Challenges in Bovine TB Diagnostics – Cattle & Camelid blood tests in Great Britain

Shelley Rhodes

TB Research Group/TB Test Consultant

Animal and Plant Health Agency

shelley.rhodes@apha.gsi.gov.uk

APHA Weybridge, Surrey [TB Research]

APHA Starcross, Devon [Camelid Serology]

APHA Sutton Bonington, Nottinghamshire [Cattle Gamma]

Challenges;

Cattle main focus - control & eventual eradication of BTB

Camelids introduction of improved testing for animals that are still

largely unregulated, untested & increasingly known to be a significant [& mobile] reservoir of BTB

Main Challenge – BTB in Cattle

1978 <0.2% herd incidence

1992 – Protection of Badgers Act

1988 – BSE Order – Restrictions

served where BSE suspected

1996 – BSE – Restrictions &

Passports

2001 – FMD – Complete halt

on livestock movement

1975 - Change of Tuberculin for

skin testing

1993 - Change from Annual to 2, 3 or 4-year skin testing (some

areas)

1950’s - Removal of clinical cases

and annual whole herd skin testing

Using the host immune response to diagnose BTB in cattle

Modifed from S. Neill

Test % Sensitivity % Specificity

Tuberculin skin test 81.0 99.98

IFNg blood test 90.0 96.5

Tuberculin (PPD) Skin Test (single intradermal comparative cervical tuberculin test)

Avian PPD

Bovine PPD

Cattle testing for bovine tuberculosis – IFNg as an ancillary test

GB IFNg data: Sp: GB Specificity Trial (n=874); Se: 431 diseased cattle GB skin test data: Sp: Goodchild et al, 2014, in prep.: Se: Karolemeas et al., 2012, PLOSone, 7[8].

IFNg test detects additional infected cattle in BTB breakdown herds that are skin test-negative

In GB since IFNg test rollout (Oct 2006) to Dec 2014; 324,920 skin-test-negative cattle in TB-infected herds tested using IFNg

21,560 IFNg-positive cattle identified These IFNg-positive TB-infected cattle would have been missed without the use of the IFNg test, leading to: - persistent resurgence of TB in the herd - spreading of infection via sales

Strategic use of Skin and Gamma Testing

Low Risk Areas – 4 year skin test, mandatory blood test all new confirmed breakdowns

Edge Area – Annual skin test, mandatory blood test for confirmed TB herds, discretionary for suspected TB herds

Keeping Clean Areas clean - move towards TB-free status in North and East (as Scotland)

Contain infection & push south and west

High Risk Areas – Annual skin test, discretionary use blood test for chronic herds

Wales – Annual skin test, mandatory blood test for new breakdowns (and 2xIRs), discretionary use blood test for chronic herds,

Is there a role for antibody tests?

e.g. detection of later stage, or anergic BTB infection?

Modifed from S. Neill

Maybe – but the data so far suggests that antibody test-positivity actually increases with IFNg-positivity and the presence of VL

Whelan, C et al., 2008. CVI 15(12):1834-38 Multiplex immunoassay for serological

diagnosis of Mycobacterium bovis infection in cattle

Sensitivity 93% Specificity 98%

Whelan, C et al., 2010. CVI 17(5): 813-17 Performance of Enferplex TB assay with cattle in

GB and assessment of suitability as a test to distinguish infected and vaccinated cattle.

Sensitivity 77-86.5% Specificity 100-77.6%

Waters, WR et al., 2011, IAI 18(11): 1882-1888 Development and evaluation of an

enzyme-linked immunosorbent assay for use in the detection of bovine TB in cattle

Sensitivity 63% Specificity 98%

Published evidence for use of serology for BTB testing in cattle:

Limitations of these studies (for GB):

None were aimed at the population that an ancillary test has to target in GB

(infected, skin test-negative)

None compared serology test with current ancillary test (BOVIGAM)

in the target population

Antibody tests for BTB in cattle: comparison with cell-based testing

Lateral Flow [cassettes] – STAT-PAK (Chembio, USA) ELISA - IDEXX (Idexx Laboratories, USA) - Enferplex (Enfer Scientific, Ireland)

