Field assessment of Orientia tsutsugamushi infection in small mammals and its association with the occurrence of human scrub typhus in Taiwan.

We conducted an extensive study in Taiwan of Orientia tsutsugamushi (OT) infection in small wild mammals. Field trapping was carried out at six districts in eastern and western Taiwan as well as various offshore islands during the period 2006-2010. A total of 1061 specimens representing 11 rodent species were captured. The presence of OT infection was assessed by indirect immunofluorescence assay and polymerase chain reaction assays of 56-kDa type-specific antigen gene. The chigger infestation rate among the animals was 35% (371/1061). Among these, OT was detected in 64% (238/371) of the chiggers from the infested animals and in the spleens from 273 (34.3%) of 797 animals. Excluding animals in the Suncus murinus group, the antibody positive rate of scrub typhus was 69.1% (477 of 690 of serum samples). The prevalence of OT infection in animals from areas with a low incidence of human cases of scrub typhus was significantly lower than that in rodents obtained from regions with a high incidence of human cases of the disease (44.4%±4.0% vs. 71.2%±9.7%, p<0.001). In Taiwan, the prevalence of OT infection in wild rodents is considerably high and appears to correlate positively with the occurrence of scrub typhus in humans.

Suspension bead array of the single-stranded multiplex polymerase chain reaction amplicons for enhanced identification and quantification of multiple pathogens.

Rapid identification of single and multiple infectious agents is vital in clinical settings and during biothreat attack. This study assesses the assay of single-stranded multiplex polymerase chain reaction (PCR) amplicons by suspension bead array (SSMP-SBA) for multiple pathogens identification in a single-tube reaction. A 15-plex assay for identification of 11 highly infectious pathogens was developed to evaluate the performance of SSMP-SBA. Pathogen-specific amplicons were obtained by sequential amplification of genomic DNAs using gene-specific primers tagged with artificial unique sequences and unique primers of which the reverse primer was modified by biotin and phosphorothioate. The SSMP products generated by T7 exonuclease-mediated DNA hydrolysis were hybridized to 15 sets of beads coupled with gene-specific and control oligonucleotide probes for pathogen identification and quantification by flow cytometry. This method was validated via assessment of 57 reference strains and one clinical bacterial isolate. All 11 pathogens can be detected by the 15-plex SSMP-SBA assay, and this design significantly enhanced the signal-to-noise ratio and improved the assay performance. This assay achieves similar sensitivity to our in-house real-time PCR system with the limit of detection equivalent to 5-100 genome copies and a linear dynamic range crossing three to five logs. In the validation assay, a 100% accuracy rate was achieved when the pathogens were among the target species. Notably, the species of pathogens were accurately identified from the samples with multiple infections. SSMP-SBA presents superior performance with multiplexing capability in a single-tube reaction and provides a new approach for detection and species identification of multiple pathogen infections.

Genetic typing, based on the 56-kilodalton type-specific antigen gene, of Orientia tsutsugamushi strains isolated from chiggers collected from wild-caught rodents in Taiwan.

Orientia tsutsugamushi is the etiological agent of scrub typhus, a mite-borne, febrile illness that occurs in the Asia-Pacific region. We conducted strain characterization of O. tsutsugamushi isolates from chiggers obtained from rodents based the nucleotide sequence of the 56-kDa outer membrane protein gene. With the use of PCR, a total of 68 DNA sequences of 56-kDa antigen genes were amplified. Phylogenetic analysis revealed that there were at least six definable clusters among the 68 isolates: 37% Karp-related strains (25/68), 27% TA763 strains (18/68), 12% JG-related strains (8/68), 19% Kato-related strains (13/68), 4% divergent strains (3/68), and 1% representing a Gilliam prototype strain (1/68). Overall, the O. tsutsugamushi genotypes exhibited a high degree of diversity, similar to that seen in strains from the rest of the areas where scrub typhus is endemic. Moreover, the 56-kDa protein sequence similarity between O. tsutsugamushi isolates from mites and those from human patients (H. Y. Lu et al., Am. J. Trop. Med. Hyg. 83:658-663, 2010) were striking, thus highlighting potential risk factors for this emerging zoonotic disease.

Role of amphotericin B upon enhancement of protective immunity elicited by oral administration with liposome-encapsulated-Japanese encephalitis virus nonstructural protein 1 (NS1) in mice.

