• View in gallery
    Figure 1.

    Polymerase chain reaction (PCR) products derived from the amplification of (A) the full-length and (B) variable domains I–III 56-kDa TSA gene of O. tsutsugamushi from 12 infected Leptotrombidium colonies.

  • View in gallery
    Figure 2.

    Neighbor-joining (NJ) tree derived from partial 56-kDa TSA genes (variable domains I–III, 498 bp) of O. tsutsugamushi. The analysis involved 46 nucleotide sequences of O. tsutsugamushi-infected chigger lines (Lc-1, Li-1, Ld-1), O. tsutsugamushi-infected wild-caught rodent and chigger along with reference sequences retrieved from the GenBank database as shown in Table 5. The NJ tree was constructed using the p-distance method of nucleotide substitution model. The bootstrap P values > 50% are indicated at major nodes (2,000 replicates). Tree is rooted at its midpoint. Scale bar represents substitutions per site.

  • 1.

    Rai J, Bandopadhyay D, 1978. Vertical transmission in chigger borne rickettsiosis. Indian J Med Res 68: 3138.

  • 2.

    Rapmund G, Dohany AL, Manikumaran C, Chan TC, Chan TC, 1972. Transovarial transmission of Rickettsia tsutsugamushi in Leptotrombidium (Leptotrombidium) arenicola Traub (Acarina: Trombiculidae). J Med Entomol 9: 7172.

    • Search Google Scholar
    • Export Citation
  • 3.

    Rapmund G, Upham RW Jr, Kundin WD, Manikumaran C, Chan TC, 1969. Transovarial development of scrub typhus rickettsiae in a colony of vector mites. Trans R Soc Trop Med Hyg 63: 251258.

    • Search Google Scholar
    • Export Citation
  • 4.

    Takahashi M, Murata M, Nogami S, Hori E, Kawamura A Jr, Tanaka H, 1988. Transovarial transmission of Rickettsia tsutsugamushi in Leptotrombidium pallidum successively reared in the laboratory. Jpn J Exp Med 58: 213218.

    • Search Google Scholar
    • Export Citation
  • 5.

    Urakami H, Takahashi M, Hori E, Tamura A, 1994. An ultrastructural study of vertical transmission of Rickettsia tsutsugamushi during oogenesis and spermatogenesis in Leptotrombidium pallidum. Am J Trop Med Hyg 50: 219228.

    • Search Google Scholar
    • Export Citation
  • 6.

    Takahashi M, Murata M, Misumi H, Hori E, Kawamura A Jr, Tanaka H, 1994. Failed vertical transmission of Rickettsia tsutsugamushi (Rickettsiales: Rickettsiaceae) acquired from rickettsemic mice by Leptotrombidium pallidum (Acari: trombiculidae). J Med Entomol 31: 212216.

    • Search Google Scholar
    • Export Citation
  • 7.

    Walker JS, Chan CT, Manikumaran C, Elisberg BL, 1975. Attempts to infect and demonstrate transovarial transmission of R. tsutsugamushi in three species of Leptotrombidium mites. Ann N Y Acad Sci 266: 8090.

    • Search Google Scholar
    • Export Citation
  • 8.

    Traub R, Wisseman CL Jr, Jones MR, O'Keefe JJ, 1975. The acquisition of Rickettsia tsutsugamushi by chiggers (trombiculid mites) during the feeding process. Ann N Y Acad Sci 266: 91114.

    • Search Google Scholar
    • Export Citation
  • 9.

    Nakayama K, Yamashita A, Kurokawa K, Morimoto T, Ogawa M, Fukuhara M, Urakami H, Ohnishi M, Uchiyama I, Ogura Y, Ooka T, Oshima K, Tamura A, Hattori M, Hayashi T, 2008. The whole-genome sequencing of the obligate intracellular bacterium Orientia tsutsugamushi revealed massive gene amplification during reductive genome evolution. DNA Res 15: 185199.

    • Search Google Scholar
    • Export Citation
  • 10.

    Rosenberg R, 1997. Drug-resistant scrub typhus: paradigm and paradox. Parasitol Today 13: 131132.

  • 11.

    Yamashita T, Kasuya S, Noda S, Nagano I, Ohtsuka S, Ohtomo H, 1988. Newly isolated strains of Rickettsia tsutsugamushi in Japan identified by using monoclonal antibodies to Karp, Gilliam, and Kato strains. J Clin Microbiol 26: 18591860.

    • Search Google Scholar
    • Export Citation
  • 12.

    Shirai A, Wisseman CL Jr, 1975. Serologic classification of scrub typhus isolates from Pakistan. Am J Trop Med Hyg 24: 145153.

  • 13.

    Qiang Y, Tamura A, Urakami H, Makisaka Y, Koyama S, Fukuhara M, Kadosaka T, 2003. Phylogenetic characterization of Orientia tsutsugamushi isolated in Taiwan according to the sequence homologies of 56-kDa type-specific antigen genes. Microbiol Immunol 47: 577583.

    • Search Google Scholar
    • Export Citation
  • 14.

    Ohashi N, Koyama Y, Urakami H, Fukuhara M, Tamura A, Kawamori F, Yamamoto S, Kasuya S, Yoshimura K, 1996. Demonstration of antigenic and genotypic variation in Orientia tsutsugamushi which were isolated in Japan, and their classification into type and subtype. Microbiol Immunol 40: 627638.

    • Search Google Scholar
    • Export Citation
  • 15.

    Ohashi N, Nashimoto H, Ikeda H, Tamura A, 1992. Diversity of immunodominant 56-kDa type-specific antigen (TSA) of Rickettsia tsutsugamushi. Sequence and comparative analyses of the genes encoding TSA homologues from four antigenic variants. J Biol Chem 267: 1272812735.

    • Search Google Scholar
    • Export Citation
  • 16.

    Shirai A, Tanskul PL, Andre RG, Dohany AL, Huxsoll DL, 1981. Rickettsia tsutsugamushi strains found in chiggers collected in Thailand. Southeast Asian J Trop Med Public Health 12: 16.

    • Search Google Scholar
    • Export Citation
  • 17.

    Ruang-Areerate T, Jeamwattanalert P, Rodkvamtook W, Richards AL, Sunyakumthorn P, Gaywee J, 2011. Genotype diversity and distribution of Orientia tsutsugamushi causing scrub typhus in Thailand. J Clin Microbiol 49: 25842589.

    • Search Google Scholar
    • Export Citation
  • 18.

    Rodkvamtook W, Ruang-Areerate T, Gaywee J, Richards AL, Jeamwattanalert P, Bodhidatta D, Sangjun N, Prasartvit A, Jatisatienr A, Jatisatienr C, 2011. Isolation and characterization of Orientia tsutsugamushi from rodents captured following a scrub typhus outbreak at a military training base, Bothong district, Chonburi province, central Thailand. Am J Trop Med Hyg 84: 599607.

    • Search Google Scholar
    • Export Citation
  • 19.

    Phasomkusolsil S, Tanskul P, Ratanatham S, Watcharapichat P, Phulsuksombati D, Frances SP, Lerdthusnee K, Linthicum KJ, 2009. Transstadial and transovarial transmission of Orientia tsutsugamushi in Leptotrombidium imphalum and Leptotrombidium chiangraiensis (Acari: Trombiculidae). J Med Entomol 46: 14421445.

    • Search Google Scholar
    • Export Citation
  • 20.

    Horinouchi H, Murai K, Okayama A, Nagatomo Y, Tachibana N, Tsubouchi H, 1996. Genotypic identification of Rickettsia tsutsugamushi by restriction fragment length polymorphism analysis of DNA amplified by the polymerase chain reaction. Am J Trop Med Hyg 54: 647651.

    • Search Google Scholar
    • Export Citation
  • 21.

