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Email received 15th February 2011 from Roger Breeze re http://fazd.tamu.edu/files/2011/01/AST-Workshop-Report-FAZD-Center-FINAL-1.2011.pdf (For brief extracts, see below Dr Breeze's email.)

Hi Mary:

It is good to see USDA APHIS putting high priority on defining the needs and requirements for Agricultural screening tools, almost 150 years after Abraham Lincoln established the Department of Agriculture in 1862 to protect America against dangerous foreign animal infections like Rinderpest and foot and mouth disease (amongst other purposes).

I have five sons and a daughter so I will add a codicil to my will, tasking a great-great-grandchild to check in on APHIS' progress in 2061. Maybe with a ouija board he or she can update me or chisel the answer on my tombstone.

It probably escapes readers that the real time PCR tests referred to at this meeting were all developed in 2000-2001by USDA ARS at Plum Island (and in Athens, Georgia for AI and NDV) and validated genomically and in experimentally infected animals at that time to a degree never previously achieved for a US veterinary diagnostic.

In 2002 Senator Roberts ensured that USDA received $320 million for emergency responses to the threat of agricultural terrorism in the aftermath of the anthrax mail attacks. "Validating" these PCR tests so they could be used by APHIS to protect the American livestock and poultry industry was said to be one of the top priorities for use of the money by USDA and the new Department of Homeland Security.

APHIS had never previously developed and validated any PCR test for animal or plant diagnostic use in the US, and had never licensed and approved any commercial PCR test for any animal or plant disease in 2001- not even the PCR tests developed and used by APHIS itself for dangerous foreign animal and plant diseases.

As the Head of APHIS Veterinary Services said in 2001 "APHIS does not need to validate and license tests it uses itself, we are grandfathered in." Whatever the excuses in 2001 it is surely pitiful that a decade and tens of millions of dollars later the PCR tests are still not validated and in daily use, even though the OIE Terrestrial Animal Health Manual describes PCR protocols as routine procedures for the rest of the world.

This is particularly serious for Rinderpest, once the world's most dangerous animal disease and now globally eradicated - which means that global vaccine stocks and their production capability no longer exist. Rinderpest is the smallpox of livestock biological weapons and a deliberate release would be truly catastrophic for the world.

Previously in your pages I misquoted Winston Churchill to say that "Never in the field of foreign animal disease diagnostics has so much been spent by so many to do so little for so few." I say it again in a more dangerous world.

The differential test that discriminates animals previously vaccinated against foot and mouth from those that have recovered from infection was developed at Plum Island in 1993 by Juan Lubroth, then an APHIS employee and a PhD student at Yale, and Fred Brown. Juan Lubroth has gone on to a distinguished career and is now Chief Veterinarian for the United Nations Food and Agriculture Organization. Years ago a Swedish company adapted the test into a commercial product that is now widely used around the world. But USDA has still not licensed it for use to diminish economic losses for American agriculture and, from the conference narrative, has apparently forgotten that this critical tool for modern FMD control was developed two decades ago specifically for US emergency use when I was Plum Island Director and with the support of APHIS's Alex Thierman, now Deputy Director of OIE.

In the US there is a national chain of hardware stores - Home Depot - that has almost every tool a builder might want. But when you buy a tool there it is best to know in advance what you intend to do. If you need to plane a plank of wood it is not useful to come home with a nail gun that fastens shingles to a roof. Even though the nail gun is fully validated for its purpose and approved by Underwriters Laboratories for sale in the US.

The Washington conference was fatally flawed because it did not start with a clear statement of how the US intends to respond to accidental or deliberate outbreaks of these dangerous animal and poultry diseases: if you do not know how you will respond, you do not know what tools will be needed.

For example, the pen side dipstick tests for FMD and CSF are terrific in less developed countries of the world with difficult transport conditions and few human or fiscal resources. But they are surely irrelevant to the US, which pioneered on farm portable real time PCR testing for these diseases a decade ago to provide laboratory standard state of the art detection linked to a Command and Control System via the wireless internet.

