The reporting of a Bacillus anthracis B-clade strain in South Africa after more than 20 years
Lekota et al. BMC Res Notes
The reporting of a Bacillus anthracis B-clade strain in South Africa after more than 20 years
K. E. Lekota 0 3
A. Hassim 0
P. Rogers 2
E. H. Dekker 1
R. Last 4
L. de Klerk‑Lorist 1
H. van Heerden 0
0 Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria , Onderstepoort , South Africa
1 State Veterinary Services, Department of Agriculture, Forestry and Fisheries , Skukuza , South Africa
2 Provet Wildlife Services, Raptors Safari Junction , Main Road, Hoedspruit , South Africa
3 College of Agriculture and Environmental Sciences, University of South Africa , Christiaan De Wet/Pioneer Dr., Florida , South Africa
4 Vetdiagnostix-Veterinary Pathology Services , 257 Boshoff Street, Pietermaritzburg , South Africa
Objectives: Anthrax is a disease with an age old history in Africa caused by the Gram‑ positive endospore forming soil bacterium Bacillus anthracis. Epizootics of wild ungulates occur annually in the enzootic region of Pafuri, Kruger National Park (KNP) in the Limpopo Province of South Africa. Rigorous routine surveillance and diagnostics in KNP, has not revealed these rare isolates since the 1990s, despite unabated annual outbreaks. In 2011 a cheetah was diagnosed as anthrax positive from a private game reserve in Limpopo Province and reported to State Veterinary Services for further investigation. Isolation, molecular diagnostics, whole genome sequencing and comparative genomics were carried out for B. anthracis KC2011. Results: Bacteriological and molecular diagnostics confirmed the isolate as B. anthracis. Subsequent typing and whole genome single nucleotide polymorphisms analysis indicated it clustered alongside B. anthracis SA A0091 in the B.Br.010 SNP branch. Unlike B. anthracis KrugerB strain, KC2011 strain has unique SNPs and represents a new branch in the B‑ clade. The isolation and genotypic characterisation of KC2011 demonstrates a gap in the reporting of anthrax outbreaks in the greater Limpopo province area. The identification of vulnerable and susceptible cheetah mortalities due to this strain has implications for conservation measures and disease control.
Bacillus anthracis; Whole genome sequencing (WGS); Single nucleotide polymorphisms (SNPs)
Anthrax is a zoonotic disease with an age old history in
], causing acute mortalities. It primarily affects
ungulates with episodic spill over into humans and
]. The causative agent of this disease is the
Gram positive, endospore forming, exotoxin producing
soil bacterium Bacillus anthracis [
]. Sporulation is
triggered by nutrient scarcity, the presence of bicarbonate
and oxygen exposure [
]. The virulence factors of this
bacterium are on the two plasmids, pXO1 (181 kilobases)
and pXO2 (94 kilobases), encoding the toxin and capsule
genes respectively [
]. The toxin complex is composed
of three components namely, lethal toxin (LF) made up
of the protective (PA) and lethal factor (EF) [
capsule consists of a five gene operon (capBCADE) that
produces a poly-gamma-d-glutamic acid (PGA) on the
bacterial cell membrane that protects the vegetative cells
from phagocytosis [
Anthrax was widespread across southern Africa in
the early 20th Century, until the development and
South African state implementation of the Sterne
vaccine in 1937 [
]. While livestock cases have since
been infrequent due to vaccination practices; outbreaks
are still common amongst wildlife in endemic regions
of South Africa and game reserves across the continent
]. Limpopo province covers 125,754 km2
bordering Botswana and Zimbabwe to the north. The northern
half of the Kruger National Park (KNP) makes up the
entire eastern border of the Limpopo Province bordering
Mozambique. The anthrax endemic region in KNP falls
between the Limpopo and Luvhuvhu rivers [
distribution of outbreaks and the animal species affected by
anthrax have been described extensively in KNP in the
last 35 years [
]. The genetic population structure of
B. anthracis consists of the global A, B and C clades .
The B clade isolates identified in South Africa were
confined to the northern tip of KNP in Limpopo Province
due to it being an ideal soil environment for spore
]. The A clade isolates, in contrast, have a wide
distribution, predominating and prevailing over the rarer
B clade isolates during outbreaks since the late 1980s.
