Molecular detection of anthrax sporeson animal fibres�

K. Levi1, J.L. Higham2, D. Coates3 and P.F. Hamlyn1

1BTTG, Shirley House, Wilmslow Road, Didsbury, Manchester M20 2RB, UK, 2School of Applied Sciences, University of Northumbria,

Newcastle upon Tyne, NE1 8ST, UK and 3School of Biology, University of Leeds, Leeds LS2 9JT, UK

2002/379: received 9 December 2002, revised 10 February 2003 and accepted 19 February 2003

ABSTRACT

K. LEVI , J .L . H IGHAM, D. COATES AND P.F . HAMLYN. 2003.

Aims: To develop a rapid, specific and sensitive diagnostic test for the detection of the spores of Bacillus anthracis

on commercial samples of animal fibres (e.g. wool and cashmere).

Methods and Results: Extraction of DNA from spores using a mechanical disruption method based on bead

beating was evaluated but subsequently abandoned as it compromised the sensitivity of the overall protocol.

A multiplex PCR and two nested amplification reactions designed for B. anthracis were developed during this study.

Conclusions: A simple selective incubation step in combination with multiplex PCR was found to be more

effective than generic DNA extraction coupled to a sensitive nested amplification reaction.

Significance and Impact of the Study: The rapid diagnostic test could be applied to the analysis of commercial

fibre samples for the detection of anthrax as required by health and safety legislation resulting in considerable

savings in time and expense.

Keywords: Animal fibres, anthrax, Bacillus anthracis, cashmere, multiplex PCR, spores, wool.

INTRODUCTION

Anthrax primarily affects herbivores, which become infected

through ingestion of virulent spores in grazing vegetation or

contaminated feed. The bacilli that cause the infection

produce resistant spores that can survive for many years.

Before the recent bioterrorism incidents, anthrax was mainly

a disease of those having close contact with infected animals

or contaminated animal products. Although outbreaks of

anthrax are now extremely rare in the UK, the disease is still

prevalent in many countries from which raw wool and

cashmere are imported. The spores of Bacillus anthracis, the

causative agent of anthrax, can be carried on raw fibre

samples posing an occupational risk of cutaneous or inhala-

tion anthrax for transportation and textile workers. In the

textile industry activities in the early stages of processing

such as blending, carding, combing and handsorting of fibres

carry the greatest risks of infection (Crook et al. 1996).

Under the Anthrax Prevention Order (APO) of 1971 wool

and cashmere imported into the UK from certain ‘high risk’

countries are currently tested for the presence of B. anthracisspores using conventional microbiological techniques. The

procedure takes several days during which time consign-

ments of fibre are held in quarantine and deliveries are

delayed. The APO is currently under review and may be

revoked in the next few years. Responsibility for controlling

and assessing fibres for anthrax contamination would be

transferred to importers under the Control of Substances

Hazardous to Health (COSHH) Regulations 1999 and the

Health and Safety at Work Act (HSWA) 1974.

The aim of this study has been to reduce the timescale and

provide an accessible system for the detection of spores of

B. anthracis on animal fibres. A generic DNA extraction

�Note: All work with Bacillus anthracis was carried out at the Health and Safety

Laboratory, Health and Safety Executive, Broad Lane, Sheffield, S3 7HQ, UK.

Some preliminary experiments not involving B. anthracis were carried out at BTTG

and the University of Leeds.

Correspondence to: P.F. Hamlyn, BTTG, Shirley House, Wilmslow Road, Didsbury,

Manchester M20 2RB, UK (fax: +44 (0)161 434 9957; e-mail: pfhamlyn@

bttg.co.uk).

ª 2003 The Society for Applied Microbiology

Letters in Applied Microbiology 2003, 36, 418–422

method, based on bead beating, has been evaluated and a

multiplex polymerase chain reaction (PCR) test developed

for the detection of B. anthracis.

MATERIALS AND METHODS

Bacterial strains and culture conditions

Type strains of B. anthracis (NCTC 10340, Vollum), B. cereus

(NCTC 2599), B. mycoides (NCTC 926), B. thuringiensis

(NCTC 9134), B. subtilis (NCTC 3610), B. licheniformis(NCTC 10341) and B. subtilis var. globigii (NCTC 10073)

were obtained from the National Collection of Type Cultures

(Public Health Laboratory Service (PHLS), London). Bac-

terial cultures were grown on nutrient agar (Lab M, Bury,

UK) at 37�C overnight. Bacillus anthracis is classified in

Hazard Group 3 (Advisory Committee on Dangerous

Pathogens) therefore all work with this organism was

conducted at Containment Level 3.

Spore removal from fibre samples

This procedure is the one used to detect anthrax bacilli in

fibre samples by microbiological culture at the PHLS and is

carried out under Containment Level 3 conditions. Approx.

