Appendix 47. Wilna Vosloo, David Paton, Emiliana Brocchi, Kris de Clercq, Don King, Samia Metwali

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1 Wilna Vosloo, David Paton, Emiliana Brocchi, Kris de Clercq, Don King, Samia Metwali MEASUREMENT 2 Active surveillance for infected animals (including preclinical cases) FMD virus in blood 1 Rapid confirmation of clinical signs Clinical lesions 3 Sero-surveillance for FMDV exposed animals antibody response DAYS Alexandersen et al., 2003 and Unpublished data from IAH, slide by D. King Review of serotype O experimental infections and set of regression models used Latent phase: days Sub-clinical phase: days Incubation: days Infectious period: days Mardones et al, 2010 Factors that impacted on the duration of phases: Species duration of infectiousness is longer in small ruminants than cattle Latent-subclinical and incubation periods longer for small ruminants than cattle Longer incubation times when exposed to infected animals from different species Duration of latency and sub-clinical period longer in pigs exposed to ruminants Shorter time to clinical disease in small ruminants exposed to pigs Specific virus strain Policies needed for notifiable diseases Control over sales and distribution Used by competent persons Fit for purpose Rules on notification for pos and neg results Rules on submission of samples to labs Record keeping (species, age, epidemiological info, etc.) Validation in different countries/regions

2 Detection of FMD antigen One device for 6 serotypes Separate device for SAT-2 Ferris et al, 2009; 2010 Retrieval from LFD for further characterisation Genomic material found to be stable over 1 month Potential for retrieval of larger regions to sequence Real-time RT-PCR (CT value) D 1W 1M LP WS AbB Data provided by D. King and B. Bankowski Detection of FMD antibodies Abs to structural proteins Abs to NSPs Detection of antibodies to FMD NSPs Device made with peptide within 3B-2 Limited validation: Sp 100%, Se 95% Sp comparison to Ceditest and UBI ELISA: 98.6% and 96.6% IgA detection in saliva or nasal swabs? Yang et al., 2010 Comparable to a dry ELISA for ag detection BD Directigen TM Flu A+B Test is performed on membrane Test time ~15 minutes Suitable for clinical samples Negative results to be confirmed Enigma have licence to use TaqMan PCR technology basis for routine molecular assays (FMDV, ASFV, BTV, BVDV, CSFV etc ) : Evaluation of machine s capability at IAH wet assay with MagNA PURE reagents equivalent performance to routine rrt-pcr assays used in the WRL Results in < 60 minutes Future project to develop a drydown consumable for FMDV? (D. King & M. Madi, IAH) Conc and detection on microfluidic chip with integratred nanoporous membrane Method relies on separation of labelled ab from ab/ag complex and detection via laser-induced fluorescence Assay for swine flu where virus is concentrated and separated More sensitive Reduced assay complexity and time Could include secondary abs 6 minute reaction time 50ul sample volume Reichmuth et al, 2008 IRT could assist in selecting infected animals for further testing Decrease # of lab tests Decrease # of animals handled and restrained Images can be sent remotely for decisions Used in combination with POC assays Rainwater-Lovett et al, 2009

3 IRT detected increased foot temps in infected cattle IRT was less successful in detecting vaccinated and infected animals in the pre-clinical phase with lower temp increases Difficulties in determining the baseline that impacts on Se and Sp Limitations Rainwater-Lovett et al, 2009 Measuring the temperatures of normal cattle feet with a temperature recording button and an infra red digital camera Temperature recording button Profile over 24 hr: Snapshot from digital camera: Thermograph C C Foot temperatures are greatly affected by ambient temperature and activity (e.g. lying down on straw) In temperate climates, thermal image derived hoof temperatures can only be used to indicate an inflammatory condition such as FMD by reference to the other feet of an animal and its herd-mates and not on the basis of a simple comparison to a threshold for normality Poster by J. Gloster et al., IAH Recombinant reagents expressed in E. coli (3ABC and rec abs) Alkaline phosphatase (AP) reporter gene genetically fused to the scfv to generate a new FM27-AP construct celisa worked well with limited number of sera, further validation is needed Stable immortalised reagents, safe, cheap, consistent, well characterised, unlimited quantities, eliminate need for animals Rec ab can be mutated (affinity maturation, altered specificity, reporter genes) 2-step reagent Epitope-tag Reporter 1-step reagent Reporter enzymefusion Muller et al., 2010 Provided by H. Heine, AAHL

