Comparison of Ehrlichia chaffeensis recombinant proteins for serologic diagnosis of human monocytotropic ehrlichiosis

Abstract

Diagnosis of human monocytotropic ehrlichiosis (HME) generally depends on serology that detects the antibody response to immunodominant proteins of Ehrlichia chaffeensis. Protein immunoblotting was used to evaluate the reaction of the antibodies in patients' sera with the recombinant E. chaffeensis 120- and 28-kDa proteins as well as the 106- and the 37-kDa proteins. The cloning of the genes encoding the latter two proteins is described in this report. Immunoelectron microscopy demonstrated that the 106-kDa protein is located at the surfaces of ehrlichiae and on the intramorular fibrillar structures associated with E. chaffeensis. The 37-kDa protein is homologous to the iron-binding protein of gram-negative bacteria. Forty-two serum samples from patients who were suspected to have HME were tested by immunofluorescence (IFA) using E. chaffeensis antigen and by protein immunoblotting using recombinant E. chaffeensis proteins expressed in Escherichia coli. Thirty-two serum samples contained IFA antibodies at a titer of 1:64 or greater. The correlation of IFA and recombinant protein immunoblotting was 100% for the 120-kDa protein, 41% for the 28-kDa protein, 9.4% for the 106-kDa protein, and 0% for the 37-kDa protein. None of the recombinant antigens yielded false-positive results. All the sera reactive with the recombinant 28- or the 106-kDa proteins also reacted with the recombinant 120-kDa protein.

FIG. 1

DNA sequence of the 106- and 37-kDa protein genes of E. chaffeensis. The first ORF is the 106-kDa protein gene, and the second ORF is the 37-kDa protein gene. Arrows indicate the sequences and directions of primers that were used to amplify the DNA fragments to express the genes. The predicted signal sequences of amino acids at the beginning of each protein are underlined.

FIG. 2

Southern blotting revealed that the 106-kDa protein gene probe (lane 106) and the 37-kDa protein gene probe (lane 37) hybridized with Alw26I and EcoRI double-digested E. chaffeensis genomic DNA. Lane M, Digoxigenin-labeled DNA marker (in kilobases).

FIG. 3

DNA sequence homology of the E. chaffeensis 37-kDa protein gene (E.ch) with the S. marcescens iron-binding protein (S.ma).

FIG. 4

Protein immunoblotting. (A) Rabbit antiserum to the recombinant 37-kDa protein reacted with E. canis (ca) and E. chaffeensis (ch) antigens. (B) Rabbit antiserum to the recombinant 106-kDa protein (106) and normal preimmunization rabbit serum (NR) reacted with E. chaffeensis antigens. (C) Rabbit antiserum to the recombinant 120-kDa protein reacted with E. chaffeensis Arkansas (ark) and E. chaffeensis Sapulpa (sap) antigens. Arrows indicate the ehrlichial proteins that reacted with rabbit antisera to each recombinant protein. H, heated antigen; N, non-heated antigen.

FIG. 5

Immunoelectron microscopic visualization of 106- and 37-kDa proteins in ultrathin sections of E. chaffeensis-infected DH82 cells. Bar, 0.5 μm. (A) The 106-kDa protein is located at the surfaces of ehrlichiae (arrows) and in intramorular fibrils originating from the ehrlichial cell surfaces (arrowheads). Gold particle label is also seen on the membrane limiting ehrlichial inclusions (morulae). n, host cell nucleus. (B) Gold particle label with antibodies to the 37-kDa protein is localized in the ehrlichial cell cytoplasm (arrowhead) and in the periplasmic space (arrows).

FIG. 6

Protein immunoblotting of HME patients’ sera with recombinant E. chaffeensis 120-, 28-, and 106-kDa proteins. Asterisks indicate the weak bands.