    Lerdthusnee K, Nigro J, Monkanna T, Leepitakrat W, Leepitakrat S, Insuan S, Charoensongsermkit W, Khlaimanee N, Akkagraisee W, Chayapum K, Jones JW, 2008. Surveys of rodent-borne disease in Thailand with a focus on scrub typhus assessment. Integr Zool 3: 267273.

    • Search Google Scholar
    • Export Citation
  • 22.

    Jiang J, Chan TC, Temenak JJ, Dasch GA, Ching WM, Richards AL, 2004. Development of a quantitative real-time polymerase chain reaction assay specific for Orientia tsutsugamushi. Am J Trop Med Hyg 70: 351356.

    • Search Google Scholar
    • Export Citation
  • 23.

    Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG, 1997. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25: 48764882.

    • Search Google Scholar
    • Export Citation
  • 24.

    Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S, 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28: 27312739.

    • Search Google Scholar
    • Export Citation
  • 25.

    Chao CC, Zhang Z, Wang H, Alkhalil A, Ching WM, 2008. Serological reactivity and biochemical characterization of methylated and unmethylated forms of a recombinant protein fragment derived from outer membrane protein B of Rickettsia typhi. Clin Vaccine Immunol 15: 684690.

    • Search Google Scholar
    • Export Citation
  • 26.

    Sonthayanon P, Peacock SJ, Chierakul W, Wuthiekanun V, Blacksell SD, Holden MT, Bentley SD, Feil EJ, Day NP, 2010. High rates of homologous recombination in the mite endosymbiont and opportunistic human pathogen Orientia tsutsugamushi. PLoS Negl Trop Dis 4: e752.

    • Search Google Scholar
    • Export Citation
  • 27.

    Shirai A, Dohany AL, Ram S, Chiang GL, Huxsoll DL, 1981. Serological classification of Rickettsia tsutsugamushi organisms found in chiggers (Acarina: Trombiculidae) collected in Peninsular Malaysia. Trans R Soc Trop Med Hyg 75: 580582.

    • Search Google Scholar
    • Export Citation
  • 28.

    Shirai A, Huxsoll DL, Dohany AL, Montrey RD, Werner RM, Gan E, 1982. Characterization of Rickettsia tsutsugamushi strains in two species of naturally infected, laboratory-reared chiggers. Am J Trop Med Hyg 31: 395402.

    • Search Google Scholar
    • Export Citation
  • 29.

    Shirai A, Robinson DM, Brown GW, Gan E, Huxsoll DL, 1979. Antigenic analysis by direct immunofluorescence of 114 isolates of Rickettsia tsutsugamushi recovered from febrile patients in rural Malaysia. Jpn J Med Sci Biol 32: 337344.

    • Search Google Scholar
    • Export Citation
  • 30.

    Tamura A, Yamamoto N, Koyama S, Makisaka Y, Takahashi M, Urabe K, Takaoka M, Nakazawa K, Urakami H, Fukuhara M, 2001. Epidemiological survey of Orientia tsutsugamushi distribution in field rodents in Saitama Prefecture, Japan, and discovery of a new type. Microbiol Immunol 45: 439446.

    • Search Google Scholar
    • Export Citation
  • 31.

    Jiang J, Paris DH, Blacksell SD, Aukkanit N, Newton PN, Phetsouvanh R, Izzard L, Stenos J, Graves SR, Day NPJ, Richards AL, 2013. Diversity of the 47 kDa HtrA nucleic acid and translated amino acid sequences from 17 recent human isolates of Orientia. Vector Borne Zoonotic Dis 13: 367375.

    • Search Google Scholar
    • Export Citation
  • 32.

    Chattopadhyay S, Richards AL, 2007. Scrub typhus vaccines: past history and recent developments. Hum Vaccin 3: 7380.

Past two years Past Year Past 30 Days
Abstract Views 0 0 0
Full Text Views 270 101 23
PDF Downloads 94 31 6
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 

 

 

Characterization Based on the 56-Kda Type-Specific Antigen Gene of Orientia tsutsugamushi Genotypes Isolated from Leptotrombidium Mites and the Rodent Host Post-Infection

Ratree TakhampunyaDepartment of Entomology, United States Army Medical Component - Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand; Viral Diseases Branch, Walter Reed Army Institute of Research, Silver Spring, Maryland; Mahidol Oxford Tropical Medicine Research Unit, Mahidol University, Bangkok, Thailand; Viral and Rickettsial Diseases Department, Naval Medical Research Center, Silver Spring, Maryland; Entomology Branch, Walter Reed Army Institute of Research, Silver Spring, Maryland

Search for other papers by Ratree Takhampunya in
Current site
Google Scholar
PubMed
Close
,
Bousaraporn TippayachaiDepartment of Entomology, United States Army Medical Component - Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand; Viral Diseases Branch, Walter Reed Army Institute of Research, Silver Spring, Maryland; Mahidol Oxford Tropical Medicine Research Unit, Mahidol University, Bangkok, Thailand; Viral and Rickettsial Diseases Department, Naval Medical Research Center, Silver Spring, Maryland; Entomology Branch, Walter Reed Army Institute of Research, Silver Spring, Maryland

Search for other papers by Bousaraporn Tippayachai in
Current site
Google Scholar
PubMed
Close
,
Sommai PromsathapornDepartment of Entomology, United States Army Medical Component - Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand; Viral Diseases Branch, Walter Reed Army Institute of Research, Silver Spring, Maryland; Mahidol Oxford Tropical Medicine Research Unit, Mahidol University, Bangkok, Thailand; Viral and Rickettsial Diseases Department, Naval Medical Research Center, Silver Spring, Maryland; Entomology Branch, Walter Reed Army Institute of Research, Silver Spring, Maryland

Search for other papers by Sommai Promsathaporn in
Current site
Google Scholar
PubMed
Close
,
Surachai LeepitakratDepartment of Entomology, United States Army Medical Component - Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand; Viral Diseases Branch, Walter Reed Army Institute of Research, Silver Spring, Maryland; Mahidol Oxford Tropical Medicine Research Unit, Mahidol University, Bangkok, Thailand; Viral and Rickettsial Diseases Department, Naval Medical Research Center, Silver Spring, Maryland; Entomology Branch, Walter Reed Army Institute of Research, Silver Spring, Maryland

Search for other papers by Surachai Leepitakrat in
Current site
Google Scholar
PubMed
Close
,
Taweesak MonkannaDepartment of Entomology, United States Army Medical Component - Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand; Viral Diseases Branch, Walter Reed Army Institute of Research, Silver Spring, Maryland; Mahidol Oxford Tropical Medicine Research Unit, Mahidol University, Bangkok, Thailand; Viral and Rickettsial Diseases Department, Naval Medical Research Center, Silver Spring, Maryland; Entomology Branch, Walter Reed Army Institute of Research, Silver Spring, Maryland

Search for other papers by Taweesak Monkanna in
Current site
Google Scholar
PubMed
Close
,
Anthony L. SchusterDepartment of Entomology, United States Army Medical Component - Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand; Viral Diseases Branch, Walter Reed Army Institute of Research, Silver Spring, Maryland; Mahidol Oxford Tropical Medicine Research Unit, Mahidol University, Bangkok, Thailand; Viral and Rickettsial Diseases Department, Naval Medical Research Center, Silver Spring, Maryland; Entomology Branch, Walter Reed Army Institute of Research, Silver Spring, Maryland

Search for other papers by Anthony L. Schuster in
Current site
Google Scholar
PubMed
Close
,
Melanie C. MelendrezDepartment of Entomology, United States Army Medical Component - Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand; Viral Diseases Branch, Walter Reed Army Institute of Research, Silver Spring, Maryland; Mahidol Oxford Tropical Medicine Research Unit, Mahidol University, Bangkok, Thailand; Viral and Rickettsial Diseases Department, Naval Medical Research Center, Silver Spring, Maryland; Entomology Branch, Walter Reed Army Institute of Research, Silver Spring, Maryland

