Novel Rickettsia in Ticks, Tasmania, Australia | CDC EID

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Volume 15, Number 10–October 2009
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Novel Rickettsia in Ticks, Tasmania, Australia
Leonard Izzard, Stephen Graves, Erika Cox, Stan Fenwick, Nathan Unsworth, and John Stenos
Author affiliations: Australian Rickettsial Reference Laboratory, Geelong, Victoria, Australia (L. Izzard, S. Graves, J. Stenos); Murdoch University, Murdoch, Western Australia, Australia (L. Izzard, S. Fenwick, J. Stenos); Launceston General Hospital, Launceston, Tasmania, Australia (E. Cox); and Texas A&M Health Science Center, College Station, Texas, USA (N. Unsworth)

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Abstract
A novel rickettsia was detected in Ixodes tasmani ticks collected from Tasmanian devils. A total of 55% were positive for the citrate synthase gene by quantitative PCR. According to current criteria for rickettsia speciation, this new rickettsia qualifies as Candidatus Rickettsia tasmanensis, named after the location of its detection.

In Australia, 4 rickettsial species are known to cause disease in humans; none of these species has been identified in Tasmania. However, 3 cases of human rickettsial infections in Tasmania have been documented (1–3). Ixodes tasmani ticks are of particular interest because they are known to be vectors for other rickettsial species in Australia (4) and are also the most common tick species in Tasmania (5). In addition, because these ticks bite humans, they are candidates for rickettsial transmission in Tasmania.

Although Candidatus Rickettsia tasmanensis, a proposed new species of rickettsiae, has not been associated with human disease, the possible virulence of this rickettsia cannot be disregarded. Some initially identified rickettsiae were later found to cause disease in humans. For example, R. parkeri was discovered in 1939 (6) but was only confirmed as a human pathogen in 2004 (7). To investigate infections in Tasmania, we collected ticks from Tasmanian devils (Sacrophilus harrissi) and analyzed them for rickettsial species.

The Study
Forty-four I. tasmani ticks were collected from Tasmanian devils from various sites in Tasmania during 2005–2006; 36 were engorged females, 5 were nymphs, and 3 were males. Each tick was washed in 70% ethanol, rinsed in sterile phosphate-buffered saline, and homogenized. Homogenates were then subjected to DNA extraction by using a QIAmp DNA Blood Mini Kit (QIAGEN, Hilden, Germany). The presence of a rickettsial agent was detected by real-time PCR (8). Characterization of novel rickettsial species was achieved by comparing sequences of genes as described (9).

Amplification and sequencing of 1,096-, 3,005-, 588-, and 4,918-bp products for the citrate synthase (gltA), surface cell antigen (sca4), outer membrane protein A (ompA), and ompB genes, respectively, were conducted by using primers previously described (9). The 16S rRNA (rrs) gene was not amplified because cell culture isolation was not performed. Amplicons were cloned by using the TA Cloning Kit (Invitrogen, Carlsbad, CA , USA) and extracted by using a QuickLyse Mini Prep Kit (QIAGEN).

Big Dye sequencing was performed by using a GeneAmp PCR System 2400 thermocycler (Applied Biosystems, Foster City, CA, USA). Resulting products were analyzed at the Australian Genomic Research Facility by using an ABI Prism 3730xl DNA Analyzer (Applied Biosystems).

Sequences were assembled and edited by using the SeqMan Pro program (DNASTAR, Inc., Madison, WI, USA) and evaluated by using neighbor-joining and maximum-parsimony methods in MEGA 4 (10) and the maximum-likelihood method in PHYLIP (11). Results were confirmed by using BLAST analysis software (http://blast.ncbi.nlm.nih.gov/Blast.cgi). All sequences have been deposited in GenBank (Table).

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