before 2006 remains unclear. In this study, we investigated the presence of anti-SFTSV antibodies in a total of 521 serum samples from nine wild animal species collected from 11 prefectures in central and eastern Japan between 1980 and 2000. All samples yielded negative results for antibodies to SFTSV, suggesting that there had been few or no SFTSV infections before 2000 in the sampled areas.

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.

Complete Genome Sequences of Two Strains of Francisella tularensis subsp. holarctica bv. japonica.

Francisella tularensis, a highly infectious bacterium, is the etiological agent of the zoonotic disease tularemia. It is widely distributed in the Northern Hemisphere, including Japan. Here, we have determined the complete genome sequences of two strains of F. tularensis subsp. holarctica bv. japonica isolated from hares in 2008 and 2009.

Effective methods for the inactivation of Francisella tularensis.

Francisella tularensis (F. tularensis) is highly pathogenic to humans and must be handled under biosafety level 3 conditions. Samples used for the diagnosis and experimental analysis must be completely inactivated, although methods for the inactivation of F. tularensis are limited. In this study, effective methods for the inactivation of F. tularensis SCHU P9 and five other strains were determined by comparisons of colony-forming units between treated and control samples. The results showed that F. tularensis SCHU P9 was denatured by heat treatment (94°C for 3 min and 56°C for 30 min), filtration with a 0.22 µm filter, and the use of various solutions (i.e. >70% ethanol, methanol, acetone, and 4% paraformaldehyde). F. tularensis SCHU P9 remained viable after treatment with 50% ethanol for 1 min, filtration with a 0.45 µm filter, and treatments with detergents (i.e. 1% lithium dodecyl sulfate buffer, 1% Triton X-100 and 1% Nonidet P-40) at 4°C for 24 h. Additionally, F. tularensis SCHU P9 suspended in fetal bovine serum in plastic tubes was highly resistant to ultraviolet radiation compared to suspensions in water and chemically defined medium. The methods for inactivation of F. tularensis SCHU P9 was applicable to the other five strains of F. tularensis. The data presented in this study could be useful for the establishment of guidelines and standard operating procedures (SOP) to inactivate the contaminated samples in not only F. tularensis but also other bacteria.

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.

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.

Pullulanase Is Necessary for the Efficient Intracellular Growth of Francisella tularensis.

Pullulanase, an enzyme that catalyzes the hydrolysis of polysaccharides, has been identified in a broad range of organisms, including bacteria, yeasts, fungi, and animals. The pullulanase (pulB; FTT_0412c) of F. tularensis subspecies tularensis Schu S4 is considered to be a homologue of the type I pullulanase (pulA) of the other Francisella subspecies. The significance of Francisella pullulanase has been obscure until now. In the present study, we characterized a recombinant PulB of F. tularensis SCHU P9, which was expressed as a his-tagged protein in Escherichia coli. The recombinant PulB was confirmed to be a type I pullulanase by its enzymatic activity in vitro. A pulB gene knockout mutant of F. tularensis SCHU P9 (ΔpulB) was constructed using the TargeTron Knockout system and plasmid pKEK1140 to clarify the function of PulB during the growth of F. tularensis in macrophages. The intracellular growth of the ΔpulB mutant in murine macrophage J774.1 cells was significantly reduced compared with that of the parental strain SCHU P9. Expression of PulB in ΔpulB, using an expression plasmid, resulted in the complementation of the reduced growth in macrophages, suggesting that PulB is necessary for the efficient growth of F. tularensis in macrophages. To assess the role of PulB in virulence, the knockout and parent bacterial strains were used to infect C57BL/6J mice. Histopathological analyses showed that tissues from ΔpulB-infected mice showed milder lesions compared to those from SCHU P9-infected mice. However, all mice infected with SCHU P9 and ΔpulB showed the similar levels of bacterial loads in their tissues. The results suggest that PulB plays a significant role in bacterial growth within murine macrophage but does not contribute to bacterial virulence in vivo.

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.

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.

Serosurveillance for Francisella tularensis among wild animals in Japan using a newly developed competitive enzyme-linked immunosorbent assay.

