© 2008 ubio. All rights reserved. 1 FOOT-AND-MOUTH DISEASE IMPLEMENTING EFFECTIVE DISEASE CONTROL PROGRAMS AND ROLE OF NSP ANTIBODY TESTING V I Bishor & Faisal Siyavudeen ubio Biotechnology Systems Pvt. Ltd Biotechnology Incubation center, Kinfra Hitech Park, Kalamassery, Kochi 2010

© 2008 ubio. All rights reserved. 2 Proportions of FMD Virus Serotypes Isolated by the WRLFMD 64% 7% 23% 4%2% O A C SAT1 SAT2 SAT3 ASIA1 (n=311)

© 2008 ubio. All rights reserved. 3 Foot and Mouth Disease Control in Kerala Kerala’s Animal Disease Control Project (ADCP) is lauded as the model for the entire country. The vaccination program of ADCP has led to significant reduction in the occurrence of FMD in the state. Continuous monitoring and re ‑ vaccinations have kept the disease away from the state for years altogether. Recent our breaks : factors – Increased movement of slaughter animals into the state – Introduction of new dairy animals from infected areas into the state, – Lack of continuous disease monitoring among livestock populations in the state that slows down outbreak detection, emergency response and epidemic prevention. The FMD control program has to be refined by incorporating control measures that consider these new factors, so that we sustain and improve the FMD disease status across the state.

© 2008 ubio. All rights reserved. 4 FMD Disease control in Kerala: Proposed measures. 1. Clinical Surveillance 2. Migration Control and Monitoring 3. Control of cross-border livestock trade 4. Early detection measures and epidemic control

© 2008 ubio. All rights reserved. 5 Clinical surveillance 1. Regular random sampling of serum from across the state 2. Testing the sample for the presence of infection 3. Identification of infected carriers or new infections 4. Quarantine and control measures including emergency vaccination

© 2008 ubio. All rights reserved. 6 Migration Control and Monitoring Purchase of new livestock animals under various schemes from other states – While this is a welcome development from a demographical point of view, it has a serious negative impact on disease control programs. – Most areas adjoining our state have higher incidence of FMD with less developed disease control systems, there is a high chance that FMD carriers may be imported through inter-state purchase of new dairy animals. A program where every new dairy animal imported into the state under government schemes has to be tested and certified free of FMD before the purchase is made has to be considered.

© 2008 ubio. All rights reserved. 7 Control of Cross-Border livestock trade. Slaughter animals imported into the state through our border check-posts is huge. spend just a few hours in the state before they are slaughtered: so risk of infections from these animals lesser in a general sense. However, this does not hold for FMD, as the disease is so contagious that even animals that live near the road through which infected animals are transported are at risk of infection. A screening program at border check-posts to screen and exclude infected animals has to be implemented Ideally, every animal should be checked for infection and the physical or financial responsibility to dispose of the infected animal should be fixed on the importer. The tagging system at the check-posts should be extended to retain test data and associate it with individual animals. A good vigilance program that randomly screens animals at slaughter- houses can then ensure accountability and assure efficacy of the screening program by cross-checking results from the check-post.

© 2008 ubio. All rights reserved. 8 Early detection measures and epidemic control The most important step in FMD epidemic control is to quickly establish an infection perimeter. All susceptible animals in infected and adjoining areas and areas with high risk of contagion be screened to identify early infections whenever an epidemic is reported. This data should be used to establish the infection perimeter quickly and accurately. This perimeter should then be used to implement quarantine measures in the infected area, and emergency vaccination should be performed around the perimeter to block spread, as is done today. Once the epidemic is over, all the animals in the area should be screened again for the presence of carriers so that follow- up outbreaks do not occur.

© 2008 ubio. All rights reserved. 9 FMD control measures: Implementation Challenges. A clinical surveillance scheme that aims to detect silent FMD carriers cannot rely on clinical examination, and would require laboratory diagnosis. – Laboratory facility is limited. – Difficulty in sample management: need additional infrastructure and manpower Migration control and monitoring programs that require ‘FMD-free’ certification of dairy animals before purchase would need a testing method, which would allow the veterinary doctor to test the animal at the point of purchase. Similar challenges also exist for check-post screening. – A typical animal carriage vehicle will spend minutes at the check-post for animal tagging and clearance. It is impossible to screen all the animals in the carriage within this time using conventional methods. Serum collection and off-site lab diagnosis is not an option here, as infected animals would have carried the infection into the state by the time the results are available. In the case of disease screening to establish infection perimeters, quick results are very important In all these measures, it is important to make sure that vaccinated, non-infected animals

© 2008 ubio. All rights reserved. 10 Rapid FMD NSP Testing All the FMD control measures outlined above involve fast, reliable, de-centralized screening of the disease. Requirements for the ideal diagnostic technique – Quick results should available in just a few minutes. – Should require no instrumentation, instruments should be portable if present. – Should be easy to use. – Should allow decentralized implementation (should not require serum transport) – Should require no, or low, capital expenditure. – Should be able to execute the test and read result in the field itself. – Should not require refrigerated storage. – Should differentiate between vaccinated and infected animals. The only diagnostic technique available today that satisfies these requirements for FMD diagnosis is the FMD Non-structural-protein (NSP) rapid lateral flow assay.

