RAPID ON-SITE DETECTION OF FOOT AND MOUTH DISEASE VIRUS
Those puzzled and frustrated by the seeming lack of technological breakthroughs in control of foot and mouth and other foreign animal diseases should not be discouraged by recent lacklustre results reported for foot and mouth virus detection by real-time polymerase chain reaction (PCR) on a portable device (Donaldson et al Vet Rec 149 6 Oct 2001). This technology is now so well developed for high-consequence pathogen detection in defence, public health, law enforcement and agriculture in the United States (U.S.) that it is clear that those indifferent results can be explained entirely by the inappropriate choice of reagents and assay conditions. Furthermore, the caveats raised about potential limitations on applicability of the technology have not been found valid in experienced hands.
To understand the current frontiers of high-consequence pathogen detection elsewhere in the world, British government officials, livestock owners, practitioners and other interested parties should know the pedigree of this technology. It was essentially driven by the needs of the U. S. Defense Department, to detect biological threat agents quickly, in the field and with such a high level of confidence that there would be time to take protective measures (Higgins et al., 1999). To solve these problems, the Defense Department initiated revolutionary approaches to pathogen detection. One of these is the portable real-time PCR machine, which is now widely deployed. The term "real-time" refers to the ability to monitor all stages of the reaction and identification as it proceeds, rather than to have to wait until the end for a result. "Portable" means that the device is taken to the site of the problem and operated there by the military - it is not confined to a central laboratory staffed by trained microbiologists.
Recently, as U. S. public health and law enforcement officials have come to realize that these same biological threat agents might be employed against civilian populations, they too have had to confront the problem of detecting high-consequence pathogens, outside the comfort of a reference laboratory and have again adopted portable real-time PCR devices as a way to provide fast, accurate answers wherever the pathogen might be present.
Veterinary medicine is also deeply involved in high-consequence pathogen detection - for control of infectious diseases moving swiftly through international boundaries and perhaps also, deliberately introduced diseases for the purpose of overwhelming conventional defences. Hence, the Agricultural Research Service (ARS) of the U.S. Department of Agriculture (USDA) has begun to develop a uniform system of animal, plant and zoonotic pathogen detection, identification and epidemic response. There are two components:
1. Rapid on-site tests that detect and identify important animal and plant pathogens.
2. An Internet-based information technology programme that incorporates these assays into an integrated management scheme to focus assets for rapid outbreak control in real time.
This system is fully compatible with that used by U. S. military, public health and law enforcement agencies, which obviously maximises its utility. This is not a new test system that is an incremental improvement over the past - it is a fundamentally new way to control highly-infectious disease epidemics.
The rapid detection and identification tests are performed with standardized reagents designed to work on a common device platform for all assays. Several devices are already commercially available and others are close to production. The most popular devices are made by Idaho Technology Inc. (www.idahotech.com) and Cepheid Inc. (www.cepheid.com). These devices both work in the same way. Future models from these and other manufacturers are expected to become smaller, quicker and cheaper to purchase and operate - hand held machines are already available. The devices cost between £15,000 and£30,000.
Real-time PCR assays are performed on a small, portable computer controlled device operating on mains or car battery power. The device is specifically designed to be taken to or near the site of the problem and used there by a person with limited training. An integral global positioning system can identify the exact location of the device. A wireless Internet connection provides world-wide communication, so that distant experts can "look over the shoulder" of the person conducting the assay, to offer advice, expert analysis and validation of assay performance as it proceeds. Instead of taking the sample to the expert, at a distant site for analysis, the sample is analysed on the spot and the data sent electronically to the expert and back to the operator with whatever advice is needed. This immediately saves one or two or more days. If time is gained, multiple alternate courses of action become possible for those charged with taking action to control the disease outbreak. This is the true significance of the technology (Breeze, 2001).
Sample preparation is minimal and can be automated. The process of preparation inactivates infectious agents, including foot and mouth disease virus. All necessary reagents are contained in a single-assay sealed tube in a freeze-dried form stable under a wide range of environmental conditions for over a year. Reagents are produced to ISO 9000 standards and weekly updates of quality control and quality assurance data for each batch are available over the Internet. Sample collection, preparation and the assay itself can be completed in 90 minutes after arriving on site, but positive results can be obtained much earlier.
Real-time PCR assays for foot and mouth disease and classical swine fever virus detection and identification were described and demonstrated by scientists from the USDA at the 105th Annual Meeting of the U.S. Animal Health Association/44th Annual meeting of the American Association of Veterinary Laboratory Diagnosticians held in November 2001 in Hershey, Pennsylvania. Members of the FMD Forum were in the audience and had the opportunity to see the research results and watch assays being conducted.
Briefly, data were presented to show that the real-time PCR assay for foot and mouth disease virus detects all seven FMD virus serotypes and differentiates this virus from other RNA viruses of animals and man and specifically from three viruses that cause almost identical diseases in livestock, namely swine vesicular disease, vesicular exanthema and vesicular stomatitis viruses. The assay detects as few as 10 virus particles, well below the number required to establish an infection and is more sensitive than cell culture. Significantly, this has been found to be a preclinical test: in experimentally infected cattle, sheep and pigs, foot and mouth virus can be detected 24 to 48 hours before the onset of clinical signs of disease. A single assay costs about £5.
The classical swine fever real time PCR assay detects all the strains of this virus and differentiates these from similar viruses, such as border disease and bovine viral diarrhoea viruses. This test is also more sensitive than cell culture and has again been found to be preclinical - detecting infected animals several days before the onset of clinical signs.
Once a positive identification is made on-site, the information technology part of the system allows those responsible to take immediate action in cooperation with other parties who must become involved. Since the device location is known by global positioning, officials can immediately see electronically a map of the area around the infection, predict where infection may have been spread by recent wind, map this spread according to geography and topography, identify quarantine zones, set up control measures (such as road blocks) and identify farms at risk where animals should be tested immediately to detect any infection. The system is designed to coordinate Government officials, academia and private industry, cooperatively, to focus all available resources on immediately stamping out such an introduction through quick, targeted and science-based interventions.
Consequentially, in future, the British people - once aware that such amazing and proven science is available from our friends across the water - will not tolerate the little Englander attitude and medieval approach, adopted for the control of this outbreak
1. Breeze, R.G. Foot and mouth disease preparedness -USA. Promed-mail, 20010520.0981, May 20, 2001.
2. Donaldson, A. L., Hearps, A., and Alexandersen, S. Evaluation of a portable, "real-time" PCR machine for FMD diagnosis, Veterinary Record,149, 430, 2001.
3. Higgins, J.A., Ibrahim, M.S., Knauert, F.K., Ludwig, G.V., Kijek, T.M., Ezzell, J.W., Courtney, B.C., and Henchal, E.A.. Sensitive and rapid identification of biological threat agents. In "Food and Agricultural Security: Guarding against natural threats and terrorist attacks affecting health, national food supplies and agricultural economics". Editors Frazier, T.W. and Richardson, D.C. Annals of the New York Academy of Sciences, 894, 130-148, 1999.