Tularemia, a highly infectious zoonotic disease caused by Francisella tularensis, occurs sporadically in Japan. However, little is known about the prevalence of the disease in wild animals. A total of 632 samples obtained from 150 Japanese black bears, 142 Japanese hares, 120 small rodents, 97 rats, 53 raptors, 26 Japanese monkeys, 21 Japanese raccoon dogs, 20 masked palm civets, and three Japanese red foxes between 2002 and 2010 were investigated for the presence of antibodies to F. tularensis by competitive enzyme-linked immunosorbent assay (cELISA) and the commonly used microagglutination (MA) test. Seropositive cELISA and MA results were obtained in 23 and 18 Japanese black bears, three and two Japanese raccoon dogs, and two and one small rodents, respectively. All MA-positive samples (n=21) were also positive by cELISA. Six of seven samples that were only positive by cELISA were confirmed to be antibody-positive by western blot analysis. These findings suggest that cELISA is a highly sensitive and useful test for serosurveillance of tularemia among various species of wild animals. Because this is the first study to detect F. tularensis-seropositive Japanese raccoon dogs, these could join Japanese black bears as sentinel animals for tularemia in the wild in Japan. Further continuous serosurveillance for F. tularensis in various species of wild animals using appropriate methods such as cELISA is important to assess the risks of human exposure and to improve our understanding of the ecology of F. tularensis in the wild.

Role of pathogenicity determinant protein C (PdpC) in determining the virulence of the Francisella tularensis subspecies tularensis SCHU.

Francisella tularensis subspecies tularensis, the etiological agent of tularemia, is highly pathogenic to humans and animals. However, the SCHU strain of F. tularensis SCHU P0 maintained by passaging in artificial media has been found to be attenuated. To better understand the molecular mechanisms behind the pathogenicity of F. tularensis SCHU, we attempted to isolate virulent bacteria by serial passages in mice. SCHU P5 obtained after 5th passages in mice remained avirulent, while SCHU P9 obtained after 9th passages was completely virulent in mice. Moreover, SCHU P9 grew more efficiently in J774.1 murine macrophages compared with that in the less pathogenic SCHU P0 and P5. Comparison of the nucleotide sequences of the whole genomes of SCHU P0, P5, and P9 revealed only 1 nucleotide difference among P0, P5 and P9 in 1 of the 2 copies of pathogenicity determinant protein C (pdpC) gene. An adenine residue deletion was observed in the pdpC1 gene of SCHU P0, P5, and P9 and in the pdpC2 gene of SCHU P0, and P5, while P9 was characterized by the wild type pdpC2 gene. Thus, SCHU P0 and P5 expressed only truncated forms of PdpC protein, while SCHU P9 expressed both wild type and truncated versions. To validate the pathogenicity of PdpC, both copies of the pdpC gene in SCHU P9 have been inactivated by Targetron mutagenesis. SCHU P9 mutants with inactivated pdpC gene showed low intracellular growth in J774.1 cells and did not induce severe disease in experimentally infected mice, while virulence of the mutants was restored by complementation with expression of the intact PdpC. These results demonstrate that PdpC is crucial in determining the virulence of F. tularensis SCHU.

In vitro antibiotic susceptibility of Francisella tularensis isolates from Japan.

The antibiotic susceptibilities of 36 isolates of Japanese Francisella tularensis, an etiological agent of the zoonotic disease tularemia, were analyzed using the E test. All the isolates were susceptible to ciprofloxacin, doxycycline, erythromycin, and gentamicin but resistant to benzylpenicillin and cephalothin. The susceptibility to seven other ß-lactams (aztreonam, cefotaxime, cefoxitin, ceftriaxone, cefuroxime, imipenem, and meropenem) varied among the isolates. These findings suggest that the guidelines for the antibiotic treatment of tularemia issued by the World Health Organization are appropriate for Japanese tularemia patients.

Identification of the source of Francisella tularensis infection by multiple-locus variable-number tandem repeat analysis.

