Four strains of mice with known susceptibility to various SFG Rickettsia strains were examined for susceptibility to infection, and pathological parameters were assessed. Of the four strains used, R. parkeri DNA was detectable in all tissues examined only from the C3H/HeJ mice, suggesting susceptibility to sustained infection. Additionally, although most models for rickettsioses have been based upon abnormal routes of infection (intravenous and intraperitoneal), the more natural route of intradermal inoculation of the C3H/HeJ mice resulted in several of the mice developing eschar lesions characteristic of human infection, which is a novel finding in mice utilized for SFG Rickettsia modeling. Therefore, although animal models of rickettsial infection appear multivariate, this study provides an experimental model of R. parkeri rickettsiosis which can be utilized to elucidate the mechanisms of infection and pathogenesis.
Based on the temporality of the experiments and the quantities of Rickettsia recovered by qPCR, the resulting values are indicative of rickettsial replication. In the mouse strains which showed no gross pathology, for example, the rickettsial concentrations were much lower than those in the C3H/HeJ mice and were actually undetectable in most of the tissues. While impaired clearance may be an explanation for the greater concentrations of rickettsial DNA recovered from the C3H/HeJ mice, the extended time course of the consequent studies would have been expected to eliminate this factor, as the rickettsial DNA should have degraded much faster in the absence of the homeostatic environment of the living pathogen. To further support this, research enumerating the growth at the lesion must be undertaken, along with studies which consider the effect of tick feeding on rickettsial replication at the site of inoculation.
Animal models have proven vital to expanding our knowledge of the pathogenesis of infectious diseases. Within Rickettsia
, murine models have elucidated the importance of TLR4 signaling and subsequent dendritic cell activation to the clearance of pathogenic Rickettsia
). Mouse models have also revealed that R. prowazekii
lesions are independent of humoral immune response (1
). These discoveries highlight the importance of an accurate animal model, and the identification of the C3H/HeJ strain as a viable model for R. parkeri
rickettsiosis provides a platform for future revelations.
This animal model may also prove beneficial in ways that some other model systems for rickettsial diseases do not. The C3H/HeN strain of mouse is used for modeling R. conorii
infection; however, this system results in a fatal disease, which lacks consistency with the human illness (9
). In many ways this model remains more than adequate for studying the pathogenesis of this rickettsiosis, but the terminal nature of the infection precludes the use of long-term studies into the progression of and eventual recuperation from disease. In contrast, the C3H/HeJ model of R. parkeri
rickettsiosis has not yet proven fatal even in prolonged studies, and it shows promise as a model of progression and resolution of eschars.
TLR4 has been shown to be of great importance in the immune response to rickettsial infection (9
). Mice with competent TLR4 signaling are more resistant to rickettsial infection (9
). This is primarily because of the stimulation of dendritic cells and subsequent production of gamma interferon (IFN-γ), followed by the expansion of the population of activated natural killer cells (10
). This provides a probable explanation for the observed susceptibility of C3H/HeJ mice to R. parkeri
. The lack of stimulation of the Th1 response secondary to TLR4 signaling also leads to the expansion of T regulatory cells, which may result in the suppression of proinflammatory immune responses (9
). These deficiencies in the immune regulation of the C3H/HeJ mice in response to rickettsial infection warrant further study, including comparisons to the TLR4-competent C3H/HeN strain, to elucidate the mechanisms of rickettsial recognition and clearance by the vertebrate host.
The facial edema, which developed in the intravenously inoculated C3H/HeJ strain, was an unexpected finding. Gross edema is not a common finding in R. parkeri rickettsiosis; however, edema is frequently observed histopathologically in the immediately adjacent tissues surrounding inflamed vessels. The mechanism of this edema is related to the inflammation and necrosis of the infected vessels. The distribution of the edema is intriguing, as the head and neck are areas commonly associated with tick infestation. Although not tested in the current study, it is possible that a distribution pattern of the heavier infection of the vessels of the head and neck increases the likelihood of uptake by and infection of the tick vectors.
