The Enferplex Bovine TB Antibody test Validation to Eradication Neil Watt 1 , Amanda O’Brien 2 , Clare Whelan 2 , John Clarke 2 , Alastair Hayton 3 & Gordon Harkiss 1 1 MV Diagnostics Ltd, Roslin Innovation Centre, Edinburgh, U.K., 2 Enfer Scientific Ltd, M7 Business Park, Naas, Ireland, 3 Synergy Farm Health Ltd, Crewkerne, UK.
Immunology of diagnostic testing for Bovine TB Cell Mediated Antibody Immunity Immunity T T IFN g cytokine, IL-21, IL -4 cytokines helper Intracellular pathogens Extracellular pathogens helper cell cell SICCT M. bovis antigen release Enferplex IFN g Bovine TB M. bovis B cell infected M Ø
Enferplex Bovine TB Antibody test Light emission localised to spots HRP Chemiluminescent conjugate substrate Sample antibodies Antigen 1 2 3 1 2 3 spots
Enferplex Bovine TB Antibody test • A positive response to 2 or more antigens is required before the Spot ID RLU sample is regarded as POSITIVE Ag1 60,119 Ag2 15,469 Ag3 52,118 • Increasing the individual antigen Ag4 3,685 positive thresholds makes the test Ag5 29,121 MORE SPECIFIC Ag6 10,098 Ag7 54,434 High Specificity setting Ag8 58,912 Ag9 22,551 • Reducing the individual antigen Ag10 41,573 positive threshold makes the test Ag11 65,232 MORE SENSITIVE Blank 300 High Sensitivity setting
OIE-approved purposes for use of the Enferplex Bovine TB Antibody test Fit for the detection of antibody to Mycobacterium bovis in cattle serum samples, to be used as an ancillary test in conjunction with other methods for serological prevalence surveys, or diagnosis and management of M. bovis infection within herds, for the following purposes: To confirm, but not negate, diagnosis of suspect or clinical cases, including confirmation of positive screening tests in individual animals and in herds with infection prevalence ranging from very low to high, based on detection of antibodies in bovine serum. To detect Mycobacterium bovis infected animals not positive by single intradermal comparative cervical tuberculin (SICCT) or interferon gamma release assay (IFNɣ) tests, based on detection of antibodies in bovine serum. To confirm, but not negate, infection in animals giving inconclusive reactions in the SICCT, based on detection of antibodies in bovine serum. As a screening test, to identify animals most likely to have visible lesions by scoring the number of M. bovis antigens recognised by seropositive animals with bovine tuberculosis.
Diagnostic Specificity of Enferplex Bovine TB Antibody test High Sensitivity High Specificity Country of bTB-free animals Statistical Variable* setting setting N 4258 4258 ALL Countries DSp 98.4% 99.7% UK, IE, USA, CH/LE, NL, NO, ES CI 98.0 – 99.0 99.5 – 99.8 2268 UK N 2268 99.8% DSp 98.1% Herd SICCT negative for 8 years and more than 10 Km from 99.6-100 nearest herd with bTB in previous 12 months, or OTF status CI 97.5-98.6 of region (Scotland). 504 N 504 IE 99.8% DSp 98.2% Long term evidence of no bTB in herd 99.4-100 CI 97.1-99.4 *N = number; DSp – diagnostic specificity; CI – 95% Confidence Interval
‘Boosting’ effect of PPDb on antibody levels Injection of PPDb increases (‘ boosts ’) antibody levels in bTB -infected animals but not in uninfected animals Lightbody et al (1998 – Northern Ireland), Thom et al (2004 - England), Casal et al (2014 – Spain), Waters et al (2015 - USA) This can be used to optimise the sensitivity of serological tests for bTB Studies in 1883 SICCT-positive animals showed the optimum time to take the blood sample is 4-30 days after TT1 Studies over five SIT cycles in bTB-free animals showed no generation of antibodies in response to repeat PPDa/b injections (n = 156-160 animals, Sp 98-100%)
Repeat PPDa/PPDb injection does not induce antibodies over 5 SIT cycles in bTB free animals Cycle 1: 100% specificity at both TT2 and TT20 (161 animals) Cycle 2: 98.1% specificity at both TT2 and TT20 (160 animals) Cycle 3: 99.4% specificity at both TT2 and TT20 (159 animals) Cycle 4: 99.4% specificity at both TT2 and TT20 (156 animals) Cycle 5: 100% specificity at both TT2 and TT20 (158 animals)
Diagnostic sensitivity in bTB infected animals bTB infection status, Samples and Statistical High Sensitivity High Specificity POSITIVE by: sources Variable* setting setting N 208 208 DSn 94.2% 94.2% Boosted M. bovis culture UK, IE CI 91.1-97.4 91.1-97.4 N 1237 1237 RSn 95.8% 94.1% Boosted VL UK, IE CI 94.7-96.9 84.8-88.6 N 2076 2076 RSn 92.6% 89.6% Boosted SICCT UK, IE CI 91.5-93.8 88.2-90.9 N 1220 1220 RSn 86.7% 82.7% Boosted IFNg UK, IE CI 84.8-88.6 80.6-84.8 *N = number; DSp – diagnostic specificity; DSe – diagnostic sensitivity; CI – 95% Confidence Interval