Tularemia is a zoonotic disease caused by Francisella tularensis. Most patients in Japan have reportedly acquired such infections through direct contact with infected Japanese hares. We recently encountered a patient who contracted tularemia after skinning and butchering a dead hare. Because the remains of the hare were available, we attempted to determine whether the patient actually contracted infection by handling the carcass. F. tularensis-specific sequences were successfully amplified by PCR from the patient specimens as well as from the remnants of discarded hare carcass. PCR amplification of the ISFtu2 and RD1 regions indicated infection by F. tularensis subsp. holarctica, which was considered as a prevalent strain in Japan. Furthermore, high-resolution multiple-locus variable-number tandem repeat analysis (MLVA) showed that the combination of repeat numbers in sequences from the patient and hare were indistinguishable, thus indicating that the patient had been infected with F. tularensis strain that had also infected the hare. These findings demonstrated that MLVA is a useful epidemiological investigational tool to identify possible sources of certain zoonotic diseases such as tularemia.

Distribution of antimicrobial-resistant lactic acid bacteria in natural cheese in Japan.

To determine and compare the extent of contamination caused by antimicrobial-resistant lactic acid bacteria (LAB) in imported and domestic natural cheeses on the Japanese market, LAB were isolated using deMan, Rogosa and Sharpe (MRS) agar and MRS agar supplemented with six antimicrobials. From 38 imported and 24 Japanese cheeses, 409 LAB isolates were obtained and their antimicrobial resistance was tested. The percentage of LAB resistant to dihydrostreptomycin, erythromycin, and/or oxytetracycline isolated from imported cheeses (42.1%) was significantly higher than that of LAB resistant to dihydrostreptomycin or oxytetracycline from cheeses produced in Japan (16.7%; P=0.04). Antimicrobial resistance genes were detected in Enterococcus faecalis (tetL, tetM, and ermB; tetL and ermB; tetM) E. faecium (tetM), Lactococcus lactis (tetS), Lactobacillus (Lb.), casei/paracasei (tetM or tetW), and Lb. rhamnosus (ermB) isolated from seven imported cheeses. Moreover, these E. faecalis isolates were able to transfer antimicrobial resistance gene(s). Although antimicrobial resistance genes were not detected in any LAB isolates from Japanese cheeses, Lb. casei/paracasei and Lb. coryniformis isolates from a Japanese farm-made cheese were resistant to oxytetracycline (minimal inhibitory concentration [MIC], 32 µg/mL). Leuconostoc isolates from three Japanese farm-made cheeses were also resistant to dihydrostreptomycin (MIC, 32 to >512 µg/mL). In conclusion, the present study demonstrated contamination with antimicrobial-resistant LAB in imported and Japanese farm-made cheeses on the Japanese market, but not in Japanese commercial cheeses.

Detection of Francisella tularensis-specific antibodies in patients with tularemia by a novel competitive enzyme-linked immunosorbent assay.

A novel competitive enzyme-linked immunosorbent assay (cELISA) was developed and evaluated for detection of antibodies against Francisella tularensis in humans. The assay is based on the ability of serum antibodies to inhibit the binding of monoclonal antibodies (MAbs) directed against F. tularensis lipopolysaccharide antigens. The assay was evaluated using serum samples of tularemia patients, inactivated F. tularensis-immunized rabbits, and F. tularensis-infected mice. Antibodies against F. tularensis were successfully detected in serum samples of tularemia patients as well as the immunized and infected animals. The cELISA method was compared to indirect ELISA (iELISA) and the commonly used microagglutination test (MA) using serum samples of 19 tularemia patients and 50 healthy individuals. The sensitivity and specificity of cELISA were 93.9 and 96.1%, respectively, in comparison to the iELISA. MA was less sensitive than cELISA with a sensitivity and specificity of only 81.8 and 98.0%, respectively. A high degree of correlation (R(2) = 0.8226) was observed between cELISA and iELISA results. The novel cELISA developed in this study appears to be highly sensitive and specific for serodiagnosis of human tularemia. The potential of the MAb-based cELISA to be used in both human and animal samples emphasizes its usefulness for serological survey of tularemia among multiple animal species.

Altered specificity of single-chain antibody fragments bound to pandemic H1N1-2009 influenza virus after conversion of the phage-bound to the soluble form.

