Utility of ultra-rapid real-time PCR for detection and prevalence of Rickettsia spp. in ticks.

Background: Rickettsia spp. are important tick-borne pathogens that cause various human and animal diseases worldwide. A tool for rapid and accurate detection of the pathogens from its vectors is necessary for prevention of Rickettsioses propagation in humans and animals, which are infested by ticks. Therefore, this study was conducted to evaluate a molecular tool, ultra-rapid real-time PCR (UR-qPCR), for rapid and accurate detection of Rickettsia spp. from 5644 ticks in 408 pools collected from livestock and their surrounding environments in Gangwon and Jeju province in South Korea.

Results: The UR-qPCR of Rickettsia DNA showed a limit of detection of 2.72 × 10 copies of Rickettsia DNA and no cross reaction with other tick-borne pathogens, namely Anaplasma phagocytophilum, Ehrlichia chaffeensis, E. canis, Toxoplasma gondii, and Borrelia burgdorferi. In addition, the PCR assay also showed possibility of various Rickettsia species detection including R. monacensis, "Candidatus R. longicornii", R. japonica, R. roultii, and R. tamurae. The collected ticks were identified with major species belonged to Haemaphysalis longicornis (81.62%), followed by H. flava (15.19%), and Ixodes nipponensis (3.19%). Rickettsia detection from tick samples using the UR-qPCR showed that the minimum infection rate (MIR) of Rickettsia in collected ticks was 1.24‰ and that all positive pools contained H. longicornis, equal to the MIR of 1.39‰ of this species. Additionally, MIR of Rickettsia spp. detected in ticks collected in Gangwon and Jeju was 1.53‰ and 0.84‰, respectively. Furthermore, the sequencing results of the 17 kDa protein antigen gene and ompA gene showed that Rickettsia spp. sequences from all pools were related to "Candidatus R. longicornii" and "Candidatus R. jingxinensis".

Conclusions: The UR-qPCR system was demonstrated to be useful tool for accurate and rapid detection of Rickettsia from its vector, ixodid ticks, within 20 min. The data on Rickettsia spp. in ticks detected in this study provide useful information on the distribution of Rickettsia in previously unstudied Korean provinces, which are important for the prevention and control of the spread of rickettsioses in both animals and humans in the country.