A serological assay to detect and differentiate rodent exposure to soft tick and hard tick relapsing fever infections in the United States

Abstract

Human cases of relapsing fever (RF) in North America are caused primarily by Borrelia hermsii and Borrelia turicatae, which are spread by argasid (soft) ticks, and by Borrelia miyamotoi, which is transmitted by ixodid (hard) ticks. In some regions of the United States, the ranges of the hard and soft tick RF species are known to overlap; in many areas, recorded ranges of RF spirochetes overlap with Lyme disease (LD) group Borrelia spirochetes. Identification of RF clusters or cases detected in unusual geographic localities might prompt public health agencies to investigate environmental exposures, enabling prevention of additional cases through locally targeted mitigation. However, exposure risks and mitigation strategies differ among hard and soft tick RF, prompting a need for additional diagnostic strategies that differentiate hard tick from soft tick RF. We evaluated the ability of new and previously described recombinant antigens in serological assays to differentiate among prior exposures in mice to LD, soft or hard tick RF spirochetes. We extracted whole-cell protein lysates from RF Borrelia cultures and synthesized six recombinant RF antigens (Borrelia immunogenic protein A (BipA) derived from four species of RF Borrelia, glycerophosphodiester phosphodiesterase (GlpQ), and Borrelia miyamotoi membrane antigen A (BmaA)) to detect reactivity in laboratory derived (Peromyscus sp. and Mus sp.) mouse serum infected with RF and LD Borrelia species. Among 44 Borrelia exposed mouse samples tested, all five mice exposed to LD spirochetes were correctly differentiated from the 39 mice exposed to RF Borrelia using the recombinant targets. Of the 39 mice exposed to RF spirochetes, 28 were accurately categorized to species of exposure (71%). Segregation among soft tick RF species (Borrelia hermsii, Borrelia parkeri and Borrelia turicatae) was inadequate (58%) owing to observed cross-reactivity among recombinant BipA protein targets. However, among the 28 samples accurately separated to species, all were accurately assigned to soft tick or hard tick RF type. Although not adequately specific to accurately categorize exposure to soft tick RF species, the recombinant BipA protein targets from soft and hard tick RF species show utility in accurately discriminating mouse exposures to LD or RF Borrelia, and accurately segregate hard tick from soft tick RF Borrelia exposure.

Keywords: Borrelia; Recombinant proteins; Relapsing fever; Serology.

Fig. 1.

The percent identities of amino acid sequences for BipA proteins from B. turicatae 91E135 (row and column A), B. hermsii DAH (row and column B), B. parkeri HR1 (row and column C) compared with the sequence of the BipA homolog identified in B. miyamotoi LB-2001 (row and column D). (A) Alignment of BipA amino acid sequences from B. turicatae 91E135 (ADN26518.1), B. parkeri HR1 (AHF45615.1), B. hermsii DAH (ACS27065.1), and the BipA homolog identified in B. miyamotoi LB-2001 (WP_070401628.1) (B). Identical amino acids in the sequences of proteins from the four RF Borrelia species are highlighted by shading. The lightest shade of gray denotes identical amino acids between at least two species, the medium shade indicates identical amino acids between at least three species, and the darkest shade indicates amino acids shared between all four of the species shown above.

Fig. 2.

Serum from RF Borrelia exposed mice was tested with membranes containing Borrelia whole-cell lysate and recombinant antigen combinations. Tube A contained B. hermsii, B. parkeri, B. turicatae, and B. miyamotoi whole-cell lysates. Tubes B-D contained each of the following recombinant proteins: tube B: B. hermsii rBipA (72 kDa) and B. miyamotoi rBipA(43 kDa), tube C: B. parkeri rBipA (72 kDa) and rGlpQ (43 kDa), and tube D: B. turicatae rBipA (72 kDa) and rBmaA (43 kDa). Molecular weight is indicated in kilodaltons on the left of the blot. A negative control serum sample (A) and a positive control sample containing equal amounts of serum from individual mice infected with either B. hermsii, B. parkeri, B. turicate, or B. miyamotoi (B) were used to probe membranes each time samples were tested. Blots were probed with serum from individual mice infected with B. parkeri (C), B. hermsii (D), B. turicatae (E), and B. miyamotoi (F), were evaluated for reactivity with rBmaA and rGlpQ, and pathogen calls were made by the consensus of blinded raters based on the presence or absence of bands for species-specific rBipA targets.