Screening of tick-borne pathogens in European ticks using High-throughput qpcr

Screening of tick-borne pathogens in European ticks using High-throughput qpcr Sara Moutailler ANSES - Animal Health Laboratory UMR BIPAR Vectotiq Team 6es Journées d étude du pôle Santé & Société Agents infectieux et vaccins chez l Homme et l Animal- Maisons-Alfort- 30/31th March 2016

Context : Ticks and transmission of pathogens Ticks: excellent vectors : - Different ways of infection: saliva, faeces, coxal glands - Several hosts - Transport over long distances by their hosts emergence or extension of many pathogens - Wide geographic repartition Source: Sarah Bonnet Ticks transmit more pathogens (bacteria, parasites and viruses) than other arthropod vectors

Context : Ticks and transmission of pathogens Human and animal bacteriosis - Borreliosis (Lyme disease) : Borrelia spp - Mediterranean spotted fever: Rickettsia conorii - Tularemia : Francisella tularensis - Q fever: Coxiella burnetti - Bartonellosis : Bartonella henselae - Ehrlichiosis: Ehrlichia chaffensis, E. canis Borrelia burgdorferi spirochètes (Source: CDC) Coxiella burnettii (Source: vetnext) Human and animal protozoosis - Babesiosis : Babesia divergens, B. EU1, B. microti - Theileriosis : Theileria equi, T. annulata, T. parva Babesia divergens (Source: EUCALB) Human and animal arbovirosis - Encephalitis : Tick-borne encephalitis, Louping ill, Powassan - Hemorragic fever: CCHF, Omsk, Kyasanur TBE virus (Source: Sciencephoto)

Context : Epidemiological surveillance of TBP Research of pathogens: - Amplification by PCR, RT-PCR or qpcr of a specific gene (Babesia/Theileria spp., Anaplasma spp., Rickettsia spp., Borrelia sl., TBE virus) - Identification or confirmation of species by sequencing Limits: Low number of target due to the low volume of DNA or RNA Probable under-estimation of pathogens present in ticks

Objectives High-throughput screening of tick-borne pathogens in Ixodes ricinus collected in 5 European countries. Be able to detect a high number of tick-borne pathogens in a high number of ticks in one experiment Develop a high-throughput real-time PCR chip (BioMark TM dynamic arrays) Screen of 19,474 I. ricinus nymphs for the presence of 59 pathogens

Targets: pathogens Bacteria : 25 species (Anaplasma phagocytophilum, A. marginale, A. platys, A. ovis, A. centrale, Bartonella henselae, B. quintana, Borrelia burgdorferi ss, B. garinii, B. afzelii, B. valaisiana, B. lusitaniae, B. spielmanii, B. bissettii, B. miyamotoi, Coxiella burnetii, Francisella tularensis, Ehrlichia chaffeensis, E. ruminantium, E. canis, Neoehrlichia mikurensis, Rickettsia conorii, R. slovaca, R. massiliae, R. helvetica), 1 general group (spotted fever group), and 1 genus (Borrelia spp.) Parasites : Francisella tularensis (Source: CDC) 12 species (Babesia divergens, B. microti, B. venatorum (EU1), B. canis, B. vogeli, B. bovis, B. caballi, B. bigemina, B. major, B. ovis, Theileria equi, T. annulata) Borrelia burgdorferi spirochètes (Source: CDC) Babesia divergens (Source: EUCALB)

Targets: pathogens Viruses (25 species): Asfarviridae : Genus Asfivirus = African swine fever virus (ASFV) Orthomyxoviridae : Thogoto virus, Dhori virus and Bourbon virus Reoviridae : Genus Orbivirus = Kemerovo virus Genus Coltivirus = Colorado tick fever virus, Eyach virus Bunyaviridae : Genus Nairovirus = Crimean-Congo Hemorragic fever virus (CCHF), Dugbe virus, Nairobi sheep disease virus ; Genus Phlebovirus = Uukuniemi virus, Heartland virus, SFTS virus Genus Orthobunyavirus = Schmallenberg Flaviviridae : Genus Flavivirus : Tick-Borne Encephalitis virus (3 subtypes), Louping Ill virus, Powassan virus, Deer tick virus, Omsk hemorragic fever virus, Kyasanur forest disease virus, Langat virus, West Nile virus, Meaban virus