IDEXX ELISA Enferplex ELISA STAT-PAK

Animal groups

Sensitivity*

Specificity**

Serology

STAT-PAK

Skin test +ve

41-54 %

93.6 %

Skin test -ve

20 %

IFN-g

Bovigam

(B-A)

Skin test +ve

89.7 %

96.5 %

Skin test -ve

91.4 %

*Culture positive and/or VL, naturally-infected cattle

**TB-free cattle

Antibody tests have a lower sensitivity than IFNg

SE3263: Evaluation of the ENFERplex and IDEXX serological

tests for BTB in cattle

IFNg-positive

(100%)

ENFERplex-positive

n % [95%CI]

IDEXX-positive

n % [95%CI]

VL 54/94 57.5% [47.5-67.4] 38/94 40.4% [30.5-50.4]

NVL 233/774 30.01% [26.8-33.2] 163/774 21.1% [18.2-23.9]

- Antibody-positivity increased with the presence of VL - Antibody tests failed to detect 40-60% of (IFNg+) VL animals - Overall, ~ 80% of antibody-positive animals were NVL

http://www.defra.gov.uk/animal-diseases/a-z/bovine-tb/research/

Outbreak of bovine tuberculosis featuring anergy to the skin test, udder lesions and milkborne disease in young calves

M. G. Houlihan, MVB, MSc, MRCVS1, F. W. Dixon, BVM&S, MRCVS2 and N. A. Page, BVSc, MRCVS2

1 Veterinary Laboratories Agency, Kendal Road, Harlescott, Shrewsbury SY1 4HD 2 DEFRA Animal Health, Worcester Division, Whitehall, Monk Moor Road, Shrewsbury SY2 5AJ

The Veterinary Record, September 20, 2008

Cow negative to SICTT eleven times 6 Calves all SCITT-positive – 1 calf VL Cow = IFNg-positive PM;

Rocio Abad Veterinary Officer

Gloucester

udder mesenteric

retropharyngeal bronchomediastinal

IFNg test has proven track record for detecting skin test-unresponsive BTB-infected cattle

Cell-mediated tests better at identifying

advanced infection. e.g. IL-2

Sensitivity

(n=64 SICCT-/IFNg+)

Specificity

(n=64 TB-free)

VL

(n=37)

NVL

(n=27)

TB-free

14/37 37.8% 5/27 18.5%

0/64 100%

- IL-2-positivity increased with the presence of VL - IL-2 test failed to detect 60% (IFNg+) VL animals - Overall, 74% of IL-2+ animals were VL - IFNg+/IL-2+ animal ~3X more likely to be VL than NVL Rhodes et al., 2014, CVI, 21(1):39

DEFRA Conclusions on Serological Testing in Cattle: (Veterinary & Science Advisory Team, TB Program, DEFRA, Nov. 2012)

- Antibody tests cannot replace the BOVIGAM IFNg test

- More logical to expand IFNg testing to its full potential in areas

where currently not used (Eradication Plan), and

- Tap into the limited BTB antibody testing currently available at

APHA as and when required (lateral flow, Idexx)

Camelids: a growing industry………….

Eddie

Cattle Herd BTB breakdowns 2012 Alpaca Herd Locations (Nov.2012)

Camelids are kept in areas of GB that are Endemic for BTB in Cattle

Map courtesy BAS)

Map: DEFRA Strategy for achieving OBTF Status for England

Pathology in infected camelids can be extensive and severe

Lesions of the tracheal mucosa in an apparently healthy,

skin-test-negative alpaca euthanized as a direct contact

of a herd mate with a cough.

Photograph courtesy James Barnett, APHA.

Skin test-negative alpaca with a cough & continuous hiccups.

No other symptoms. Liquefactive flowing abscess in the

Lung.

