A better understanding of the pathogenesis of rickettsial disease requires elucidation of mechanisms governing host defense during infection. TLRs are primary sensors of microbial pathogens that activate innate immune cells, as well as initiate and orchestrate adaptive immune responses. However, the role of TLRs in rickettsia recognition and cell activation remains poorly understood. In this study, we examined the involvement of TLR2 and TLR4 in recognition of Rickettsia akari, a causative agent of rickettsialpox. Transfection-based complementation of TLR2/4-negative HEK293T cells with human TLR2 or TLR4 coexpressed with CD14 and MD-2 enabled IκB-α degradation, NF-κB reporter activation, and IL-8 expression in response to heat-killed (HK) R. akari. The presence of the R753Q TLR2 or D299G TLR4 polymorphisms significantly impaired the capacities of the respective TLRs to signal HK R. akari-mediated NF-κB reporter activation in HEK293T transfectants. Blocking Ab against TLR2 or TLR4 markedly inhibited TNF-α release from human monocytes stimulated with HK R. akari, and TNF-α secretion elicited by infection with live R. akari was reduced significantly only upon blocking of TLR2 and TLR4. Live and HK R. akari exerted phosphorylation of IRAK1 and p38 MAPK in 293/TLR4/MD-2 or 293/TLR2 stable cell lines, whereas only live bacteria elicited responses in TLR2/4-negative HEK293T cells. These data demonstrate that HK R. akari triggers cell activation via TLR2 or TLR4 and suggest use of additional TLRs and/or NLRs by live R. akari.

There is insufficient evidence to support screening of various tick-borne diseases (TBD) related microbes alongside Borrelia in patients suffering from TBD. To evaluate the involvement of multiple microbial immune responses in patients experiencing TBD we utilized enzyme-linked immunosorbent assay. Four hundred and thirty-two human serum samples organized into seven categories followed Centers for Disease Control and Prevention two-tier Lyme disease (LD) diagnosis guidelines and Infectious Disease Society of America guidelines for post-treatment Lyme disease syndrome. All patient categories were tested for their immunoglobulin M (IgM) and G (IgG) responses against 20 microbes associated with TBD. Our findings recognize that microbial infections in patients suffering from TBDs do not follow the one microbe, one disease Germ Theory as 65% of the TBD patients produce immune responses to various microbes. We have established a causal association between TBD patients and TBD associated co-infections and essential opportunistic microbes following Bradford Hill’s criteria. This study indicated an 85% probability that a randomly selected TBD patient will respond to Borrelia and other related TBD microbes rather than to Borrelia alone. A paradigm shift is required in current healthcare policies to diagnose TBD so that patients can get tested and treated even for opportunistic infections.

In general, cultivation and purifi cation of intracellular pathogenic rickettsiae represents a risk for laboratory personnel due to exposure to highly infectious aerosol or accidental inoculation during these procedures. In this study, we describe an alternative, eff ective and time saving technique for rickettsial purifi cation using digitonin to release intracellular bacteria from host cell without physical disruption. No signifi cant diff erences were noted in yield and infectivity between digitonin treated rickettsiae and rickettsiae purifi ed by sonication. Th is is the fi rst report of using digitonin in purifi cation of pathogenic rickettsiae and this approach might be eff ective for other intracellular pathogenic bacteria.

Summary. -to date, only three rickettsial species have been found in ticks in Slovakia by serological and/ or molecular-biological techniques, namely Rickettsia slovaca, Candidatus rickettsia IrS, and Rickettsia raoultii. recently, we succeeded in isolation of the forth species, Rickettsia helvetica from Ixodes ricinus, the most frequent tick in Slovakia. The isolation, positive for 10% of tested ticks, was performed on Xtc cells by the shell-vial technique, Gimenez staining and light microscopy. The infected cell cultures contained rod-shaped particles morphologically identical to rickettsiae. The isolation was confirmed by direct detection of a fragment of the R. helvetica gene for citrate synthase in the positive ticks by Pcr and its subsequent cloning, sequencing and comparison with the database.