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Human/Animal Discrimination by F RNA specific bacteriophages Application on Elorn river Available method of numeration of genotyping Data on specificity and sensibility of genogroups Results on a natural site in Finistere Conclusions and

  1. Human/Animal Discrimination by F RNA specific bacteriophages Application on Elorn river Available method of numeration of genotyping Data on specificity and sensibility of genogroups Results on a natural site in Finistere Conclusions and perspective Colloque TSM 29 th October, 2010 Marie Paule Caprais

  2. Available method of numeration Analysis of F-specific RNA bacteriophages by reference method norm NF- ISO 10705-1 2 cases : contaminated sample : direct analysis sligtly contaminated sample : analysis after concentration Choice of concentration procedure No consensus on what procedure can be adopted - Centrifugation Ultrafiltration by Centricon system of Millipore - Concentration par filtration après ajout de MgCl 2 Obtention of isolates on WG 49 ( Salmonella typhimurium )

  3. Available method of genotyping (1) 1-Genotyping by RT PCR on isolates (with at least 20 isolates)  System primers and probe Taqman described by Ogorzaly and Gantzer, 2006  Kit « QuantiTect Probe RT-PCR Kit » (QIAGEN)  q RT PCR instrumentation (MX3000 de Stratagène) Determination of genogroup on more than 90% of isolates

  4. Available method of genotyping (2) 2- Genotyping by q RT PCR (without culture) Choice of extraction kit of nucleic acids  Magnetic beads (Nuclisens Biomerieux) for effluents and waters  RNA viral mini kit (Qiagen) for faeces Choice of amplification kit for q RT-PCR of nucleic acids  System primers and probe Taqman described by Ogorzaly and Gantzer, 2006  Ultrasens kit (Invitrogen) One Step q RT-PCR

  5. 2- Genotyping by q RT PCR (without culture) Detection limit: 20 copies Detection limit: 24 copies GA Genogroup MS2 Genogroup • Set up standard curves with specific plasmids for genogroup I (supposed animal) et genogroup II (supposed human)

  6. Culture-Genotyping of FRNAPH from faeces and effluents Samples Nb. Nb. positive samples samples (%) 42 3 (7.1) Bovine manure Pig faeces + 73 41 (56) slurries 32 2 (6.2) Bovine faeces 12 1 (8.3) Ovine faeces 32 11 (34) Bird faeces Total animal 191 58 (30.3) Human faeces 4 0 (0.0) 31 31 (100.0) WWTE Total human 35 31 (91.2)

  7. Culture-Genotyping of FRNAPH from faeces and effluents Samples Nb. Nb. positive samples samples (%) Bovine manure 42 3 (7.1) 73 41 (56) Pig faeces + slurries Bovine faeces 32 2 (6.2) 12 1 (8.3) Ovine faeces Weak presence of FRNAPH in ruminants 32 11 (34) Bird faeces Total animal 191 58 (30.3) Difficulty to track Human faeces 4 0 (0.0) ruminant contamination by these markers marqueurs 31 31 (100.0) WWTE Total human 35 31 (91.2)

  8. Culture-Genotyping of FRNAPH from faeces and effluents Samples Nb. Nb. positive samples samples (%) 42 3 (7.1) Bovine manure Pig faeces + slurries 73 41 (56) Bovine faeces 32 2 (6.2) Ovine faeces 12 1 (8.3) Bird faeces 32 11 (34) Total animal 191 58 (30.3) 4 0 (0.0) Human faeces Presence of FRNAPH WWTE 31 31 (100.0) in less than 33% of Total human 35 31 (91.2) total animal

  9. Culture-Genotyping of FRNAPH from faeces and effluents Samples Nb. Nb. positive samples samples (%) 42 3 (7.1) Bovine manure 73 41 (56) Pig faeces + slurries 32 2 (6.2) Bovine faeces Presence of FRNAPH in more than 90% of 12 1 (8.3) Ovine faeces human effluent 32 11 (34) Bird faeces 191 58 (30.3) Total animal More easier to track 4 0 (0.0) Human faeces contamination from human 31 31 (100.0) WWTE 35 31 (91.2) Total human

