Abstract
Anthrax is an acute and highly infectious disease of almost all warm-blooded animals including humans. It occurs most frequently as an epizootic or enzootic disease of herbivores that acquire spores from direct contact with contaminated soil. The disease can be prevented in animals by using Anthrax vaccine. The vaccine (Sterne strain F-34) has been used in cattle, buffaloes and small ruminants in Bangladesh for more than five decades. There is little work describing the efficacy of the vaccine, genomic properties of the vaccine strain and field isolates of B. anthracis in Bangladesh. This study was, therefore, aimed to characterize Anthrax vaccine (F-34 Sterne strain) and virulent field isolates by biological and gene based assays. The vaccine efficacy was evaluated in cattle and mice model by immunoassay and protection efficacy respectively. A total of thirteen outbreaks of anthrax were investigated and samples were collected to isolate bacteria on culture. Anthrax F-34 strain was isolated from vaccine vials. Out of 13 field samples examined, 5 samples and vaccine isolate showed growth of Anthrax bacteria on PLET agar. The bacteria on sheep blood agar media showed typical rough, sticky, white-gray non hemolytic growth. The vaccine efficacy in cattle was evaluated by ELISA using both the vaccine and field isolates of B. anthracis as plate coating antigen. The sera were collected on day 0, 30, 60, 120, 240 and 365 of immunization. The anti anthrax antibody response (OD value in ELISA) was detected on day 30 of immunization (0.647±0.275) while reached its peak on day 60 (1.147±0.251). The anti anthrax immune response was detected until day 365 of immunization (0.571±0.193). Results of slide agglutination test showed that the vaccine bacteria and field isolates clumped with bovine anti anthrax antisera at 1:20 dilution as seen in naked eye. Under microscopic investigation of slide agglutination test, the reaction was read up to 1:100 dilutions. Male mice immunized with the Anthrax Sterne vaccine strain and then challenged with virulent field isolates (2x105 bacteria), the vaccinated mice conferred solid protection. The sugar fermentation test, nitrate reduction test and gelatin liquefaction test appeared inconclusive for confirmatory detection of B. anthracis. PCR amplifications of Cap protein gene of B. anthracis detected fragments of pX02 gene (1035bp) from field isolates (n=05) but not from the vaccine strain. The fragments of pX01 and pX02 genes were sequenced and analyzed with Basic Local Alignment Search Tool. The nucleic acid sequences were aligned with other related sequences retrieved from the GenBank and phylogenetic trees were constructed. The present Bangladeshi field isolates of B. anthracis were named as FI GHA 2015 PX02, FI S.MON 2015 pX02, FI T. SADAR 2014 pX02, FI SPUR 2014 pX02, FI D.BARI 2014 pX02 and grouped in Lineage A3 together with the vaccine strain. In pX02 gene, mutation was seen in a Bangladeshi isolate (B. anthracis FI SPUR 2014 pX02), where, Cytonine (C) at the nucleotide position 4 was replaced by Guanine (G). At amino acid level the change was seen in amino acid position 2, where Arginine (Arg) was replaced by Glysine (Gly). The effect of mutation in pX02 gene was not evaluated in this study. The Anthrax Sterne vaccine strain induces anti anthrax immunity in cattle and persists up to 365 days of immunization. The vaccine is protective in mice model and anti anthrax antibody clumps both the vaccine and field isolates. It requires evaluating protective efficacy of the vaccine in farm animal model.