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Originally Published IVD Technology May 2005

Beyond Clinical Diagnostics

Tailored assays for the detection of foreign disease pathogens in animals

Recent advances in assay development have led to high-quality, rapid tests for agriculture.

Mary T. McBride, Sharon M. Messenger, Thomas R. Slezak, and Paula M. Imbro

Oral swabs from dairy cattle are collected and placed in media for transport to the laboratory for testing. The development of high-throughput multiplexed assays will enable researchers to quickly diagnose both endemic and exotic diseases in animals from a single sample.

Since September 11, 2001, the United States has significantly improved its ability to prevent, detect, respond to, and recover from terrorist attacks. Although gaps remain, investments in bioterrorism preparedness, training, and response have led to the development of critical infrastructure protection against attacks ranging from conventional bombings to chemical, biological, radiological, and nuclear incidents.
However, agroterrorism is one area that needs more attention. Agroterrorism is defined as the use, or threatened use, of biological, chemical, or radiological agents against some component of agriculture to harm the agricultural industry, the economy, or the food-consuming public.¹

Agriculture is a major sector of the U.S. economy, accounting for more than 13% of the gross domestic product and employing more than 15% of the U.S. population.² Cattle and dairy farmers alone earn between $50 billion and $54 billion a year through meat and milk sales, and roughly $50 billion is raised every year through farm-related exports. In 2001, overall livestock sales exceeded $108 billion.³

An agroterrorism attack in the United States could cause devastating economic consequences, not only for the affected agribusinesses but also for allied industries and services, disrupting food supplies, trade, and tourism. Moreover, the structure of American agribusiness (e.g., highly concentrated herds with frequent movement, suboptimal animal tracking systems, minimal farm security/surveillance) leaves the agricultural and food industries particularly vulnerable to such an attack.

The agricultural community currently views a potential outbreak of foot-and-mouth disease (FMD) in the United States as one of its greatest concerns. FMD is a severe, highly communicable viral disease that infects cattle, other ruminants, and swine. FMD is enzootic to many countries in the world, and the virus is easy to obtain. But because FMD does not pose a direct threat to human health, there is no need for elaborate containment procedures or personal protective equipment while handling or preparing the virus. Even so, it can cause significant financial burden. Recent estimates associated with the 2001 FMD outbreak in the United Kingdom place economic losses at greater than $30 billion.⁴

Current Shortcomings

The Animal and Plant Health Inspection Service (APHIS), a branch of the U.S. Department of Agriculture (USDA; Washington, DC), is charged with protecting the nation’s livestock and poultry from the introduction of foreign animal diseases (FADs) and for coordinating the response to an agricultural disease outbreak. The system for detecting a FAD such as FMD typically involves the following procedures: observations by veterinary practitioners and livestock owners, who likely will be the first to suspect and report a FAD case; investigation of suspect cases and submission of samples to USDA/APHIS at the Plum Island Animal Disease Center (PIADC; Suffolk County, NY); and diagnostic workup of tissues at the Plum Island Foreign Animal Diagnostic Disease Laboratory (FADDL). Currently, all testing for FMD is conducted, by law, at FADDL, which averages about 300 investigations per year. During a ma-jor outbreak, demand could rise to 100 investigations per week, far exceeding analysis capacity. Authorities would have to resort to subjective clinical observations to determine whether herds must be destroyed.

A critical problem facing the USDA/APHIS and state agriculture departments in preparing for a potential FMD outbreak is the lack of rapid, validated diagnostic assays for detecting and identifying the disease. At PIADC, FAD detection methods include agar-gel immunodiffusion assays, enzyme-linked immunosorbent assays (ELISA), serum neutralization assays, virus isolation in tissue culture, direct fluorescent antibody tests, electron microscopy, and animal inoculation. These methods are often time consuming and labor intensive. Rapid polymerase chain reaction (PCR) assays have been used on a limited basis, and a few rapid diagnostic tests are currently undergoing validation, but these tests are not yet widely available.

APHIS also lacks rapid, validated diagnostic assays that can differentiate FMD from the many look-alike diseases indigenous to the animal agricultural industries (e.g., bovine viral diarrhea virus, contagious ecthyma, bluetongue). These FADs induce symptoms that are similar to FMD: drooling, blisters, or lameness. In the absence of an FMD outbreak, these look-alike diseases could instill a sense of complacency in practitioners and producers, who may confuse them with common enzootic diseases.

Figure 1. Overview of the tailored assay pipeline process (click to enlarge).

When a practitioner notifies a regulatory agency of a suspicious disease, animal samples are sent to PIADC for FAD diagnosis only, not for diagnosis of indigenous diseases. Consequently, without the ability to offer a timely diagnosis, practitioners and producers might disregard important disease signs or decide not to undertake a FAD investigation of their animals at all.