Search for other papers by Melanie C. Melendrez in
Current site
Google Scholar
PubMed
Close
,
Daniel H. ParisDepartment of Entomology, United States Army Medical Component - Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand; Viral Diseases Branch, Walter Reed Army Institute of Research, Silver Spring, Maryland; Mahidol Oxford Tropical Medicine Research Unit, Mahidol University, Bangkok, Thailand; Viral and Rickettsial Diseases Department, Naval Medical Research Center, Silver Spring, Maryland; Entomology Branch, Walter Reed Army Institute of Research, Silver Spring, Maryland

Search for other papers by Daniel H. Paris in
Current site
Google Scholar
PubMed
Close
,
Allen L. RichardsDepartment of Entomology, United States Army Medical Component - Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand; Viral Diseases Branch, Walter Reed Army Institute of Research, Silver Spring, Maryland; Mahidol Oxford Tropical Medicine Research Unit, Mahidol University, Bangkok, Thailand; Viral and Rickettsial Diseases Department, Naval Medical Research Center, Silver Spring, Maryland; Entomology Branch, Walter Reed Army Institute of Research, Silver Spring, Maryland

Search for other papers by Allen L. Richards in
Current site
Google Scholar
PubMed
Close
, and
Jason H. RichardsonDepartment of Entomology, United States Army Medical Component - Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand; Viral Diseases Branch, Walter Reed Army Institute of Research, Silver Spring, Maryland; Mahidol Oxford Tropical Medicine Research Unit, Mahidol University, Bangkok, Thailand; Viral and Rickettsial Diseases Department, Naval Medical Research Center, Silver Spring, Maryland; Entomology Branch, Walter Reed Army Institute of Research, Silver Spring, Maryland

Search for other papers by Jason H. Richardson in
Current site
Google Scholar
PubMed
Close

Characterization of the 56-kDa type-specific antigen (TSA) genes of Orientia tsutsugamushi (OT) from three naturally infected, laboratory-reared mite colonies comprising three species (Leptotrombidium deliense [Ld], Leptotrombidium imphalum [Li], and Leptotrombidium chiangraiensis [Lc]) has revealed the presence of single and coexisting OT genotypes found in individual chiggers. The Karp genotype was found in all of the chiggers examined, whereas Gilliam and UT302 genotypes were only observed in combination with the Karp genotype. From analysis of these OT genotypes after transmission from chiggers to mice it was determined that with the Lc and Li mites, the OT genotype composition in the rodent spleens post-infection had not changed and therefore resembled that observed in the feeding chiggers. However, only the Karp genotype was found in rodents after feeding by Ld chiggers carrying Karp and Gilliam genotypes. The current findings reveal a complex association among the host, pathogen, and vector.

Introduction

Orientia tsutsugamushi, the causative agent of scrub typhus, is an obligate intracellular Gram-negative bacterium vectored by larval Leptotrombidium mites (chiggers). Mites serve as reservoirs and the bacterium is maintained in successive mite generations by transovarial transmission.15 The chigger infects a rodent or human host when feeding on tissue fluid.68 With no vaccine, scrub typhus prevention is a major challenge and infections may be fatal if untreated.9 Orientia tsutsugamushi-infected mites are widely distributed throughout many Asian countries, northern Australia, and the western Pacific islands thereby placing an estimated one billion people at risk for scrub typhus, which averages one million cases annually.10

Orientia tsutsugamushi has been characterized through serotyping on the basis of reactivity to hyperimmune serum raised against prototype strains (Karp, Gilliam, Kato, TA716)11,12 and phylogenetic analysis using 56-kDa type-specific antigen (TSA) sequences from outer membrane protein-encoding genes.13 The 56-kDa TSA of O. tsutsugamushi is the immunodominant protein14 for which the conserved domain regions of the 56-kDa TSA are responsible for cross-reactivity of antisera against diverse serotypes, whereas the variable domains I–IV can be used for serotype identification.15

A past study showed the most prevalent O. tsutsugamushi serotype found in rodents and vectors in Thailand was Karp, whereas other serotypes (Gilliam, Kato, TA716, and TA763) were proportionately less common.16 However, recent genotyping based on the 56-kDa TSA gene sequence has shown significant diversity in the nucleotide sequence of O. tsutsugamushi isolates from patients and rodent hosts in many parts of Thailand. The resulting genotypes exhibited much lower similarity to the prototype strains than previously described.17,18 In the current study, we also use the 56-kDa TSA gene sequence (700 basepairs [bp]) to characterize O. tsutsugamushi from 12 naturally infected, laboratory-reared Leptotrombidium colonies (comprising three Leptotrombidium species: L. deliense, L. imphalum, and L. chiangraiensis) located at the Armed Forces Research Institute of Medical Sciences in Bangkok, Thailand (AFRIMS). As knowledge regarding the transmission dynamics of O. tsutsugamushi and chigger-host interactions is limited, we evaluated the degree to which the composition of O. tsutsugamushi genotype(s) found in an individual chigger is maintained in the rodent host post-infection. In addition, O. tsutsugamushi were characterized by phylogenetic analysis of the 56-kDa TSA gene sequences from field-collected rodents and chiggers to supplement our findings.

Materials and Methods

Laboratory-reared O. tsutsugamushi-infected mites.

Twelve O. tsutsugamushi-infected mite colonies (four lines of L. chiangraiensis, seven lines of L. imphalum, and one line of L. deliense) are maintained in an animal biosafety level-3 (ABSL-3) facility at AFRIMS (Table 1).19 Larval mites were fed the ears of Institute of Cancer Research (ICR) mice from a Charles River Technology (BioLASCO, Taiwan) colony maintained by the Department of Veterinary Medicine, AFRIMS, whereas nymphs and adults were fed a diet of springtail eggs (protocol #09-11, Maintenance of the Leptotrombidium larval mite colonies: chigger feeding on ICR mice [Mus musculus]).

Table 1

Origins of 12 Orientia tsutsugamushi-infected Leptotrombidium mite colonies maintained at Armed Forces Research Institute of Medical Sciences (AFRIMS), Bangkok, Thailand

Colony Date established Mite species Host Location
Ld-1 9 Sep 1992 L. deliense Tupaia glis Bangkruai, Nonthaburi
Li-1 24 Sep 1993 L. imphalum Rattus koratensis Ban Thungha, Chiang Rai
Li-2 25 Nov 1993 L. imphalum Rattus rattus Ban Pagook, Chiang Rai
Li-3 25 Nov 1993 L. imphalum Rattus rattus Ban Pagook, Chiang Rai
Li-4 18 May 1994 L. imphalum Rattus rattus Ban Nongkrok, Chiang Rai
Li-5 19 Oct 1995 L. imphalum Rattus rattus Ban Maesad, Chiang Rai
Li-6 19 Jun 1996 L. imphalum Rattus rattus Ban Nongkrok, Chiang Rai
Li-7 19 Jun 1996 L. imphalum Rattus rattus Ban Nongkrok, Chiang Rai
Lc-1 13 Jan 1994 L. chiangraiensis Rattus losea Ban Pagook, Chiang Rai
Lc-2 18 Oct 1995 L. chiangraiensis Rattus rattus Ban Pagook, Chiang Rai
Lc-4 15 Jul 1998 L. chiangraiensis Rattus rattus Ban Pagook, Chiang Rai
Lc-5 15 Jul 1998 L. chiangraiensis Rattus rattus Ban Pagook, Chiang Rai

Extraction of genomic DNA from O. tsutsugamushi-infected mites.