BACKGROUND: In 2009, a novel influenza A/H1N1 virus (H1N1pdm) quickly spread worldwide and co-circulated with then-existing seasonal H1N1 virus (sH1N1). Distinguishing between these 2 viruses was necessary to better characterize the epidemiological properties of the emergent virus, including transmission patterns, pathogenesis, and anti-influenza drug resistance. This situation prompted us to develop a point-of-care virus differentiation system before entering the 2009-2010 influenza season. Aiming to establish H1N1pdm-specific detection tools rapidly, we employed phage display libraries to select H1N1pdm-specific single-chain variable fragments (scFvs). FINDINGS: Human single-fold scFv libraries (Tomlinson I + J) underwent selection for the ability to bind H1N1pdm virus particles. Three rounds of panning brought 1152 phage-bound scFvs, of which 58 clones reacted with H1N1pdm specifically or preferentially over sH1N1 in an enzyme-linked immunosorbent assay (ELISA). After conversion of the scFvs to soluble form, 7 clones demonstrating high/stable expression were finally obtained. However, all the soluble scFvs except No. 29 were found to have lost their specificity/preference for H1N1pdm in ELISA. The specificity/preference of No. 29 was also confirmed by immunofluorescence assay and immunoprecipitation, and the viral nucleoprotein was identified by ELISA as its target protein. The change in specificity associated with scFv conversion from phage-bound to soluble form could be due to loss of phage scaffold pIII protein, which likely provides structural support for the scFv antigen-binding site. It is also possible that the similar antigenic properties of H1N1pdm and sH1N1 led to the observed alterations in scFv specificity. DISCUSSION: Using a phage display library, we obtained 7 soluble scFv clones reactive against H1N1pdm; however, only 1 showed specificity/preference toward H1N1pdm. Our results confirmed that using phage display libraries was highly advantageous for the rapid development of molecules to detect target antigens. However, our results also indicated that this strategy might not have been effective for selecting H1N1pdm-specific antibodies during the 2009 pandemic, where the co-circulating sH1N1 virus shared similar antigenic properties. This suggests that it might be advisable to use a synthetic scFv phage display library by strategically considering the characteristics of target antigens and the potential situations.

Survival of avian H5N1 influenza A viruses in Calliphora nigribarbis (Diptera: Calliphoridae).

In a previous study, the highly pathogenic avian influenza (HPAI) H5N1 viruses were isolated from blow flies collected at the Tamba Town of Kyoto prefecture during the outbreak period in March 2004. In this study, we carried out virus exposure experiments to investigate whether the H5N1 virus would survive in a blow fly, Calliphora nigribarbis. The virus exposure experiments showed that the H5N1 influenza virus was isolated from the crop and intestine of C. nigribarbis for at least 24 h, and the viruses remained viable with titers ranging from 0.5 to 4.63 TCID50. This result suggests that C. nigribarbis could possibly transport the H5N1 virus over a distance of 2 km, which is the distance they can migrate within 24 h.

Pathological and microbiological studies of Japanese Hare (Lepus brachyurus angustidens) naturally infected with Francisella tularensis subsp. holarctica.

An adult male hare (Lepus brachyurus angustidens) was discovered in a moribund condition in the bush in the mountains of Aomori prefecture in Japan. Upon gross inspection, many ticks were found on the neck and the external ear regions, and more than half the ticks contained blood in the intestine. The skin around the tick bite wounds was alopecic and mildly thickened. At necropsy, enlargement of the cervical lymph nodes and spleen were observed. Histologically, acute necrotizing splenitis, lymphadenitis, hepatitis, pneumonia, myelitis, adrenalitis, and encephalitis with bacterial organisms were observed. The cutaneous lesions were chronic and cysts had formed in the areas marked by tick bites. Immunohistochemically, the organisms in the skin, liver, spleen, lymph nodes, lungs, adrenal glands, brain, bone marrow, and ticks were positive for F. tularensis antigen. Microbiological and polymerase chain reaction results were consistent with F. tularensis subsp. holarctica. Because the cutaneous lesions were more chronic than those in the visceral organs and F. tularensis was detected in the ticks, we inferred that F. tularensis was transmitted to the hare via tick bites.