INTERNATIONAL JOURNAL OF SYSTEMATIC BACTERIOLOGY, Apr. 1997, p. 446-452 Copyright 0 1997, International Union of Microbiological Societies 0020-7713/97/$04.00 + 0 Vol. 47, No. 2 Rickettsia peacockii sp. nov., a New Species Infecting Wood Ticks, Demacentor andersoni, in Western Montana? MARK L. NIEBYLSKI," MERRY E. SCHRUMPF, WILLY BURGDORFER, ELIZABETH R. FISCHER, KENNETH L. GAGE,$ AND TOM G. SCHWAN Laboratory of Microbial Structure and Function, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana 59840 Rickettsia peacockii, a new species of spotted fever group rickettsiae, was identified from Rocky Mountain wood ticks (Dermacentor andersoni) collected in the Sapphire Mountain Range on the eastern side of Bitterroot Valley, Montana. DNA from R. peacockii SkalkahoT (T = type strain) in naturally infected tick tissue was amplified by a PCR assay with primer sets derived from eubacterial 16s ribosomal DNA (rDNA), rickettsial citrate synthase, and 190-kDa surface antigen (rOmpA) genes. Partial 16s rDNA and rOmpA gene sequences exhibited levels of similarity of 99.7 and 93.2%, respectively, with the sequences of the spotted fever agent Rickettsia rickettsii R. By using GimCnez staining, fluorescent antibody tests, a PCR assay, and a restriction fragment length polymorphism analysis, 76 of 115 female ticks (minimal field infection rate, 66.1%) collected between 1992 and 1995 were found to be infected. The organism is passed transstadially and transovarially (minimal vertical transmission rate, 73.3%), and infections are localized in ovarial tissues. Attempts to cultivate R. peacockii were unsuccessful. Rickettsiae are gram-negative bacteria that are maintained in nature by replication in cells of small mammals and arthro- pods (5, 7, 23, 32, 47). At least 23 Rickettsia species (alpha subdivision of the Proteobacteria, family Rickettsiaceae) have been or are being described (40, 43), while undoubtedly more remain to be discovered. Of these species, Rickettsia rickettsii, the etiological agent of Rocky Mountain spotted fever (RMSF), is the most significant public health threat in the western hemisphere. Human RMSF cases were first recog- nized in 1873 in Bitterroot Valley in western Montana (34). Through a series of classic experiments begun in 1906, H. T. Ricketts and coworkers clearly established that the etiology of RMSF involves R. rickettsii in Rocky Mountain wood ticks (Demacentor andersoni) (39). Transmission to humans oc- curs principally through bites of infected ticks. Between 1981 and 1992, 4,217 confirmed human RMSF cases (minimum case/fatality ratio, 4.0%) were reported in the United States Since the pioneering work of Ricketts, several other species of rickettsiae have been recognized in Bitterroot Valley. A survey of 3,705 adult D. andersoni ticks collected in the valley during 1977 revealed four distinct serotypes of hemocyte-asso- ciated rickettsiae that were later identified as R. rickettsii,Rick- ettsia rhipicephali, Rickettsia montana, and Rickettsia bellii (33). Remarkably, only 2 of the 309 isolates found came from the 1,138 ticks collected on the eastern side of the valley, and both of these isolates were nonvirulent R. rhipicephali strains (33). With only one exception, spotted fever group (SFG) rickettsiae virulent for guinea pigs have never been recovered from east side wood ticks (32). These data correspond to the RMSF disease focality in the valley, where most human cases result (9). * Corresponding author. Mailing address: Rocky Mountain Labora- tories, NIAID-NIH, 903 S. Fourth St., Hamilton, MT 59840. Phone: (406) 363-9376. Fax: (406) 363-9204. E-mail: mark-niebylski@nih.gov. T This paper is dedicated to Mort G. Peacock in recognition of his lifelong contributions to rickettsiology, inimitable cell culture talents, and help provided to us and many others in studying rickettsiae. $ Present address: Division of Zoonotic Diseases, Centers for Dis- ease Control and Prevention, Ft. Collins, CO 80522. from exposure to west side ticks (8, 32). In 1992, a PCR analysis of hemolymph samples from 226 west side D. ander- soni adult ticks revealed that 4.0% of the samples were infected with one of three species of previously described rickettsiae (14). No dual rickettsial infections were reported in either the 1977 tick survey or the 1992 tick survey (14, 33). Despite the low prevalence of hemocyte-associated rickett- siae in east side wood ticks, examination of the tissues of these ticks revealed that a high proportion are infected with rickett- siae. Parker and Spencer determined that 36% of wood ticks collected during 1925 from the eastern side of Bitterroot Val- ley harbored nonvirulent rickettsiae (29). In 1981, Burgdorfer et al. found that a nonvirulent rickettsia was transstadially and transovarially maintained in approximately 70% of the east side wood ticks examined but was uncommon in west side ticks (8). This organism was designated the east side (ES) agent. The ES agent has not been found in hemocytes, and, given the high natural infection rate, it seems likely that the ES agent went undetected in the 1977 and 1992 tick hemolymph surveys. Burgdorfer et al. postulated that the ES agent interfered with the stable maintenance of virulent rickettsiae in nature, thereby providing a plausible explanation for the focal distri- bution of RMSF in Bitterroot Valley (8). Unfortunately, the inability to cultivate the ES agent and distinguish it from other rickettsiae precluded further study at the time. In this study, we identified the ES agent as a unique member of the genus Rickettsia which infects D. andersoni ticks. Stain- ing, indirect fluorescent antibody (IFA) tests, and a PCR assay of tick tissues revealed that this organism is an apparent en- dosymbiont that can be distinguished from previously de- scribed rickettsiae. The results of DNA sequencing and a re- striction fragment length polymorphism (RFLP) analysis, techniques which have been successful in helping to character- ize rickettsiae and other bacteria associated with hematopha- gous arthropods (1, 3, 10, 14, 20, 38, 41, 46), indicated that the organism is a unique SFG rickettsial species. The sequence results suggest that strain SkalkahoT (T = type strain) either lacks or has a truncated form of the major rickettsial 190-kDa surface antigen (rOmpA). Unsuccessful attempts to cultivate the bacterium are also described below. 446