Detection and Characterization of Rickettsia tsutsugamushi (Rickettsiales: Rickettsiaceae) in Infected Leptotrombidium (Leptotrombidium) fletcheri Chiggers (Acari: Trombiculidae) with the Polymerase Chain Reaction

Abstract

We developed a method for detecting and characterizing the DNA of Rickettsia tsutsugamushi in chiggers (larval trombiculid mites) by polymerase chain reaction (PCR). Three procedures for extracting DNA from frozen chiggers were compared by evaluating the yield of PCR amplicand obtained with nine oligonucleotide primer pairs derived from the rickettsial 22 kD, 47 kD, groESL, 56 kD, and 110 kD antigen genes. Although extracts and primer pairs differed in amplification efficiency, R. tsutsugamushi DNA was successfully detected in extracts of colonized infected Leptotrombidium (Leptotrombidium) fletcheri (Wormersley & Heaslip) chiggers and in uninfected chigger extracts seeded with known amounts of Karp-strain rickettsiae. The 22 kD gene restriction fragment length polymorphisms (RFLP) observed in PCR amplicands from five rickettsial isolates obtained from the infected chigger colony over a 26-yr period were identical to those of PCR amplicands derived directly from infected chiggers taken from the same colony. This suggests that stable transmission of R. tsutsugamushi occurs in mites (62 generations), and isolates encompass the full genetic heterogeneity found in the chigger. PCR/RFLP analysis is an important new tool for investigating the complex epidemiology of scrub typhus rickettsiae in their mite vectors.

Dot-ELISA Rapid Test Using Recombinant 56-kDa Protein Antigens for Serodiagnosis of Scrub Typhus

“…23 Other immunological techniques for the diagnosis of scrub typhus have been developed, including a passive hemagglutination assay, 24 ELISAs, 25 dot-blot immunoassays, 26 and PCR. 27 However, none of these methods is routinely used on a widespread basis. Scrub typhus is an endemic infectious disease in rural areas of Asia, and diagnosis is the biggest challenge.…”

The Body Louse as a Vector of Reemerging Human Diseases

“…More recently, the association of amplification of specific fragments and analysis of the PCR products has been shown to be effective to obtain rapid and specific identification of the suspected bacterial agent in arthropods [72][73][74][75][76]. Different methods have been described for the extraction of bacterial DNA from arthropods: boiling triturated arthropods in saline buffer or in PCR extraction buffer [77], boiling hemolymph [78], phenol-chloroform extraction [79], or other recent extraction techniques [80,81]. In our laboratory, we have used columns (QIAamp Tissue Kit; QIAGEN, Hilden, Germany) to extract DNA from different arthropods, including lice, ticks, fleas, and ladybugs.…”

Early Diagnosis of Scrub Typhus with a Rapid Flow Assay Using Recombinant Major Outer Membrane Protein Antigen (r56) of Orientia tsutsugamushi

“…demonstrated to be a reliable method for diagnosing scrub typhus (14,17). Furthermore, different genotypes associated with different Orientia serotypes can be identified by analysis of variable regions of this gene without isolation of the organism (10,13,14,16,17,25,34).…”

“…Furthermore, different genotypes associated with different Orientia serotypes can be identified by analysis of variable regions of this gene without isolation of the organism (10,13,14,16,17,25,34). However, gene amplification requires a sophisticated instrument and labile reagents which are generally not available in most rural medical facilities.…”