Pathogenicity Of Rickettisa Heilongjiangensis Infection Of BALB/c Mice

Rickettsia heilongjiangensis is a member of the spotted fever group of rickettsiae and the etiologic agent of Far-eastern spotted fever (FESF). R. heilongjiangensis is a small obligate intracellular Gram-negative organism that is maintained in its tick host through transovarial transmission. Infection with R. heilongjiangensis occurs through the bite of an infected tick. FESF has been diagnosed in patients in the northeastern area of China, far-eastern area of Russian, and Japan. Besides isolated from patients, R. heilongjiangensis has been isolated from different ticks in these areas. Far-eastern spotted fever has been suggested to be an important emerging infectious disease in northeastern Asia,In the present research, plaque assay was applied to clone R. heilongjiangensis, and Vero cells was used as host cells to multiply this cloned organism. The multiplied rickettsiae were purified by Renografin density gradient centrifugation. The purified viable organisms were used to infect BALB/c mice by injection of retro-orbital venous plexus (iv route). At days 1, 3, 6, and 9 post-infection (p.i.), 5 mice in each group were sacrificed and their blood was collected in EDTA tubes and liver, spleen, lung, and brain tissues were aseptically excised, respectively.DNA was extracted from blood and organ tissues of each mouse and the rickettsial DNA copies were measured using quantitative PCR (qPCR). DNA-free RNA samples extracted from mouse tissues were used to produce cDNA with the Oligo d(T)18 primers of IFN-γ, TNF and RANTES and reverse transcriptase in qPCR analysis. The antigen-coated slides were used to detect the antibodies to R. heilongjiangensis in mouse sera by indirect immunofluorescence assay (IFA).BALB/c mice infected with 105 viable organisms showed decreased activity and ruffled fur by day 2 to 5 post-infection (p.i.) and they returned to normal physical appearance and activity at day 6 p.i. However, BALB/c mice inoculated with 105 heat-inactivated organisms survived without any signs of illness during the course of the experiment. The rickettsial DNA copies were detectable in whole blood and tissues from mice infected with viable organisms at day 1 p.i., significantly increased by day 3 p.i.; the highest levels were observed at day 6 p.i., significantly decreased by day 9 p.i. The rickettsial DNA copies in spleen and lung tissues were significantly higher than that in liver and brain tissues, respectively. Pathological lesions were observed in liver, lungs, and brain of the R. heilongjiangensis-infected mice at day 6 p.i. and the lesions decreased at day 9. In contrast to live organisms, heat-inactivated organisms did not induce any lesion in hepatic, pulmonary and cerebral tissues in mice.IFN-γ, TNF and RANTES transcripts in organs of R. heilongjiangensis-infected mice were assessed by qPCR. Their transcripts were significantly increased in liver, spleen, lungs, and brain of viable organism-infected mice at day 3 and decreased at day 6. Though their transcripts were also observed in these organs of mice inoculated with heat-inactivated organisms, but the levels were significantly lower than that of viable organism-infected mice.The specific antibodies were detected in sera from mice infected with viable organisms at day 7 p.i. and progressively increased up to day 21. The specific antibodies were also detected in sera from mice inoculated with heat-inactivated organisms, but the antibody levels were significantly lower than that of viable organism-infected mice.In this study, via intravenous inoculation, R. heilongjiangensis established disseminated intracellular infection and caused pathological lesions and inflammatory cytokine expression in major organs similar to what is observed in human spotted fever. Our results reveal that the pathological lesions in the infected organs are associated with body's inflammatory response induced by R. heilongjiangensis. The high level of specific antibodies in the mice during the late phase of the rickettsial infection is associated with the protective immunological response of mice to resist the rickettsial organisms.