Eschars are common to most SFG Rickettsia
and form at the site of tick or mite inoculation of Rickettsia
as a result of local dermal and epidermal necrosis with marked vasculitis (23
). The eschars that developed in this murine model are especially important, as this is a prominent clinical sign of R. parkeri
rickettsiosis as well as two of its closest relatives, R. conorii
and R. africae
). The ability to recapitulate these lesions in a murine model provides a basis for comparison to the effects of these closely related SFG rickettsiae. Eschars from patients with Mediterranean spotted fever express high mRNA levels of tumor necrosis factor (TNF), IFN-γ, interleukin-10 (IL-10), RANTES, indoleamine-2,3-dioxygenase, and inducible nitric oxide synthase (3
). The lesions from R. parkeri
-infected C3H/HeJ mice can be used to determine if a similar pattern of immunological response develops for this rickettsiosis, while another potential source of future research into eschars is evaluating the differences in pathogenesis at the site of tick inoculation between eschar-associated rickettsioses and those that do not typically cause eschars. Although the non-eschar-associated agent of Rocky Mountain spotted fever, R. rickettsii
, is the most commonly implicated etiologic agent of rickettsial disease in the United States, there are at least seven eschar-associated rickettsioses encountered by clinicians in the United States, including R. parkeri
rickettsiosis, rickettsialpox, 364D rickettsiosis, cat flea-associated rickettsiosis, African tick bite fever, Mediterranean spotted fever, and R. massiliae
). It would be very interesting to establish the differences in pathogenesis between Rickettsia
associated with these distinct disease manifestations.
The histopathology of the eschars is similar to that reported in humans, with epidermal necrosis and perivascular dermatitis being the prominent features (2
). The inflammatory infiltrates associated with this murine model eschar are also analogous to the inflammation described in human patients with R. parkeri
), suggesting a similar immunological response, but this will require further investigation to confirm. Rickettsiae were present within the vascular lesions as assessed by immunohistochemistry. As described for patients with R. parkeri
rickettsiosis, the SFG Rickettsia
strains were found predominantly in the cytoplasm of mononuclear cells (macrophages) and endothelial cells (13
). These similarities in necrosis at the site of inoculation, inflammatory pattern, and infiltrate and distribution of Rickettsia
support the concept that the infection process is similar for the murine model of infection and the human infection at least at the site of inoculation, which further supports the use of the C3H/HeJ mouse as a model for R. parkeri
rickettsiosis. The absence of these eschar-like lesions at the intradermal inoculation site at the nape of the neck suggests that additional factors are necessary to stimulate infection at this site. It is likely that the tick vector plays a significant role in the ability of R. parkeri
to infect its vertebrate host, and future studies are necessary to elucidate this process.
The presence of rickettsiae within the hepatic tissue of the BALB/c strain of mice is interesting when the lack of inflammation observed histologically is considered. Possible explanations for this discrepancy include differences in sampling sites utilized for the nucleic acid extraction and histopathology and contamination during sample acquisition, nucleic acid extraction, or qPCR preparation. The latter seem unlikely for various reasons. Contamination during sample acquisition should have resulted in similar findings in other organs from the BALB/c mice, while the nucleic acid extraction and qPCR preparations were repeated with similar results, suggesting that either no contamination occurred or that very similar contaminants were present at two separate time points. Sampling error appears to be the most probable cause for the discrepancy and is a commonly encountered issue in diagnostic settings. It is probable that rickettsial lesions were present in different foci of the hepatic tissue, including that sampled for nucleic acid extraction, but the same was not true for the sections evaluated by histopathology.
With the increase in TBRD recognized in humans, further study into the transmission of the responsible organisms and the pathogenesis of the diseases has become of paramount importance. R. parkeri serves as an elegant example of this, as the first confirmed case of human disease was reported less than a decade ago. The relatively recent recognition of this organism as a pathogen has deeper implications, considering research into its biology remains in its infancy compared to that for other rickettsiae. To elevate the knowledge base regarding R. parkeri and its disease, a commonly available inbred mouse strain with strong potential for use as a model for research into the pathogenesis of R. parkeri rickettsiosis has been identified in the current study. The C3H/HeJ inbred mouse strain was found to be susceptible to infection with R. parkeri, while typical gross and histopathological features of human disease developed at the site of intradermal inoculation, and also in concert with findings in humans with R. parkeri rickettsiosis, the infection has thus far proven nonfatal. This work identifies a new tool for investigators in subsequent studies of the pathogenesis of R. parkeri rickettsiosis, which can now include the detailed characterization of the immune response to the bacteria along with incorporating the effects of the tick vector on the establishment and maintenance of infection.