Host species differ in their susceptibility to B.
anthracis. Wild ungulates are more susceptible to the disease
than carnivores [
]. Even amongst herbivores, roan
antelope (Hippotragus equinus) are far more susceptible
to anthrax than other antelope species . Similarly,
cheetahs (Acinonyx jubatus) are more susceptible to the
toxins produced by the bacterium than other
carnivorous felids and canids [
]. It is hypothesised that
this could be due to the immune response dictated by
the monomorphic genetic structure of southern African
] or by the feeding habits of free roaming
]. In either event, it is of importance to
cheetah conservation practices since these felids are
considered vulnerable (bordering on endangered) [
This status has led to more stringent investigations of
cheetah mortalities in both captive and free roaming
animals. In 2011 a cheetah mortality from a private game
reserve in Limpopo Province was investigated by the
local veterinarian. The cheetah was observed to have
substantial facial oedema with unclotted blood around the
kidneys (Fig. 1A, B). Biological samples from the
cheetah were submitted for bacteriologic diagnostics (Fig. 1C,
D). Colony morphology indicative of B. anthracis then
confirmed the initial diagnosis (Fig. 1D). The isolate was
then submitted by Skukuza State Veterinary Services
for further analysis on behest of the Anthrax Advisory
Isolation and identification
A Giemsa smear stain from the oedema fluid was
performed for identification of B. anthracis. Pure culture
isolation of KC2011 isolate was obtained by culturing
biological samples directly onto 5% sheep blood agar
after 24 h incubation at 37 °C characteristics including
colony and cell morphology, gamma-phage and penicillin
sensitivity were evaluated as described in OIE [
Capsule formation was observed by visualization using light
microscopy of Giemsa stained fluid and colonies.
DNA extractions and qPCR
Genomic extraction of B. anthracis KC2011 was carried
out using a DNA Blood Mini Kit (Qiagen, Germany)
according to manufacturer’s instructions. The DNA was
quantified using Qubit™ fluorometric quantitation
(Invitrogen™, USA). Quality of the extracted DNA was
visualized on 0.8% agarose gel electrophoresis. The diagnostic
qPCR for B. anthracis was performed with 2.5 µL DNA in
1× FastStart™ Taq DNA Polymerase mastermix (Roche®,
Germany) and 0.5 μM of each primer small acid soluble
proteins (SASP), Bacillus anthracis protective antigen
(BAPA), and capsule region (capC) along with 0.2 μM
of probe for each chromosomal and plasmid target pairs
with fluorescein on the one and LCRed640 on the other
(Tib MolBiol GmbH, Germany) in a final volume of 20 µL
as described in Turnbull [
]. The PCR conditions on a
LightCycler™ Nano (Roche®, Germany) were used as
described in OIE [
The PCR single nucleotide polymorphism (SNP) assay
was performed for the oligonucleotide markers A/B.
Br.001, B.Br.003, B.Br.004, B.Br.002, B.Br.001, A.Br.007
and A.Br.002 as described in Birdsell et al. [
reaction included 2.5 µL DNA diluted in 1× FastStart DNA
Green Master (Roche®, Germany) with an ancestral
forward and a derived forward SNP target primer
(GCclamp: no-GC-clamp) and a common reverse primer
with a starting concentration of 0.2 µM depending on
the ratio indicated which allowed for separation of melt
peaks by at least 5 °C. Thermocycling parameters on the
LightCycler™ Nano (Roche®, Germany) were conducted
as described in Birdsell et al. [
Whole genome sequencing and Bioinformatics analysis
Sequence libraries of the DNA isolate was generated
using the Nextera DNA Sample Prep Kit (Illumina, USA)
protocol. Sequence reads of paired end library was
performed on a HiSeq 2500 sequencer (Illumina, USA).