Orientia tsutsugamushi-infected chiggers from different lines of Leptotrombidium spp. were individually subjected to genomic DNA extraction using a modified tissue protocol from the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). An infected chigger was placed in a 1.5-mL microcentrifuge tube and punctured with a fine needle. Ninety microliters of ATL lysis buffer was added and the sample was either processed immediately or stored at −70°C. Ten microliters of Proteinase K solution (20 mg/mL) was added and the sample was incubated at 56°C for 3 h. Subsequently, 100 μL of AL buffer was added and the sample mixed by pulse-vortexing for 15 s followed by incubation at 70°C for 10 min. One hundred microliters of absolute ethanol was added and the sample mixed by pulse-vortexing for 15 s. The sample was then applied to a QIAamp spin column and DNA was eluted in 50 μL AE buffer and stored at −20°C until amplification.

Amplification and sequencing of the 56-kDa TSA.

The 56-kDa TSA gene was amplified by nested polymerase chain reaction (nPCR) using previously described primers20 and in-house primers designed from published sequences (GenBank accession nos. AY956315, M33004). The first amplification step was performed using in-house designed primers; CG56F (5′- TTA CAA TGG ATA AAA CGC TTT GAA-3′) and CG56R (5′-AGA AAA ACC TAG AAG TTA TAG CGT ACA-3′) and the second step used previously described primers RTS8 and RTS9.20 A volume of 2.5 μL of extracted DNA was used as template in the first amplification step. One-tenth of the total volume from the first amplification was used for the second amplification. A total of 25 μL of the PCR reaction mixture contained template DNA, 400 nM of each primer, 200 μM of dNTP, 1× PCR buffer, and 0.5 units of iProof High-Fidelity DNA polymerase (Bio-Rad, Hercules, CA). Amplification was performed using a DNA thermal cycler (GeneAmp PCR System 2700, Applied Biosystems, Foster City, CA) under the following conditions: initial denaturation at 98°C for 2 min; 40 cycles of 98°C for 10 s (denaturation); 53°C for 20 s (annealing); 72°C for 1 min (extension); and 72°C for 5 min. The second amplification step was carried out following the same protocol above except that the annealing step in the cycle was 55°C for 15 s and the extension period was shortened to 45 s. The PCR product was run on agarose gel (1.5%) using electrophoresis and visualized with GelStar Nucleic Acid Gel Stain (Lonza, Basel, Switzerland). The image of the gel was documented using a Gel imaging system-U:Genius (Syngene, Cambridge, UK).

Characterization of O. tsutsugamushi genotype.

The PCR product of 56-kDa TSA gene (700 bp) was purified by Qiagen DNA purification kit (Qiagen) for removal of salts and primer dimers. Purified PCR fragment of about 700 bp was then cloned into pCR2.1-TOPO vector (Invitrogen, Foster City, CA) and grown in Escherichia coli (DH5α-T1R). Seventeen to 20 E. coli colonies carrying a plasmid successfully harboring 56-kDa insert were randomly selected for sequencing. Plasmids were purified from E. coli using QIAprep Spin Miniprep Kit (Qiagen) and sent for DNA sequencing (Sanger method) at AITBiotech (Singapore).

Transmission of O. tsutsugamushi from the chigger to the rodent host.

A single chigger from each of three O. tsutsugamushi-infected chigger lines (L. chiangraiensis [Lc-1], L. imphalum [Li-1], and L. deliense [Ld-1]) was individually placed on the ear of an anesthetized O. tsutsugamushi-naive ICR mouse (4 to 10 weeks of age). Mice were restrained in cages for 3 to 4 d, removed, and then transferred to a polycarbonate shoebox cage. The chigger was removed from each mouse, placed in a 1.5-mL tube, and punctured with a fine needle. Ninety microliters of ATL lysis buffer (Qiagen) were added and the sample either processed immediately or stored at −70°C. Mice were clinically observed for 30 d for signs and symptoms of O. tsutsugamushi infection. Mice infected with O. tsutsugamushi were euthanized when they exhibited signs of moribund illness. The liver, spleen, and kidneys were dissected and stored at −70°C. Mice not developing signs of clinical scrub typhus infection at the end of the 28-d observation period were euthanized, and blood and organs collected for determination of O. tsutsugamushi presence using nPCR.20 The procedures described previously were approved by the AFRIMS Institutional Animal Care and Use Committee (IACUC) under protocol number 09-11 entitled, “Maintenance of the Leptotrombidium larval mite colonies: chigger feeding on ICR mice (Mus musculus).”

Extraction of genomic DNA from infected mice.

Extraction of genomic DNA from the spleens of infected mice obtained from the transmission experiment described previously was performed using the Wizard Genomic DNA Purification Kit (Promega, Madison, WI). Samples of infected spleen were placed into a 1.5-mL tube containing 600 μL of chilled nucleic lysis solution and incubated at 65°C for 15–30 min. Proteinase K solution (20 mg/mL) was added to reach a final concentration of 580 μg/mL and the solution incubated at 55°C for 3 h. Three microliters of RNase solution were added and the mixture incubated at 37°C for 15–30 min. Undesirable protein was precipitated out by first adding 200 μL of protein precipitation solution. The solution was vortexed, placed on ice for 5 min, and spun at 13,000 rpm for 4 min at room temperature. The supernatant was transferred to a sterile tube and 600 μL of isopropanol was added to precipitate the DNA. The DNA was recovered by centrifugation at 13,000 rpm for 1 min at room temperature, briefly washed with 70% ethanol, air-dried, and resuspended in 100 μL of DNA rehydration solution. The PCR, product purification, and cloning were carried out as described previously.

Field-collected rodents and chiggers.

Rodent and chigger collections were conducted during the wet season (August 2010) in Buri Ram and Chachoeng Sao provinces, Thailand. Field-collected rodents were identified to species and the blood obtained by cardiac puncture and organs collected following dissection as previously described.21 Chiggers were removed from the ears and stored in 70% ethanol. Animal collection procedures were approved by the AFRIMS IACUC under protocol number 09-02 entitled, “Field sampling of small mammal populations to support zoonotic disease surveillance and ectoparasite collection.”

Quantitative real-time PCR (qPCR) screening for O. tsutsugamushi from field-collected rodents/chiggers.

Field-collected rodents and chiggers were screened for O. tsutsugamushi by a qPCR assay.22 The primers and probe were designed to detect a portion of the 47-kDa HtrA gene. The reaction mixture (25 μL) contained 12.5 μL 2× Platinum Quantitative PCR SuperMix-UDG (Invitrogen), 0.2 μM probe, and 0.2 μM each of primers. The qPCR assay, Otsu47, was performed by incubating the sample at 95°C for 2 min followed by 45 cycles of 95°C for 15 s, and 60°C for 1 min using a chromo4 Real-time PCR detector (Bio-Rad). Rodents or chiggers found to be positive were further characterized by amplification and sequencing of the 56-kDa TSA as described previously.

Sequence and phylogenetic analysis.

Sequences were edited and assembled using Sequencer program v4.1.4 (Applied Biosystems) and compared with sequences in the BLASTN database (National Center for Biotechnology Information). Multiple sequence alignment was performed using the CLUSTALW program.23 The Neighbor-Joining (NJ) tree with bootstrapping (2,000 replicates) was constructed using the p-distance model of nucleotide substitution (56-kDa gene variable domains I-III) in the MEGA5 program.24

Determination of serological reactivity to O. tsutsugamushi 56-KDa recombinant proteins by enzyme-linked immunosorbent assay (ELISA).

