INL BIOLOGICAL SCIENCES

Science

Detection and Forensic Analysis of

Wildlife and Zoonotic Disease

The INL is developing a

variety of assays and

techniques that will

facilitate detection and

molecular fingerprinting of

high consequence pathogens.

Current work is focused on

the detection and forensic

analysis of Brucella spp.,

which are animal and human

pathogens responsible for

disease in a broad spectrum of

hosts. Concern exists over

the possible use of Brucella

spp. as agents of biological

warfare directed towards

humans and/or domestic

animals, specifically cattle.

In addition, this research will

contribute to understanding

the potential for natural

transmission of brucellosis

from bison and elk

populations – in which the

disease is endemic – domesticated animals (cattle) in the

Greater Yellowstone Area.

Detection and

Forensic Analysis of

Wildlife and Zoonotic

Disease

The reagents developed and

refined in our work will have

value not only to national

biodefense, but also to

national and regional animal

husbandry, and wildlife

management issues that

impact U.S. agricultural

security.

The project will

generate a unique set

of validated (against

real world diagnostic

and environmental

samples) DNA

signatures for the

closely related Category

B select agents, Brucella

abortus, B. melitensis, and B.

suis. A variety of techniques

will be employed. INL

researchers have developed a

real-time (fluorescence-based)

Polymerase Chain Reaction

(PCR) test that allows

detection of active Brucella

abortus infection in bison,

other wildlife, and cattle in

approximately 30 minutes.

This is an improvement over

conventional (gel-based) PCR

which typically requires about

3 hours for assay results.

INL has a field-portable realtime PCR instrument allowing

the assay to be run in the field

at trap sites. Additional realtime PCR assays are being

developed and validated to

target other species, incorporate internal control, and

allow multiplexing (detection

of more than one target in a

single reaction). While realBrucellosis is endemic to the

bison and elk populations within

the Greater Yellowstone Area.

Distribution of the northern (green) and central (yellow) bison herds

within Yellowstone National Park. Red indicates seasonal migration

outside of the park boundaries. Green triangles indicate sites where

samples have been taken for real-time PCR and cultivation analyses.
INL BIOLOGICAL SCIENCES

05-GA50193-89

For more information

Continued from front

Deborah Newby, Ph.D.

Lead Researcher

(208) 526-7779

Deborah.Newby@inl.gov

Don Maiers,

Manager

Biological Sciences

(208) 526-6991

Donald.Maiers@inl.gov

time PCR is rapid and sensitive, it may not afford a

suitable platform to perform

strain typing, particularly

within the Brucella, which are

genetically very homogeneous

across species and strains.

Accordingly, scientists are

developing appropriate

methods and instrumentation

to perform rapid, highthroughput microbial forensic

analysis of samples for

identification at the strain or

isolate level. By combining

sets of highly-discriminatory

primers to amplify and label

repetitive or unique sequences

from the target organism’s

DNA, with high-throughput,

high resolution capillary

electrophoresis, a means of

handling large numbers of

samples will be established.

Strain typing of pathogenic

strains by molecular methods

is important to epidemiological and forensic studies.

Methods include pulsed field

gel electrophoresis of large

chromosomal restriction

fragments, insertion element

number and restriction

fragment length polymorphisms (RFLP), rRNA RFLP

patterns (ribotyping),

arbitrary fragment length

polymorphisms (AFLP), and

Field-portable real-time PCR

instrument called the Ruggedized

Advanced Pathogen Identification

Device (RAPID).

B. melitensis AFLP Analysis. Stars indicate positions of unique

fragments with potential for use in high-resolution typing.

analysis of variable-number

tandem repeats (VNTR).

Newby, D.T., T.L. Hadfield,

and F.F. Roberto. 2003 Realtime PCR detection of Brucella

abortus: a comparative study

of SYBR Green I, 5’-exonuclease, and hybridization probe

assays. Appl. Environ.

Microbiol. 69:4753-4759.

INL is a U.S. Department of Energy

national laboratory operated by

Battelle Energy Alliance