Quality of the genome sequenced reads were assessed
using FastQC software 0:10.1 [
]. Trimmomatic [
was used to remove the ambiguous nucleotide reads.
De novo assembly of the B. anthracis KC2011 was
carried out using the CLC Genomics Workbench version
7.5 (CLC, Denmark). The assembled contigs were aligned
with BLASTn [
] using B. anthracis Ames ancestor
(GenBank: AE017334.2, AE017336.2, and AE017336.2) as
a reference. Mauve tool [
] was used to align and order
the assembled contigs using B. anthracis Ames ancestor
as a reference. The genome was annotated using PGAAP
at NCBI [
The trimmed reads of B. anthracis KC2011 were
aligned to B. anthracis Ames ancestor using the
BurrowsWheeler Aligner (BWA) [
]. SAMtools [
] was used
to sort and index the aligned sequenced reads. Unified
genotyper in GATK [
] was used to call for SNPs. In
order to construct WGS- SNPs tree, complete and draft
genomes from different clades of B. anthracis available
in NCBI Genbank (http://www.ncbi.nom.nih.gov) were
included in this study (Additional file 1: Table S1). SNPs
positioning sets were deducted from the aligned genomes
of B. anthracis Ames ancestor using molecular
evolutionary genetics analysis (MEGA) 7 [
]. SNPs with
informative sites (core SNPs) in all genome sequences were used
for the phylogenetic tree construction using MEGA 7
The genome sequence of B. anthracis KC2011 was
deposited in the Genbank genome database under the
accession number: NJGK00000000.
Isolation and identification
A Giemsa stained impression smear from the oedema
fluid revealed encapsulated, square ended bacilli
pathognomonic for B. anthracis (Fig. 1C). The microbiological
characteristics confirmed the bacterium to be B.
anthracis on blood smear with typical B. anthracis colony
morphology and structure on 5% sheep blood agar [
qPCR and Melt‑MAMA analysis
The B. anthracis KC2011 isolate was confirmed
positive for the presence of B. anthracis BAPA, SASP and
capC. B. anthracis KC2011 was further typed using
Melt-MAMA and amplified the derived markers for
A/B.Br.001, B.Br.003 and B.Br.002, whilst amplifying the
B.Br.001 ancestral marker. This SNP profile indicated
KC2011 to be a B clade (B.Br.001/002), but not the rare
Genome assembly and phylogeny
De novo assembly was carried out which resulted in 45
contigs (Table 1). The total genome contigs contributed
to a 5.42 MB, with a GC content of 35%. A total of 6 047
coding sequences (CDSs) was determined in the KC2011
strain (Table 1). Comparative genome alignment of the B.
anthracis KC2011 genome with Ames ancestor revealed
no evidence of novel genes (Additional file 3: Fig. S1).
The genome coverage of B. anthracis KC2011 was 99%
covered to B. anthracis Ames ancestor (Additional file 1:
Table S1; Additional file 2: Table S2). High copy number of
pXO1 was observed represented by 725 coverage (725×).
A high resolution tree of global B. anthracis strains
was constructed to visualise the SNP grouping of the B.
anthracis KC2011 (Fig. 2). This yielded 3 247 parsimony
informative SNPs that were used to construct the
phylogram. The B. anthracis KC2011 grouped in the B-clade
separate from the KrugerB strain. The closest related
branch was SA B. anthracis A0091.
Strain KC2011 is the first B. anthracis identified as a
B-clade lineage bacterium in over 20 years in South
Africa and represents a new subclade in the B-clade.
About 90% of the anthrax outbreaks reported
worldwide arise from the A-clade, while less than 10% have
been reported from the B-clade (KrugerB subclade) [
The majority of the B. anthracis strains isolated from the
northern part of KNP from 1970 to 1981 grouped in the
B-clade (KrugerB subclade) but this clade became rare
since the 1990s [
]. Only one B strain reported between
1982 and 1997 [
] to the extent that it was no longer
isolated from 1997 until this report in South Africa.
