Development and Evaluation of IgM ELISA for the detection of FMDV specific IgM antibodies in bovine and ovine sera
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1 Development and Evaluation of IgM ELISA for the detection of FMDV specific IgM antibodies in bovine and ovine sera Usman Waheed 1&2, Debi Gibson 1, David J Paton 1, Qaiser M Khan 2 and Satya Parida 1 * 1-Institute for Animal Health, Pirbright, UK 2-National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan.
2 Clinical Signs
3 Aims Whether IgM test has potential to discriminate between recent and historic infection in field out breaks, particularly in unvaccinated populations of sheep where lesions are very difficult to find out? Whether the IgM test has potential to identify infection in vaccinated populations as a DIVA screening or confirmatory test?
4 Design of experiment Assay development 27 negative serum (cattle+ sheep) samples were analysed Serum samples from two cattle vaccine challenge experiments were evaluated Serum samples from two sheep vaccine challenge experiments were evaluated 87 serum samples were analysed from 21 outbreak
5 Frequency distribution Frequency Distribution 12 1 Frequency <.1.1<.2.2<.3.3<.4.4<.5.5<.6 optical density
6 Results from vaccine challenge expt-i (Parida et al., 26, Vaccine) VI positive-black blocks; IgA positive (a) ; Animal dead ( ); RT-PCR positive- Double lined rectangle; CEDI positive (c)
7 Results from vaccine challenge expt-ii VI positive-black blocks; IgA positive (a) ; Animal dead ( ); RT-PCR positive- Double lined rectangle; CEDI positive (c) (Parida et al., 26, Vaccine)
8 IgM antibody detection in unvaccinated animals in vaccine I & II experiments Experimental Control Animals Unvaccinated Unvaccinated 1 Experim ental Control Anim als.8 UV22 UV23 UV24 UV25 UV UY93 UY94 UY95 UY96 UY97 5 DPV 14DPV 2 dpc 7 dpc 12 dpc 16 dpc 28 dpc 42 dpc 56 dpc 7 dpc 84 dpc 98 dpc 112 dpc 126 dpc 14 dpc 154 dpc 168 dpc ODPV 5DPV 14DPV Days Days DPV OD Value OD Values
9 Detection of IgM antibody in vaccinated carrier animals Carrier Animals Vaccine I Carrier animals vaccine II 1.2 UV2 UV14 UV5 UV1 UV13 UV11 UV19 UV9 UV dpc 12 dpc 16 dpc 28 dpc 42 dpc 56 dpc 7 dpc 84 dpc 98 dpc 112 dpc 126 dpc 14 dpc 154 dpc 168 dpc ODPV 5DPV 14DPV Days Days UY76 UY83 UY9 2 dpc 14DPV 5 DPV DPV OD Values OD Values
10 Detection of IgM in vaccinated and transiently infected cattle No or transient Infection UV6 UV7 UV8 UV15 UV3 UV4 UV18 UV16 UV2 UV21 UV12 OD Values DPV 5 DPV 14DPV 2 dpc 7 dpc 12 dpc 16 dpc 28 dpc 42 dpc 56 dpc 7 dpc 84 dpc 98 dpc 112 dpc 126 dpc 14 dpc 154 dpc 168 dpc Days
11 Clinical and virological results of vaccinated and unvaccinated control sheep following Groups 1 days vaccination 4 days vaccination indirect aerosol challenge Sheep VR2v1 3-7 dpc 8-28 dpc 35 to 39 dpc VR21v1 VR22v1 VR23v1 VR24v1 VR25v1 VR26v1 VR27v1 VR28v1 VR29v1 VR3s1 VR31s1 VR32s1 VR33s1 VR34v4 VR35v4 VR36v4 VR37v4 VR38v4 VR39v4 VR4v4 VR41v4 VR42v4 VR43v4 VR44s4 VR45s4 VR46s4 VR47s4 Controls VR48c VR49c VR5c VR51c VR52c VR53c VR54c VR55c Parida et al., 28, Vaccine Clinical signs Fever >39.5C RNA / Virus NSP Clinical signs Fever >39.5C RNA / Virus NSP Clinical signs Fever >39.5C RNA / Virus NSP
12 Detection of IgM in unvaccinated and clinically infected sheep Control Unvaccinated OD value VR 48 VR49 VR5 VR51 VR52 VR53 VR54 VR55-1 DPI 7 DPI 1 DPI 14 DPI 21 DPI 28 DPI 35 DPI 4 DPI DPI
13 Detection of IgM in 1 days vaccinated and aerosol challenged sheep 1 Days vaccinated VR 2 VR21 OD values VR22 VR23 VR24 VR25 VR26 VR27.2 DPV 9 DPV VR28 VR29 Days
14 Detection of IgM in 4 days vaccinated and aerosol challenged sheep 4 days vaccinated 1.2 OD values VR34 VR35 VR36 VR37 VR38 VR39 VR4 VR41 DPV 3 DPV VR41 VR42 VR43 Days
15 Detection of infection by IgM test in 21 Outbreak serum samples 521 bovine and ovine serum samples originated from in and around the farms where clinical lesions were found-early submission- 16 samples were found positive 147 samples from originated from in and around the farms where clinical lesions were found-late submission- No positive in IgM test Out of 16 serum samples ~2 were found positive in SP tests. They were retested for NSP and IgM antibodies-almost all are positive for NSP, but only 3 are positive for IgM
16 Conclusions After FMD vaccination or transient infection, IgM persists for approximately 2-3 weeks. IgM test has potential to discriminate between recent and historic infection in field out breaks, particularly in unvaccinated populations of sheep where lesions are very difficult to find out Persistence of IgM has been associated with persistence of other pathogens and now the same is shown for FMDV. This requires further study. Preliminary findings indicate that though emergency vaccine induces IgM, the antibody does not persist but reappears if animals become infected. Therefore the IgM test has potential to identify infection in vaccinated populations as a DIVA screening or confirmatory test.
17 Acknowledgements Paul Barnett Sarah Cox Nigel Ferris Funding Defra, UK EU- IMPROCON HEC, Pakistan
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