The experiment was performed as previously described.25 The ELISA plates were coated with 0.3 μg/well/100 μL of Karp, Gilliam, Kato, and TA763 56-KDa recombinant protein overnight at 4°C. Briefly, coated plates were first rinsed three times with 1 × phosphate-buffered saline (PBS) containing 0.1% Tween 20 (1 × PBST), and then blocked with 200 μL/well of 10% milk in 1 × PBST, and incubated for 1 h at room temperature. Mouse serum was serial diluted by a factor of 4 (1:100, 1:400, 1:1,600) in 5% milk in 1 × PBST. Plates were washed after blocking and incubated with diluted mouse serum for 1 h at room temperature. The plates were then washed again and incubated with Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Santa Cruz Biotechnology, Santa Cruz, CA) at a 1:4,000 dilution for 1 h at room temperature. At the end of incubation, plates were washed and substrate (Kirkegaard and Perry Laboratories, Gaithersburg, MD) was added. The plates were incubated at room temperature for 15–30 minutes in the dark and then read the optical density (OD) at 405–650 nm on an UVmax kinetic microplate reader (Molecular Devices, Sunnyvale, CA). The mean of the negative controls plus two times the standard deviation was used as the cutoff value for determining a positive result. The cutoff value was 0.1 for mouse serum samples.

Animal care and use.

The experiments reported herein were conducted in compliance with the Animal Welfare Act and in accordance with the principles set forth in the “Guide for the Care and Use of Laboratory Animals,” Institute of Laboratory Animals Resources, National Research Council, National Academy Press, 1996.

Results

Characterization of O. tsutsugamushi from colonized Leptotrombidium lines.

The full-length 56-kDa TSA DNA gene (1.8 kb) and ∼700-bp nested PCR product covering variable domains I–III were amplified from individual chigger from each of 12 distinct Leptotrombidium lines maintained in colony at AFRIMS (Figure 1A and B). We selected one line from each Leptotrombidium species (Lc-1, Li-1, Ld-1) for further characterization of O. tsutsugamushi strains/genotypes maintained by that species. Sequence analysis of the partial 56-kDa gene (700 bp) of O. tsutsugamushi from 17 to 20 sequences/clones randomly selected from each chigger (Leptotrombidium) species showed the presence of O. tsutsugamushi genotypes closely related to Karp, UT302, or Gilliam (Table 2, Figure 2). Alignment of 19 sequences of the 56-kDa genes selected from one Lc-1 resulted in one identical genotype closely related to Karp-related genotypes with 94.2% similarity to the reference Karp genotype (Table 2, Figure 2). However, the alignment of 56-kDa TSA sequences derived from one Li-1 and from one other Ld-1 chigger revealed different results.

Figure 1.
Figure 1.

Polymerase chain reaction (PCR) products derived from the amplification of (A) the full-length and (B) variable domains I–III 56-kDa TSA gene of O. tsutsugamushi from 12 infected Leptotrombidium colonies.

Citation: The American Society of Tropical Medicine and Hygiene 90, 1; 10.4269/ajtmh.13-0393

Table 2

Sequence distance of 56-kDa TSA gene sequences (Variable Domains I–III, 640 bp) of three Orientia tsutsugamushi-infected Leptotrombidium mite colonies (Lc-1, Li-1, Ld-1), references O. tsutsugamushi Karp, Gilliam, Kato, TA763, and other Genotypes

  Lc-1 Li-1a Li-1b Ld-1a Ld-1b Karp Gilliam Kato TA763 UT302 Taitung4
Lc-1 (19*) *** 81.3 96.9 83.2 97.5 94.2 84.3 81.2 83.8 82.1 81.0
Li-1a (15/20*) 21.6 *** 79.9 76.5 80.3 78.0 79.2 77.1 85.5 98.5 99.1
Li-1b (5/20*) 3.2 23.5 *** 82.5 95.4 93.2 83.3 80.3 83.8 80.6 79.6
Ld-1a (11/17*) 19.1 28.3 19.9 *** 20.1 82.1 93.0 79.8 80.4 77.2 76.2
Ld-1b (6/17*) 2.6 22.9 4.7 82.4 *** 93.2 84.5 79.6 82.6 81.6 79.9
Karp 6.1 26.1 7.2 20.5 7.1 *** 83.7 77.8 81.9 78.6 78.0
Gilliam 17.7 24.5 18.9 7.5 17.3 18.4 *** 79.8 82.7 78.9 79.0
Kato 21.8 27.4 23.0 23.7 24.0 26.5 23.7 *** 78.4 77.4 76.6
TA763 18.3 16.2 18.3 22.7 19.9 20.7 19.8 25.7 *** 85.9 85.1
UT302 20.5 1.5 22.5 27.2 21.2 25.3 24.8 27.0 15.7 *** 2.2
Taitung4 22.0 0.9 23.9 28.7 23.5 26.1 24.7 28.3 16.7 97.9 ***

Percent similarity shows in upper triangle and percent divergence in lower triangle. Letter “a” and “b” indicate the difference of sequence type. Highest identity with reference genotypes are shown in bold type.

Number of bacterial colonies with a 56-kDa type-specific antigen (TSA) insert, matching reference genotype per total number of colonies evaluated.

Figure 2.
Figure 2.

Neighbor-joining (NJ) tree derived from partial 56-kDa TSA genes (variable domains I–III, 498 bp) of O. tsutsugamushi. The analysis involved 46 nucleotide sequences of O. tsutsugamushi-infected chigger lines (Lc-1, Li-1, Ld-1), O. tsutsugamushi-infected wild-caught rodent and chigger along with reference sequences retrieved from the GenBank database as shown in Table 5. The NJ tree was constructed using the p-distance method of nucleotide substitution model. The bootstrap P values > 50% are indicated at major nodes (2,000 replicates). Tree is rooted at its midpoint. Scale bar represents substitutions per site.

Citation: The American Society of Tropical Medicine and Hygiene 90, 1; 10.4269/ajtmh.13-0393

The alignment of 20 sequences from Li-1 and 17 sequences from Ld-1 lines resulted in two distinct genotypes found in single chigger, designated “a” and “b” in both lines. For the Li-1 line, the majority of sequences (15 of 20 sequences) clustered with the UT302-related genotypes consisting of TA763, TA716, Taitung-4, UT302, and CB19 with 99.1% and 98.5% sequence similarity to genotypes Taitung-4 and UT302, respectively (Figure 2, Table 2). This sequence type was designated as Li-1a. The remaining five sequences from the Li-1 line (designated Li-1b) grouped with Karp-related genotypes with 93.2% sequence similarity to the reference Karp and 98.8% similarity to a nearby cluster of genotypes that included UT76 and UT332 (Figure 2). For the Ld-1 line, 11 of 17 sequences were identical and clustered with the Gilliam group (designated Ld-1a) with 93.0% sequence similarity to the reference Gilliam and 97.8% sequence similarity to a nearby group of genotypes that included UT125 and UT196 (Figure 2). The remaining six sequences (designated Ld-1b) fell into Karp-related genotypes with 93.2% sequence similarity to reference Karp and 98.0% to the TW45R genotype. The sequence similarity among Karp-related genotypes found in Lc-1, Li-1b, and Ld-1b was relatively high (95.4% to 97.5%). Although Lc-1 and Ld-1b grouped together in the same cluster, Li-1b formed a separate group distant from but shared common ancestor with Lc-1/Ld-1b types.

