Molecular Diagnosis of Bovine Tuberculosis and Genotypes of Mycobacterium bovis in Chile

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Molecular Diagnosis of Bovine

Tuberculosis and Genotypes of

Mycobacterium bovis in Chile

Dra. Ana María Zárraga

Instituto de Bioquímica, Facultad de Ciencias,

Universidad Austral de Chile

International Seminar on Bovine Tuberculosis

IICA_ SAG

Santiago, enero 2010

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1. BTB remains as a significant disease of cattle and

other species.

2. BTB is a zoonotic threat in developing countries due to

consumption of unpasteurized milk and dairy products.

3. 5-10 % of global human tuberculosis is due to M.

bovis.

4. Most countries do not distinguish cases caused by

non-human mycobacteria .

5. Non- tuberculous mycobacteria has been isolated from

cattle and wildlife animals.

2 Background

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-

Essential to the eradication of bovine tuberculosis is the

epidemiologic surveillance of animals during routine sanitary

inspection.

-

Fast, sensitive and reliable diagnostic tools are critical to

confirm the existence of the disease in lesions taken in

abattoir, during regular sanitary inspection.

-

Molecular typing for pathogenic mycobacteria is also

needed because it defines prevalent strains, the origin of

infection, outbreak tracing, and relates genotypes with

virulence.

3 Molecular Diagnosis and genotypíng

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Our Research Aims

Development of a National

Geographic map of Mycobacterium

bovis isolates

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PARTICIPANTS

5 Laboratorios Servicio Agrícola y Ganadero

Plantas Faenadoras

Corporación de la Carne

Asociación Gremial de Productores de Leche

Instituto de Patología Animal, Facultad de

Medicina Veterinaria, UACH.

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1.Design a strategy of isolation of strains with recorded traceability

to ensure adequate representation of strains by region.

2. Identify areas of high and low prevalence of bovine tuberculosis,

persistence and outbreaks of disease.

3. Confirm the identity of the strains collected by real-time PCR

4. Genotype M. bovis isolates using spoligotyping and VNTRs

3. Generate a genetic-geographic map of M. bovis prevalent in the

country, linked to the epidemiological data of each strain.

SPECÍFIC OBJETIVES

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STRATEGY

Selection of infected tissues by real time PCR to M. bovis

Registration of the geographical region of animal origin

and epidemiological data.

Culture of tissues carrying compatible BTB lesions.

Genotyping: Spoligotypes, VNTR

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Real Time PCR for M bovis Identification

Confirmatory

Procedure

Postmortem diagnosis:

Laboratory

DNA extraction

8 Gold Standard:

2 months

3 hours

•nodules afected

•ready for diagnosis

BoviMan:

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negativas

postivias

Validation Real Time PCR for M- bovis

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BoviMan

Cultivo

Positivo

266

38

304 Negativo

23

881

904 Total

289

919 1208

Sensibilidad: 266/304 = 87,5%

Especificidad: 881/904 = 97.5%

Positivo

Negativo

Total

Validation of real time PCR for M bovis

directly in fresh tissue lesions

Note: Half of thelesion for culture, other half for qPCR

Sensitivity increased to 95% using whole lesion. Reference SAG

laboratory.

