Correction: 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.

[This corrects the article DOI: 10.1371/journal.pone.0205986.].

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.

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.

ANTXR-1 and -2 independent modulation of a cytotoxicity mediated by anthrax toxin in human cells.

Several animal models have shown that anthrax toxin (ATX) elicits a cytotoxic effect on host cells through anthrax toxin receptor (ANTXR) function. In this study, compared with mouse cells, cells obtained from humans exhibited low sensitivity to ATX-mediated cytotoxicity, and the sensitivity was not correlated with expression levels of ANTXRs. ATX treatment also induced a cytotoxic effect in other cultured human cells, human embryonic kidney (HEK) 293 cells, that express ANTXRs at undetectable levels. Furthermore, ectopic expression of ANTXRs in HEK293 cells did not affect the sensitivity to ATX treatment. These findings suggest that there is an ANTXR-independent cytotoxic mechanism in human cells.

Bacillus cereus from the environment is genetically related to the highly pathogenic B. cereus in Zambia.

To follow-up anthrax in Zambia since the outbreak in 2011, we have collected samples from the environment and the carcasses of anthrax-suspected animals, and have tried to isolate Bacillus anthracis. In the process of identification of B. anthracis, we collected two isolates, of which colonies were similar to B. anthracis; however, from the results of identification using the molecular-based methods, two isolates were genetically related to the highly pathogenic B. cereus, of which clinical manifestation is severe and fatal (e.g., pneumonia). In this study, we showed the existence of bacteria suspected to be highly pathogenic B. cereus in Zambia, indicating the possibility of an outbreak caused by highly pathogenic B. cereus.

A novel multiplex PCR discriminates Bacillus anthracis and its genetically related strains from other Bacillus cereus group species.

Anthrax is an important zoonotic disease worldwide that is caused by Bacillus anthracis, a spore-forming pathogenic bacterium. A rapid and sensitive method to detect B. anthracis is important for anthrax risk management and control in animal cases to address public health issues. However, it has recently become difficult to identify B. anthracis by using previously reported molecular-based methods because of the emergence of B. cereus, which causes severe extra-intestinal infection, as well as the human pathogenic B. thuringiensis, both of which are genetically related to B. anthracis. The close genetic relation of chromosomal backgrounds has led to complexity of molecular-based diagnosis. In this study, we established a B. anthracis multiplex PCR that can screen for the presence of B. anthracis virulent plasmids and differentiate B. anthracis and its genetically related strains from other B. cereus group species. Six sets of primers targeting a chromosome of B. anthracis and B. anthracis-like strains, two virulent plasmids, pXO1 and pXO2, a bacterial gene, 16S rRNA gene, and a mammalian gene, actin-beta gene, were designed. The multiplex PCR detected approximately 3.0 CFU of B. anthracis DNA per PCR reaction and was sensitive to B. anthracis. The internal control primers also detected all bacterial and mammalian DNAs examined, indicating the practical applicability of this assay as it enables monitoring of appropriate amplification. The assay was also applied for detection of clinical strains genetically related to B. anthracis, which were B. cereus strains isolated from outbreaks of hospital infections in Japan, and field strains isolated in Zambia, and the assay differentiated B. anthracis and its genetically related strains from other B. cereus group strains. Taken together, the results indicate that the newly developed multiplex PCR is a sensitive and practical method for detecting B. anthracis.