Francisella tularensis subspecies tularensis, the etiological agent of tularemia, is highly pathogenic to humans and animals. However, the SCHU strain of F. tularensis SCHU P0 maintained by passaging in artificial media has been found to be attenuated. To better understand the molecular mechanisms behind the pathogenicity of F. tularensis SCHU, we attempted to isolate virulent bacteria by serial passages in mice. SCHU P5 obtained after 5th passages in mice remained avirulent, while SCHU P9 obtained after 9th passages was completely virulent in mice. Moreover, SCHU P9 grew more efficiently in J774.1 murine macrophages compared with that in the less pathogenic SCHU P0 and P5. Comparison of the nucleotide sequences of the whole genomes of SCHU P0, P5, and P9 revealed only 1 nucleotide difference among P0, P5 and P9 in 1 of the 2 copies of pathogenicity determinant protein C (pdpC) gene. An adenine residue deletion was observed in the pdpC1 gene of SCHU P0, P5, and P9 and in the pdpC2 gene of SCHU P0, and P5, while P9 was characterized by the wild type pdpC2 gene. Thus, SCHU P0 and P5 expressed only truncated forms of PdpC protein, while SCHU P9 expressed both wild type and truncated versions. To validate the pathogenicity of PdpC, both copies of the pdpC gene in SCHU P9 have been inactivated by Targetron mutagenesis. SCHU P9 mutants with inactivated pdpC gene showed low intracellular growth in J774.1 cells and did not induce severe disease in experimentally infected mice, while virulence of the mutants was restored by complementation with expression of the intact PdpC. These results demonstrate that PdpC is crucial in determining the virulence of F. tularensis SCHU.

A novel competitive enzyme-linked immunosorbent assay (cELISA) was developed and evaluated for detection of antibodies against Francisella tularensis in humans. The assay is based on the ability of serum antibodies to inhibit the binding of monoclonal antibodies (MAbs) directed against F. tularensis lipopolysaccharide antigens. The assay was evaluated using serum samples of tularemia patients, inactivated F. tularensis-immunized rabbits, and F. tularensis-infected mice. Antibodies against F. tularensis were successfully detected in serum samples of tularemia patients as well as the immunized and infected animals. The cELISA method was compared to indirect ELISA (iELISA) and the commonly used microagglutination test (MA) using serum samples of 19 tularemia patients and 50 healthy individuals. The sensitivity and specificity of cELISA were 93.9 and 96.1%, respectively, in comparison to the iELISA. MA was less sensitive than cELISA with a sensitivity and specificity of only 81.8 and 98.0%, respectively. A high degree of correlation (R 2 ‫؍‬ 0.8226) was observed between cELISA and iELISA results. The novel cELISA developed in this study appears to be highly sensitive and specific for serodiagnosis of human tularemia. The potential of the MAb-based cELISA to be used in both human and animal samples emphasizes its usefulness for serological survey of tularemia among multiple animal species.

Objective Abdominal aortic aneurysm is caused by the accumulation of inflammatory cells in the aortic wall. Our recent studies demonstrated that inhibition of Notch signaling attenuates abdominal aortic aneurysm formation by shifting the macrophage balance towards anti-inflammatory (M2) phenotype. Using mouse model of M2-predominant macrophages (IL12p40−/−; interleukin 12 p40) mice, we investigated their effects in the development of abdominal aortic aneurysm. Approach and Results Male (8–10 week-old) wild-type and IL12p40−/− mice (n=15) on C57BL/6 background were infused with Ang II (angiotensin II, 1000 ng/kg per minute) by implanting osmotic pumps subcutaneously for 28 days. In the IL12p40−/− mice, Ang II significantly increased the maximal intraluminal diameter (9/15) as determined by transabdominal ultrasound imaging. In addition, IL12p40-deletion significantly increased aortic stiffness in response to Ang II as measured by pulse wave velocity and atomic force microscopy. Histologically, IL12p40−/− mice exhibited increased maximal external diameter of aorta and aortic lesions associated with collagen deposition and increased elastin fragmentation compared with wild-type mice infused with Ang II. Mechanistically, IL12p40 deficiency by siRNA augmented the Tgfβ2-mediated Mmp2 expression in wild-type bone marrow-derived macrophages without affecting the expression of Mmp9. No such effects of IL12p40 deficiency on MMP2/MMP9 was observed in human aortic smooth muscle cells or fibroblasts. Depletion of macrophages in IL12p40−/− mice by clodronate liposomes significantly decreased the maximal external diameter of aorta and aortic stiffness in response to Ang II as determined by imaging and atomic force microscopy. Conclusions IL12p40 depletion promotes the development of abdominal aortic aneurysm, in part, by facilitating recruitment of M2-like macrophages and potentiating aortic stiffness and fibrosis mediated by Tgfβ2.