Diagnostic tools to detect pathogens causing tuberculosis in cattle and prevent their transmission through dairy products to humans

Diagnostic tools to detect pathogens causing tuberculosis in cattle and

prevent their transmission through dairy products to humans

The Role of Agricultural Biotechnologies in Sustainable Food Systems and Nutrition

Etiology • Mycobacterium tuberculosis Complex (MTC)

• Mycobacterium tuberculosis• Mycobacterium africanum• Mycobacterium bovis • Mycobacterium microti• Mycobacterium caprae• Mycobacterium pinnipedii• Mycobacterium mungi

Importance of bTB• Mycobacterium bovis is a zoonotic organism • Should be treated as a risk/hazard group III organism • Affects cattle, other domesticated animals and certain free

or captive wildlife species• Costs associated with bTB testing; condemnation of

carcasses at slaughter; decrease in milk production; movement restrictions; compensation and insurance payments, etc

Foodborne illnesses associated with M. bovis

• Prior to milk pasteurization in many countries, M. bovis accounted for ˜ 25% of tuberculosis cases in children

• Great Britain, human consumption of infected cows’ milk led to an estimate of 2,500 deaths and more than 50,000 new cases of TB per year in the early 1900s

• In Latin America, M. bovis is involved in 2% pulmonary and 8% extrapulmonary human tuberculosis cases

Foodborne illnesses associated with M. bovis

• Infection associated with consumption of infected raw milk and soft fresh cheeses

• Artisanal cheeses made with raw milk, following by a ripening: risk?

• In some parts of Brazil, consumption of fat from raw milk

Intradermal tuberculin test

• Measures dermal swelling due to a cell-mediated immune response (CMI) 72 hs after intradermal injection of purified protein derivative (PPD)

• PPD tuberculin is prepared after a heat-treatment and lysis of M. bovis AN5 (bovine PPD) and M. avium D4ER or TB56 (avian PPD)

• Consists of a mixture of small water-soluble proteins

Caudal fold test

Comparative Intradermal Tuberculin Test (CITT)

Interpretation of CITT

Advantages of the intradermal tuberculin test

• Low cost• Low logistical demands• Well-documented use• Many countries have used this test in eradication programs• Recognised by the World Organisation of Animal Health

(OIE) and the European Commission as the primary screening test for detection of tuberculosis in cattle

Limitations of the intradermal tuberculin test

• Difficulties in administration and interpretation of results


• Need for a second-step visit• Low degree of standardisation and imperfect test

accuracy PPD UK

67PPD BR

1237

Proteins present in the PPDs from Brazil and UK(Borsuk et al. 2009).


• False-negative reactions are also a concern, since infected cattle may remain on herds


Limitations of the intradermal tuberculin test • In Brazil, evidence of resurgence of bTB in accredited-free

herds due to infected cattle not responsive on CITT


• When results of intradermal tests are inconclusive, it is necessary to wait at least 60 days before confirmatory tests

• This mandatory interval requires cattle to be kept in quarantine

• Increased risk of spreading the disease to herd and potentially to humans

Limitations of the intradermal tuberculin test • Kept in quarantine?

Limitations of the intradermal tuberculin test • Interference of NTM reactions on CITT

ID SITT PPD-B PPD-A CITT CULTURE

352889 3,7 0,5 INC POS

353601 5,2 3,2 INC POS7624 4,5 2,5 INC POS

346680 19 16,4 INC POS325002 POS 10,5 10 NEG POS325005 10,8 10,2 NEG POS

325006 9,8 9,8 NEG POS

351263 INC 14 14 NEG POS351273 POS 3 2,6 NEG POS353584 POS 14,6 15,2 NEG POS353600 INC 13 14 NEG POS353603 12,5 15 NEG POS

384162 17,5 16,4 NEG POS

Interferon-gamma tests• Lymphocytes from M. bovis infected cattle produce IFN-

gamma in vitro in response to PPD

• IFN-gamma detected by antigen capture ELISA

Interferon-gamma testsAdvantages of IFN-gamma tests

• Objective and reproducible results

• One visit

• No interference on the immune system of the animal

• Re-testing of suspected unspecific reactors to the skin test

Interferon-gamma tests• BOVIGAM as a confirmatory test

Interferon-gamma testsLimitations of IFN-gamma tests

• High costs in some countries

• Short time between collection of blood and testing

• Need to incubate blood + PPD in culture plates (Bovigam)

