[PPT] - The Enferplex Bovine TB Antibody test Validation to Eradication Neil PowerPoint Presentation

SLIDE 1

The Enferplex Bovine TB Antibody test Validation to Eradication

Neil Watt1, Amanda O’Brien2, Clare Whelan2, John Clarke2, Alastair Hayton3 & Gordon Harkiss1

1MV Diagnostics Ltd, Roslin Innovation Centre, Edinburgh, U.K., 2Enfer Scientific Ltd, M7 Business Park, Naas, Ireland, 3Synergy Farm Health Ltd, Crewkerne, UK.

SLIDE 2

Cell Mediated Immunity

IFNg cytokine, Intracellular pathogens

SICCT IFNg Antibody Immunity

IL-21, IL -4 cytokines Extracellular pathogens

Enferplex Bovine TB T helper cell T helper cell

Immunology of diagnostic testing for Bovine TB

B cell

M. bovis

infected MØ

M. bovis antigen release

SLIDE 3

Sample antibodies HRP conjugate Antigen 1 2 3 spots 1 2 3 Chemiluminescent substrate

Enferplex Bovine TB Antibody test

Light emission localised to spots

SLIDE 4

Enferplex Bovine TB Antibody test

A positive response to 2 or more

antigens is required before the sample is regarded as POSITIVE

Increasing the individual antigen

positive thresholds makes the test MORE SPECIFIC

High Specificity setting

Reducing the individual antigen

positive threshold makes the test MORE SENSITIVE

High Sensitivity setting

Spot ID RLU Ag1 60,119 Ag2 15,469 Ag3 52,118 Ag4 3,685 Ag5 29,121 Ag6 10,098 Ag7 54,434 Ag8 58,912 Ag9 22,551 Ag10 41,573 Ag11 65,232 Blank 300

SLIDE 5

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.

SLIDE 6

Diagnostic Specificity of Enferplex Bovine TB Antibody test

Country of bTB-free animals Statistical Variable* High Sensitivity setting High Specificity setting ALL Countries UK, IE, USA, CH/LE, NL, NO, ES

N

DSp

CI 4258

98.4%

98.0 – 99.0 4258

99.7%

99.5 – 99.8

UK

Herd SICCT negative for 8 years and more than 10 Km from nearest herd with bTB in previous 12 months, or OTF status

f region (Scotland).

N

DSp

CI 2268

98.1%

97.5-98.6 2268

99.8%

99.6-100

IE

Long term evidence of no bTB in herd

N

DSp

CI 504

98.2%

97.1-99.4 504

99.8%

99.4-100

*N = number; DSp – diagnostic specificity; CI – 95% Confidence Interval

SLIDE 7

‘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%)

SLIDE 8

Repeat PPDa/PPDb injection does not induce antibodies

ver 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)

SLIDE 9

Diagnostic sensitivity in bTB infected animals

bTB infection status, POSITIVE by: Samples and sources Statistical Variable* High Sensitivity setting High Specificity setting

M. bovis culture

SLIDE 10

Relative Diagnostic Sensitivity in bTB infected animals missed by SICCT and IFNg tests

bTB infection status Statistical Variable*

RSn

187

87.7%

7.9% VL/M bovis positive

*N = number; RSn – relative diagnostic sensitivity;

SLIDE 11

Relationship between the number of antigens recognized and VL in SICCT-positive cattle from the UK and IE

aNumber of

Antigens Recognised Total number (n = 2056 ) Percentage with VL Percentage

f total VL

bOdds

ratio for 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.

SLIDE 12

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)

SLIDE 13

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?

SLIDE 14

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

SLIDE 15

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 variable All Samples Boosted samples Non-Boosted samples SICCT negative IFNg positive ‘In contact’ with bTB infected animals

N

% positive

CI 1283

44.8%

42.1-47.5 294

89.8%

86.3-93.3 240

40.8%

34.6-47.0

SICCT negative IFNg negative ‘In contact’ with bTB infected animals

N

% positive

CI 1238

21.7%

19.4-24.0 226

67.2%

61.1-73.4 226

22.3%

15.8-28.8

SLIDE 16

Fate of animals negative by SICCT and IFNg but positive by Enferplex Bovine TB Antibody test over the six months following detection.

