Insight of diagnostic performance using B-cell epitope antigens derived from triple P44-related proteins of Anaplasma phagocytophilum.

Human granulocytic anaplasmosis (HGA) is caused by Anaplasma phagocytophilum. Indirect immunofluorescence assay (IFA) is generally used for HGA serodiagnosis. A. phagocytophilum immunodominant P44 major outer membrane proteins are encoded by p44/msp2 multigene family, responsible for IFA reactivity. However, because multiple P44-related proteins may involve immunoreactivity in IFA, the available diagnostic antigens remain obscure. In this study, we identified 12 B-cell epitopes on triple P44-related proteins using peptide array that reacted with 4 HGA patients' sera. Then, peptide spot immunoassay using 14 synthetic peptides derived from those 12 epitopes as antigens was applied for the detection of antibody to A. phagocytophilum from patients with fever of unknown origin. The sensitivities and diagnostic efficiencies of this immunoassay were higher than those of Western blot analysis using 3 recombinant proteins previously developed. Thus, the immunoassay using our epitope-derived antigens, which has higher diagnostic performances, may have significant benefit for HGA serodiagnosis.

Predominant Shift of Different P44-Expressing Anaplasma phagocytophilum in Infected HL-60, THP-1, NB4, and RF/6A Cell Lines.

Anaplasma phagocytophilum, an agent of human granulocytic anaplasmosis, is an obligatory intracellular bacterium that dominantly produces P44 outer membrane proteins encoded by the p44/msp2 multigene family, which are major antigens for serodiagnosis. However, A. phagocytophilum antigens from cultures with different cell lines seem to have varying reactivities with sera. In this study, we performed RNA-seq to investigate the P44 expression of A. phagocytophilum propagated in 4 cell lines. In infected HL-60 cells, the P44-2b transcript was predominant in the first RNA-seq analysis (HL-60.1). However, the P44-23 transcript was predominant in the second RNA-seq analysis at 1 month after additional passages (HL-60.2). We further analyzed the P44 expression of A. phagocytophilum cultured in THP-1, NB4, and RF/6A cells through consecutive passages in the same cell lines for 1 year after transferring A. phagocytophilum from infected HL-60 cells to the respective cell lines. In the long-term cultures, P44-18, P44-78, and P44-51 were predominantly transcribed in infected THP-1, NB4, and RF/6A cells, respectively. Therefore, the predominant shifts of different P44-expressing transcripts of A. phagocytophilum might occur during cell culture even in the same cell line at different time points of sample harvest (HL-60.1 and HL-60.2), which may be attributed to host cell adaptation/selection/interaction.