diagnostic tools to detect pathogens causing tuberculosis in cattle and prevent their transmission...
TRANSCRIPT
Diagnostic tools to detect pathogens causing tuberculosis in cattle and
prevent their transmission through dairy products to humans
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
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
Limitations of the intradermal tuberculin test
• 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).
Limitations of the intradermal tuberculin test
• 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
Limitations of the intradermal tuberculin test
• 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 • 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 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)
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. 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
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)