Development and validation of a 3ABC antibody ELISA in Australia for foot and mouth disease A Colling, a * C Morrissy, a J Barr, a G Meehan, a L Wright, a W Goff, a LJ Gleeson, a B van der Heide, a S Riddell, a M Yu, a D Eagles, a R Lunt, a S Khounsy, b Ngo Than Long, c Pham Phong Vu, c Nguyen Than Phuong, c Nguyen Tung, d W Linchongsubongkoch, e J Hammond, f M Johnson, f WO Johnson, g H Unger, h P Daniels a and JR Crowther h Objective To measure the diagnostic performance of an Australian-developed ELISA for the detection of antibodies against the non-structural proteins (NSP) 3ABC of the foot and mouth disease (FMD) virus. Design Test development and validation study. Methods The diagnostic specificity was determined using 2535 sera from naïve animals and 1112 sera from vaccinated animals. Diagnostic sensitivity was calculated from the data for 995 sera from experimentally and field-infected animals from FMD-endemic countries in South East Asia. A commercial ELISA detecting antibod- ies against FMD virus NSP was used as the reference test to establish relative sensitivity and specificity. Bayesian latent class analysis was performed to corroborate results. The diagnostic window and rate of detection were determined at different times using sera from cattle, sheep and pigs before and after infection, and after vaccina- tion and subsequent infection. Repeatability and reproducibility data were established. Results At 35% test cut-off, the 3ABC ELISA had an overall diag- nostic sensitivity of 91.5% and diagnostic specificity of 96.4%. The diagnostic sensitivity in vaccinated and subsequently infected cattle was 68.4% and diagnostic specificity in vaccinated cattle was 98.0%. Conclusions The 3ABC ELISA identified field and experimentally infected animals, as well as vaccinated and subsequently infected animals. Diagnostic sensitivity and specificity estimates for other FMD NSP tests are comparable with the results obtained in this study. This NSP ELISA was found to be ‘fit for purpose’ as a screening assay at the herd level to detect viral infection and also to substan- tiate absence of infection. Keywords 3ABC ELISA; foot and mouth disease; screening tests Abbreviations ASe, analytical sensitivity; ASp, analytical specific- ity; CI, confidence interval; CV, coefficient of variation; dpi, days post infection; DSe, diagnostic sensitivity; DSp, diagnostic specificity; FMD, foot and mouth disease; NSP, non-structural protein; PI, percentage of inhibition; ROC, receiver-operating characteristic; SD, standard deviation; SPCE, solid-phase competition ELISA Aust Vet J 2014;92:192–199 doi: 10.1111/avj.12190 F oot and mouth disease (FMD) must be differentiated from other vesicular diseases, so rapid laboratory confirmation of a suspected case is imperative. Direct diagnosis is by virus iso- lation or demonstration of antigen or nucleic acid in samples of tissue or fluid. Detection of antibodies to FMD viral non-structural proteins (NSPs) is an indicator of infection, irrespective of vaccination status, 1 and is particularly useful in countries where vaccination is used or in mild cases or where epithelial tissue cannot be collected. Most of the commercial tests for the detection of antibodies to NSPs use recom- binant expressed NSP 3ABC target antigens. 2 Antibodies against NSP indicate past or present infection with any of the seven serotypes of FMD virus, even if animals have been vaccinated. Recent publications indicate that the sensitivity and specificity of such assays for the detection of carriers among vaccinated cattle populations are appro- ximately 90% and 99%, respectively. 1,3-8 The use of NSP-ELISAs to substantiate freedom from FMD infection after emergency vaccina- tion of cattle has become an important option in Europe in recent years. 9 Vaccine purity is an important consideration when using these assays because trace amounts of NSP in formulations may result in false- positive reactions in animals that are repeatedly vaccinated. 1,10 Con- versely, antibodies against NSPs are not detected by some assays 2,11 in some FMD-vaccinated animals that have been subsequently chal- lenged with infectious virus and then confirmed to be persistently infected. Paradoxically, in some vaccinated cattle that later become infected, there is a delayed antibody response against NSP compared with the response in unvaccinated-and-infected cattle. 2,5 Some studies have shown that anti-NSP tests can identify carriers in the absence of any previous clinical signs when virus is not detectable by reverse transcriptase polymerase chain reaction, suggesting that anti-NSP testing, even of individual animals, may detect infections that may be missed by other tests. 2,12 For these reasons, NSP assays are best used at the herd level to detect FMD virus circulation in vaccinated populations. For FMD-free countries such as Australia, it is important to develop and validate test platforms based on a differentiating-infected-from- vaccinated-animals (DIVA) strategy and to be prepared for FMD incursions by having an independent supply of reagents. The aim of this study was to assess an Australian-developed 3ABC ELISA for *Corresponding author. a CSIRO Livestock Industries, Australian Animal Health Laboratory, Geelong, Victoria, Australia; axel.colling@csiro.au b Northern Region Sustainable Livelihoods through Livestock Development Project, Ministry of Agriculture, DLF Regional Office, Luang Prabang, Lao PDR c Regional Animal Health Office No. 6, Ho Chi Minh City (RAHO6-HCMC), Vietnam d National Centre for Disease Control, Hanoi, Vietnam e Foot and Mouth Disease Centre, Department of Livestock Development, Pakchong, Thailand f Institute for Animal Health, Pirbright Laboratory, Pirbright, Woking, Surrey, UK g Department of Statistics, University of California at Irvine, USA h Joint FAO/IAEA Division, Vienna, Austria PRODUCTION ANIMALS PRODUCTION ANIMALS © 2014 Australian Veterinary Association Australian Veterinary Journal Volume 92, No 6, June 2014 192