And finally, it is truly laughable to see validated real time PCR tests for these livestock viral diseases described as "screening tests." Presumably the screening tests are followed by a "definitive" test somewhere else, in this case Plum Island and Ames, Iowa. If the PCR test being used is not definitive, it is time to read the literature or ask an expert to tell you one that is. Amazingly for 2011, the regulatory error persists that viral culture is the "gold standard" for diagnosis. Not true, and not true for at least a decade (and demonstrated in Taiwan in 1999 by a FMD virus that did not grow on bovine cells).

As the conference summary reveals, APHIS lacks definitive modern diagnostic tools that are capable of QUICKLY and accurately identifying dangerous pathogens, mixtures of dangerous pathogens, and new variants of dangerous pathogens. .

Two that come to mind, (that already exist - all APHIS has to do is buy them) are the Tessarae re-sequencing microarray and the IBIS 5000. The Tessarae microarray identifies any of some 200 different viral and bacterial agents (and mixtures of these) in a single 5 hour assay that also provides about 1500 bases of the actual genomic sequence of any pathogen present, including new variants not seen before.

The IBIS 5000 identifies any of hundreds of pathogens, including new strains not seen before, again in a single 5 hour assay. (Both these assays are performed at biological safety level 2 so there is no need for a high containment diagnostic laboratory like Plum Island or Pirbright or for live virus in the test sample.) The first child identified with swine flu in the US in 2010 was fortunate enough to have his respiratory samples tested with an IBIS 5000 at a US Navy Lab in San Diego California: the analysis revealed the presence of an influenza A virus never previously seen that had elements of human, avian and swine influenza viruses. This was before anyone cultured and sequenced the virus.

Unfortunately for U.S. Agriculture, the first California cow with foot and mouth or Rinderpest would not be so lucky.

California has the eighth largest economy in the world (http://en.wikipedia.org/wiki/Economy_of_California) and is the leading U.S. dairy state, employing over 400,000 people and generating over $47 billion/year from this activity alone. Rinderpest or foot and mouth would stop this industry overnight - and successive introductions of more than one viral agent or strain could halt it for years.

It is truly astonishing that in 2011 this industry is not supported by definitive diagnostic capability in California for the world's most dangerous livestock diseases.

What is it going to take to stop procrastinating and wasting time on pen side tests and thermal imaging of hot feet?

If foot and mouth or Rinderpest were to occur in California tomorrow, what could one say to the hundreds of thousands of people whose lives would be devastated? And if we can apologize tomorrow, why can't we act today? Have those responsible no sense of shame?

All the best,

Roger

Extracts from the paper "Protecting Agricultural Infrastructure: Defining the Needs and Requirements for Agricultural Screening Tools " Status, Gaps, and Requirements November 1-2, 2010 | Washington, DC

• Screening Tools That Rely on Agent, Antigen and/or Nucleic Acid Detection:

Real-time RT-PCR (rRT-PCR) assays for FMD, CSF, END and AI have been deployed to the NAHLN laboratories. These assays were validated and intended for use in early detection and response (providing national surge capacity). More specifically, they were validated for defined specimen matrices (rRT-PCR for FMD [epithelium and oral and nasal swabs] and rRT-PCR for CSF [nasal swabs and tonsil scrapings]) and intended to be used as a screening tool within the NAHLN laboratories for identification of suspect cases of FMD, CSF, END and/or AI. Standard operating procedures, operational and response plans and policy for use have been established for each of these assays. The real time PCR assays for rinderpest (RP) and African swine fever (ASF) are currently undergoing the final stages of validation by NVSL, in collaboration with the NAHLN, and expected to be deployed within the next year. Like their FMD, CSF and AI counterparts, the ASF and RP assays’ intended use will be for early detection and response.