© 2008 ubio. All rights reserved. 11 Practical issues of NSP testing The 3ABC polyprotein is concluded to be the single most reliable indicator of infection (Concerted Action CT , 1998). The 3ABC protein is not serotype specific. A positive result of NSP test indicates an infection with the FMD virus, irrespective of the serotype. This not contain any infectious material and therefore does not, contrary to a VN test, have to be performed in high containment. In case of an outbreak large numbers of samples can be processed in the field. Waiting time for the test is minimal, since the test provides results within minutes. the test is tested and validated in local cattle. The NSP marker-test is recommended for herd diagnosis rather than for individual diagnosis.

© 2008 ubio. All rights reserved. 12 EU FMD Committee Recommendation Using conventional diagnostic techniques – Not possible to distinguish FMD infected animals from purely vaccinated animals – In vaccinated areas, have limited possibilities to monitor virus presence or circulation NSP-free or NSP-reduced vaccines in combination with a NSP-test lead to a so- called marker-system – Polyprotein Nonstructural Protein(NSP) 3ABC is the single most reliable indicator of infection – Modern, state of the art-vaccines are based upon highly purified antigens, which are free from NSP of the FMD virus. – Animals, vaccinated with highly purified( NSP-free vaccines), produce antibodies against the Structural Proteins (SP) but not against NSP

© 2008 ubio. All rights reserved. 13 OIE Recommendation (2006) Schematic representation of laboratory tests for determining evidence of FMDV infection through or following serological surveys

© 2008 ubio. All rights reserved. 14 Vaccine Quality control Ideal FMD vaccine…. Safe and efficient (FMD control) Fast & long lasting clinical and virological protection High potency (minimum number of dose to get protection) DIVA test (distinction between vaccinated and infected animals): means vaccine should be free from NSP Storage (room temperature) Economical issue (cheap) OIE and EU recommends the implementation of additional purification steps in the manufacturing process to remove NSP. EU allows only NSP free vaccines for effective screening and surveillance programs.(EMEA/CVMP/775/02-FINAL) This week (5/3/10) IVRI has decided to modify their FMD vaccine as per this OIE standard with no/reduced NSP antigens. ( http :// ) http ://

© 2008 ubio. All rights reserved. 15 Differentiation Infected and Vaccinated Animal Results serology Conventional test(LPB,VN) FMDV NSP test VaccinatedInfected++ VaccinatedNot infected+- Not vaccinatedInfected++ Not vaccinatedNot infected--

© 2008 ubio. All rights reserved. 16 Immune response of infected and vaccinated animals (cattle) +ve -ve Result Days after vaccination or infection SP antibodies ( Infected) SP antibodies (Vaccinated) NSP antibodies ( Infected ) NSP antibodies (Vaccinated)

© 2008 ubio. All rights reserved. 17 Assay Overview and Usage Rapid FMD NSP test (available as ‘ubio quickVET FMD NSP’) uses recombinant FMD 3ABC antigen and to detect the disease. FMD 3ABC antigen on test line Anti-NSP antibody in serum react with colloidal gold conjugated detection antibody on conjugate pad This labeled antigen-antibody complex is captured by the test area forming a red line. Test is suitable for Bovine, Ovine, Caprine and swine.

© 2008 ubio. All rights reserved. 18 Test procedure NEGATIVE Only control band visible POSITIVE Control and test bands visible INVALID No control line, test is invalid Collect whole blood or Serum Separate serum if testing in a lab Use whole blood if testing in Field Take out the test card from the aluminium foil pouch and place it on a horizontal surface. Add 2µl serum or 5 µl Whole Blood into sample hole ‘S’ Take 2µl serum or 5 µl Whole Blood Add 2 drops of buffer provided with the kit Read the results in minutes DO NOT READ RESULTS AFTER 20 MINUTES

© 2008 ubio. All rights reserved. 19 ubio QUICKVET FMD NSP test Rapid, Immuno-chromatography based. One step 10 minutes test. Can be done directly on serum. Easy to do in the field. Products – FMD-NSP for Bovines – FMD-NSP for Porcines,

© 2008 ubio. All rights reserved. 20 Recommendation of Kerala Post vaccination sero-surveillance for NSP antibodies. Border control of slaughter animals. Screening of animals brought for dairy purpose.

© 2008 ubio. All rights reserved. 21 Program flow chart for Screening Dairy Animals

© 2008 ubio. All rights reserved. 22 Program Flow Chart for Screening Slaughter Animals The practical screening scenario 40 animals/Truck With 3 staff (veterinary doctor and/or livestock inspectors) the total time for one vehicle: 57 minutes. In addition to inspection, the check-post authority will also note animal identification numbers on the issued certificate and on individual animals. ubio can provide custom kits with paired random number identification stickers for this purpose

© 2008 ubio. All rights reserved. 23 Vigilance program

© 2008 ubio. All rights reserved. 24 Thank you ubiquitous. biology