4 Improved assays to ensure pan serotype PCR picks up all variants Multiplex assay with 6 3D and 2 3C targets (Tam et al., 2009) More sequences needed in databases Serotyping assays still needed Lower sens with assays covering all topotypes Region specific assays most promising Primary cells for virus isolation BTY most sensitive Laborious and expensive Batch variation Cell lines preferable in some labs Usual lines BHK, IBRS2 Fetal goat tongue cell line (ZZ- R 127) LF-BK (bovine kidney) and MVPK (porcine kidney) cell lines showed promising performance when compared to 2 O LK and IBRS-2. Further evaluation needed (PIADC- FADDL) Microarrays represent large quantities of sequence data Large regions of the genome can be sequence characterized using a low density, multiplex array 8 individual samples can be tested on a single microarray slide Doesn t require specific primers for amplification - prior knowledge of sequence unnecessary Brehm et al, 2009 PIADC- FADDL Roger Barrette et al., unpublished Β-galactosidase allosteric biosensor Decreased enzymatic activity when foreign peptides are inserted Reactivated/increased activity in the presence of Mabs or sera from infected pigs Reaction conditions to perform DIVA Advantages of biosensors as diagnostic tools Homogenous nature of assay Short reaction time Potential for automation No need for species specific reagents Potential for POC devices Sanchez-Aparicio et al. 2009

5 Designed a functionalised material using MIP that interacts noncovalently with the analyte - an artificial antibody to the virus that recognises the whole virus particle Used MIP on HRV and found it could distinguish HRV serotypes and FMD Jenik et al, 2009 Artificially expanded genetic information system (AEGIS) bind to each other and not to natural DNA by different hydrogenbond patterns 6 letter PCR Self-avoiding molecular-recognition system (SAMRS) binds to natural DNA, but not to other members of the same SAMRS species (Yang et al, 2010) (Hoshika et al, 2010) Vaccinated and infected and naïve infected cattle Only carriers, whether vaccinated or not, tested positive for IgA Saliva gave more consistent results than probang and nasal fluids IgA in saliva correlated with persistence of virus or viral RNA in OP fluids Parida, IAH, presentation Parida et al, 2006 Responses post vaccination Vaccinated pigs had a low IgA response correlated to ag dose (> dose = > response) Reponses post challenge: IgA response depended on immune status Lasted longer in non-vaccinated and lower ag dose Vaccinated and infected pigs Vaccinated pigs were negative and specific IgA could only be detected PI Pacheco et al, 2010 Eble et al, 2007

6 OIE ad-hoc group on Principles and methods of diagnostic test validation, OIE Paris January 2010 OIE manual now has only one chapter on test validation that includes principles and methods for test validation and 7 annexes as best practice documents Antibody detection tests (Ab) Antigen detection tests (Ag) Nucleic acid detection tests (NAD) Statistical approaches to test validation including modern Non- Gold Standard methods (NGS), such as Bayesian stats Method comparison or equivalence testing Measurements of uncertainty International evaluation of NSP tests (reference panel) USA (APHIS) when using the Prionics ELISA Nonspecific reactivity on sera of small ruminants acutely infected with parapox virus (Orf) Low specificity on U.S. cattle population (94%) Info provided by Axel Colling, AAHL Ready to use ELISA kits for serotype specific ab detection (O, A and Asia-1) Brocchi et al, poster Ag detection kits for O, A, C and Asia-1 Grazioli et al, presentation Tests needed for different PCP stages Free regions - early warning system Stage 1 determine level of virus circulation Serological assays and especially NSP Typing of circulating viruses Stages 2 4: control measures with improvement of labs All the above assays RT-PCR Further characterisation Confirmation of the index case in QA environment POC devices may become more prevalent Labs role to confirm negative/inconclusive results Developing and validating devices and making recommendations Surveillance high throughput (post outbreak and vaccine monitoring) Characterisation of disease agents Responsible for reagent stockpiles Responsible for validation, determining uncertainty of measurement and precision Lab needs to adjust its role POC devises need improvement, further validation and policies on use Ensure fitness for purpose High throughput needed in PCP stages More sensitive assays to detect infection in latent phase