Human/Animal Discrimination by F RNA specific bacteriophages - - PowerPoint PPT Presentation

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Human/Animal Discrimination by F RNA specific bacteriophages - - PowerPoint PPT Presentation

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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

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SLIDE 1

Human/Animal Discrimination by F RNA specific bacteriophages Application on Elorn river

Colloque TSM 29th October, 2010

Available method of numeration

  • f genotyping

Data on specificity and sensibility of genogroups Results on a natural site in Finistere Conclusions and perspective

Marie Paule Caprais

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SLIDE 2

Analysis of F-specific RNA bacteriophages by reference method norm NF- ISO 10705-1 Available method of numeration Obtention of isolates on WG 49 (Salmonella typhimurium)

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 MgCl2

2 cases : contaminated sample : direct analysis sligtly contaminated sample : analysis after concentration

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SLIDE 3

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

Available method of genotyping (1)

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SLIDE 4

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

Available method of genotyping (2)

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SLIDE 5
  • Set up standard curves with specific plasmids

for genogroup I (supposed animal) et genogroup II (supposed human)

MS2 Genogroup GA Genogroup

Detection limit: 20 copies Detection limit: 24 copies

2- Genotyping by q RT PCR (without culture)

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SLIDE 6

Culture-Genotyping of FRNAPH from faeces and effluents

Samples Nb. samples Nb. positive samples (%) Bovine manure

42 3 (7.1)

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)

Human faeces

4 0 (0.0)

WWTE

31 31 (100.0) Total human 35 31 (91.2)

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SLIDE 7

Culture-Genotyping of FRNAPH from faeces and effluents

Samples Nb. samples Nb. positive samples (%)

Bovine manure

42 3 (7.1)

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)

Human faeces

4 0 (0.0)

WWTE

31 31 (100.0)

Total human

35 31 (91.2)

Weak presence of FRNAPH in ruminants Difficulty to track ruminant contamination by these markers marqueurs

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SLIDE 8

Samples Nb. samples Nb. positive samples (%)

Bovine manure

42 3 (7.1)

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)

Human faeces

4 0 (0.0)

WWTE

31 31 (100.0)

Total human

35 31 (91.2)

Presence of FRNAPH in less than 33% of total animal

Culture-Genotyping of FRNAPH from faeces and effluents

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SLIDE 9

Samples Nb. samples Nb. positive samples (%)

Bovine manure

42 3 (7.1)

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)

Human faeces

4 0 (0.0)

WWTE

31 31 (100.0)

Total human

35 31 (91.2)

Presence of FRNAPH in more than 90% of human effluent More easier to track contamination from human

Culture-Genotyping of FRNAPH from faeces and effluents

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SLIDE 10

Samples Nb. samples Nb. positive samples (%) Total isolates

Bovine manure

42 3 (7.1) 74

Pig faeces + slurries

73 41 (56) 719

Bovine faeces

32 2 (6.2) 24

Ovine faeces

12 1 (8.3) 24

Bird faeces

32 11 (34) 259

Total animal

191 58 (30.3) 1100

Human faeces

4 0 (0.0)

WWTE

31 31 (100.0) 707

Total human

35 31 (91.2) 707

Culture-Genotyping of FRNAPH from faeces and effluents

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SLIDE 11

Samples

Nb. samples Nb. positive samples (%) Total isolates FRNAPH I (%) FRNAPH II (%) FRNAPH III (%) FRNAPH IV (%) Genotypes not identified (%)

Bovine manure 42 3 (7.1) 74

38.0 11.0 0.0 0.0 51.0

Pig faeces + slurries 73 41 (56) 719

69.1 0.0 0.0 20.6 10.3

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

Bird faeces 32 11 (34) 259

72.6 10.4 8.2 8.8

Total animal 191 58 (30.3) 1100

66.4 4.28 0.0 5.76 23.6

Human faeces 4 0 (0.0)