15 g of the fibre sample was weighed and placed into a

sterile container to which 100 ml sterile 1/4 strength

Ringers solution (Oxoid, Basingstoke, UK) was then added.

The sample was compressed and agitated in this solution

until thoroughly wetted and left for 2 h, with further

agitation after 1 h. After soaking 10 ml of the sample

solution was removed, heat treated at 70�C for 10 min to kill

vegetative cells, serially diluted and incubated on non-

selective medium at 37�C for 18–20 h.

Multiplex PCR and nested amplifications

Universal bacterial 16S rDNA primers adapted from Relman

(1993), were 926F (5¢-AAACTYAAAKGAATTGACGG-

3¢) and 1492R (5¢-CGGYTACCTTGTTACGAC-3¢). Spe-

cies-specific primers for the Ba813 chromosomal region of

B. anthracis (Patra et al. 1996) were AR1 (5¢-TTAATTCAC-

TTGCAACTGATGGG-3¢) and AR2 (5¢-AACGATAG-

CTCCTACATTTGGAG-3¢). Specific primers targeted to a

region of the trans-activator of encapsulation acpA (Vietri

et al. 1995) on virulence plasmid pX02 were acpA3

(5¢-TGGTTCACGCTTTTTGAGTTAGA-3¢) and acpA4

(5¢-TGTGCTTTCCCCCTCTTTGTA-3¢).Multiplex PCR amplification was carried out in a reaction

mixture (50 ll) containing MgCl2 (2 mMM), dNTPs (200 lMM)

and 0Æ2 lMM of each primer. 1Æ25U Taq (Promega) was added

after the hot start. The thermal cycler (Hybaid Omnigene)

program was as follows: 1 · 95�C for 5 min; 30 cycles of

(95�C for 1 min; 57�C for 1 min; 72�C for 1 min); 1 · 72�Cfor 7 min.

Nested primers were designed using Oligo 5Æ0 software

(NBI) to amplify products within the acpA3 and acpA4 and

the AR1 and AR2 amplicons. The nested Ba813 primers

were AR3 (5¢-AGGGAATACAGCAAACACAG-3¢) and

AR4 (5¢-ACCTGGCATTAAAAGACTCAT-3¢) and the

nested acpA primers were acpA7 (5¢-AATTCGGTTTA-

TCTTTGGAA-3¢) and acpA8 (5¢-AAGGCCATTCTT-

CTTTTATCA-3¢). The optimal conditions for acpA7 and

acpA8 were 1 lMM of each primer and 2 mMM MgCl2.

Bead beating

Mechanical disruption studies were carried out using the

FastDNA SPIN Kit for Soil in conjunction with a FastPrep

FP120 instrument (Bio101, Vista, CA, USA). Efficiency of

the disruption matrices at different speeds and for varying

periods was measured by the reduction in viability of the

spores as indicated by direct colony counts.

RESULTS

Multiplex PCR and nested amplifications

A multiplex PCR was designed for the detection of

B. anthracis DNA composed of universal bacterial 16S

rDNA primers (positive control), the Ba813 primers of

Patra et al. (1996), and oligonucleotides targeted to a

region of the trans-activator of encapsulation acpA (Vietri

et al. 1995) on virulence plasmid pX02. The Ba813

chromosomal region has been claimed to be specific for

B. anthracis whilst amplification of a region of the virulence

plasmid pX02 would enable differentiation between viru-

lent and avirulent strains. The presence of pX02 indicates

a virulent strain of B. anthracis as no environmental or

clinical pX01)/pX02+ isolates have been identified

(Turnbull et al. 1992). Therefore, the combination of the

three primer sets would allow monitoring of the DNA

extraction and amplification processes, and differentiation

between benign and pathogenic strains of B. anthracis. The

multiplex was optimized by varying Taq DNA polymerase

(Promega), primer and MgCl2 concentrations (results not

shown).

Two nested amplification reactions were designed for

B. anthracis within the acpA3 and acpA4 amplicons on

virulence plasmid pX02 and the AR1 and AR2 amplicons for

the Ba813 chromosomal region. The specificity of the pX02

nested amplification was assessed with the four members of

the B. cereus group. DNA was extracted by lysis from a

single overnight colony of B. anthracis, B. cereus, B. mycoidesand B. thuringiensis. Each extract was amplified with the 16S

rDNA primers to demonstrate that DNA had been

DETECTION OF B. ANTHRACIS ON FIBRES 419

ª 2003 The Society for Applied Microbiology, Letters in Applied Microbiology, 36, 418–422

successfully isolated, as well as being added to the nested

reactions (Fig. 1). The nested primers only amplified DNA

extracted from B. anthracis. The Ba813 nested primers were

tested with DNA extracts from the same B. cereus group.