High Sensitivity setting of Enferplex

Enferplex bTB test status Reason for cull Numbers culled Percentage culled

POSITIVE SICCT-R 3 x IR TOTAL 21/248 30/248 51/248* 8.5% 12.1% 20.6% NEGATIVE SICCT-R 3 x IR TOTAL 21/806 11/806 32/806* 2.6% 1.4% 4.0% *Odds ratio: 6.26 95% CI: 3.92-10.0

SLIDE 17

Proportion of Enferplex positives (%)* Number of Holdings (%) Time Under Restrictions in Days from first SICCT reactor to issue of TB10 (95%CI)

62 (37%) 361

(222-500)

1-25 36 (21%) 885

(533-1237)

26-50 28 (17%) 842

(525-1159)

51-75 25 (15%) 1237

(682-1793)

76-100 16 (10%) 1007

(350-1663)

Time under restriction increases with proportion of Enferplex Bovine TB Antibody test positives

* 2-50 IFNg tested animals sampled for Enferplex, not a statistical sample of each holding

SLIDE 18

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 6th 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 16th SIT
OTF status regained July 2019

Investigation of SICCT negative, Enferplex positive animals in an

ngoing bTB outbreak, Farm A

SLIDE 19

Investigation of SICCT negative, Enferplex positive animals in ongoing bTB outbreaks Farm History % Enferplex positives

(High sensitivity setting)

Comments

A

500 cows, bTB summer 2016, 20 SICCT, 80 IFNg, 2 VL over three years

20% after 6th SIT 35% after 16th SIT 9/10 NVL M. bovis PCR positive Co-infection with BIV

B

300 cows, bTB summer 2016, 17 SICCT, 85 IFNg, 8 VL over three years

23% after 7th SIT 62 Enferplex positives private IFNg test, 16% positive 4/4 NVL

SLIDE 20

Proposed Scheme to Integrate Enferplex Bovine TB Antibody testing with SICCT and IFNg testing to Optimise Control and Eradication of Bovine TB

Whole Herd SICCT 1 Plus optional IFNg test at TT2 SICCT or IFNg POSITIVE SICCT or IFNg NEGATIVE Enferplex POSITIVE Enferplex NEGATIVE CULL

All positives

r

high risk animals based on the number of antigens recognised

Enferplex test

4 – 30 days post SICCT

Repeat SICCT (plus optional IFNg test at TT2) 60 days after SICCT 1 and then Enferplex test 4 – 30 days later and cull positives until consecutive tests show that OTF status regained Number of test cycles needed will depend on specific herd factors

SLIDE 21

Enferplex Bovine TB Antibody test Summary

OIE approved for control of Bovine TB, see OIE website
Works on serum and milk
Detects bTB-infected animals missed by SICCT and IFNg tests
Detects over 95% of ‘true positive’ animals as defined by SICCT, Visible Lesions or culture of Mycobacterium

bovis

Sensitivity and specificity can be tailored to meet user requirements - specificity can be maximised to 99.8%

in a ‘true negative’ population

Numbers of positive antigens is well correlated with the presence of lesions, culling could be focused on the

animals most likely to be shedding M bovis.

On average the >170 bTB-infected herds we have studied contained ~20% Enferplex positives but this varied

markedly between herds

These were not whole herd tests
Not all samples were ‘Boosted’

SLIDE 22

Acknowledgements

Paul Upton, APHA TB Database Manager – herd and individual bTB test histories APHA and farmers for access to on-going bTB outbreaks Prof Jayne Hope, Roslin Institute – M bovis PCRs Prof Linda Scobie, Glasgow – BIV PCRs

SLIDE 23

SLIDE 24

SLIDE 25

OIE-approved purposes for use of the Enferplex Bovine TB Antibody test

To detect antibodies to Mycobacterium bovis (M. bovis), and other mycobacteria belonging to the Mycobacterium tuberculosis complex, in serum from cattle, to be used as a supplemental test in conjunction with other methods for diagnosing and managing M. bovis infection. In particular, the test can be used for the following specific 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.

