Investigation on the infection of spotted fever group Rickettsiae and Rickettsia mooseri of wild rodents in the field of plague foci in Western Yunnan / 中华地方病学杂志

Objective:To investigate the infection of spotted fever group Rickettsiae (SFGR) and Rickettsia mooseri ( R.mooseri) of wild rodents in the field of plague foci in Western Yunnan. Methods:The DNA of liver samples of 2 512 wild rodents captured from the plague foci in Lianghe County, Jianchuan County and Yulong County in Western Yunnan from 2015 to 2016 was extracted by magnetic bead method, and the heat shock protein groEL gene primers were used for nested PCR amplification. Gene sequence splicing and Blast homology comparison were performed using DNAStar 7.1 software and GenBank of the National Center for Biotechnology Information (NCBI) of the United States, respectively, and DNAStar 7.1 and MEGA 6.0 softwares were used to construct phylogenetic trees.Results:The wild rodents infected with SFGR were Mus pahari, Rattus steini, Crocidura attenuata and Suncus murinus (one for each), with a total infection rate of 0.16% (4/2 512); no R.mooseri infection was detected. The SFGR infection rates of wild rodents in the plague foci of Lianghe County and Jianchuan County were 0.49% (3/611) and 0.10% (1/1 029), respectively; no SFGR infection was detected in the wild rodents in the plague foci of Yulong County. The homology analysis showed that the homology between SFGR positive samples and reference sequences was 95.45%-100.00%; some of the groEL gene sequences were highly similar among the four positive samples, and the homology was 89.60%-97.40%. Sequence evolution analysis showed that the sequences of three SFGR positive samples from the plague focus in Lianghe County were clustered in the same branch, and the homology reached 94.40%-97.40%; one positive sample sequence from the plague focus in Jianchuan County was clustered in one branch. Conclusion:SFGR infection rate of wild rodents in the field of plague foci in Western Yunnan is low, and no R.mooseri infection is found.

Rapid Identification of Rickettsiae using the Real-Time PCR

In this study, new real-time PCR method based on the groEL gene was developed and investigated. Four spotted fever group (SFG) strains, four typhus group (TG) strains, and four scrub typhus group (STG) strains were easily differentiated as a distinct entity. This PCR assay was applied to detect Rickettsia DNA from 100 ticks. Twelve Haemaphysalis longicornis ticks were found positive and identified as spotted fever group Rickettsia. This real-time PCR method could simultaneously perform the rapid identification of rickettsiae and the differential diagnosis of SFG, TG, and STG in a single reaction.

Prevalence of Spotted Fever Group Rickettsia from Haemaphysalis Ticks in Chungju Province

A total of 190 ticks collected from the Chungju area of Korea was examined for the presence of Spotted Fever Group(SFG) Rickettsia using a PCR assay. Twenty-five (13.2%) Haemaphysalis ticks were found positive of the groEL gene of SFG Rickettsia. The prevalence rate of R. japonica in these 25 Haemaphysalis ticks was 72% (18 out of 25 SFG Rickettsia). The prevalence rate of R. conorii and new SFG rickettsia in these 25 Haemaphysalis ticks was 4% (1 out of 25 SFG Rickettsia) and 24% (6 out of 25 SFG Rickettsia), respectively. These results suggest that R. japonica was the highest infection frequency among in Haemaphysalis ticks SFG Rickettsia, and that R. conorii and new SFG Rickettsia are also present in the Chungju area.

groEL Gene Analysis of Borrelia afzelii Isolated in Korea

Eleven Borrelia afzelii strains, isolated from Ixodes nipponensis and Apodemus agrarius in Korea, were characterized by groEL gene analysis. Results from previous studies suggested that the groEL gene, which encodes the 60-kDa heat shock protein GroEL, was useful for the differentiation of B. burgdorferi sensu lato. The B. afzelii isolates could be divided into two groups by the phylogenetic tree constructed by UPGMA method and Tsp509 I PCR-RFLP analysis. The result suggested that the groEL gene is useful for identification and characterization of B. burgdorferi sensu lato though a short DNA fragment (310 bp) of the gene was sequenced and compared each other, and that Korean B. afzelii strains are heterogeneous genotypically.

groEL Gene Analysis of Borrelia afzelii Isolated in Korea

Eleven Borrelia afzelii strains, isolated from Ixodes nipponensis and Apodemus agrarius in Korea, were characterized by groEL gene analysis. Results from previous studies suggested that the groEL gene, which encodes the 60-kDa heat shock protein GroEL, was useful for the differentiation of B. burgdorferi sensu lato. The B. afzelii isolates could be divided into two groups by the phylogenetic tree constructed by UPGMA method and Tsp509 I PCR-RFLP analysis. The result suggested that the groEL gene is useful for identification and characterization of B. burgdorferi sensu lato though a short DNA fragment (310 bp) of the gene was sequenced and compared each other, and that Korean B. afzelii strains are heterogeneous genotypically.

Development of Rickettsia Specific Nested PCR Method Based on groEL Gene Sequences

To detect Rickettsia, we have developed a nested PCR method amplifying the groEL gene. Rickettsia strains were successfully amplified by this PCR method but the microorganisms causing other febrile diseases, such as Orientia tsutsugamushi, Coxiella burnetii, Ehrlichia sennetsu, Borrelia burgdorferi sensu lato, Borrelia hermsii, and Leptospira interrogans were not amplified. This PCR assay was applied to detect Rickettsia DNA from 100 ticks. Sixteen Haemaphysalis longicornis ticks were positive by this PCR assay. These results suggest that the new nested PCR method might be sensitive and useful for discrimination between Rickettsia and other febrile disease-causing microorganisms.