O. tsutsugamushi genotype characterization from laboratory-reared rodents and chiggers post-infection.

The previous results show that Lc-1, Li-1, and Ld-1 lines maintain O. tsutsugamushi Karp-related, Karp-related/UT302, and Karp-related/Gilliam genotypes, respectively. We determined whether the composition of O. tsutsugamushi genotype(s) found in the rodent post-infection reflected the pattern observed in the chigger that fed on the rodent. Mice challenged with Lc-1 and Li-1 showed signs of illness within 10 d post-infection, whereas the mouse challenged with Ld-1 did not develop signs of infection within the 28-d period of observation, although spleen enlargement and O. tsutsugamushi-specific antibodies were observed (data not shown). Spleens from mice challenged with Lc-1, Li-1, and Ld-1 were positive for O. tsutsugamushi as determined by nPCR assay. Analysis of 56-kDa sequences showed that the composition of O. tsutsugamushi genotype(s) in spleens from infected mice generally mimicked the pattern observed in the Lc-1 and Li-1 chiggers, which fed on the mice (Table 3). Twenty sequences from the Lc-1 chigger that fed on an O. tsutsugamushi-naïve rodent were found to be a member of the Karp-related genotype (94.2% similarity) and this same pattern was observed in the rodent spleen post-transmission (Table 3). The sequence alignment shows almost no variability in sequences within and between the chigger and the rodent (data not shown). The UT302-related sequences (98.5% similarity) comprised the majority of sequences (12 of 18) in Li-1 and a similar majority (17 of 19) was maintained in the rodent post-infection (98.5% similarity) (Table 3). Karp-related genotype sequences comprised a minority of sequences in the Li-1 chigger (6 of 18; 5 with 93.2% similarity and 1 with 99.8% similarity) and a similar minority was maintained in the rodent (2 of 19 with 93.2% similarity) post-infection. However, the pattern exhibited in the Ld-1 chigger differed from that observed in the rodent spleen. Even though Karp-related (7 of 18 sequences with 93.2% similarity) and Gilliam (11 of 18 sequences with 93.0% similarity) genotypes were found in the Ld-1 chigger, only the Karp-related genotype (20 of 20 sequences with 93.2% similarity) was found in the mouse spleen post-infection.

Table 3

Similarities of 56-kDa TSA gene sequences of the variable domains I–III from chiggers (Lc-1, Li-1, Ld-1) and the rodent post-infection to reference genotypes (Karp, Gilliam, UT302)

Mouse feeding experiment Sample Sequence type Sequence similarity to reference genotypes*
Karp (AY956315) UT302 (EF213095) Gilliam (DQ485289)
Lc-1 Chigger Lc-1 20/20 (94.2)
Mouse spleen Lc-1 20/20 (94.2)
Li-1 Chigger Li-1a 12/18 (98.5)
Li-1b 5/18 (93.2)
N/A 1/18 (99.8)
Mouse spleen Li-1a 17/19 (98.5)
Li-1b 2/19 (93.2)
Ld-1 Chigger Ld-1a 11/18 (93.0)
Ld-1b 7/18 (93.2)
Mouse spleen Ld-1b 20/20 (93.2)

Number of bacterial colonies with a 56 kDa type-specific antigen (TSA) gene insert matching reference genotype/total number of colonies evaluated. Numbers in parentheses represent percent sequence similarity to corresponding reference genotype.

O. tsutsugamushi genotype characterization from field-collected rodents and chiggers.

The data of O. tsutsugamushi characterization reported previously are solely from the experiments using laboratory-reared Leptotrombidium chiggers, even though the original mite colonies were established from chiggers collected from wild-caught rodents in Thailand, however, the mite colonies have been maintained in a laboratory environment for over 10 years. To confirm the coexistence of O. tsutsugamushi genotypes in wild chigger and rodent populations, we examined chiggers and rodents collected from field sites in Thailand during the rodent-borne diseases surveillance in 2010. One O. tsutsugamushi-positive rodent from each province was selected for 56-kDa TSA sequence analysis. Bandicota indica from Buri Ram (BA0185) contained 14 of 20 Karp-related genotype sequences that were most similar to Lc-1 (Table 4, Figure 2). Five of 20 sequences were Gilliam-related genotype sequences most similar to Ld-1a. Finally, 1 of 20 sequences was most similar to the UT302-related genotype particularly to Taitung-4 and Li-1a. No chiggers from BA0185 (total N = 16 chiggers) were positive for O. tsutsugamushi. The rodent captured in Chachoeng Sao (BA0344) contained only Gilliam-related genotype sequences (15 of 15 sequences) that were most similar to UT125, UT196, and UT144. Two of 7 chiggers from this same rodent were positive for O. tsutsugamushi. Characterization of one of the two chiggers showed coexistence of Gilliam-related (4 of 18 sequences) and UT302-related (14 of 18 sequences) genotypes. The Gilliam-related genotype sequences were most similar to Ld-1a and the UT302-related genotype sequences were most similar to Taitung-4 and Li-1a.

Table 4

Characterization of Orientia tsutsugamushi obtained from field-collected rodents and a chigger in Thailand based on 56-kDa TSA sequence homology to reference genotypes (Karp, Gilliam, UT302)

Location Rodent species No. of infected chiggers/total collected* Similarity to reference genotypes (no. of 56-kDa TSA sequences matching reference genotype/total sequences analyzed)
Rodent Chigger
Buri Ram (Forest) Bandicota indica (BA0185) 0/16 Gilliam (5/20)
Karp (14/20)
UT302 (1/20)
Chachoeng Sao (Rice field) Bandicota indica (BA0344) 2/7 Gilliam (15/15) Gilliam (4/18)
UT302 (14/18)

Determined by Otsu47 quantitative real-time polymerase chain reaction (qPCR) assay on DNA sample extracted from an individual chigger.

Number of bacterial colonies with a 56-kDa type-specific antigen (TSA) insert matching reference genotype/total number of colonies evaluated.

Characterization performed on one infected chigger.

Table 5

Descriptions of Orientia tsutsugamushi isolates and reference genotypes used in this study

Isolate Source Country Accession no.
CB19 Rattus rattus Thailand GU068058
CB62 Bandicota indica Thailand GU068055
FPW2016 Human Thailand EF213085
Gilliam Human Japan DQ485289
Hirahata Leptotrombidium pallidum Taiwan AF201835
Je-cheon Human Taiwan AF430143
Karp Human Taiwan AY956315
Kato Human Japan AY836148
Kawasaki Human Japan M63383
Kuroki Japan M63380
LA-1 Leptotrombidium arenicola Japan AF173049
LF-1 Japan AF173050
Shimokoshi Thailand M63381
TA678 Rattus rattus Thailand U19904
TA686 Tupaia glis Thailand U80635
TA716 Menetes berdmorei Thailand U19905
TA763 Rattus rajah Taiwan U80636
Taitung-4 Taiwan AY787232
TW45R Rattus losea Taiwan AY222632
TW461 Rattus rattus Taiwan AY222631
TWyu81 Leptotrombidium pallidum Thailand AY222640
UT76 Human Thailand EF213078
UT125 Human Thailand EF213096
UT144 Human Thailand EF213091
UT167 Human Thailand EF213080
UT176 Human Thailand EF213081
UT177 Human Thailand EF213084
UT196 Human Thailand EF213079
UT219 Human Thailand EF213100
UT302 Human Thailand EF213095
UT332 Human Thailand EF213083
Yeojoo Human South Korea AF430144
Yonchon South Korea U19903
Young-world Human South Korea AF430141

Discussion

Characterization of O. tsutsugamushi based on the 56-kDa TSA gene sequence revealed the coexistence of genotypes (Karp/Gilliam or Karp/UT302) in naturally infected, laboratory-reared mites (L. imphalum and L. deliense) and in one field-collected rodent (Gilliam/Karp/UT302) and chigger, Leptotrombidium spp. (Gilliam/UT302). Further evaluation revealed that the composition of O. tsutsugamushi genotype(s) observed in an individual chigger during feeding on an O. tsutsugamushi-naive rodent does not always mimic that which is found in the rodent post-infection. As such, these findings show a complex association between the host, pathogen, and vector.