Neither B. cereus, B. mycoides nor B. thuringiensis DNA was

amplified by this reaction. In sensitivity studies with serially

diluted genomic DNA isolated from B anthracis the pX02

nested amplification improved the sensitivity of the overall

reaction 102-fold giving a detection limit of 10 fg DNA

(Fig. 2) which we have estimated to equate to 2Æ5 cells (Levi

1999). A similar result (not shown) was obtained with the

Ba813 nested reaction.

DNA extraction from spores by bead beating

Initially, B. cereus spores were used as a model for

B. anthracis. Following the manufacturer’s instructions, 103

and 104 CFU ml)1 spores were disrupted for 5–30 s at

speeds of 4, 4Æ5, 5 and 5Æ5 m s)1. Although after 30 s bead

beating at 5Æ5 m s)1 the viability of 103 CFU ml)1 spores

was reduced to 6Æ5%, the equivalent figure when

104 CFU ml)1 spores were disrupted was 28Æ6%. Ten gram

samples of wool and cashmere previously tested at the Health

and Safety Laboratories (Sheffield, UK) have contained from

104 to 105 spores (A. Bowry, personal communication). The

FastDNA SPIN Kit for Soil (Bio101) is designed to extract

DNA from soil-borne organisms, including Gram positive

bacteria. Samples of 106 CFU ml)1 B. cereus spores were

processed at the maximum speed of 6Æ5 m s)1 for 30 s to

3 min. After 3 min processing the viability of the spores,

assessed by direct colony counting, was reduced by 99Æ6%.

Since a 15 g representative fibre sample would be expected

to contain up to 105 spores of various species the overall

detection protocol must be able to differentiate B. anthracis

spores from other contaminating species even if the former

only constitute a small proportion of the total population.

Therefore B. anthracis spores were combined with B. cereusspores in various combinations (Table 1) to test the sensi-

tivity of the bead beating extraction method. The samples

were processed using the FastDNA SPIN Kit for Soil

(Bio101) and the DNA extracts were amplified with the

universal 16S rDNA primers. However, in most cases, very

little amplification occurred and no amplification was

achieved for these samples with the nested B. anthracis

chromosomal reaction although the positive control of

700 bpouterproduct

600 bp 16SrDNA product

1 2 3 4 5 6 7 8 9 10 1112 15 161413

200 bpnestedproduct

Fig. 1 Evaluation of the specificity of the pX02 nested amplification,

using DNA extracted from the four members of the Bacillus cereus

group. Lanes 1 and 16: 100 bp ladder (Promega); lane 2: B. anthracis

lysate + outer pX02 primers; lane 3: B. anthracis lysate + nested pX02

primers. Lanes 4–6: B. cereus lysate, 4: B. cereus extract + 16S rDNA

primers, 5: B. cereus extract + outer pX02 primers, 6: B. cereus

extract + nested pX02 primers. Lanes 7–9: B. mycoides lysate, 7:

B. mycoides extract + 16S rDNA primers, 8: B. mycoides ex-

tract + outer pX02 primers, 9: B. mycoides extract + nested pX02

primers. Lanes 10–12: B. thuringiensis lysate, 10: B. thuringiensis

extract + 16S rDNA primers, 11: B. thuringiensis extract + outer pX02

primers, 12: B. thuringiensis extract + nested pX02 primers. Lane 13:

10 ng E. coli DNA (Sigma) +16S rDNA amplified during nested pX02

reaction; lane 14: no template control, outer pX02 reaction; lane 15: no

template control, nested pX02 reaction

200 bpnestedproduct

800 bpouterproduct

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Fig. 2 Evaluation of the sensitivity threshold of the pX02 nested amplification reaction. Lanes 1 and 20: 100 bp ladder (Promega). Lanes 2–11:

outer primer amplification: 2: 16S rDNA primers + 10 ng Bacillus anthracis DNA, 3: no template control, 4: 10 ng B. anthracis DNA, 5: 1 ng

B. anthracis DNA, 6: 100 pg B. anthracis DNA, 7: 10 pg B. anthracis DNA, 8: 1 pg B. anthracis DNA, 9: 100 fg B. anthracis DNA, 10: 10 fg

B. anthracis DNA, 11: 1 fg B. anthracis DNA. Lanes 12–19: nested primer amplification: 12: 100 fg B. anthracis DNA product, 13: 10 fg B. anthracis

DNA product, 14: 1 fg B. anthracis DNA product, 15: 100 fg B. anthracis DNA product diluted 1 : 10, 16: 10 fg B. anthracis DNA product diluted 1 :

10, 17: 1 fg B. anthracis DNA product diluted 1 : 10, 18: 16S rDNA primers + 10ng B. anthracis DNA, 19: no template control

420 K. LEVI ET AL.

ª 2003 The Society for Applied Microbiology, Letters in Applied Microbiology, 36, 418–422

Escherichia coli DNA (Sigma) was successfully amplified in

each PCR amplification (results not shown). The data

indicated that, as a result of increasing the bead-beating

period to 3 min to overcome the resistant nature of the

Bacillus spores, the released DNA was sheared to a point,

which seriously compromised the sensitivity of the PCR test.