  10. Culture-Genotyping of FRNAPH from faeces and effluents Samples Nb. Nb. Total samples positive isolates samples (%) 42 3 (7.1) 74 Bovine manure Pig faeces + 73 41 (56) 719 slurries Bovine faeces 32 2 (6.2) 24 Ovine faeces 12 1 (8.3) 24 32 11 (34) 259 Bird faeces 191 58 (30.3) 1100 Total animal Human faeces 4 0 (0.0) 0 31 31 (100.0) 707 WWTE Total human 35 31 (91.2) 707

  11. Culture-Genotyping of FRNAPH from faeces and effluents Samples Nb. Total FRNAPH FRNAPH FRNAPH FRNAPH Genotypes not Nb. samples isolates I (%) II (%) III (%) IV (%) identified (%) positive samples (%) Bovine 42 3 (7.1) 74 38.0 11.0 0.0 0.0 51.0 manure 69.1 0.0 0.0 20.6 10.3 Pig faeces + 73 41 (56) 719 slurries 52.1 0.0 0.0 0.0 47.9 Bovine 32 2 (6.2) 24 faeces Ovine faeces 12 1 (8.3) 24 100 0.0 0.0 0.0 0.0 72.6 10.4 0 8.2 8.8 Bird faeces 32 11 (34) 259 66.4 4.28 0.0 5.76 23.6 Total animal 191 58 1100 (30.3) Human 4 0 (0.0) 0 0.0 0.0 0.0 0.0 0.0 faeces 8.9 62.2 17.6 5.5 5.5 WWTE 31 31 707 (100.0) Total human 35 31 707 8.9 62.2 17.6 5.5 5.5 (91.2)

  12. Culture-Genotyping of FRNAPH from faeces and effluents Samples Nb. Total FRNAPH FRNAPH FRNAPH FRNAPH Genogroups Nb. samples isolates I (%) II (%) III (%) IV (%) not identified positive (%) samples (%) Bovine 42 3 (7.1) 74 38.0 11.0 0.0 0.0 51.0 manure 69.1 0.0 0.0 20.6 10.3 Pig faeces + 73 41 (56) 719 slurries 52.1 0.0 0.0 0.0 47.9 Bovine 32 2 (6.2) 24 faeces Ovine faeces 12 1 (8.3) 24 100 0.0 0.0 0.0 0.0 Bird faeces 32 11 (34) 259 72.6 10.4 0 8.2 8.8 Total animal 191 58 1100 66.4 4.28 0.0 5.76 23.6 (30.3) 0.0 0.0 0.0 0.0 0.0 Human 4 0 (0.0) 0 faeces 8.9 62.2 17.6 5.5 5.5 WWTE 31 31 707 (100.0) Total human 35 31 707 8.9 62.2 17.6 5.5 5.5 (91.2)

  13. Culture-Genotyping of FRNAPH from faeces and effluents Samples Nb. Total FRNAPH FRNAPH FRNAPH FRNAPH Genotypes not Nb. samples isolates I (%) II (%) III (%) IV (%) identified (%) positive samples (%) Bovine 42 3 (7.1) 74 38.0 11.0 0.0 0.0 51.0 manure 69.1 0.0 0.0 20.6 10.3 Pig faeces + 73 41 (56) 719 slurries 52.1 0.0 0.0 0.0 47.9 Bovine 32 2 (6.2) 24 faeces Ovine faeces 12 1 (8.3) 24 100 0.0 0.0 0.0 0.0 Bird faeces 32 11 (34) 259 72.6 10.4 0 8.2 8.8 Total animal 191 58 1100 66.4 4.28 0.0 5.76 22.7 (30.3) 0.0 0.0 0.0 0.0 0.0 Human 4 0 (0.0) 0 faeces 8.9 62.2 17.6 5.5 5.5 WWTE 31 31 707 (100.0) Total human 35 31 707 8.9 62.2 17.6 5.5 5.5 (91.2)