Our results show isolates genetically related to members of the Karp genotype based on the 56-kDa TSA to be the most prevalent O. tsutsugamushi among the naturally infected mites maintained in our ABSL-3 laboratory. Each L. chiangraiensis line contained sequences belonging to Karp-related genotype, whereas L. imphalum and L. deliense each contained a member of the Karp genotype in coexistence with a member of the UT302 and Gilliam genotypes, respectively. These latter findings are consistent with our results from field-collected rodents and a chigger, and complement a previous report, which showed polytypic infections of O. tsutsugamushi in patient samples from Udon Thani province, Thailand using multi-locus sequence typing (MLST).26 Additionally, although the antigenic combination of O. tsutsugamushi strains has been previously observed in chiggers27,28 and human isolates from Malaysia using direct immunofluorescence staining and the complement fixation test,29 cross-reactivity was observed for type-specific monoclonal antibodies (mAbs) that recognize the 56-kDa antigen.30 Indeed, it is widely assumed that the 56-kDa TSA undergoes strong selection pressure from the host immune response and therefore the protein sequences change rapidly for bacteria to escape the immune response, resulting in antigenic diversity and the creation of new immunotypes based on the 56-kDa TSA gene. Therefore, 56-kDa TSA gene sequencing or MLST are preferable approaches to characterize O. tsutsugamushi isolates. In this study, it was possible to test only a single chigger from each line because of logistical considerations; however, future studies will be performed to show if most of any given hatch would yield identical rickettsial strain patterns.

To our knowledge, this is the first report to characterize O. tsutsugamushi genotype transmission from the chigger to the rodent host. In the case of Lc-1 and Li-1, the composition of genotypes found in the mouse spleen 14 d post-feeding was the same as that observed in the chigger during feeding. However, only the Karp genotype was found in the mouse that was fed upon by an Ld-1 chigger that carried multiple genotypes (Gilliam and Karp genotypes). The mechanism responsible for this latter observation is unknown. Interestingly, the Ld-1-infected mouse did not develop signs of illness and the antibody titer to the Karp prototype antigen, determined by ELISA assay, was found to be very high, whereas mice fed upon by either Lc-1 or Li-1 chiggers developed symptoms 10 d post-feeding with antibody titers lower than those observed in the mouse associated with the Ld-1 chigger (data not shown).

We clearly show significant variability in the 56-kDa gene of O. tsutsugamushi among chiggers. However, analysis of nucleotide sequences from the complete ORF 47-kDa gene from our 12 infected lines found no significant differences in the alignment of 47-kDa sequences among bacteria from O. tsutsugamushi-infected chiggers and those from reference genotypes (bootstrap P value ≤ 25) with sequence similarities ranging from 96.9% to 99.4% (data not shown). Such data, similar to recently described limited sequence variation of the 47-kDa gene found among recent human O. tsutsugamushi isolates and reference strains,31 provide justification for using the 47-kDa protein as an immunogen in the development of a scrub typhus vaccine as has been suggested previously.32

The host response observed after feeding by L. deliense in which the Gilliam genotype was not transmitted to the host, provides an impetus to better characterize the immune response of the host to infection. Our results highlight the need to better understand the transmission dynamics of O. tsutsugamushi, chigger-host interactions, and the host immune response in an effort to develop an effective vaccine against scrub typhus.

ACKNOWLEDGMENTS

We thank the following individuals AFRIMS: Kriangkrai Lerdthusnee for his assistance in acquiring funding for this project, Warinpassorn Leepitakrat for primer design, Achareeya Korkusol for conducting a preliminary data analysis, Ampornpan Kengluecha for performing the experiment throughout the study, and Brian P. Evans for editing and proofreading the first draft of this manuscript. Special thanks are extended to Wei-Mei Ching from the Naval Medical Research Center (Silver Spring, MD) for her review of this manuscript and performing ELISA assay.

  • 1.

    Rai J, Bandopadhyay D, 1978. Vertical transmission in chigger borne rickettsiosis. Indian J Med Res 68: 3138.

  • 2.

    Rapmund G, Dohany AL, Manikumaran C, Chan TC, Chan TC, 1972. Transovarial transmission of Rickettsia tsutsugamushi in Leptotrombidium (Leptotrombidium) arenicola Traub (Acarina: Trombiculidae). J Med Entomol 9: 7172.

    • Search Google Scholar
    • Export Citation
  • 3.

    Rapmund G, Upham RW Jr, Kundin WD, Manikumaran C, Chan TC, 1969. Transovarial development of scrub typhus rickettsiae in a colony of vector mites. Trans R Soc Trop Med Hyg 63: 251258.

    • Search Google Scholar
    • Export Citation
  • 4.

    Takahashi M, Murata M, Nogami S, Hori E, Kawamura A Jr, Tanaka H, 1988. Transovarial transmission of Rickettsia tsutsugamushi in Leptotrombidium pallidum successively reared in the laboratory. Jpn J Exp Med 58: 213218.

    • Search Google Scholar
    • Export Citation
  • 5.

    Urakami H, Takahashi M, Hori E, Tamura A, 1994. An ultrastructural study of vertical transmission of Rickettsia tsutsugamushi during oogenesis and spermatogenesis in Leptotrombidium pallidum. Am J Trop Med Hyg 50: 219228.

    • Search Google Scholar
    • Export Citation
  • 6.

    Takahashi M, Murata M, Misumi H, Hori E, Kawamura A Jr, Tanaka H, 1994. Failed vertical transmission of Rickettsia tsutsugamushi (Rickettsiales: Rickettsiaceae) acquired from rickettsemic mice by Leptotrombidium pallidum (Acari: trombiculidae). J Med Entomol 31: 212216.

    • Search Google Scholar
    • Export Citation
  • 7.

    Walker JS, Chan CT, Manikumaran C, Elisberg BL, 1975. Attempts to infect and demonstrate transovarial transmission of R. tsutsugamushi in three species of Leptotrombidium mites. Ann N Y Acad Sci 266: 8090.

    • Search Google Scholar
    • Export Citation
  • 8.

    Traub R, Wisseman CL Jr, Jones MR, O'Keefe JJ, 1975. The acquisition of Rickettsia tsutsugamushi by chiggers (trombiculid mites) during the feeding process. Ann N Y Acad Sci 266: 91114.

    • Search Google Scholar
    • Export Citation
  • 9.

    Nakayama K, Yamashita A, Kurokawa K, Morimoto T, Ogawa M, Fukuhara M, Urakami H, Ohnishi M, Uchiyama I, Ogura Y, Ooka T, Oshima K, Tamura A, Hattori M, Hayashi T, 2008. The whole-genome sequencing of the obligate intracellular bacterium Orientia tsutsugamushi revealed massive gene amplification during reductive genome evolution. DNA Res 15: 185199.

    • Search Google Scholar
    • Export Citation
  • 10.

    Rosenberg R, 1997. Drug-resistant scrub typhus: paradigm and paradox. Parasitol Today 13: 131132.

  • 11.

    Yamashita T, Kasuya S, Noda S, Nagano I, Ohtsuka S, Ohtomo H, 1988. Newly isolated strains of Rickettsia tsutsugamushi in Japan identified by using monoclonal antibodies to Karp, Gilliam, and Kato strains. J Clin Microbiol 26: 18591860.

    • Search Google Scholar
    • Export Citation
  • 12.

    Shirai A, Wisseman CL Jr, 1975. Serologic classification of scrub typhus isolates from Pakistan. Am J Trop Med Hyg 24: 145153.

  • 13.

    Qiang Y, Tamura A, Urakami H, Makisaka Y, Koyama S, Fukuhara M, Kadosaka T, 2003. Phylogenetic characterization of Orientia tsutsugamushi isolated in Taiwan according to the sequence homologies of 56-kDa type-specific antigen genes. Microbiol Immunol 47: 577583.

    • Search Google Scholar
    • Export Citation
  • 14.

    Ohashi N, Koyama Y, Urakami H, Fukuhara M, Tamura A, Kawamori F, Yamamoto S, Kasuya S, Yoshimura K, 1996. Demonstration of antigenic and genotypic variation in Orientia tsutsugamushi which were isolated in Japan, and their classification into type and subtype. Microbiol Immunol 40: 627638.

