by Samples of liver, spleen, peritoneal exudate, peripheral blood mononuclear cells (PBMCs), and plasma were harvested and stored at ?80C. Plasma and PBMC isolation. toxic shock-like illness (4, 10). The bacterium is transmitted by the tick vector How host defense is mediated towards the ehrlichiae is not well understood, but T cells appear to play an essential role in the immune response (9, 14, 39), and immunodeficient individuals are particularly susceptible to serious infections (28). However, we and others have also demonstrated that antibodies can be effective during this intracellular infection (27, 39). Our previous studies demonstrated that passive transfer of Paritaprevir (ABT-450) antibodies could provide long-term protection to susceptible SCID mice (22, 39). Although not a model for humoral immunity in immunocompetent mice or humans, SCID mice provide a means to investigate the function of antibodies in the absence of cellular responses, which can obscure the activity of antibodies. Antibodies were found to be highly effective in SCID mice even when the antibodies were administered after infection had Mouse monoclonal to CD40 been well established, and the effects of the antibodies were often evident as early as 24 to 48 h after administration (39). Highly effective antibodies were found to recognize immunodominant outer membrane proteins. The outer membrane proteins in and related ehrlichiae comprise families of related proteins that exhibit antigenic variation (13, 26, 27, 31, 41). During infection of cattle by the ehrlichial pathogen infection might reveal unexpected features of the bacterium’s life cycle in the host. In the current study, we speculated that ehrlichiae were exposed to antibodies in the host extracellular milieu, perhaps during intercellular Paritaprevir (ABT-450) spreading, and so we investigated whether bacteria could Paritaprevir (ABT-450) be found outside of host cells during infection in vivo. Our findings reveal that significant numbers of infectious bacteria can be found outside of host macrophages, providing a possible mechanism to explain the susceptibility of these bacteria to antibodies. Our studies also led to the unexpected observation that retains a limited capacity to persist and replicate outside of the environment of the host cell, a finding that may have relevance for our understanding of ehrlichial microbiology and host-to-vector transmission. MATERIALS AND METHODS Mice and infections. BALB/c-and BALB mice were obtained from the Jackson Laboratory (Bar Harbor, Maine) or bred in the Wadsworth Center Animal Care Facility under institutional guidelines for animal care and use. BALB/c-mice were infected with 1 106 to 2 106 infected SCID splenocytes or similar numbers of infected DH82 cells via the peritoneum, as described previously (38). Quantitative PCR analyses revealed that this inoculum contained approximately 108 bacterial genomes. The mice were sacrificed when moribund, typically at day 21 postinfection. Morbidity was characterized by a lack of mobility, hunched posture, ruffled fur, and a pronounced loss of 30% of initial body weight (21). Blood samples were taken by cardiac puncture and collected in tubes containing EDTA as an anticoagulant. Tissue samples were harvested and stored at ?80C for further analyses. For antibody treatment, mice were administered three doses of 200 g of the outer membrane protein-specific monoclonal antibody (MAb) Ec56.5 (22) or an isotype-matched control antibody, KJ1-26 (16), via the peritoneum on days 6, 12, and 18 postinfection, and the mice were harvested on day 22 postinfection. Samples of liver, spleen, peritoneal exudate, peripheral blood mononuclear cells (PBMCs), and plasma were harvested and stored at ?80C. Plasma and PBMC isolation. Blood samples were mixed with anticoagulant during collection and immediately centrifuged at 230 .