Targets: ticks Ticks : - Ixodes ricinus, I. hexagonus et I. persulcatus - Dermacentor reticulatus, D. marginatus I. ricinus D. reticulatus I. hexagonus D. marginatus I. persulcatus

Material Samples BioMark TM Dynamic Array Targets - Taqman probe (FAM BHQ1): Tm 70 C - Primers: Tm 60 C - qpcr program: Pre-incubation 95 C 5 min Amplification 95 C 10 s (45 cycles) 60 C 15 s

Material 96x96 Chip: 9216 qpcr reactions Or 48x48 Chip : 2304 qpcr reactions

Material Advantage of BioMark TM Dynamic Array Dynamic array qpcr 96.96 48.48 384 wells 96 wells Mastermix 240 µl 120 µl 46 ml 184 ml Primer/probe 240 µl 120 µl 4.6 ml 18.4 ml Plates 1 4 24 96 Time 3 h 8 h 8 days 32 days Pipetting 192 384 18432 73728 Yield 96.96 chip 9216 reactions 48.48 chip 2304 reactions Speed : time cost 30 min Cost : 4.5 à 9.11 centimes/reactions

Methods Design primers and probe for each pathogen - adaptation from the literature - new design based on reference sequences (Genbank) In silico analysis to test the specificity of primers and probes (BLAST) Test of the sensitivity of primers and probes on a dilution range of a reference DNA or RNA sample on a LightCycler 480

Results Dilution range on Lightcycler 480 Anaplasma phagocytophilum, DNA from tick, msp2 gene TBEV European (Flaviviridae, Flavivirus) TBE virus, Hypr strain, European subtype RNA from cell culture, E gene Candidatus Neoehrlichia mikurensis, DNA from rodent, RNA 16S gene Borrelia valaisiana, culture of VS116 strain, ospe gene

Results Comparison before and after preamp on Lightcycler 480 Group 1 Bartonella henselae, culture of B1 strain, pap31 gene Group 2 Borrelia burgdorferi ss, culture of B31 strain, rpob gene Group 3 Borrelia afzelii, culture of NE632 strain, fla gene

Methods Design primers and probe for each pathogen - adaptation from the literature - new design based on reference sequences (Genbank) In silico analysis to test the specificity of primers and probes (BLAST) Test of the sensitivity of primers and probes on a dilution range of a reference DNA sample on a LightCycler 480 Test of the specificity of primers and probes on the BioMark dynamic array 48.48 system

Results BioMark TM Dynamic array 48.48: specificity 1/ Bacteria and parasites array 48 assays (primers and probes) 46 reference samples and 2 negative controls (Michelet et al., Frontiers in Cellular and Infection Microbiology 2014)

Results BioMark TM Dynamic array 48.48: specificity 2/ Viruses array 22 Assays (primers and probe) TBEV European TBEV Far Eastern TBEV Siberian Langat Louping ill Deer Tick Powassan Meaban West Nile Kyasanur Omsk ASFV Eyach Colorado tick fever Kemerovo Dhori Thogoto CCHF Dugbe Nairobi sheep Disease Uukuniemi Schmallenberg I. persulcatus I. ricinus E. coli TBEV European (RNA) TBEV Siberian (plasmid) TBEV Far Eastern (plasmid) Langat (RNA) Louping ill (RNA) Deer Tick (RNA) 22 reference samples and 2 negative controls Powassan (RNA) Meaban (RNA) West Nile (RNA) Kyasanur (plasmid) Omsk (plasmid) ASFV (DNA) Thogoto (plasmid) Dhori (plasmid) Kemerovo (plasmid) Colorado tick fever (RNA) Eyach (RNA) CCHF (plasmid) Dugbe (RNA) Nairobi sheep Disease(RNA) Uukuniemi (RNA) Schmallenberg(RNA) Water