0.0 0.0 0.0 0.0 0.0

WWTE 31 31 (100.0) 707

8.9 62.2 17.6 5.5 5.5

Total human 35 31 (91.2) 707

8.9 62.2 17.6 5.5 5.5

Culture-Genotyping of FRNAPH from faeces and effluents

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SLIDE 12

Samples

Nb. samples Nb. positive samples (%) Total isolates FRNAPH I (%) FRNAPH II (%) FRNAPH III (%) FRNAPH IV (%) Genogroups not identified (%)

Bovine manure 42 3 (7.1) 74

38.0 11.0 0.0 0.0 51.0

Pig faeces + slurries 73 41 (56) 719

69.1 0.0 0.0 20.6 10.3

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

Bird faeces 32 11 (34) 259

72.6 10.4 8.2 8.8

Total animal 191 58 (30.3) 1100

66.4 4.28 0.0 5.76 23.6

Human faeces 4 0 (0.0)

0.0 0.0 0.0 0.0 0.0

WWTE 31 31 (100.0) 707

8.9 62.2 17.6 5.5 5.5

Total human 35 31 (91.2) 707

8.9 62.2 17.6 5.5 5.5

Culture-Genotyping of FRNAPH from faeces and effluents

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SLIDE 13

Samples

Nb. samples Nb. positive samples (%) Total isolates FRNAPH I (%) FRNAPH II (%) FRNAPH III (%) FRNAPH IV (%) Genotypes not identified (%)

Bovine manure 42 3 (7.1) 74

38.0 11.0 0.0 0.0 51.0

Pig faeces + slurries 73 41 (56) 719

69.1 0.0 0.0 20.6 10.3

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

Bird faeces 32 11 (34) 259

72.6 10.4 8.2 8.8

Total animal 191 58 (30.3) 1100

66.4 4.28 0.0 5.76 22.7

Human faeces 4 0 (0.0)

0.0 0.0 0.0 0.0 0.0

WWTE 31 31 (100.0) 707

8.9 62.2 17.6 5.5 5.5

Total human 35 31 (91.2) 707

8.9 62.2 17.6 5.5 5.5

Culture-Genotyping of FRNAPH from faeces and effluents

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SLIDE 14

Samples

Nb. samples Nb. positive samples (%) Total isolates FRNAPH I (%) FRNAPH II (%) FRNAPH III (%) FRNAPH IV (%) Genotypes not identified (%)

Bovine manure 42 3 (7.1) 74

38.0 11.0 0.0 0.0 51.0

Pig faeces + slurries 73 41 (56) 719

69.1 0.0 0.0 20.6 10.3

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

Bird faeces 32 11 (34) 259

72.6 10.4 8.2 8.8

Total animal 191 58 (30.3) 1100

66.4 4.28 0.0 5.76 23.6

Human faeces 4 0 (0.0)

0.0 0.0 0.0 0.0 0.0

WWTE 31 31 (100.0) 707

8.9 62.2 17.6 5.5 5.5

Total human 35 31 (91.2) 707

8.9 62.2 17.6 5.5 5.5

Culture-Genotyping of FRNAPH from faeces and effluents 72.2%

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SLIDE 15

Samples

Nb. samples Nb. positive samples (%) Total isolates FRNAPH I (%) FRNAPH II (%) FRNAPH III (%) FRNAPH IV (%) Genotypes not identified (%)

Bovine manure 42 3 (7.1) 74 38.0 11.0 0.0 0.0 51.0 Pig faeces + slurries 73 41 (56) 719 69.1 0.0 0.0 20.6 10.3 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 8.2 8.8 Total animal 191 58 (30.3) 1100 66.4 4.28 0.0 5.76 22.7 Human faeces 4 0 (0.0) 0.0 0.0 0.0 0.0 0.0 WWTE 31 31 (100.0) 707 8.9 62.2 17.6 5.5 5.5 Total human 35 31 (91.2) 707 8.9 62.2 17.6 5.5 5.5

