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 Rick ettsia tsutsugamushi in chiggers (larval trombiculid mites) by polymer ase chain reaction (PCR). Three procedures for extracting DNA from fro zen chiggers were compared by evaluating the yield of PCR amplicand ob tained with nine oligonucleotide primer pairs derived from the rickett sial 22 kD, 47 kD, groESL, 56 kD, and 110 kD antigen genes. Although e xtracts and primer pairs differed in amplification efficiency, R. tsut sugamushi DNA was successfully detected in extracts of colonized infec ted 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 lengt h polymorphisms (RFLP) observed in PCR amplicands from five rickettsia l isolates obtained from the infected chigger colony over a 26-yr peri od were identical to those of PCR amplicands derived directly from inf ected chiggers taken from the same colony. This suggests that stable t ransmission 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 com plex epidemiology of scrub typhus rickettsiae in their mite vectors.