DISCUSSION

Two targets were employed for the detection of B. anthracis:

the Ba813 chromosomal region of Patra et al. (1996) and a

region of the virulence plasmid pX02. This enabled

identification of B. anthracis from other species and the

differentiation of virulent and avirulent strains. It has been

demonstrated that the equivalent of 2Æ5 B. anthracis genome

copies can be detected using nested amplification reactions.

Currently, when a consignment of fibre is to be tested for

the presence of anthrax spores, grab samples are obtained

from fibre bales and sent for microbiological testing.

Suspicious colonies are subjected to secondary tests to

confirm the presence of B. anthracis (Levi 1999). No

previous work has been published on the molecular

detection of B. anthracis spores on animal fibres. We have

found that this sample type raised similar problems to the

isolation of the anthrax agent from environmental samples;

that is, the sensitive amplification of a small number of

B. anthracis-specific targets from a large contaminating spore

population. As such perhaps the most important part of a

detection protocol for B. anthracis is not the PCR ampli-

fication process but the sample preparation stage.

Owing to the extreme resistance of the spores, one of the

main problems to be overcome in the development of a

molecular test for B. anthracis is the extraction of DNA. It

was initially envisaged that a mechanical disruption method,

based on bead beating, would be suitable for the lysis of fibre-

contaminating spores and subsequent DNA extraction. This

method has been reported to be a rapid method for the

extraction of DNA from the spores of B. anthracis prior to

PCR. Both Johns et al. (1994) and Reif et al. (1994)

compared mechanical disruption with germination and

found that the procedures resulted in equal PCR sensitivity.

The technique successfully reduced the viability of samples

of 105 B. anthracis spores by over 99%. However, this

approach was shown to compromise the sensitivity of the

overall protocol and failed to match the sensitivity of the

current microbiological method for fibre testing, even when

combined with nested PCR. Therefore, it is recommended

that the current detection technique is adapted to replace

morphological identification and secondary testing for

B. anthracis with a multiplex PCR test as traditional

microbiological methods are more time-consuming and

difficult to interpret. Contaminating spores would be incu-

bated overnight on a selective medium (PLET), suspicious

colonies lysed and subjected to the PCR test allowing rapid

identification of isolates reducing testing time from at least

1 week to just 2 days.

Several multiplex PCR tests have now been developed for

anthrax detection. The multiplex developed during this

study combines detection of the Ba813 region and the pX02

plasmid sequence with 16S rDNA primers as a positive

control. The multiplex reaction of Ramisse et al. (1996),

which combines the Ba813 primers used in this work with

four pairs of primers specific for regions on both virulence

plasmids, has been utilized during an outbreak of anthrax in

France (Patra et al. 1998). Shangkuan et al. (2001) have also

developed a multiplex assay, which targets two virulence

factor genes together in the same reaction mixture. Recent

reports have suggested that the Ba813 chromosomal region of

B. anthracis is less specific than was originally proposed.

Ramisse et al. (1999) have evaluated the distribution of the

Ba813 DNA sequence. Ba813 was identified from 47 strains

or isolates of B. anthracis tested, thus indicating its reliability

as a tracer for this species. However, from 60 strains of

closely related Bacillus species four were found to harbour

Ba813. Qi et al. (2001) have developed a more specific

chromosomal target by taking advantage of the unique

nucleotide sequence of the B. anthracis rpoB gene. A PCR

assay based on the rpoB gene was specific for 144 B. anthracisstrains from different geographical locations and cross-

reaction only occurred with one of 175 related bacilli.

ACKNOWLEDGEMENTS

The authors would like to thank Brian Crook, Health and

Safety Laboratory, Health and Safety Executive, Sheffield

for making available a Containment Level 3 laboratory and

other facilities. KL was in receipt of a Postgraduate Training

Partnership award from the EPSRC when she was working

on this project. PFH and KL are grateful to Celsis PLC for

providing financial support towards the costs of the project.

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Sample

B. anthracis

spores CFU ml)1

B. cereus spores

CFU ml)1

1 103 102

2 102 103

3 101 104

4 100 105

DETECTION OF B. ANTHRACIS ON FIBRES 421

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