Interferon-gamma tests

Gamma-tubes Ellie lab

Performances of CMI tests

Test SE SP

SITT 80.2% to 100% 55.1% to 99%

CITT 52% to 100% 99.5%

CF 80.4% to 93% 89.2% to 95.2%

IFN-g 73% to 100% 85% to 99.6%

Reviewed by Bezos et al. (2014)

Detection of antibodies to M. bovis• ELISA com chimera MPB70/MPB83/ESAT-6

Souza et al. (2012)

Detection of antibodies to M. bovisID SITT PPD-B PPD-A CITT ELISA * CULTURE

353603 12.5 15 NEG 273 POS353601 5.2 3.2 INC 195 POS352889 3.7 0.5 INC 172 POS325002 POS 10.5 10 NEG 131 POS353600 INC 13 14 NEG 128 POS353584 POS 14.6 15.2 NEG 103 POS351263 INC 14 14 NEG 77 POS351273 POS 3 2.6 NEG 77 POS7624 4.5 2.5 INC 64 POS

346680 19 16.4 INC 53 POS384162 17.5 16.4 NEG 33 POS 325005 10.8 10.2 NEG 23 POS325006 9.8 9.8 NEG 15 POS

*Normalized OD

Post-mortem diagnosis

• Inspection of carcasses for bTB gross lesions in

abattoirs is an important tool for eradication

programs

Post-mortem diagnosis

• The isolation of mycobacteria on selective culture media and their subsequent identification by cultural and biochemical tests or DNA techniques, such as PCR, confirms infection (OIE)

Post-mortem diagnosis• Microbiological diagnosis of M. bovis is an

extremely slow procedure

• Takes as long as 2 to 3 months

• Additional 2 to 3 weeks are required for the biochemical identification of isolates

Post-mortem diagnosis• Increasing pressure from beef markets for a definitive

diagnosis of bTB in lesions compatible with tuberculosis (LCT)

• In Brazil, farms with cases of bovine/bubaline tuberculosis cannot export beef to the Customs Union of Belarus, Kazakhstan, and Russia

• All lots of animals from a farm with suspicious animals are sequestered and the LCT are submitted to an official laboratory for etiological diagnosis

Post-mortem diagnosis• Nested-PCR TbD1

Nested-PCR rv2807

Carvalho et al. 2015

Detection of M. bovis on milk

• Excretion of mycobacteria in milk is intermittent

• Up to 30% of infected cows eliminate by milk

• PCR in bulk tank - INTA, Argentina

Detection of M. bovis on milk

• This method has been incorporated since 2012 in the Plan of Control and Eradication of Tuberculosis of Santa Fe Province, Argentina, which produces 41% of the total milk production of Argentina

96C 3min96C 1min 65C 1min 30 cycles72C 2min 72C 8min

96C 3min96C 1min 72C 1min -1 C/cycles 8 cycles72C 1min 96C 1min 65C 1min 30 cycles72C 2min 72C 8min

Detection of Mycobacterium bovis in milk

Amplification of IS6110

C+ 1 2 3 4 5 C- C+ 1 2 3 4 5 C-

245bp

Touch-DownConvencional

PCR in milk of individual cows

PCR and sanitary condition of the herd

PPD+ PPD- Total

PCR+ 58 (54,7%) 6 (4,6%) 64

PCR- 48 (45,3%) 124 (95,4%) 172

Total 106 130 236

Cultures were negative

PCR in milk tankPCR and sanitary condition of the herd

Cultures were negative

PPD+ PPD- Total

PCR+ 24 (39.4%) 29 (19.7%) 53

PCR- 37 (60.6%) 118 (80.3%) 155

Total 61 147 208

(Zumárraga et al., 2012)

Near future

• Eradication programs

• Prevalences dropping

• Need to trace back new cases

Genomic analysis Brazil x Argentina x USA

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Education

Diagnostic tools to detect pathogens causing tuberculosis in cattle and prevent their transmission through dairy products to humans