Genetic Identification of Rickettsiae Isolated from Ticks in Japan

Abstract

Following the description in Japan of Japanese spotted fever, caused by Rickettsia japonica, a search for the vector of this disease led to the isolation of several rickettsiae from various tick species. Sixty-three rickettsial isolates were obtained from six different tick species, and six type strains were described by PCR and monoclonal antibody testing. We identified these six strains by amplification and sequencing of the genes encoding 16S rRNA and citrate synthase. We confirmed that the isolates from Dermacentor taiwanensis and Haemaphysalis flava ticks were R. japonica isolates. In Ixodes ovatus, Ixodes persulcatus, and Ixodes monospinosus, we identified a Rickettsia identical or closely related to Rickettsia helvetica, a species that is pathogenic for humans and that to date has only been found in Europe. Finally, we identified a new genotype of unknown pathogenicity, genotype AT, that was isolated from Amblyomma testudinarium ticks and that is closely related to a Slovakian genotype obtained from Ixodes ricinus ticks.Rickettsioses are emerging infectious diseases caused by rickettsiae, which are obligate intracellular gram-negative bacteria associated with arthropod parasites. These arthropod vectors (ticks, fleas, mites, and insects) can transmit rickettsiae to mammals and humans. The rickettsioses share characteristic clinical features, including fever, headache, rash, and sometimes eschar (tache noire) formation at the site of the tick bite. The number of representatives of the genus Rickettsia and the number of newly described rickettsioses have increased in recent decades as a result of the development of an improved cell culture isolation technique and the extensive use of bacterial detection and identification based on molecular biology techniques (14). Comparison of the sequences of PCR-amplified fragments of the genes encoding 16S rRNA (ribosomal DNA [rDNA]) (16), citrate synthase (gltA) (17), or the rOmpA outer membrane protein (ompA) has become a reliable method for the identification of Rickettsia spp. (15).The discovery of Japanese spotted fever by Mahara (9) in 1984 began the study of spotted fever group (SFG) rickettsioses in Japan. The causative agent was identified and named Rickettsia japonica (22). The bacterium was apparently detected in three genera and six species of ticks (9). This stimulated the search for rickettsiae in ticks (19); and since 1993, 63 strains have been isolated from six tick species: Amblyomma testudinarium (25 isolates) (5, 25), Dermacentor taiwanensis (1 isolate) (20, 24), Haemaphysalis flava (1 isolate) (6), Ixodes monospinosus (1 isolate) (4), Ixodes ovatus (33 isolates), and Ixodes persulcatus (2 isolates) (4). These isolates were tested by using two monoclonal antibodies (MAbs) and the immunoperoxidase reaction. One of the two MAbs, which had a broad spectrum of reactivity, reacted with all SFG rickettsiae, and the other MAb was specific for R. japonica (12). These isolates were also tested by PCR with primer sets derived from gltA, ompA, and ompB. The glt...

Tick- and flea-borne rickettsial emerging zoonoses

“…R. helvetica is also suspected to be a human pathogen in Europe [45] and Asia [46,61,107], but this needs confirmation. Interestingly, out of the nine tick-borne SFG rickettsiae recently found to be pathogens for people after 1984, six were first isolated from ticks and later found to be pathogenic to humans.…”

“…Between 1984 and 2004, nine more species or subspecies of tick-borne spotted fever rickettsiae were identified as emerging pathogens throughout the world, including, R. japonica in Japan [6,46,65,71,72,[82][83][84]146]; "R. conorii caspia" in Astrakhan [35,38,39,143], Africa [47] and Kosovo [48]; R. africae in sub-Saharan Africa and the West Indies [62,63]; R. honei in the Flinders Island, offshore of Australia [9,57,140,141], the Island of Tasmania, Australia [153], Thailand [73], and possibly in the USA [13]; R. slovaca in Europe [29,74,101,122]; "R. sibirica mongolotimonae" in China [157], Europe [44,118] and Africa [106,113]; R. heilongjiangensis in China [42,49]; R. aeschlimannii in Africa [11,112,121] and Europe [43]; and finally R. parkeri in the USA [104]. R. helvetica is also suspected to be a human pathogen in Europe [45] and Asia [46,61,107], but this needs c...…”

Characterization of Ehrlichia Species from Ixodes ovatus Ticks at the Foot of Mt. Fuji, Japan

“…HF565 and Anan were obtained from I. ovatus, while E. muris strains were isolated from wild mice (Eothenomys kageus or Apodemus speciosus) and from Haemaphysalis flava ticks. H. flava is also common throughout Japan (6). Among these monocytic ehrlichiosis agents in Japan, the Shizuoka isolates from I. ovatus ticks found in this study appear to be most closely related to E. chaffeensis Arkansas T .…”

“…In Japan, I. ovatus ticks are known to transmit other bacteria such as Borrelia japonica (14) and Rickettsia helvetica-like spotted-fever rickettsiae (6). B. japonica as well as the ehrlichiae is found in I. ovatus ticks in Shizuoka prefecture, but the spotted-fever rickettsiae has not been found so far.…”

“…Thus, Ixodes ticks seem to transmit both ehrlichia and anaplasma. Ticks of I. ovatus are common throughout Japan and I. persulcatus ticks live both in northern parts of Japan and in the mountain areas of central Japan more than 1,000 m above sea level (6,32). Two kinds of Ehrlichia sp.…”