Growth Characteristics of Rickettsia Species LON Strains Closely Related to Rickettsia japonica Isolated from Haemaphysalis longicornis Ticks in Mouse Derived L929 and Human-Derived THP-1 Host Cell Lines.

Non-pathogenic Rickettsia species LON strains closely related to an agent of Japanese spotted fever (JSF), R. japonica, were isolated in Japan from Haemaphysalis longicornis ticks in 2001. However, the biological properties of LONs in mammalian host cells are poorly understood. In this study, microscopic analysis showed that LONs in a mouse-derived L929 host cell line were rod shaped with sizes of 0.3-0.5 × 0.5-2.0 µm. Molecular analysis revealed the existence of a LON-specific disrupted open reading frame in R. japonica-related group-specific DNA regions. Growth kinetics of LON-2 and LON-13 strains analyzed by a quantitative real-time PCR showed 100-fold or more increment of LONs cultured in L929 host cells at 30°C and slightly less increment at 33°C, and 25-fold increment in human-derived THP-1 host cells at 35°C on day 7 (168 h) post infection. The generation times of the two LON strains cultured in L929 and THP-1 were estimated to be 9.4-12.9 h and 9.6-10.9 h, respectively. To our knowledge, this is the first report on the biological characteristics of Rickettsia sp. LON strains in mammalian cells, which may provide significant information for the experimental approaches for other rickettsiae.

Predominant Shift of Different P44-Expressing Anaplasma phagocytophilum in Infected HL-60, THP-1, NB4, and RF/6A Cell Lines.

Anaplasma phagocytophilum, an agent of human granulocytic anaplasmosis, is an obligatory intracellular bacterium that dominantly produces P44 outer membrane proteins encoded by the p44/msp2 multigene family, which are major antigens for serodiagnosis. However, A. phagocytophilum antigens from cultures with different cell lines seem to have varying reactivities with sera. In this study, we performed RNA-seq to investigate the P44 expression of A. phagocytophilum propagated in 4 cell lines. In infected HL-60 cells, the P44-2b transcript was predominant in the first RNA-seq analysis (HL-60.1). However, the P44-23 transcript was predominant in the second RNA-seq analysis at 1 month after additional passages (HL-60.2). We further analyzed the P44 expression of A. phagocytophilum cultured in THP-1, NB4, and RF/6A cells through consecutive passages in the same cell lines for 1 year after transferring A. phagocytophilum from infected HL-60 cells to the respective cell lines. In the long-term cultures, P44-18, P44-78, and P44-51 were predominantly transcribed in infected THP-1, NB4, and RF/6A cells, respectively. Therefore, the predominant shifts of different P44-expressing transcripts of A. phagocytophilum might occur during cell culture even in the same cell line at different time points of sample harvest (HL-60.1 and HL-60.2), which may be attributed to host cell adaptation/selection/interaction.

Evaluation of Diagnostic Assay for Rickettsioses Using Duplex Real-Time PCR in Multiple Laboratories in Japan.

Tsutsugamushi disease and Japanese spotted fever are representative rickettsioses in Japan, and are caused by infection with Orientia tsutsugamushi and Rickettsia japonica, respectively. For molecular-based diagnosis, conventional PCR assays, which independently amplify respective rickettsial DNA, are usually used; however, this approach is time-consuming. Here, we describe a new duplex real-time PCR assay for the simultaneous detection of O. tsutsugamushi and spotted fever group rickettsiae, and its evaluation using several PCR conditions in 6 public health laboratories. The detection limit of the assay was estimated to be 102 copies and the sensitivity was almost identical to that of 3 conventional PCR methods. A total of 317 febrile patients were selected as clinically suspected or confirmed cases of rickettsioses. The detection efficiency of this assay for O. tsutsugamushi from blood or skin (eschar) specimens appeared to be almost the same as that of the conventional PCR method, even when performed in different laboratories, whereas the efficiency for spotted fever group rickettsiae tended to be higher than that of the 2 traditional double PCR assays. Our duplex real-time PCR is thus a powerful tool for the rapid diagnosis of rickettsioses, especially at the acute stage of infection.

Case of Human Infection with Anaplasma phagocytophilum in Inner Mongolia, China.

Anaplasma phagocytophilum is an obligate intracellular bacterium that causes febrile illness in humans and livestock. A 49-year-old woman was suffering from feverish symptoms, fatigue, arthralgia, general body pain, and anorexia for 2 weeks. Later, she visited the Bayannur Centers for Disease Control and Prevention Hospital in Inner Mongolia, China. Molecular-based diagnostic analysis of the patient's blood revealed that A. phagocytophilum p44 DNA was positive, but Brucella omp31, spotted fever group Rickettsia gltA, Orientia tsutsugamushi 16S rDNA, and Ehrlichia p28 were negative. The amino acid sequences of 9 A. phagocytophilum p44 clones obtained from the patient shared 44-100% similarity among them and were closely related to those of previously identified p44 clones from Canis familiaris (accession no. KJV64194) and from Ixodes persulcatus tick (no. BAN28309). Serological tests using the patient's serum showed that immunoglobulin M (IgM) and IgG titers to A. phagocytophilum antigens were 160 and 20, respectively, determined using indirect immunofluorescence assay, and the reaction to recombinant P44 proteins (rP44-1, rP44-18ES, and/or rP44-47) was confirmed using Western blot analysis. Thus, the results obtained in this study strongly suggest that the patient was infected with A. phagocytophilum. To our knowledge, this is the first case of human anaplasmosis infection in the Inner Mongolia Autonomous Region.