DIAGNOSIS

Suspicion of anthrax arises from the observation of clinical signs, the pathological and epidemiological findings. The ecology of the bacterium limits the distribution of the disease, such that it is almost always confined to well- defined territories in which sporadic outbreaks, usually involving a few animals, occur.

LABORATORY DIAGNOSIS

When taking a sample from a suspected anthrax case, one needs to take precautions to prevent human infection, and environmental contamination. The optimum sample is a cotton swab dipped in blood. Putrefaction quickly destroys vegetative B. anthracis, which can therefore be difficult to isolate from carcasses just 48 hours after the death, espe­cially in hot weather.

Microscopic Test

A preliminary examination of an unstained fresh blood smear will highlight the presence of stick forms or typical ‘bamboo canes’. The organisms are immobile and well capsulated. The slide may be fixed and stained with Gram stain (B. anthracis is violet in colour), Giemsa stain (the bacilli are purple and the capsule a characteristic red mauve), MacFadyean stain (pink capsule) or Loffler stain, which uses methylene blue (bacterial bodies stain blue and the capsule reddish).

Cultural Test

Bacillus anthracis grows well in ordinary medium under aerobic or microaerophilic conditions, at temperatures between 12 and 44°C, but optimal growth occurs around 37°C and at a pH of 7.0 to 7.4. On nutrient agar it forms white colonies 3—4 mm in diameter with a rough surface, called ‘glass beads’ and with irregular margins with medusa head appearance at low magnification. On blood agar it does not cause haemolysis. It is normally sensitive to peni­cillin. Animal samples for culture include blood, exudates and organs.

Polymerase chain reaction (PCR) is the method of choice as a parallel diagnostic test, whether performed directly on clinical samples after non-selective enrichment of mixed cultures, or as a confirmation test for suspect colonies. To identify virulent B. anthracis strains, and for the differentiation of non-virulent strains, the presence of both of the plasmids pXO1 (toxins) and pXO2 (capsule formation) must be confirmed.

Molecular Characterization

Bacillus anthracis represents one of the most genetically homogeneous bacteria, with strains sharing more than 99% of homologous nucleotide sequences. The high- resolution typing assay par excellence is that of the multiple­locus variable number tandem repeat analysis (MLVA), as it seeks to identify specific genomic regions known as vari­able number tandem repeats (VNTR). This technique, initially with 8 VNTR, was able to identify 89 genotypes from 400 isolates from around the world, whereas the 15 VNTR assay increased this to 221 genotypes with 1033 isolates. This method has now been increased up to 25 loci, which allows an excellent organism discrimination with this high genetic homology.