• Serological Screening Tools:

Assays for use during recovery (serological assays which can help prove freedom from disease and are vital for re-gaining trade status) are not currently approved for use in the NAHLN laboratories. NVSL is in the final stages of validating a serological assay (Prionics FMD 3-ABC test) for use during the recovery phase of an FMD outbreak.

This assay has herd-level DIVA capabilities. However, it is time consuming (requires overnight incubation), is not adapted to high throughput/robotic platforms, and lacks the required specificity (currently produces an unacceptable number of false positive results). NVSL has also begun pilot testing of a commercially available CSF serological assay (Ceditest ®, ID- Lelystad) in two of the NAHLN laboratories. Serological assays for other high priority FADs have not yet been developed, validated or deployed to the NAHLN. Currently, none of the serology tests produced by foreign manufacturers are approved for importation, sale and distribution by USDA CVB.

Additional screening tools under development, evaluation and/or validation:

  • On-site "Penside" Screening Tools: At present, the US does not have validated pen-side and/or on farm/premise technologies available for screening individual animals, herds and/or animal products for any of the high-priority diseases.

    A commercialized lateral flow antigen detection device is available that is capable of rapid-penside detection of FMD and has sensitivity equivalent to that of the vesicular antigen ELISA.

    Initial studies by USDA APHIS at Plum Island have shown that this device is adequate for use with epithelial samples from experimentally challenged cattle. Initial recommendations for use of this assay include: 1) triage tool for prioritizing samples arriving at the regional or reference laboratory and 2) "ruling-in" (not ruling-out) FMD when clinical signs are present in a herd.

    • Other technologies:

    Infrared thermography (IRT) has been assessed as a means of identification of animals for further testing to confirm FMD infection. Initial work performed at Plum Island using this technology in a highly controlled experimental setting indicated that infrared thermography is a promising screening technology for quickly detecting potentially infected animals prior to onset of clinical lesions for subsequent confirmatory diagnostic testing during FMD outbreaks.

    Further evaluation of this technology is needed to develop a practical prototype for further evaluation under field conditions, and to determine the value and feasibility of IRT in screening for FMDV-infected animals with mild clinical signs or sub- clinical disease.

    3-3. Identification of gaps, future needs and requirements

    Although validation and deployment of several real time RT-PCR assays to the NAHLN greatly boosted the nation’s preparedness status, a significant amount of additional work remains. Specifically, business continuity planning for the different agricultural sectors calls for the capability to move animals and animal products from within disease a buffer zone (BZ) and control area (CA) during a high consequence disease event. The Fast Egg and Secure Milk Supply (draft in progress) plans are two examples of business continuity documents under development by the federal, state and industry stakeholders to help alleviate unnecessary destruction of animals and products during an FAD outbreak response. The overall goals of these plans are to allow safe movement of agricultural products from, into, or within a control zone without endangering flocks and/or herds; support a continuous supply of objectives will require: 1) additional sample/specimen matrix validation and 2) evaluation and validation of pooled samples with the currently deployed rRT-PCR assays.

    Once a high consequence disease has been introduced into the US, focus will quickly be shifted to controlling, eradicating, and subsequently proving freedom from disease in the impacted region and/or entire country. The earlier these actions occur, the lower the biological and economic tolls. As stated previously, if FMD or other high consequence diseases were introduced in the US, there will be severe economic and societal ramifications. The country could lose many of its key trading partners, as shown during the 2009 nH1N1 human influenza virus pandemic. After the initial identification of nH1N1 human influenza cases in people in the US, over 22 countries placed a full or partial ban on US pork imports.

    Some countries, such as China and Russia, did not lift the ban for months after initial human cases were detected.

    With an outbreak of FMD or other FAD, extensive testing would have to be done among susceptible animals to prove freedom of disease and re-establish trade. Requirements for testing during the recovery phase of an outbreak are projected to significantly escalate as regions and individual states work to regain freedom status. During the response and recovery phases, high- throughput molecular and serological-based assays will be critically needed. At present, the US has not deployed within the NAHLN a serological assay for FMD or for other high consequence diseases. Serological tests are currently required for demonstrating freedom of disease under the internationally-recognized OIE standards.