 To detect M. bovis infected animals not positive by Single Intradermal Comparative Cervical Tuberculin or Interferon

Gamma tests.



To confirm, but not negate, infection in animals giving inconclusive reactions in the SICCT.

 To identify animals most likely to have visible lesions (VL) by scoring the number of M. bovis antigens recognised by

seropositive Bovine TB-infected animals.

SLIDE 26

Potential uses of the Enferplex Bovine TB antibody test

OIE approved uses

To detect antibodies to Mycobacterium bovis (M. bovis), and other mycobacteria belonging to the Mycobacterium tuberculosis complex, in serum from cattle, to be used as a supplemental test in conjunction with other methods for diagnosing and managing M. bovis infection. In particular, the test can be used for the following specific 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.

 To detect M. bovis infected animals not positive by Single Intradermal Comparative Cervical Tuberculin or Interferon

Gamma tests.



To confirm, but not negate, infection in animals giving inconclusive reactions in the SICCT.

 To identify animals most likely to have visible lesions (VL) by scoring the number of M. bovis antigens recognised by

seropositive Bovine TB-infected animals.

SLIDE 27

Topics

 Rationale for using serology – brief outline of immunology  Enferplex technology – brief outline  OIE validation - extracts of relevant data  Description of some field examples  Proposals for use of Enferplex in control and eradication of bTB

SLIDE 28

Enferplex Bovine TB antibody test Summary

The test works on serum and milk making enhanced control and surveillance options

feasible in both dairy and beef herds

A combined use with a tuberculin test will provide the optimal combination for

removal of confirmed infection within infected herds as it will significantly improve sensitivity of detection over tuberculin tests alone.

Test approved by OIE for the purposes stated, see OIE website

SLIDE 29

Potential uses of the Enferplex Bovine TB antibody test

OIE approval to be sought

 To detect antibody in bulk tank milk

 to screen for infection in dairy herds  to detect residual infection after SICCT/IFNg reactors have been removed

 To detect antibody in slaughterhouse material eg blood/tissue fluid

 to improve sensitivity of SLH detection in surveillance

 To validate Enferplex on plasma samples taken for IFNg testing  To detect infection as a primary herd test as an alternative to SICCT

SLIDE 30

Why are so many SICCT negative but Enferplex positive? Why so many SICCT, IFNg or Enferplex positive but very few VL?

Repeat PPD injection at SITs de-sensitising SICCT?
PPDb dose for SICCT in cow = 100ug (?500x more than human dose)
PPDa contains cross reactive epitopes so effective dose >100ug
Repeat PPDb after SICCT positive may lead to it becoming

SICCT negative ?IL-10

Does injecting repeat doses of PPDb when infected but still

SICCT negative prevent animals from becoming SICCT positive?

‘Stress’ of being a high yielding dairy cow?
Pregnancy, lactation, nutrition, environmental
BIV suppressing DTH and other immune responses which are basis of SICCT and

lesion formation?

Happens in HIV, FIV, MVV
Also in other intercurrent infections….Parasites? ParaTB?.....not BVD

Coad et al (2010) Vet Res 41: 14-26

SLIDE 31

Investigation of SICCT negative, Enferplex positive animals in an

ngoing bTB outbreak, Farm A
Generalised lymphadenopathy
Lymphocytic folliculitis and marked follicular hyperplasia

with large, bizarre-shaped germinal centres

Thin mantle zone
CD3+ cells in clusters and scattered in GC especially at

light-dark zone interface

Bovine Immunodeficiency Virus detected by PCR in animals

with histopathological changes

22/50 seropositive for BIV, gp41 TM1 antigen
??Significance
Lentiviruses depress DTH response so SICCT less sensitive?
??immunodeficiency contributing to mastitis
GB survey 450+ animals in 2000
60% dairy and beef herds seropositive, 1-5% within herd