No significant anthrax outbreaks were noted outside
the endemic regions of KNP (northern part of KNP) in
2011 according to state veterinary surveillance data. The
isolate KC2011 was only identified because of the
delicate conservation status of cheetah in Africa and because
it was within the context of the broader Limpopo
province area (i.e. outside KNP). Cheetahs are especially
susceptible to the B. anthracis exotoxins, however, they
do respond well to vaccination with the Sterne vaccine
]. This study highlighted the importance of
vaccinating cheetah, where logistically possible, in conservancies
across Limpopo province.
The phylogenetic structure of B. anthracis determined
in this study (Fig. 2) is similar to other reported
WGSSNP trees [
]. Past B-clade (1965–1990) isolates
from the State Veterinary archival collection have all
clustered in the KrugerB lineage. WGS-SNP analysis
defined KC2011 strain grouping as a distinctive sub-clade
within southern Africa B.Br.010.
Although, distant in terms of number of SNPs, the
isolate A0091 and KC2011 belong to a common albeit
broader lineage. The isolate A0091 is recorded as being
from South Africa circa 1939, but has no other
information to link it to KNP or Limpopo Province. There is also
a dearth of information from the countries (Zimbabwe
and Mozambique) bordering the South African province
across the Limpopo River. Further surveillance of
Limpopo Province is required to reveal whether deposits of
B-clade isolates are subsisting outside KNP or whether
this was an incidental mortality due to circumstances
outside the realm of current monitoring criteria.
Surveillance in the non-endemic anthrax regions is difficult as
there are private game reserves and rural livestock
farmers bordering KNP. Moreover the rural subsistence
farmers (predominantly cattle farmers) are less likely to report
mortalities and/or vaccinate their livestock thus
complicating disease monitoring and control. This report of the
B. anthracis KC2011 isolated outside the endemic region
highlights the necessity of disease surveillance in
The identification of KC2011 is significant in that
mortalities linked to the B-clade have diminished over several
decades and therefore was estimated to have disappeared
from the local environment. This suggests a possible gap
in surveillance of carcasses submitted for anthrax
diagnostics in the Limpopo province especially in the
nonendemic regions. It also has implications for the inclusion
of the Sterne vaccine as part of the conservation
measures to be included for cheetah in sanctuaries and private
game reserves across the Limpopo Province in South
The tree topology of the southern African strains was
not well characterised due to the limited number of
genomes available. Branch lengths vary according to
inclusion of number and variety genomes in the data
sets, as this explicates the informative SNPs used to
construct the tree. Isolate KC2011 will thus be the basis
for further typing of B. anthracis strains for southern
Additional file 1: Table S1. Whole genomes of Bacillus anthracis retrieved
from public database used in this study.
Additional file 2: Table S2. Genome alignment of the Bacillus anthracis
KC2011 to the B. anthracis Ames ancestor reference.
Additional file 3: Fig. S1. Alignment of Bacillus anthracis Ames ancestor
genome with B. anthracis KC2011. Each colour block indicates homolo‑
gous regions of the genome sequences. White areas in colour blocks
indicate possible nucleotide variation absence or presence within the
PGA: poly‑ gamma‑ d‑ glutamic acid; LF: lethal toxin; PA: protective; EF: lethal
factor; KNP: Kruger National Park; WGS: whole genome sequencing; SNP: sin‑
gle nucleotide polymorphism; Melt‑MAMA: melting mismatch amplification
mutation analysis; CDSs: coding sequences.
KEL and AH performed the molecular, as well as, in silico analysis and drafting
of the manuscript. RL and PR identified the mortality and performed the
pathology evaluation. LDKL and HED performed the bacterial and DNA isola‑
tions. HvH participated in the design of the study, drafting the manuscript,
revising it critically and provided funding. All authors read and approved the
We would like to thank the Anthrax Advisory Committee.
The authors declare that they have no competing interests.
Availability of data and materials
All data generated or analysed during this study are included in this published
Consent for publication
Ethics approval and consent to participate
This study was funded by the National Research Foundation (NRF) of South
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lished maps and institutional affiliations.
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