Methods Design primers and probe for each pathogen - adaptation from the literature - new design based on reference sequences (Genbank) In silico analysis to test the specificity of primers and probes (BLAST) Test of the sensitivity of primers and probes on a dilution range of a reference DNA sample on a LightCycler 480 Test of the specificity of primers and probes on the BioMark dynamic array 48.48 system Application on field samples

Evaluation on field samples: Methods 3N 91 pools 100µL Backup 25N 49 pools Lysis in 300µL DMEM + 10% FCS 100µL DNA extractions 100µL RNA extractions RT random primer Collection sites = 13 in 5 European countries Preamplification step (pool 0.2X of primers targeted bacteria and parasites species) Preamplification step (pool 0.2X of primers targeted viruses) Bacteria and Parasites array Viruses array

Evaluation on field samples: Exemple of Results Bacteria and Parasites Denmark Internal control to check potential inhibition E. coli D1 Vestskoven forest H2O D2 Gribskov forest Design for I. ricinus DNA H2O (Michelet et al., Frontiers in Cellular and Infection Microbiology 2014)

Evaluation on field samples: Exemple of Results Bacteria and Parasites The Netherlands N1 Duin en Kruidberg area N2 Austerlitz area (Michelet et al., Frontiers in Cellular and Infection Microbiology 2014)

Evaluation on field samples: Exemple of Results Bacteria and Parasites France F1 Murbach F2 Wasselonne (Michelet et al., Frontiers in Cellular and Infection Microbiology 2014)

Evaluation on field samples: Exemple of Results Viruses Denmark UK H2O Internal control to check potential inhibition E. coli Design for I. ricinus RNA (Moutailler et al., in prep)

Evaluation on field samples: Exemple of Results Viruses Sweden The Netherlands (Moutailler et al., in prep)

Evaluation on field samples: Exemple of Results Viruses France (Moutailler et al., in prep)

Discussion Capacity of the method for large-scale studies => detection several pathogens in the same time in a large number of samples Interesting tool for prevalence studies => first detection of Borrelia spielmanii, Borrelia miyamotoi and two species of Babesia in Denmark => prevalence estimation of 10 bacteria, 2 parasites, and 3 viruses (TBE, Eyach, Uukuniemi) in 5 European countries => 15 bacteria, 10 parasites and 18 viruses not detected in I. ricinus in the 13 sites Possible to detect : - expected pathogens (Borrelia burgdorferi sl, Anaplasma phagocytophilum, TBE virus) - unexpected pathogens (Borrelia miyamotoi ) - rare pathogens (Bartonella henselae, Eyach virus, Uukuniemi virus)

Perspectives Adapted to screen other kind of samples: DNA extracted from rodent spleen DNA extracted from human blood Development of new «vector-borne arrays»: Caribean tick-borne array in course, Mosquito-borne flaviviruses in course

Acknowledgment UMR BIPAR, Vectotiq Team Lorraine Michelet Elodie Devillers Mathilde Gondard Léna Yousfi Muriel Vayssier JF Cosson And the rest of the team Plateforme Identypath, ANSES Patrick Fach Sabine Delannoy Fundings: Partners Anna Aspán, Mikael Juremalm, Jan Chirico (SVA, Sweden) Rene Bødker, Kirstine Klitgaard Schou, Thomas Peter Boye Pihl (DTU, Denmark) Fimme Van Der Wal (CVI, The Netherlands) Karen Mansfield (APHA, UK) Hein Sprong (RIVM, The Netherlands) Gérald Umhang (ANSES, Nancy) Thanks to all the partners who have provided reference DNA and RNA samples! sara.moutailler@anses.fr

Results