Development and application of a Bacillus anthracis protective antigen domain-1 in-house ELISA for the detection of anti-protective antigen antibodies in cattle in Zambia

Abstract

In Zambia, anthrax outbreaks among cattle are reported on nearly an annual basis. Presently, there is a lack of serological assays and information to develop an anthrax management and control strategy. In this study, an indirect enzyme-linked immunosorbent assay (ELISA) based on recombinant protective antigen domain 1 (rPA-D1) of Bacillus anthracis was developed and used to detect anti-PA antibodies in cattle in Zambia. An antigen coating of 10 ng/well and a serum dilution of 1:100 were determined to be the optimal rPA-D1 ELISA titration conditions. The intra- and inter-assay % coefficients of variation were less than 10% and 15%, respectively. The rPA-D1 ELISA could detect seroconversion in the cattle 1 month after anthrax vaccination. In a cross-sectional study conducted in the Western Province, Zambia, 187 serum samples from 8 herds of cattle were screened for anti-PA antibodies using the rPA-D1 ELISA. The seropositive rate of the serum samples was 8%, and the mean anti-PA antibody was 0.358 ELISA units. Additionally, we screened 131 cattle serum samples from Lusaka, which is a nonendemic area, and found no significant association between the antibody levels and sampling area (endemic versus nonendemic area). Conversely, significant differences were observed between the anti-PA antibody levels and herds, anti-PA antibody levels and vaccination status and anti-PA antibody levels and vaccination timing. Collectively, these findings suggest that the rPA-D1 ELISA is a useful tool for the detection of anti-PA antibodies in cattle in Zambia. The low proportion of seropositive sera indicates that there is inadequate cattle vaccination in the Western Province and, in addition to other epidemiological factors, this may precipitate the anthrax outbreak recurrence.

Fig 1. Sampling areas in the Western Province, Zambia.

The blood samples used in this study were collected from eight herds of cattle in the Nalolo District and Luampa Districts.

Fig 2. Production and purification of rPA-D1.

(A) Cartoon of full-length PA revealing the PA-D1 gene used in antigen preparation. (B) Western blot analysis of the affinity purification and desalting process: lane 1, total cell lysate; lane 2, pellet fraction; lane 3, supernatant fraction (PA-D1-GST: 54 kDa); lane 4, beads bound; lane 5, elute after treatment with PreScission Protease (PA-D1: 28 kDa); lane 6, desalting fraction. (C and D) Coomassie brilliant blue staining and Western blot analysis of the fractions collected during the cation exchange process. Lane 1, molecular weight marker; lane 2, sample loaded onto the cation exchange chromatography column; lane 3, flow through; lane 4, Resource-S fraction. Mw; molecular weight marker (in kDa), *: truncated product.

Fig 3. Optimization of rPA-D1 ELISA.

(A) By using the checkerboard titrations, we determined the optimal concentrations of the antigen, the antibody and serum dilutions as follows: antigen, 10 ng/well; serum dilution, 1:100; second antibody dilution, 1:12,000. (B) Analysis of control serum samples using rPA-D1 ELISA under optimal conditions. NC: negative control group, (n = 53). PC: positive control group (n = 25). Red dot: pooled sample from cattle with exposure to natural infection. Error bars represent the mean and standard deviation. Horizontal dotted line: cut-off value of 0.67.

Fig 4. Association between the anti-PA antibodies and state.

Box plots for the distribution of anti-PA antibodies. (A) Herds 1–8 in the endemic area and 9–12 in the nonendemic area. (B) Vaccination timing. The boxes indicate the interquartile range, the horizontal lines indicate the median, and the lower and upper hinges indicate the minimum and maximum values, respectively (outliers not included). Horizontal dotted line: cut-off value of 0.67.