Culture-Genotyping of FRNAPH from faeces and effluents

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SLIDE 16

Samples

Nb. samples Nb. positive samples (%) Total isolates FRNAPH I (%) FRNAPH II (%) FRNAPH III (%) FRNAPH IV (%) Genotypes not identified (%)

Bovine manure 42 3 (7.1) 74 38.0 11.0 0.0 0.0 51.0 Pig faeces + slurries 73 41 (56) 719 69.1 0.0 0.0 20.6 10.3 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 8.2 8.8 Total animal 191 58 (30.3) 1100 66.4 4.28 0.0 5.76 22.7 Human faeces 4 0 (0.0) 0.0 0.0 0.0 0.0 0.0 WWTE 31 31 (100.0) 707 8.9 62.2 17.6 5.5 5.5 Total human 35 31 (91.2) 707 8.9 62.2 17.6 5.5 5.5

79.8% Culture-Genotyping of FRNAPH from faeces and effluents

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SLIDE 17

Elorn river

Upstream 8 sites 24 waters 3 campaigns

Catchment of 280 km2

Downstream

Urban Catchment

  • 57km

42km

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SLIDE 18

RNAPH in Elorn river

0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 L a n d e r n e a u V e r v i a n P e i n a n T r e i n P

  • u

l d u r i v i è r e P

  • u

l d u e s t u a i r e C a m f r

  • u

t r i v i è r e C a m f r

  • u

t e s t u a i r e L e p a s s a g e log 10 FRNA /100ml campaign I campaign II campaign III Detection limit 100 PFU/100ml

Numeration of FRNAPH in waters

Upstream Downstream

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SLIDE 19

RNAPH in Elorn river

0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 L a n d e r n e a u V e r v i a n P e i n a n T r e i n P

  • u

l d u r i v i è r e P

  • u

l d u e s t u a i r e C a m f r

  • u

t r i v i è r e C a m f r

  • u

t e s t u a i r e L e p a s s a g e log 10 FRNA /100ml campaign I campaign II campaign III

Upstream Downstream

Numeration of FRNAPH in waters

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SLIDE 20

R N A P H in E lo rn riv e r

0 ,0 0 ,5 1 ,0 1 ,5 2 ,0 2 ,5 3 ,0 3 ,5 L a n d e r n e a u V e r v i a n P e i n a n T r e i n P

  • u

l d u r i v i è r e P

  • u

l d u e s t u a i r e C a m f r

  • u

t r i v i è r e C a m f r

  • u

t e s t u a i r e L e p a s s a g e log 10 FRNA /100ml c a m p a ig n I c a m p a ig n II c a m p a ig n III

3 2 3 2 2 3 1 1 Upstream Downstream GI GII No Id.

Culture-Genotyping of FRNAPH in Elorn river

Legend

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SLIDE 21

Study by q RT-PCR after concentration of waters (500 ml by Centricon system) Extraction by Nuclisens kit (magnetic beads) q RT-PCR with Ultrasens kit

First campaign

4/8 ea detected in GA 6/8 waters detected in MS2

Second campaign

1/8 water detected in GA 0/7 eau

Third campaign

7/8 waters detected in MS2 Quantification of GA in 4/8 waters and detection in

  • thers

Numbers of copies from 400 to 900/100ml

Genotyping of FRNAPH in Elorn river by q RT-PCR

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SLIDE 22
  • 17/24 genotyped (culture-genotyping) waters 71% of success

for this study

  • Study by q RT-PCR after concentration of waters 4/24 results

17%

Conclusion and perspectives Culture genotyping seems more successful than q RT-PCR Difficulty to track ruminant contamination and more generally animal contamination Genogroup II good candidate to identify human contamination Must be used parallely to another MST marker

Merci à Michèle, Dominique, Cécile et Margot