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Genotyping Methodology

RFLP-IS6110

Spoligotyping

MIRU-VNTR

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RFLP-IS

6110

RFLP

-

IS

6110

₆₁₁₀

fingerprint 181+fingerprint DR compar 10 0 80 60 40 20 fingerprint 181 compar fi n g e rpr in t 18 1: 7. 71 E 3 pb fi n g e rpr in t 18 1: 6. 92 E 3 pb fi n g e rpr in t 18 1: 5. 92 E 3 pb fi n g e rpr in t 18 1: 4. 49 E 3 pb fi n g e rpr in t 18 1: 3 849 pb fi n g e rpr in t 18 1: 3 536 pb fi n g e rpr in t 18 1: 3 222 pb fi n g e rpr in t 18 1: 2 934 pb fi n g e rpr in t 18 1: 2 504 pb fi n g e rpr in t 18 1: 2 055 pb fi n g e rpr in t 18 1: 1 891 pb fi n g e rpr in t 18 1: 1 729 pb fi n g e rpr in t 18 1: 1 630 pb fi n g e rpr in t 18 1: 1 387 pb fi n g e rpr in t 18 1: 1 232 pb 010 071 096 099 100 101 102 103 104 095 098 012 013 014 015 051 023 026 003 005 024 040 070 077 017 065 034 039 004 009 016 018 029 036 045 068 074 076 105 011 007 008 019 Osorno UN1610 Rio Bueno Rio Bueno Temuco Osorno Osorno Rio Bueno Temuco Rio Negro Osorno Los Abedul. Castillo Sur Las Vegas Las Vegas Buenos Aires Las Vegas Las Vegas Los Abedul. Las Vegas Las Vegas Las Vegas Copiapo Coquimbo Castillo Sur Sta Isabel Osorno Buenos Aires UN 1134 Osorno Las Vegas Osorno Los Maitenes San Juan Las Vegas Sta Isabel UN1020 Temuco Pto Montt UN1098 UN65 UN645 Osorno X X X X IX X X X IX X X VIII VIII VIII VIII VIII VIII VIII VIII VIII VIII VIII IV IV VIII VIII X VIII X X VIII X VIII VIII VIII VIII X IX X X X X X 008 019 022 030 031 032 033 041 043 044 048 052 056 058 063 066 067 082 001 027 035 047 050 060 064 069 073 081 083 084 085 086 087 088 089 090 091 092 093 094 097 UN645 Osorno UN1098 Osorno Osorno Osorno Osorno Las Vegas Las Vegas Las Vegas Las Vegas UN645 Las Vegas Las Vegas Coquimbo Los Maitenes Sta Isabel Temuco Tucuman UN 645 Osorno Las Vegas Los Maitenes Osorno Copiapo Sta Isabel Osorno Castro Ovalle Osorno Pto Varas Futrono Osorno Osorno Rio Bueno Los Alamos Osorno Los Alamos La Capilla La Capilla Valdivia X X X X X X X VIII VIII VIII VIII X VIII VIII IV VIII VIII IX VIII X X VIII VIII X IV VIII X X IV X X X X X X X X X VIII VIII X

12

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“

Clusters

”

of

IS6110 fingerprints

*

13 IS1, IS2, IS3=

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Geographic distribution of IS6110 genotypes

VIII

N= 5 caprine strains

N = 33

strains

X

IX

n= 4 strains Temuco

n= 40 strains, 10 locations

IV

Coquimbo

IS2D

IS3A

Copiapó

IS2D

IS2A

Ovalle

IS2A

IS2A,

IS3B, IS2C, IS2C, IS1B. IS1A

IS1A, IS2B,

IS3A

IS1A,

IS2A,

IS2A

,

IS3A, IS2B, IS6A

IS3A

, IS2B, IS6A

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43 SPOLIGOTYPES OF M bovis

15

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SPOLIGOTYPES

13 clusters

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18 Distribution of the ETRs, MIRUs and QUB11b

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VNTR

´

s of

M. bovis

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ZOONOSIS

23 SB0920

SB0920

SB0269

SB1072

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Tabla 2. Prevalencia de TBB en rebaños lecheros y animales, por Región.

Región Número

Rebaños

Prevalencia

%

Número

Rebaño +

Número

Vacas

Prevalencia

%

Número

Vacas +

V 1.777 63.0 1.120

12.222 30.0 3.667

VI 2.568 48.0 1.233

14.979 17.3 2591

VII 4.671 40.3 1.882

22.480 15.5 3.484

VIII 8.476 11.7 992 73.112 4.4

3.217 IX 9.066 3.2 293

71.844 1.2

1

862 X 17.598 2.8 484

378.853 1.1

1

4.167 RM 720 71.8 517

31.587 29.8 9.413

Total

48.356 13.5 6.520

605.077 4.5 27.401

Fuente: INE 1997 y SAG 2003 – 2004.

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Future work

25 The number of strains to genotype consider herd

prevalence in dairy cattle and laboratory capacity

to culture.

The project proposed to genotype a total of 1200

isolates obtained from different regions of the

country.

Major challenge is to organize the flow chart to

effectively obtained traceable lesions .

The team for the project is interdisciplinary and

has the support of the BTB national program.

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26 F U N D S

FONDOSAG C5-100-10-23

CONICYT- FONDEF D02I 1111

CONICYT- FONDEF D04T2046