Amphotericin B (AmB) is an antifungal antibiotic the activity of which has been associated with modulation of pro-inflammatory cytokines expression in cultured cells. Herein we reveal that co-administration with AmB enhances the immunogenicity of oral Lip-JENS1 vaccine which derived from liposomes functionalized with DSPC (distearoylphosphatidylcholine) and cholesterol (2:1, molar ratio)-bearing JE virus NS1 protein (600 microg ml(-1)). Oral single dose of Lip-JENS1 elicited a detectable serum NS1-specific IgG antibody response from a mouse model. Remarkably, the addition of AmB (125 microg per mouse), particularly, 2 h prior to, but not simultaneously with, the administration of Lip-JENS1 significantly enhanced the systemic antigen-specific antibody response, providing superior protection against lethal JEV challenges. Further, we observed AmB-induced the transcription of cytokine expression and translocation of transcriptional factor NF-kappaB from the cytoplasm to the nucleus for the murine macrophage J774A.1. Moreover, Peyer's-patch lymphocytes (PPL) from AmB-treated mice produced high levels of IL-1beta, IL-6 and TNF-alpha expression compared to the corresponding control of cells from non-treated mice. Taken together, the results suggest that AmB exerts a profound influence upon mucosal vaccination with Lip-JENS1, possibly playing an adjuvant-augmented role to "fine-tune" humoral as well as cellular immune response, thus conferring enhanced protective immunity for immunising individuals against JE infection.

A comparison of immunogenicity and protective immunity against experimental plague by intranasal and/or combined with oral immunization of mice with attenuated Salmonella serovar Typhimurium expressing secreted Yersinia pestis F1 and V antigen.

We investigated the relative immunogenicity and protective efficacy of recombinant X85MF1 and X85V strains of DeltacyaDeltacrpDeltaasd-attenuated Salmonella Typhimurium expressing, respectively, secreted Yersinia pestis F1 and V antigens, following intranasal (i.n.) or i.n. combined with oral immunization for a mouse model. A single i.n. dose of 10(8) CFU of X85MF1 or X85V induced appreciable serum F1- or V-specific IgG titres, although oral immunization did not. Mice i.n. immunized three times (i.n. x 3) with Salmonella achieved the most substantial F1/V-specific IgG titres, as compared with corresponding titres for an oral-primed, i.n.-boosted (twice; oral-i.n. x 2) immunization regimen. The level of V-specific IgG was significantly greater than that of F1-specific IgG (P<0.001). Analysis of the IgG antibodies subclasses revealed comparable levels of V-specific Th-2-type IgG1 and Th-1-type IgG2a, and a predominance of F1-specific Th-1-type IgG2a antibodies. In mice immunized intranasally, X85V stimulated a greater IL-10-secreting-cell response in the lungs than did X85MF1, but impaired the induction of gamma-interferon-secreting cells. A program of i.n. x 3 and/or oral-i.n. x 2 immunization with X85V provided levels of protection against a subsequent lethal challenge with Y. pestis, of, respectively, 60% and 20%, whereas 80% protection was provided following the same immunization but with X85MF1.

Role of the universal stress protein UspA of Salmonella in growth arrest, stress and virulence.

Pathogenic bacteria employ a variety of mechanisms to resist a barrage of stresses they encounter during active growth in or outside the host as well as during growth stasis. An in silico screen of the Salmonella genome sequence revealed that Salmonella typhimurium LT2 possesses a homologue belonging to the universal stress protein A (UspA) family. We assessed the transcriptional profile of uspA in S. typhimurium C5 by constructing a lacZ fusion revealing that uspA is induced by metabolic, oxidative, and temperature stresses. The highest transcriptional levels occurred in cells entering stationary phase, an observation consistent with expression patterns in Escherichia coli. The protein was purified as a fusion with GST (UspA(F)) and antibodies raised against UspA(F) revealed elevated protein levels in stressed and growth-arrested cells. Inactivation of uspA in S. typhimurium C5, lead to increased susceptibility to stress conditions. Furthermore, UspA makes an important contribution to the in vivo virulence of Salmonella in mice thus highlighting the importance of stress resistance regulation in pathogenicity and survival within the host.

Enhanced immune response by amphotericin B following NS1 protein prime-oral recombinant Salmonella vaccine boost vaccination protects mice from dengue virus challenge.

A recombinant vaccine strain SL3261/pLT105 of attenuated aroA Salmonella enterica serovar Typhimurium SL3261 strain expressing a secreted dengue virus type 2 non-structural NS1 and Yersinia pestis F1 (Caf1) fusion protein, rNS1:Caf1, was generated. Immunological evaluation was performed by prime-boost vaccine regimen. Oral immunization of mice with 1 x 10(9)cfu of SL3261/pLT105 only induced low levels of NS1-specific antibody response and protective immunity following dengue virus challenge. The parenteral NS1 protein priming-oral Salmonella boosting protocol enhanced both NS1-specific serum IgG response and protective efficacy as compared to mice immunized with each type vaccine alone. Addition of an antifungal antibiotic amphotericin B (AmB) to Salmonella vaccine further enhanced the synergic effects of prime-boost vaccine regimen on the elicited NS1-specific serum IgG response and the protective efficacy. Together, the results demonstrated that the rNS1:Caf1 producing Salmonella SL3261/pLT105 strain fails to provide effective protection as an oral vaccine alone despite co-administration of AmB as an adjuvant capable of enhancing the immune responses, and moreover, the protein priming-oral Salmonella vaccine boosting approach in combination with AmB as an immunization regimen may have the potential to be further explored as an alternative approach for dengue vaccine development.