piscirickettsia salmonis workshop - bc cahs · ch10-as-i chile chiloé sur (x) ... results -...

Campbell River

31.05.16

Piscirickettsia salmonis workshop

Snapshot of Cermaq

Employees: 4 400

Cermaq

+ Mitsubishi Corporation

= #2 global salmon farmer

Revenue (2013) NOK 5.2bn

EBIT (2013) NOK 495m

142 thousand tons gwe sold in 2013

151 thousand tons gwe estimated for 2014

Canada

Chile

Norway Cermaq

head office

100% owned by

Mitsubishi Corporation

R&D within Cermaq at a glance

• R&D Manager at HQ Cermaq Group

• Central R&D Fish Health Research Group and lab

in Bergen Norway

• About 20 project leaders whereof ca 10 with

scientific background (Norway, Chile and Canada).

Dedicated resources in each of the operation

companies

• Two research centers with specialized operators

started in 2015

• Actively involved in joint public-private R&D with

public funding (FHF, RCN, CORFO, Tax-refund)

• Turnover in R&D projects (2015)

- NOK 61 mill of which 2/3 from Cermaq

- 1/3 of the projects have public co-funding

Arctic Salmon Research Centre

Finnmark, Norway

Cermaq Chile R&D farm in Colaco

Los Lagos, Chile

SRS R&D program

• SRS is currently the largest economic and environmental

sustainability challenge in Chilean aquaculture, and for

Cermaq as a group.

• This disease produces severe clinical manifestations

causing high mortalities and is the major cause of antibiotic

use in Chilean aquaculture

• Cermaq Chile initiated in 2011 the R&D project STOP SRS

Early in 2015 this activity was strengthened by developing a

SRS R&D program with overall objective to develop new

knowledge and tools to combat SRS.

• Consisting of 12 specific projects with start-up in 2015

and 2016.

• Targeting to aid:

- Vaccine development

- Production / Area management

- Treatment efficacy and optimization

Todays talk

• Characterization of Piscirickettsia salmonis from Chilean and

Canadian salmonids

• P. Salmonis challenge experiment and treatment with

Florfenicol

• Survival of two P. salmonis isolates in a active and sterile FW

and SW microcosm

Background

• Three species affected

- Atlantic salmon, Rainbow trout and Coho salmon

• Differences in clinical signs and severity in Chile

- Time

- Species

• Difference in treatment efficacy

• Exists in Canada and Europe but causes more severe

disease in Chile

• Most research and vaccine development done on the type

strain isolate

Objective

• The main objective was to characterize genotypically and

phenotypically various Piscirickettsia spp. isolates from Chile

and Canada and compare them to the type-strain

- This was done to obtain information about possible presence of

heterogeneous clades that may explain the variable vaccine effect and

the variable clinical expressions observed in the field

Materials and Methods

P. salmonis isolates (2011 – 2012)

• 18 Chilean (including LF-89)

o Atlantic salmon (12)

o Rainbow trout (4)

o Coho salmon (LF-89)

o Region X (17)

o Region XI (1)

o Mortalities from 1.9 to 21.4 %

o Subacute – acute and chronic clinic conditions

o Microbiological samples from lesions, kidney, liver, spleen, brain

• Two Canadian (same outbreak)

o Atlantic salmon

o British Columbia, Campbell River

o Mortalities < 0.03 %

o Chronic clinic condition

o Microbiological samples from kidney

1

6

1

3,4

11

5

10

2

7

9

15

8

12, 14, 16, 17 13,18

Chiloé Island

Puerto Montt

http://www.enchantedlearning.com/geography/outlinemaps/

Isolates

Isolate code Country County (Region) Sampling date Mortality (%) Host Sample tissue Clinical condition

