This project addresses two voids in Ehrlichia chaffeensis research: An animal model and a lack of geographically distributed strains (isolates) for comparative studies. The immediate objective of this project is to establish and validate a reproducible animal model for human ehrlichiosis.
Specific Aim 1 will consist of the inoculation of an early passage Ehrlichia chaffeensis into xenografted SCID-beige canine-PBL mice (SCID-ca). The initial dose experiments will involve the use of the Arkansas strain of Ehrlichia chaffeensis. Later studies will involve an anticipated isolate of Ehrlichia chaffeensis to be characterized from Missouri (human or deer). The inoculum will consist of liberated organisms from infected cells. For the dose study, blood will be collected prior to and at intervals after inoculation for PCR, reisolation, and serum for antibody determination. Sample collection will continue until 30 days after inoculation. One control group will be inoculated with 10(exp)7 non-infected DH82 cells. Another control group will be inoculated with 10(exp)7 infected DH82 cells. Animals will be observed daily for clinical signs. At the conclusion of the experiment, tissues will be collected and histopathology will be done on liver, spleen, lymph nodes, bone marrow and kidney. The dose that consistently demonstrates clinical signs will be used for pathogenesis studies. For pathogenesis studies, blood will be collected prior to inoculation, and control and inoculated mice will be necropsied at 4, 8, 12, 16, 20, 24, and 28 days. Blood and tissue will be collected for PCR and histopathology. Serum will be collected for antibody and serum chemistry. The responses and histopathology in infected animals will be compared to non-inoculated control mice.
Specific Aim 2) Isolation of a Missouri strain of Ehrlichia chaffeensis. Blood samples will be collected from suspected human cases and Missouri deer. It is estimated that the investigators will obtain from 6 to 10 human blood samples and up to 150 deer samples. The detection/presence of Ehrlichia chaffeensis infection will be established by PCR. Isolation will be done by inoculation of DH82 and HL-60 cell cultures. Isolates will be cryopreserved at an early passage.
Specific Aim 3) Characterization of a Missouri strain of Ehrlichia chaffeensis. The selected strains will be cultivated in DH82 cells and Ehrlichia organisms will be isolated for antigenic and genomic studies. Specifically, the characterization will consist of morphology based on electron microscopy of thin sections, and antigenic analysis by Western blot, and genetic analysis involving the sequencing of 16S rRNA and 120-kDa protein genes. The antigenic and genetic results of the Missouri strains will be compared initially to the Arkansas strain. Later studies will involve comparisons to other US isolates.