    • Search Google Scholar
    • Export Citation
  • 15.

    Ohashi N, Nashimoto H, Ikeda H, Tamura A, 1992. Diversity of immunodominant 56-kDa type-specific antigen (TSA) of Rickettsia tsutsugamushi. Sequence and comparative analyses of the genes encoding TSA homologues from four antigenic variants. J Biol Chem 267: 1272812735.

    • Search Google Scholar
    • Export Citation
  • 16.

    Shirai A, Tanskul PL, Andre RG, Dohany AL, Huxsoll DL, 1981. Rickettsia tsutsugamushi strains found in chiggers collected in Thailand. Southeast Asian J Trop Med Public Health 12: 16.

    • Search Google Scholar
    • Export Citation
  • 17.

    Ruang-Areerate T, Jeamwattanalert P, Rodkvamtook W, Richards AL, Sunyakumthorn P, Gaywee J, 2011. Genotype diversity and distribution of Orientia tsutsugamushi causing scrub typhus in Thailand. J Clin Microbiol 49: 25842589.

    • Search Google Scholar
    • Export Citation
  • 18.

    Rodkvamtook W, Ruang-Areerate T, Gaywee J, Richards AL, Jeamwattanalert P, Bodhidatta D, Sangjun N, Prasartvit A, Jatisatienr A, Jatisatienr C, 2011. Isolation and characterization of Orientia tsutsugamushi from rodents captured following a scrub typhus outbreak at a military training base, Bothong district, Chonburi province, central Thailand. Am J Trop Med Hyg 84: 599607.

    • Search Google Scholar
    • Export Citation
  • 19.

    Phasomkusolsil S, Tanskul P, Ratanatham S, Watcharapichat P, Phulsuksombati D, Frances SP, Lerdthusnee K, Linthicum KJ, 2009. Transstadial and transovarial transmission of Orientia tsutsugamushi in Leptotrombidium imphalum and Leptotrombidium chiangraiensis (Acari: Trombiculidae). J Med Entomol 46: 14421445.

    • Search Google Scholar
    • Export Citation
  • 20.

    Horinouchi H, Murai K, Okayama A, Nagatomo Y, Tachibana N, Tsubouchi H, 1996. Genotypic identification of Rickettsia tsutsugamushi by restriction fragment length polymorphism analysis of DNA amplified by the polymerase chain reaction. Am J Trop Med Hyg 54: 647651.

    • Search Google Scholar
    • Export Citation
  • 21.

    Lerdthusnee K, Nigro J, Monkanna T, Leepitakrat W, Leepitakrat S, Insuan S, Charoensongsermkit W, Khlaimanee N, Akkagraisee W, Chayapum K, Jones JW, 2008. Surveys of rodent-borne disease in Thailand with a focus on scrub typhus assessment. Integr Zool 3: 267273.

    • Search Google Scholar
    • Export Citation
  • 22.

    Jiang J, Chan TC, Temenak JJ, Dasch GA, Ching WM, Richards AL, 2004. Development of a quantitative real-time polymerase chain reaction assay specific for Orientia tsutsugamushi. Am J Trop Med Hyg 70: 351356.

    • Search Google Scholar
    • Export Citation
  • 23.

    Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG, 1997. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25: 48764882.

    • Search Google Scholar
    • Export Citation
  • 24.

    Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S, 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28: 27312739.

    • Search Google Scholar
    • Export Citation
  • 25.

    Chao CC, Zhang Z, Wang H, Alkhalil A, Ching WM, 2008. Serological reactivity and biochemical characterization of methylated and unmethylated forms of a recombinant protein fragment derived from outer membrane protein B of Rickettsia typhi. Clin Vaccine Immunol 15: 684690.

    • Search Google Scholar
    • Export Citation
  • 26.

    Sonthayanon P, Peacock SJ, Chierakul W, Wuthiekanun V, Blacksell SD, Holden MT, Bentley SD, Feil EJ, Day NP, 2010. High rates of homologous recombination in the mite endosymbiont and opportunistic human pathogen Orientia tsutsugamushi. PLoS Negl Trop Dis 4: e752.

    • Search Google Scholar
    • Export Citation
  • 27.

    Shirai A, Dohany AL, Ram S, Chiang GL, Huxsoll DL, 1981. Serological classification of Rickettsia tsutsugamushi organisms found in chiggers (Acarina: Trombiculidae) collected in Peninsular Malaysia. Trans R Soc Trop Med Hyg 75: 580582.

    • Search Google Scholar
    • Export Citation
  • 28.

    Shirai A, Huxsoll DL, Dohany AL, Montrey RD, Werner RM, Gan E, 1982. Characterization of Rickettsia tsutsugamushi strains in two species of naturally infected, laboratory-reared chiggers. Am J Trop Med Hyg 31: 395402.

    • Search Google Scholar
    • Export Citation
  • 29.

    Shirai A, Robinson DM, Brown GW, Gan E, Huxsoll DL, 1979. Antigenic analysis by direct immunofluorescence of 114 isolates of Rickettsia tsutsugamushi recovered from febrile patients in rural Malaysia. Jpn J Med Sci Biol 32: 337344.

    • Search Google Scholar
    • Export Citation
  • 30.

    Tamura A, Yamamoto N, Koyama S, Makisaka Y, Takahashi M, Urabe K, Takaoka M, Nakazawa K, Urakami H, Fukuhara M, 2001. Epidemiological survey of Orientia tsutsugamushi distribution in field rodents in Saitama Prefecture, Japan, and discovery of a new type. Microbiol Immunol 45: 439446.

    • Search Google Scholar
    • Export Citation
  • 31.

    Jiang J, Paris DH, Blacksell SD, Aukkanit N, Newton PN, Phetsouvanh R, Izzard L, Stenos J, Graves SR, Day NPJ, Richards AL, 2013. Diversity of the 47 kDa HtrA nucleic acid and translated amino acid sequences from 17 recent human isolates of Orientia. Vector Borne Zoonotic Dis 13: 367375.

    • Search Google Scholar
    • Export Citation
  • 32.

    Chattopadhyay S, Richards AL, 2007. Scrub typhus vaccines: past history and recent developments. Hum Vaccin 3: 7380.

Author Notes

* Address correspondence to Ratree Takhampunya, Department of Entomology, AFRIMS, 315/6 Rajvithi Rd., Bangkok 10400, Thailand. E-mail: RatreeT@afrims.org

Financial support: This work was supported by the Military Infectious Disease Research Program and the United States Army Medical Research and Materiel Command, Fort Detrick, MD.

Authors' addresses: Ratree Takhampunya, Bousaraporn Tippayachai, Sommai Promsathaporn, Surachai Leepitakrat, Taweesak Monkanna, and Anthony L. Schuster, Department of Entomology, AFRIMS, Bangkok, Thailand, E-mails: RatreeT@afrims.org, BousarapornT@afrims.org, SommaiP@afrims.org, SurachaiL@afrims.org, TaweesakM@afrims.org, and Schuster.Anthony@afrims.org. Melanie C. Melendrez, Viral Diseases Branch, Walter Reed Army Institute of Research (WRAIR), Silver Spring, MD, E-mail: melanie.c.melendrez.ctr@us.army.mil. Daniel H. Paris, Mahidol Oxford Tropical Medicine Research Unit (MORU), Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand, E-mail: parigi@tropmedres.ac. Allen L. Richards, Rickettsial Diseases Research Program, Viral and Rickettsial Diseases Department, Naval Medical Research Center, Silver Spring, MD, E-mail: allen.richards@med.navy.mil. Jason H. Richardson, Entomology Branch, Walter Reed Army Institute of Research (WRAIR), Silver Spring, MD, E-mail: Jason.H.Richardson@us.army.mil.

Save