(Linda Scobie, Glasgow Vet School)

SLIDE 32

England HRA, 300 cow dairy herd, conventional grazing, most recent bTB outbreak started summer 2016
17 SICCT (2016-2019) and 85 IFNg reactors (2018), 8 VL, over three years
At 7th SIT – 3 SICCT reactors, 23% Enferplex seropositive 15 days after that
4 SICCT negative/Enferplex positive animals with highest antibody levels PMd, all NVL, search for M bovis DNA

by PCR to be conducted

No histological evidence of BIV (serology and PCR to be done)
62 Enferplex positives tested privately by IFNg, 10 (16%) positive. These were IFNg negative in 2018, now IFNg

positive in February 2019. 4 results pending due to re-sampling

?residual undetected infection, will have more SICCT reactors at next SIT or later?....or will go OTF?

Investigation of SICCT negative, Enferplex positive animals in an

ngoing bTB outbreak, Farm B

SLIDE 33

Were the Enferplex positive, SICCT and IFNg negative ‘in contact’ animals actually bTB-infected?

 The APHA scientists who planned the study commented ‘Due to the limitation

f this study – namely that antibody-positive/IFNg-negative animals were not

to be slaughtered – we were not able to comment upon the infection status of this group of cattle from breakdown herds, nor therefore address the potential usefulness of additional antibody testing in breakdown situations’

 We looked at data on the fate of those animals with respect to their

subsequent bTB testing in the six months following the Enferplex result.

SLIDE 34

SLIDE 35

Comparison of the Enferplex Bovine TB antibody test with the IDEXX Mycobacterium bovis ELISA test using the same samples

bTB infection status Statistic Enferplex Bovine TB test High sensitivity Idexx M bovis ELISA M bovis culture positive

CI

133

97.0%

94.1-99.9 133

76.7%

69.5-83.9

bTB-free

N

DSp

CI 187

100.0%

100.0-100.0 187

99.5%

98.4-100.0

SLIDE 36

20 40 60 80 20 40 60 80 Ag1 20 40 60 80 20 40 60 80 Ag2 20 40 60 80 20 40 60 80 Ag3 20 40 60 80 20 40 60 80 Ag4

20 40 60 80

20 40 60 80 Ag5

20 40 60 80

20 40 60 80 Ag6

20 40 60 80

20 40 60 80 Ag7

20 40 60 80

20 40 60 80 Ag8

20 40 60 80

20 40 60 80 Ag9

20 40 60 80

20 40 60 80 Ag10

20 40 60 80

20 40 60 80 Ag11

R = 0.921 R = 0.981 R = 0.975 P < 0.0001 P < 0.0001 P < 0.0001 R = 0.974 R = 0.973 R = 0.936 P < 0.0001 P < 0.0001 P < 0.0001 R = 0.853 R = 0.912 R = 0.724 P < 0.0001 P < 0.0001 P < 0.0001 R = 0.865 R = 0.882 P < 0.0001 P < 0.0001

Milk relative light units x 10-3 Serum relative light units x 10-3

Correlation between paired serum and milk in SICCT positive animals

SLIDE 37

Relative sensitivity to SICCT in Inconclusive Reactor animals from UK and IE

Boosted; High sensitivity setting

SICCT 1 x IR: Relative sensitivity 73.9% (VL/M. bovis + 12.2%) (n = 111 ) SICCT 2 x IR: Relative sensitivity 87.7% (VL/M. bovis + 7.9%) (n = 187)

SLIDE 38

Investigation of SICCT negative, Enferplex positive animals in ongoing bTB outbreaks

Farm History % Enferplex positives

(High sensitivity setting)

Comments

A

500 cows, bTB summer 2016, 20 SICCT, 80 IFNg, 2 VL over three years

20% after 6th SIT 35% after 16th SIT 9/10 NVL M. bovis PCR positive Co-infection with BIV

B

300 cows, bTB summer 2016, 17 SICCT, 85 IFNg, 8 VL over three years

23% after 7th SIT 62 Enferplex positives private IFNg test, 16% positive 4/4 NVL

C

720 cows, bTB ??? ???, ??SICCT, ??IFNg, ??VL over ?? years

19% after ??SIT Any PMs?

