DETECTION AND CHARACTERIZATION OF RICKETTSIA-TSUTSUGAMUSHI (RICKETTSIALES, RICKETTSIACEAE) IN INFECTED LEPTOTROMBIDIUM (LEPTOTROMBIDIUM) FLETCHERI CHIGGERS (ACARI, TROMBICULIDAE) WITH THE POLYMERASE CHAIN-REACTION

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.