    Serological assays that allow differentiation of vaccinated from infected animals (DIVA) are the optimal choice for use in critical disease eradication programs while recognized in the international community, have not been validated or deployed in the US.

    The availability and performance characteristics of current "penside" or on- site assays and projected future technologies were discussed at length. Currently available platforms (e.g., lateral flow antigen detection devices) at present have limited use for business continuity testing due to sensitivity issues, as well as regulatory and policy concerns. Newer, more sensitive and specific portable technologies for use pen-side and/or within other animal or animal product concentration points are considered critical to supporting business continuity plans for each of the country’s agricultural sectors. Additional research into this area and subsequent development of newer more sensitive technologies is critically needed. These portable screening tools would ideally: 1) be self-contained and technically easy to use, 2) rapid (within 20-30 minutes), and 3) have high detection sensitivity. Future investment in promising technologies such as loop-mediated isothermal amplification (LAMP) and the use of fluorophores was also discussed. 20-23

    While targeting attention on the significant gaps in current status and existing gaps in assay availability, validation, and field-based performance data, the attendees identified several additional areas and topics that are critically needed for efficient screening and reporting of test results. More practical methods for sample collection, preservation, and transport are critically needed. In the event of an outbreak, accredited veterinarians affiliated with large production facilities or private clients will likely be called upon to collect samples for testing, requiring sample collection technologies that allow for enhanced preservation, decreased likelihood of cross-contamination, and ease of transport.

    Workshop attendees suggested that additional research and resources be utilized to enhance this process.

    Information management, including field data collection, sample identification, data transmission, results reporting, and analysis was identified as a serious deficiency in the current system for detecting and responding to animal agriculture threats. The methods currently available for communicating and distributing test results for disease response and regulatory purposes are insufficient for the rapid, secure transmission of large amounts of data as needed during disease outbreak and recovery phases. In order to ensure rapid screening and decision making for business continuity and/or quarantine processes, the nation must have an effective method for rapid collection of data, reporting results, and electronic communication to and between regulatory officials and laboratories in real-time. Additional resources are critically needed to enhance this system for delivery and utilization of screening tools.

    Finally, it was recognized that there are new technologies that could be utilized to enhance the speed, reliability, and cost-effectiveness of disease detection. The workshop attendees stressed the practical need for development of multiplexed assays and approaches that would support dual-purposes (e.g., exotic disease assays embedded into routine diagnostic testing). This type of technology would significantly enhance national preparedness and disease surveillance effectiveness, as deemed appropriate and warranted for early detection purposes.

    3-4. Additional requirements for development, evaluation, validation and deployment of an agricultural screening tool.

    The general consensus of the workshop discussion was that the development and validation of any screening tool for high consequence agricultural pathogens should follow a standardized process with pre- identified performance metrics based upon the intended use of the assay. It was further discussed that the process should equally inform funding proposal and grant review, support initial design and development efforts, validation, and deployment efforts. The NAHLN Methods Technical Working Group (NMTWG), established in 2006, utilizes a standardized approach for the technical review of assays and equipment platforms prior to deployment to the NAHLN. The process includes review of development and validation dossiers. The NMTWG review specifically utilizes NAHLN laboratory experts and focuses on the laboratory technical aspects of the assays and equipment during their review process. The workshop attendees agreed that the standardized NMTWG process could serve as an effective model in developing an integrated and comprehensive process for development, evaluation, validation, and deployment of agricultural screening tools from the point of design through deployment. It was envisioned that the FAZD Center could assist in developing and communicating a documented process that would facilitate involvement of experts representing animal agriculture and allied industries, regulatory officials, technical experts from both commercial and public-service enterprises, communication and information technology experts, and others, as appropriate to each stage of the development and validation design and review process. The immediate need for a published and accessible, standardized process including documented requirements associated with design, validation, and deployment of agricultural screening tools beginning at the time the tool is conceptualized through deployment to the end-user was emphasized by the meeting attendees. The process of defining "fitness for purpose," end-user, operational procedures for utilization of an assay, and documentation of standard was felt to be critical in establishing appropriate funding prioritization and development timelines.