Photograph courtesy James Barnett, APHA

www.alpacatb.com for “that cough” video

Camelids are susceptible to BTB

Photograph courtesy Camelid TB Support Group

APHA Camelid BTB tests in culture-confirmed infected herds

2011 2012 2013

New confirmed breakdowns

6 14 9

Number ante mortem tests

960 1261 1275

Total number camelids removed due to BTB

82 592 192

Courtesy Sue Bradford, APHA [no data summary yet for 2014]

BAS Report to BAS Welfare Committee 2014

(deaths due to BTB as % of registered reasons for death)

BTB is a significant cause of death among camelids

Year % BTB deaths

2010 22.6 (63/279)

2011 39.1 (116/297)

2012 62.5 (408/653)

2013 34.6 (177/511)

http://www.alpacatb.com for full table of causes of deaths

No other single identifiable infectious agent caused as many deaths

Next highest cause = old age - responsible for 23.7% deaths in 2013

Evaluation of TB Tests in Alpacas

one-year project

funded by

British Alpaca Society, British Llama Society & British Camelid Ltd

and DEFRA

Validate IFN-g and serological tests for bovine TB in camelids (alpacas)

(serological tests: TB STAT-PAK (Chembio, USA), DPP lateral flow test

(Chembio, USA), IDEXX ELISA test, (IDEXX, USA), ENFERplex ELISA

(Enfer Scientific, Ireland)

Final report submitted to BAS and DEFRA March 2012

Rhodes et al., Clin. Vaccine Immunol. 2012. 19(10: 1677-83

Antibody tests as best way forward for camelids

*Enferplex = 2-spot

Combination of Two Antibody Tests in Parallel provides Higher Sensitivity

(Parallel interpretation; positive to either test = Positive)

VL alpacas

n % Sensitivity [95%CI]

TB-free alpacas n % Specificity [95%CI]

STATPAK/IDEXX

39/48 81.3% [71-91.6] 11/257 95.8% [93.3-98.2]

STAT-PAK/ENFERplex 37/48 77.1% [65.2-89.0] 14/257 94.6% [91.8-97.4]

IDEXX/ENFERplex 37/48 77.1% [65.2-89.0] 12/257 95.3% [92.7-97.9]

No single antibody test alone could achieve this higher sensitivity

*Enferplex = 2-spot

APHA % Sensitivity % Specificity

STAT-PAK/IDEXX 56.5 99.7 Serial Test - animal must be positive to both tests = Positive)

Antibody Test Options for Higher Specificity

(e.g. export, trade, movement etc, back-tracing)

Sure Farm % Sensitivity % Specificity

ENFERplex 55.1 99.8 (4-spot)

or

Results of Camelid Study:

- An improved sensitivity test for BTB breakdown herds - High specificity tests for e.g. surveillance / movement testing

For the first time an owner can request a TB blood test on the suspicion of BTB, rather than waiting for culture-confirmation

BTB Serology testing for Cervids?

DPP VetTB (DPP®VetTB test for Cervids), Chembio, USA (available at APHA, UK, from June 2014)

ETB ELISA for BTB in cervids, in collaboration with Univ. Otago, New Zealand (for more information contact SAC Inverness, UK fiona.bannerman@sac.co.uk).

Enferplex ELISA test, Enfer Scientific (available via Sure Farm Ltd., Devon, UK, www.surefarm.co.uk )

SUMMARY – BTB in Great Britain Cattle – cell-based tests currently offer the best diagnostic for BTB in GB

Camelids – serological tests currently offer best option for BTB diganostics (though no one antibody test can produce Se equivalent to cell-based test)

Cervids - no GB data as yet, but have access to 3 tests, 2 of which have been approved in other countries and for which there is published Se/Sp data

Acknowledgements Martin Vordermeier & the TB Research Group, APHA Services (Laboratory, Veterinary, ITU). Research colleagues – Camelid TB Support & Research Group, GB Camelid owner volunteers, Scottish Agricultural College Commercial colleagues: Konstantin Lyashchenko – Chembio USA; John Lawrence – Idexx Laboratoies USA; John Clarke – Enfer Scientific, Ireland;; Frank Griffin (New Zealand) Financial Support: British Alpaca Society, British Llama Society & British Camelid Ltd; APHA, DEFRA & Waitrose