D

Perridge ?? Cows, ??calves, ??SICCT, ??IFNg, ??VL

50% of 14 SICCT negative calves 6/6 NVL but 5/6 focal lesions in RPLN

SLIDE 39

Enferplex Bovine TB antibody test detects infected animals missed by SICCT of IFNg tests

Boosted; High sensitivity setting

SICCT negative, bTB lesion positive: Relative sensitivity 83.3% (n = 137 ) IFNg negative, bTB lesion positive: Relative sensitivity 82.5% (n = 57) SICCT 1 x IR: Relative sensitivity 73.9% (VL/M. bovis + 12.2%) (n = 111 ) SICCT 2 x IR: Relative sensitivity 87.7% (VL/M. bovis + 7.9%) (n = 187)

SLIDE 40

Summary of bTB-positive field reference serum samples for OIE validation

Origin of samples Number of herds Number of samples (n = 4189) bTB infection status

#TT positive

IFNg positive Lesion positive (VL or histo)

M. bovis

culture positive UK 746 683 1311 445 213 IE 550 1393 1082 928 21 IT

Not Known

15 110 54 88 USA

Not Known

91 34 156 156 Total 1296 2182 2537 1583 478

# SICCT (UK, IE); SCIT (IT); CFT (USA)

SLIDE 41

Waters et al, (2006)

Latex binding assay – ESAT6 Day PI Number positive/total (%)

14

0/20 (0%) 27 16/20 (80%) 78 18/20 (90%) 137 8/20 (40%)

Koo et al(2005)

V1 Enferplex result Experimentally infected animals (n = 5) Weeks PI 2 5 10 17 % positive 20 60 100 100

Whelan et al (2008)

Time frame for antibody detection after infection

SLIDE 42

Relative sensitivity of Enferplex Bovine TB antibody test in milk

High sensitivity setting

SICCT positive: Relative sensitivity 91.6% (n = 107 ) bTB free: Specificity 99.8% (n = 1149)

SLIDE 43

Does Enferplex cross-react with Mycobacterium avium subsp. paratuberculosis?

 Analysed using samples from animals naturally infected with MAP from bTB

free herds in UK, Ireland, Netherlands, Spain and USA.

 Negative control samples (n = 1118) were obtained from bTB free herds in the

UK including Scotland (OTF Status) that were antibody negative by the ID.Vet ID Screen Paratuberculosis Indirect ELISA.

 Negative samples were also obtained from MAP antibody negative animals

(n = 438) from Norway

SLIDE 44

Diagnostic sensitivity in bTB infected animals

bTB infection status Samples and sources Statistical Variable* High Sensitivity setting High Specificity setting

Positive by IFNg Boosted UK, IE

52.1-65.6

SLIDE 45

10 SICCT negative/Enferplex positive animals with highest

antibody levels PMd, all NVL but M bovis DNA detected by PCR in lung lymph nodes in 9/10. Sequence confirmed as M bovis.

Most bTB seropositives in December 2017 are still positive
ver 18 months later and have not been SICCT positive
New seropositives are arising
After 16 SITs, with no SICCT reactors at last two tests, the herd

has regained OTF status…..however, 35% of the herd is still seropositive for bTB

?residual infection, will break down at VE-6M or VE-12M check tests?

Investigation of SICCT negative, antibody positive animals in an

ngoing bTB outbreak, Farm A

SLIDE 46

Origin of samples Number of Samples Number of herds UK LRA, IE 2772 123 CH/LE, NO 992 54 USA, NL, ES 494

Not Known