INEEL Scientists Developing Biological Threat Agent Detection Capability
"Yellowstone sends bison to slaughter; more captured." The March 2003 newspaper headline blared the story, but to readers in the states surrounding the first national park, the slaughter of bison was nothing new. In the winter of 1996-1997, over 1,600 bison migrating from Yellowstone National Park into Montana in search of food were killed to prevent the possible spread of brucellosis to cattle ranging near the northern boundaries of the park, thus destroying Montana’s brucellosis-free status.
Brucellosis is an infectious bacterial disease caused by the Brucella species. It is called a zoonosis because the disease can be transmitted from animals to humans, particularly cattle through contact with reproductive tissues or consumption of infected, unpasteurized milk. In animals such as cattle, bison, elk, sheep and goats - and in some rare instances, seals - the disease can cause spontaneous abortion and an inability to conceive in females, and epididymitis in males. In humans, the disease is called by a variety of names such as undulant fever, Malta fever or Bang’s disease. According to the Centers for Disease Control and Prevention, symptoms of brucellosis infection in people include fever, night sweats, undue fatigue, anorexia, headache, and arthralgia or joint pain. It is seldom fatal, but there is no effective human vaccine.
Currently, brucellosis is diagnosed with either a serological test that detects antibodies against Brucella or with cultivation. Current serological tests cannot predict whether an animal is actively infected. Occasionally, seronegative animals have been culture-positive. Blood or tissue cultures - currently the gold standard for diagnosis - come with potential negatives. Since the microorganisms can be spread by aerosol routes, additional biological containment measures must be added to control the risk of infection. And culture testing takes time.
Field Assay System
INEEL molecular biologists Frank Roberto and Deborah Newby are developing a quick, safe, accurate method to detect the brucellosis strain, B. abortus, in the field. Roberto and Newby are designing a DNA-based field assay using a field-portable, real-time polymerase chain reaction (PCR) system.
INEEL molecular biologists Frank Roberto and Deborah Newby
PCR is a technique for copying and amplifying the complementary strands of a target deoxyribonucleic acid molecule. Using a DNA sample and primers (consisting of the four chemical components that make up genetic material), the DNA target can be duplicated millions of times. It is the sequence of the nucleotides that make any species unique, or in humans, every individual unique. The INEEL scientists developed the specific primers that began the copying process for B. abortus.
PCR then detects and quantifies DNA targets by measuring an increased fluorescence in each cycle of DNA duplication. Real-time PCR produces results more rapidly than conventional PCR and has the added advantage of allowing probes to increase specificity. Real-time PCR can also differentiate multiplex reactions and is more accurate since it is based on a dynamic process rather than an endpoint measurement.
Roberto and Newby optimized and evaluated several probes that monitor fluorescence during the assay process to determine the most accurate method. All three, once optimized, detected seven orders of magnitude of genomic B. abortus DNA. But one - the hybridization method - also allowed the specificity required to narrow identification to the B. abortus most accurately. The scientists’ results have been validated through culture testing.
"Domestic bioterrorism is a real threat. We saw that with the anthrax scare," said Newby. "But with the exception of Bacillus anthracis and Yersinia pestis (plague), not much validated work has been done on the detection of other pathogenic threat agents."
A strain of brucella, B. suis, was the first microorganism developed in the former U.S. biological weapons program, primarily because of the ease with which it can be spread through aerosol routes of infection.
Brucella strains remain a concern from the biological warfare and bioterrorism perspective, even though they were recently reclassified as Category B agents. But the assay identification work done by Newby and Roberto may have other far-ranging implications. Emerging genomic information points to a commonality in virulence-related genes among a range of human, animal and plant pathogens, including the deadly plague and brucellosis. In some, such as the plant pathogen A. tumefaciens, a vir locus is responsible for recognizing the host by the bacterium, leading to induction of other genes and ultimately, infection.
Brucella strains share extensive sequence similarities with other proteobacteria and recent work has focused on using these parallels to understand the biology of host-pathogen interactions. This type of research increases our understanding of host range of Brucella and may reveal new strategies for combating disease.
"In our area, brucellosis is primarily an agricultural concern," said Roberto. "The current field methods for detecting the disease in bison, cattle or elk are not very accurate. It’s comparable to saying you have measles because you have the antibodies. You may have had it as a child and are now immune."
Roberto and Newby can prepare the DNA from cattle, bison or elk blood samples in less than an hour and using PCR, can confirm active infection of B. abortus in another 45 minutes. This method, validated through culture testing, also reduces false-negative and false-positive results.
The United States has spent an estimated $3.5 billion to eradicate bovine brucellosis, but vaccinations are not completely effective. Worldwide, it is even more of a problem, infecting cattle, sheep, goats and pigs. Regionally, the impact to wildlife garners the headlines.
Newby’s and Roberto’s research will not only lead to the development of sensitive DNA-based assays supporting national and homeland security missions, it will help address the question of whether brucellosis in wild bison and elk can lead to infection of cattle in the states surrounding Yellowstone, addressing a regional problem for agriculture and wildlife management.
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