In an actual FMD outbreak, the tendency is to err on the side of overdiagnosis. During the 2001 outbreak in the United Kingdom, field diagnoses based on clinical observations resulted in a large number of false-positives (estimates range as high as 70–80%) and the unnecessary slaughter of herds (4.3 million animals killed, according to a Royal Society report by the UK Department for Environment, Food, and Rural Affairs), with only 2023 laboratory-confirmed cases of FMD. A readily available rapid test would allow new surveillance strategies and would ensure that decisions carrying such tremendous economic impact are based on objective data.

Reduced sample-processing time would greatly improve the ability to control a FAD outbreak. Currently, it can take many days for a tissue sample to be collected in the field and tested at FADDL. In the meantime, the disease could be spreading rapidly. During the 2001 outbreak, diagnostics delays contributed significantly to the spread and magnitude of the FMD epidemic.⁵ The same delays and their consequences were also demonstrated in a simulation model of an FMD epidemic in California. The model predicted that one to two additional herds could be exposed to FMD for each hour the diagnosis was held up.⁶ Rapid diagnostic tests, conducted by local and state veterinary laboratories, could provide rule-out test results within a few hours.

Developments in Diagnostics

At the Lawrence Livermore National Laboratory (LLNL; Livermore, CA), researchers have constructed a robust technical architecture for the rapid development of highest quality nucleic acid assays, tailored to end-users’ specifications (see Figure 1). The lab’s tailored-assay pipeline has been used to provide high-confidence detection assays for many priority bioterrorism agents.

A single oral swab sample collected from a sow can be screened for multiple pathogens.

The pipeline process begins with an analysis of all available genomic sequence information, which forms the basis for the development of chromosome signatures. Next, using PCR with primer pairs, LLNL’s nucleic acid assays generate the signature fragment or fragments of interest. Once candidate signatures have been identified, they are subjected to computational screening and down-selection. This in silico screening method tests the candidate regions for uniqueness among all sequence data. It also ensures that the signatures are amenable to assay chemistry requirements and provides a rapid, low-cost initial screening of candidate signatures.^7,8 The primers that emerge are then tested against an extensive panel of DNAs and cDNAs. This bench screening ensures that the primers will detect the strain diversity of the pathogen but will not react with the nucleic acids of other organisms. Primer pairs that successfully pass the wet chemistry screening criteria advance to the assay development stage. Assay development includes the optimization of extraction and detection protocols, so that the assays perform consistently to required specifications on the prototype equipment selected.

LLNL has worked closely with the Centers for Disease Control and Prevention (CDC; Atlanta) to codevelop assays that span the range of human biological threats. The tailored-assay pipeline has delivered hundreds of nucleic acid signatures and dozens of validated assays to the CDC. These assays are used internationally and represent the gold standard for molecular detection of select agent pathogens for the public health community. The LLNL assays are also used in the U.S. Department of Homeland Security (DHS) environmental-monitoring operations such as BioWatch, in which the use of federally sanctioned reagents and methods ensures the closest possible coupling of the public health architecture to a bioterror event response. These reagents have delivered exceptional performance for end-users with no false-positives since their deployment.

Figure 2. (A) 100-plex Luminex liquid array generated by intercalating varying ratios of red and infrared dyes into polystyrene latex microspheres. Each optically encoded bead has a unique spectral address. (B) Beads are coated with capture oligos. Capture oligos are assigned probe sequences (internal PCR sequence like a Taqman probe). (C) Standard biotin-labeled PCR products from a multiplex assay are allowed to hybridize to the bead set. The fluorophore, R-phycoerythrin covalently coupled to streptavidin, is then introduced to and binds with the biotin-labeled PCR products. Beads are then individually interrogated in the flow cytometer. A red laser excites the dye molecules inside the beads, and a green laser excites the fluorescent molecules bound to the bead surfaces (click to enlarge).

LLNL is leveraging its pipeline infrastructure and its assay development expertise to construct a suite of tools for the agricultural community. Based upon the priorities identified in partnership with USDA/APHIS, DHS, and the state veterinary diagnostic laboratories (National Veterinary Services Laboratories, National Animal Health Laboratory Network [NAHLN]), the primary focus of this work is to provide field-ready, high-confidence assays for rapid rule-out of FMD. These assays are being developed in both a singleplex Taqman-based format and a multiplexed bead-based format. By multiplexing an FMD assay with endemic diseases that are clinically indistinguishable from FMD, state and local laboratories will have the ability to rapidly rule out FMD and to positively diagnose enzootic vesicular diseases, thus providing the tools to better manage regional diseases.

In addition, LLNL, in partnership with the National Centre for Foreign Animal Diseases (NCFAD) in Canada, is developing multiplexed immunoassays for the detection and differentiation of the seven serotypes and major subtypes of FMD, as well as multiplexed immunoassays that can be used to discriminate animals vaccinated for FMD from nonvaccinated animals.^9,10

The development of multiplexed assays (i.e., assays that can simultaneously test a single sample for multiple analytes) makes use of the Luminex bead-based liquid array technology. The liquid array is composed of polystyrene beads that can be individually tagged with specific antibodies, antigens, oligonucleotides, peptides, or other small molecules, enabling the development of assays for the detection of antigens, antibodies, nucleic acids, and enzymes (Figure 2). The beads are imbedded with precise ratios of red and infrared fluorescent dyes, yielding an array of 100 different beads, each with a unique spectral address. Beads with different tags can be mixed together, and up to 100 different pathogens can be tested accurately and concurrently. LLNL is transforming each Taqman-based assay described above to the multiplexed format. The final assay panel will consist of assays for FMD and 10 look-alike FADs, and will include five built-in assay controls.