  14. Culture-Genotyping of FRNAPH from faeces and effluents Samples Nb. Total FRNAPH FRNAPH FRNAPH FRNAPH Genotypes not Nb. samples isolates I (%) II (%) III (%) IV (%) identified (%) positive samples (%) Bovine 42 3 (7.1) 74 38.0 11.0 0.0 0.0 51.0 manure 69.1 0.0 0.0 20.6 10.3 Pig faeces + 73 41 (56) 719 slurries 52.1 0.0 0.0 0.0 47.9 Bovine 32 2 (6.2) 24 faeces Ovine faeces 12 1 (8.3) 24 100 0.0 0.0 0.0 0.0 72.6 10.4 0 8.2 8.8 Bird faeces 32 11 (34) 259 66.4 4.28 0.0 5.76 23.6 Total animal 191 58 1100 72.2% (30.3) Human 4 0 (0.0) 0 0.0 0.0 0.0 0.0 0.0 faeces 8.9 62.2 17.6 5.5 5.5 WWTE 31 31 707 (100.0) Total human 35 31 707 8.9 62.2 17.6 5.5 5.5 (91.2)

  15. Culture-Genotyping of FRNAPH from faeces and effluents Samples Nb. Nb. Total FRNAPH FRNAPH FRNAPH FRNAPH Genotypes not samples positive isolates I (%) II (%) III (%) IV (%) identified (%) samples (%) Bovine 42 3 (7.1) 74 38.0 11.0 0.0 0.0 51.0 manure Pig faeces + 73 41 (56) 719 69.1 0.0 0.0 20.6 10.3 slurries Bovine faeces 32 2 (6.2) 24 52.1 0.0 0.0 0.0 47.9 Ovine faeces 12 1 (8.3) 24 100.0 0.0 0.0 0.0 0.0 Bird faeces 32 11 (34) 259 72.6 10.4 0 8.2 8.8 Total animal 191 58 1100 66.4 4.28 0.0 5.76 22.7 (30.3) Human faeces 4 0 (0.0) 0 0.0 0.0 0.0 0.0 0.0 WWTE 31 31 707 8.9 62.2 17.6 5.5 5.5 (100.0) Total human 35 31 707 8.9 62.2 17.6 5.5 5.5 (91.2)

  16. Culture-Genotyping of FRNAPH from faeces and effluents Samples Nb. Nb. Total FRNAPH FRNAPH FRNAPH FRNAPH Genotypes not samples positive isolates I (%) II (%) III (%) IV (%) identified (%) samples (%) Bovine 42 3 (7.1) 74 38.0 11.0 0.0 0.0 51.0 manure Pig faeces + 73 41 (56) 719 69.1 0.0 0.0 20.6 10.3 slurries Bovine faeces 32 2 (6.2) 24 52.1 0.0 0.0 0.0 47.9 Ovine faeces 12 1 (8.3) 24 100.0 0.0 0.0 0.0 0.0 Bird faeces 32 11 (34) 259 72.6 10.4 0 8.2 8.8 Total animal 191 58 1100 66.4 4.28 0.0 5.76 22.7 (30.3) Human faeces 4 0 (0.0) 0 0.0 0.0 0.0 0.0 0.0 WWTE 31 31 707 8.9 62.2 17.6 5.5 5.5 (100.0) Total human 35 31 707 8.9 62.2 17.6 5.5 5.5 79.8% (91.2)

  17. Elorn river Catchment of 280 km 2 8 sites Urban Catchment 24 waters 3 campaigns Upstream Downstream 42km - 57km

  18. Numeration of FRNAPH in waters RNAPH in Elorn river campaign I campaign II 3,5 campaign III 3,0 log 10 FRNA /100ml 2,5 2,0 1,5 Detection limit 1,0 100 PFU/100ml 0,5 0,0 e u n e e e e n g a a r r r r i i è i e è a i a e a v i r i s n u u v r T v s r e t i t i r r s a e s V n d e e p u t a u n d t u o e n u a l u d r L i L o e f l o m u r P f P o m a P C a C Upstream Downstream

  19. Numeration of FRNAPH in waters RNAPH in Elorn river campaign I campaign II 3,5 campaign III 3,0 log 10 FRNA /100ml 2,5 2,0 1,5 1,0 0,5 0,0 e u n e e e e n g a a r r r r i i è i e è a i a e a v i r i s n u u v r T v s r e t i t i r r s a e s V n d e e p u t a u n d t u o e n u a l u d r L i L o e f l o m u r P f P o m a P C a C Upstream Downstream

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