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Multi-locus approaches for the characterization of the diversity of the Rickettsiales Ehrlichia ruminantium

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Academic year: 2021

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ER GENETIC CHARACTERIZATION BY MLST & VNTRs

Multi-locus approaches for the characterization of the diversity

of the Rickettsiales Ehrlichia ruminantium

Damien F. Meyer1, Nadia Haddad2, Ludovic Pruneau1, Angélique Saldana1, Héloïse Pilet2, Moez Berrich2, Rim Bouchouicha2, Valérie Pinarello1, Catherine Carasco-Lacombe1, Mélanie Magnan1, Hassane Adakal4, Henri-Jean Boulouis2, Dominique Martinez3, Thierry Lefrançois1 and Nathalie Vachiéry1

CONCLUSION

International Meeting on Rickettsiae and Rickettsial diseases, 5-7 June 2011, Heraklion, Crete, Greece

INTRODUCTION

Ehrlichia ruminantium, ER: agent of heartwater, a tropical fatal disease of ruminants Present in Sub-Saharan Africa, in Indian ocean islands & Caribbean islands

Lack of efficient vaccines due to high genetic diversity Genetic characterization using single genes: pCS20 & map-1

DEVELOPMENT OF VNTRs USING SINGLE & NESTED PCRs

OBJECTIVES

To develop new multi-locus approach based on VNTRs (Variable Number of Tandem Repeats) for ER To characterize the ER genetic diversity by VNTRs & MLST (Multi-Locus Sequence Typing)

METHODS

For MLST typing: 5 genes LipA, LipB, SecY, SodB & SucA

MLST loci SodB SucA SecY LipB LipA

ER strains isolated in vitro

Nested PCR for 5 MLST genes

Sequence analysis of PCR amplicons

For VNTRs typing:

Based on ER genome data from Gardel and Welgevonden strains 21 VNTRs were choosen using the tandem repeats database (http://minisatellites.u-psud.fr)

Strain A Strain B Strain C

ER strains from different areas analysed by VNTRs & MLST - Design of primers & optimization of PCR conditions for single & nested PCRs

PCR amplicon size 5

3 1

- Determination of index of discrimination (I.D.): - Obtention of allelic profile with several VNTRs

N= total number of strains

nj= number of strains with same allelic profile

Successful development of ER VNTRs using nested PCRs Similar global profiles obtained by MLST & VNTRs But VNTR more discriminatory than MLST Good epidemiological molecular tools

Extension of the study to larger panel of ER strains for further phylogenetic study

MLST Neighbor Joining tree Allelic profiles using 7 VNTRs (RU)

There was no difference of VNTR profiles between: -Virulent & attenuated strains

-Gardel &Blonde isolated in the same area 19 years apart Good stability of VNTRs

Isolate/Strain Name Passages Origin Geographical

area Gardel ERGA p18 18 Guadeloupe Caribbean ERGA p237* 237 Blonde BLONDE p8 8 Bekuy 255 ERBE p9 9 West Africa Bankouma 421 ERBAK p4 4 Burkina Faso

Banan 112 ERBA p7 7

Cameroon ERCA p9 9 Cameroon Sankat 430 ERSA p4 4 Ghana

Senegal ERSE p5 5 Senegal

ERSE p64* 64

Lutale ERLU p2 2 Zambia

South Africa

Mara ERMA p1 1 South Africa Umpala ERUM p2 2 Mozambique Welgevonden ERWE p11 11 South Africa

* Attenuated strain

ER strain RU-6 RU-11 RU-12 RU-13 RU-14 RU-15 RU-19 Origin

ERGA p18 ERGA p237* Caribbean BLONDE p5 ERBE p9 ERBAK p4 ERBA p7

ERCA p9 West Africa

ERSA p4

ERSE p5

ERSE p64*

ERLU p2

ERMA p1 South Africa

ERUM p2 ERWE p11 Nb of alleles 3 4 5 3 5 3 4 ERCA p9 ERWE p11 ERLU p2 ERGA ERSE ERBE p9 ERBAK p4 ERSA p4 ERMA p1 ERUM p2 95 100 96 99 95

3 to 5 different alleles per VNTR

1French Agricultural Research Centre for International Development (CIRAD), UMR15 CIRAD-INRA, site de Duclos Prise d’Eau, 97170 Petit-Bourg, Guadeloupe; Email: nathalie.vachiery@cirad.fr ,2UPE, Ecole nationale vétérinaire d'Alfort, UMR BIPAR, ENVA, ANSES, UPEC, USC INRA, 94703 Maisons-Alfort, France, 3CIRAD, UMR15 CIRAD-INRA,

Montpellier, France, 4CIRDES Laboratoire de Biotechnologie (URBIO), 559, 3-51 Avenue du Gouverneur Louveau, B.P. 454, Bobo-Dioulasso, Burkina Faso

No link with geographical origins of strains 16 /21 single PCRs & 7 /21 nested PCRs targeting VNTRs were succesfully developped when tested on 17 ER strains

The global ID is 0.97 either for 16 or 7 VNTRs

Gardel (Caribbean) between Lutale & Umpala (South Africa)

No discrimination by MLST for Senegal cluster

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