    FINDINGS:

    Findings are presented in order of priority as determined by the group of subject matter experts.

    1 In order to facilitate the goals set forth in sector-specific business continuity plans

    (Fast Egg and Secure Milk Supply plans (in progress)), the rRT-PCR assays currently in use in the NAHLN (FMD and CSF) must be validated for use with additional specimen matrices. Additional sample types should include those that are currently in use or can be collected as part of the animal management routine with minimal disruption to daily business practices. Specifically, the assays should be evaluated for, and where practical validated for use with:
  • Bovine bulk milk tank samples
  • Swine oral fluids
  • Bovine oral fluids (possibly drinking trough)
  • Blood

    As part of normal business operations, the milk hauler routinely collects samples from each bulk tank. These samples are tested for a variety of components, to include antibiotic residues, milk protein, lactose, and total solids. "In a highly contagious FAD outbreak, this same process could be used for disease testing and positive farm identification."

    In commercial swine operations, the sample of choice for routine surveillance is oral fluids. Other sample types collected in various operations include blood, nasal and epithelial swabs. Having the ability to utilize these sample matrices with the deployed rRT-PCR assay will greatly enhance resiliency in the agricultural community.

    2 Evaluate and where possible validate a procedure for pooling of samples with multiple specimen types (matrices).

    Pooling should be evaluated for multiple matrices, where appropriate. Pooling of samples will allow for greater surge testing capability and thus, enhance resiliency and business continuity.

    3 Complete validation and deployment of available serological assays for use in proving freedom from disease.

    a. Prionics FMD-NS test (Ceditest ® 3ABC) ELISA for FMD b. IDEXX, Inc. CSF ELISA Assay c. SVANOVIR® FMDV 3ABC-Ab ELISA Serological assays capable of establishing freedom of disease are available commercially. Advancing the speed at which these assays ar evalidated and subsequently deployed will ensure that the nation is better prepared to recover from a high consequence disease. While it was clearly recognized that improved serological assays are needed (companion DIVA tests to vaccines under development), general agreement among the group was that validation on the currently available assays should be completed as rapidly as possible and hence deployed for use.

    4 Support development of more rapid, accurate FMD DIVA ELISA assays

    (e.g., joint FAZD/USDA/TVMDL –USDA ARS/APHIS Project- "Differentiation of FMDV in infected and vaccinated animals using a competitive ELISA base on an Immunodominant B-cell epitope of the 3B protein," FADDL/DHS Project "Diagnostic Technologies for FMD (3D ELISA)")

    FMD serological assays, currently available for use and undergoing validation, are time consuming (require an overnight incubation), and lack sensitivity. A new more rapid FMD DIVA ELISA assay is required to demonstrate freedom from disease at the level of the individual animal.

    5 On-site (penside) tests

    • Continue to evaluate and if warranted, validate commercialized lateral-flow antigen detection device(s) for FMD (e.g., SVANODIP® FMDV-AG).

    • Invest in more rapid, detection-sensitive technologies for use at penside/premise and/or processing points of animal or product concentration

    Develop policy and concept of operations for on-site testing tools. The general consensus of the group was that penside assays currently available for FMD antigen detection lack sensitivity required to be utilized as a screening tool, but instead were appropriate for use to 1) triage laboratory samples and 2) "rule-in" FMD on a premise with animals demonstrating clinical signs. Additional, more sensitive assays/technologies are needed for pen-side testing. Additional Findings (not prioritized)