Fig 1.
Purification and clean-up of rGlpQ for ELISA test use.
(A) Optimization of enzyme concentration needed to cleave the fusion protein. rGlpQ was incubated with different concentrations of EKMax™ (lane 1: 0.1 Units/20μg rGLPQ, lane 2: 1 Units/20μg rGLPQ and lane 3: uncleaved rGlpQ) at 37°C overnight, and products were separated in 12% SDS-PAGE gels. (B) Standardization of the incubation time required for complete cleave of fusion protein using 0.5 units/20μg rGLPQ (lane 1: Uncleaved; lane 2: 17hrs and lane 3: 24hrs) at 37°C. (C) Purified rGlpQ after fusion protein removal and ready to use in ELISA and Immunoblot assays. (D) ELISA results of negative and positive controls using uncleaved rGlpQ and cleaved one (E). Mk: Molecular Marker used in SDS-PAGE gels.
Fig 2.
ELISA results of 878 canine serum samples tested in the study.
The cleaved rGlpQ substrate was used in this assay. The red line denotes the cut off value used in the study. Cut off was established as the average of negative controls + (3 × upper 95% CI of the negative controls). The blue dashed line reference to a primary cut off value calculated as the average of negative controls + (3 × SD of negative controls).
Table 1.
Percent of ELISA sero-reactive canine serum samples by eco-region in the state of Texas.
Fig 3.
Immunoblot confirmation assay.
Borrelia turicatae whole cell lysates were separated in 12% SDS-PAGE gels (line 1) together with rGlpQ (line 2) and stained with coomassie briliant blue (A). Western blots were run to confirm the ELISA sero-reactivity (B). S1 through S6 refers to representative positive and negative samples. Molecular weigt marker is indicated on the left side in kDa. The ELISA OD450nm value is represented under each immunoblot in parenthesis. Pos (+) and Neg (-) describes the final result for each samples.
Fig 4.
Percent sero-reactive canine samples by ELISA (white bars) and immunoblot (dark grey bars) during years 2011 and 2012.
Percentage was calculated based on the total number of samples tested each month.
Table 2.
Sero-reactive specimens to Borrelia burgdorferi and Borrelia turicatae by immunoblot assay.
Fig 5.
Lyme disease MarBlot™ Strip test was done to Tick-Borne Relapsing Fever (TBRF) sero-reactive samples.
Strips 1 and 2 correspond with serum samples from dogs considered negative for both TBRF and Lyme disease (LD). Strips 3 and 4: correspond to dogs with positive serology for TBRF and negative serology for LD. Strip 5: corresponds to a dog with negative serology for TBRF and positive serology for LD. Strip 6: corresponds to a dog with positive serology for TBRF and LD. TBRF/LD Seology refers to the combined result of ELISA and Immunoblot assay for both diseases. Strip No. Refers to the provided numbers by the manufacturer. IC refers to the internal Controls on the strip. 93, 66, 60, 58, 45, 41, 39, 34, 31, 30, 28, 23 and 18 refers to the molecular weight in kDa of each B. burgdorferi antigen immobilized on the strip. Numbers in red refer to bands that lack specificity (cross-reactive) while the green numbers correspond with antigens used for the diagnostic of LD. Gray numbers correspond with antigens that have certain cross reactivity between TBRF and LD observed during this study.
Table 3.
Reactivity of positive samples to B. burgdorferi MarBlot™ test and B. turicatae immunoblot test.
Fig 6.
Geographic distribution of sero-positive samples as per immunoblot analysis.
Localization by county of samples tested in this study. Map was generated using ArcGIS 9.0. Bb: Borrelia burgdorferi; Bt: Borrelia turicatae; WB: Western blot. Bb&Bt WB seroreactive refers to samples positive for both B. burgdorferi and B. turicatae by Western blotting.