Relative Diagnostic Sensitivity in bTB infected animals missed by SICCT and IFNg tests Enferplex Sensitivity Statistical bTB infection status at high sensitivity Variable* setting N 137 SICCT negative RSn 83.3% VL positive N 57 IFNg negative RSn 82.5% VL positive N 111 RSn 73.9% SICCT 1xIR 12.2% VL /M bovis positive N 187 SICCT 2xIR RSn 87.7% 7.9% VL/M bovis positive *N = number; RSn – relative diagnostic sensitivity ;
Relationship between the number of antigens recognized and VL in SICCT-positive cattle from the UK and IE a Number of Total b Odds Antigens number Percentage Percentage ratio for Recognised (n = 2056 ) with VL of total VL having VL 0 or 1 150 23.3 3.3 - 2 or more 1906 54.4 96.7 3.9 3 or more 1823 55.8 94.9 4.1 4 or more 1759 56.6 92.9 4.3 5 or more 1670 57.2 89.1 4.4 6 or more 1436 59.0 79.1 4.8 7 or more 1071 62.7 62.7 5.6 8 or more 804 65.5 49.2 6.2 9 or more 519 69.5 33.7 7.5 10 or more 364 74.2 25.2 9.4 11 121 78.5 8.9 12.0 a High sensitivity setting b Comparison between 0 or 1 antigens recognized and each other antigen category individually.
No cross-reactivity observed in Enferplex Bovine TB Antibody test in MAP infected and non-infected, bTB free animals High sensitivity setting MAP culture positive: Specificity 98.1% (n = 257 ) MAP ELISA positive: Specificity 98.0% (n = 251) MAP ELISA negative: Specificity 99.5% (n = 1856)
What is the significance of Enferplex positives that are negative in SICCT and/or IFNg? Are they infected with Mycobacterium bovis ? Were they ever positive in SICCT or IFNg? Do they become positive in SICCT or IFNg? How many are there in bTB-infected herds? Do they spread infection, within and/or between herds? Do they cause infection to persist?
Antibody responses in SICCT and IFNg negative ‘in contact’ animals in bTB-infected herds – DEFRA study SE3263 Samples taken from a 2012 DEFRA study SE3263 ‘to determine whether serological tests could replace the IFNg test’ Approx. 30,000 animals from 555 bTB-infected herds in Wales, England (all risk areas) and Scotland tested which yielded 1500 IFNg positives 1300 SICCT negative/IFNg positives and 1300 SICCT negative/IFNg negatives from the same herds sent for serological testing – Enferplex Bovine TB and Idexx M bovis ELISA
Enferplex Bovine TB Antibody test responses in ‘in contact’ animals that were negative by SICCT and IFNg test High sensitivity setting SICCT/IFNg status Statistical All Samples Boosted Non-Boosted variable samples samples SICCT negative 1283 N 294 240 IFNg positive 44.8% % positive 89.8% 40.8% ‘In contact’ with bTB CI 42.1-47.5 86.3-93.3 34.6-47.0 infected animals SICCT negative N 1238 226 226 IFNg negative % positive 21.7% 67.2% 22.3% ‘In contact’ with bTB CI 19.4-24.0 61.1-73.4 15.8-28.8 infected animals
Fate of animals negative by SICCT and IFN g but positive by Enferplex Bovine TB Antibody test over the six months following detection. High Sensitivity setting of Enferplex Enferplex bTB test Reason for Numbers culled Percentage culled status cull SICCT-R 21/248 8.5% POSITIVE 3 x IR 30/248 12.1% TOTAL 51/248* 20.6% SICCT-R 21/806 2.6% NEGATIVE 3 x IR 11/806 1.4% TOTAL 32/806* 4.0% * Odds ratio: 6.26 95% CI: 3.92-10.0
Time under restriction increases with proportion of Enferplex Bovine TB Antibody test positives Time Under Restrictions in Days from Proportion of Enferplex Number of Holdings first SICCT reactor to issue of TB10 positives (%)* (%) (95%CI) 361 0 62 (37%) (222-500) 885 1-25 36 (21%) (533-1237) 842 26-50 28 (17%) (525-1159) 1237 51-75 25 (15%) (682-1793) 1007 76-100 16 (10%) (350-1663) * 2-50 IFN g tested animals sampled for Enferplex, not a statistical sample of each holding
Investigation of SICCT negative, Enferplex positive animals in an ongoing bTB outbreak, Farm A • England Edge area, 500 cow dairy herd, zero grazing til 2016, got bTB summer 2016. Previous bTB in 2013 • 20 SICCT and 80 IFNg reactors, 2 VL, over three SICCT and two IFNg tests • Enferplex after 6 th SIT – 20% seropositive • February 2018, 10 SICCT negative/Enferplex positive animals with highest antibody levels PMd, positive to 8 or more antigens with high antibody load, all NVL • 35% seropositive after 16 th SIT • OTF status regained July 2019
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