Mary T. McBride, PhD, is associate program leader for biological signatures and assays in the Chemical and Biological National Security Program; Sharon M. Messenger, PhD, is a biomedical scientist; Thomas R. Slezak is informatics lead in the Chemical and Biological National Security Program; and Paula M. Imbro is deputy program leader in the Chemical and Biological National Security Program at the Lawrence Livermore National Laboratory (Livermore, CA).

This technology has already been demonstrated for the detection of multiple biological threat (BT) agents from a single sample, using both antibody-based and nucleic-acid-based methods.^11-13 Work to expand the current nucleic acid BT panel from a 12-plex assay to a 30-plex assay (consisting of assays for eight different agents, where each assay is multiloci, and four internal assays controls) is in progress.

Assay development is being conducted by LLNL, in conjunction with the California Animal Health and Food Safety Laboratory, PIADC, and NCFAD. Mature assays will be evaluated by FADDL, DHS, and the NAHLN laboratories. This model will be extended to a full multicenter evaluation exercise in late fiscal 2005, as part of the Agricultural Development and Application Demonstration.

Assay Potential

The recent availability of more than 120 full-genome sequences for FMD also makes it feasible to develop a forensic-level assay that could determine the proximity of any unsequenced outbreak strain to this reference collection. The LLNL bioinformatics team has performed a preliminary analysis of the single nucleotide polymorphisms (SNPs) that provide maximal forensic resolution across the sequenced FMD isolates (this work has not been published). The group’s analysis has generated a set of oligonucleotides that define the presence or absence of each consequential SNP, allowing a bar code to be defined for each sequenced reference isolate. If put onto a microarray or similar high-density chip platform, this forensic assay could both confirm the serotype diagnosis, as discussed above, and indicate whether the current outbreak strain was of a type that could occur naturally in its region. This technique can readily be extended to other pathogens.

References

1. TM Wilson et al., “Agroterrorism, Biological Crimes, and Biological Warfare Targeting Animal Agriculture,” in Emerging Diseases of Animals, ed. C Brown and C Bolin (Washington, DC: ASM Press, 2000), 23–57.

2. FP Horn and RG Breeze, “Agriculture and Food Security,” Annals of the New York Academy of Sciences 894 (1999): 9–17.

3. “Agro-Terrorism Still a Credible Threat,” Wall Street Journal, December 26, 2001.

4. United Kingdom, National Audit Office, The 2001 Outbreak of Foot and Mouth Disease [on-line] June 2002; available from Internet: www.nao.gov.uk/publications/nao_ reports/01-02/0102939.pdf.

5. N Ferguson, C Donnelly, and R Anderson, “The Foot-and-Mouth Epidemic in Great Britain: Pattern of Spread and Impact of Interventions,” Science 292 (2001): 1155.

6. Thomas W Bates, “Evaluation of Strategies to Control Transmission of Foot and Mouth Disease Virus in a High-Density Livestock Region of California by Use of an Epidemic Simulation Model” (PhD dissertation, University of California, Davis, 2002).

7. T Slezak et al., “Comparative Genomics Tools Applied to Bioterrorism Defense,” Briefings in Bioinformatics 4, no. 2 (2003): 133–149.

8. P Chain et al., “An Applications-Focused Review of Comparative Genomics Tools: Capabilities, Limitations, and Future Challenges,” Briefings in Bioinformatics 4, no. 2 (2003): 105–123.

9. A Clavijo et al., “Development and Use of a Biotinylated 3ABC Recombinant Protein in a Solid-Phase Competitive ELISA for the Detection of Antibodies Against Foot-and-Mouth Disease Virus,” Journal of Virological Methods 120, no. 2 (2004): 217–227.

10. A Clavijo, P Wright, and P Kitching, “Developments in Diagnostic Techniques for Differentiating Infection from Vaccination in Foot-and-Mouth Disease,” The Veterinary Journal 167, no. 1 (2004): 9–22.

11. MT McBride et al., “Multiplexed Liquid Arrays for Simultaneous Detection of Simulants of Biological Warfare Agents,” Analytical Chemistry 75, no. 8 (2003): 1924–1930.

12. MT McBride et al., “Autonomous Detection of Aerosolized Bacillus anthracis and Yersinia pestis,” Analytical Chemistry 75, no. 20 (2003): 5293–5299.

13. WJ Wilson et al., “A Multiplexed PCR-Coupled Liquid Bead Array for the Simultaneous Detection of Four Biothreat Agents,” Molecular and Cellular Probes 19, no. 2 (2005): 137–144.

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