LF-89 Chile Puerto Montt (X) 1989 na Coho salmon kidney na

Ch2-As-I Chile Chiloé Sur (X) 08.08.2012 7,8 Atlantic salmon k-l-sp-b acute

Ch3-Rt-L Chile Calbuco (X) 03.10.2012 6,7 Rainbow trout lession sub-acute


Ch5-As-I Chile Chiloé Centro (X) 18.07.2012 5,1 Atlantic salmon k-l-b sub-acute


Ch7-As-L Chile Aysén (XI) na na Atlantic salmon muscle chronic

Ch8-Rt-K Chile Chiloé Centro (X) 17.06.2011 1,9 Rainbow trout kidney sub-acute

Ch9-As-na Chile Chiloé Centro (X) 27.03.2012 2,4 Atlantic salmon na sub-acute

Ch10-As-I Chile Chiloé Sur (X) 24.07.2012 21,5 Atlantic salmon k-l-sp-b acute

Ch11-As-I Chile Chiloé Centro (X) 04.05.2012 2,2 Atlantic salmon k-l-b sub-acute

Ch12-As-I Chile Chiloé Centro (X) 07.05.2012 14,8 Atlantic salmon k-l-b acute

Ch13-As-I Chile Chiloé Centro (X) 18.04.2012 3,6 Atlantic salmon k-l-sp-b chronic

Ch14-As-I Chile Chiloé Centro (X) 13.01.2012 14,8 Atlantic salmon k-sp acute

Ch15-As-I Chile Chiloé Centro (X) 23.08.2012 5,4 Atlantic salmon k-l-b-sp sub-acute

Ch16-As-I Chile Chiloé Centro (X) June 2012 14,8 Atlantic salmon k-l-b acute

Ch17-As-I Chile Chiloé Centro (X) 23.05.2012 14,8 Atlantic salmon k-l-b acute

Ch18-As-I Chile Chiloé Centro (X) 22.06.2012 3,6 Atlantic salmon k-l-b-sp chronic

Ca19-As-I Canada British Columbia 11.12.2012 < 0,03 Atlantic salmon kidney chronic

Ca20-As-I Canada British Columbia 11.12.2012 < 0,03 Atlantic salmon kidney chronic

Methods - Genotypic study

Target

gene Primer Direction Sequence (5´- 3´) Reference

16s rDNA Eug B27F Fwd AGAGTTTGATCMTGGCTCAG [28]

Eug A1518R Rev AAGGAGGTGATCCANCCRCA [28]

ITS SRS-ITS/F Fwd GTACACACCGCCCGTCACAC Present study

SRS-ITS/R Rev CCTCACGGTACTAGTTCACTATCGG Present study

dnaK SRS-dnaK/F2 Fwd CCGTGTCGTGTGGCGCTAAAA Present study

SRS-dnaK/R2 Rev TTGAGATTGAGCCTGCTCCGC Present study

SRS-dnaK3/F1 Fwd CCGCGTGTGATTGAGAGTGC Present study

SRS-dnaK3/R1 Rev CGTCATCACCCCACCCATGG Present study

groEL SRS-groEL/F1 Fwd CTTCGGTACCGGTTCCCGTC Present study

SRS-groEL/R1 Rev TCTTGCAGTTTCTCGCGGTCG Present study

SRS-groEL/F2 Fwd GTGAAGCTCTGGCAACACTCGTC Present study

SRS-groEL/R2 Rev AGGAAGCTCTGCAACCATCGC Present study

tbpB SRS-tbpB/F1 Fwd AACTGGGCAGGCGTCACTGTT Present study

SRS-tbpB/R1 Rev CGGCGCGTCTCTAATGTTCG Present study

SRS-tbpB2/F2 Fwd CCAAGCTGGATCACCGCCAT Present study

SRS-tbpB2/R2 Rev AAAGATAGGCCCAGCCACGC Present study

mltB SRS-mltB/F Fwd ACCACTCACGCGGCATCTAA Present study

SRS-mltB/R Rev ACTCAAATCATACACCGCCATTGCA Present study

ospA SRS-ospA/F Fwd AGCCGTCAAGAAGTCGGAGCT Present study

SRS-ospA/R Rev TGCCAACGACCATCCGCTTG Present study

radA SRS-radA/F1 Fwd ATCAGTCGCCAGCCTGTTGG Present study

SRS-radA/FR1 Rev GTCCTCGTTGCACTGGACGA Present study

airA SRS-air/F1 Fwd GGGTGCGTCCGGGGATTATG Present study

SRS-rairA/R1 Rev TAAGGTGCACGCAGTGGCAT Present study

bax SRS-bax/F1 Fwd TCAAGGGATCTGGGAAGTGCT Present study

SRS-bax/R1 Rev ACCACTGCCTATCTTGCTCAACA Present study

tnpA SRS-tnpA/F1 Fwd ACCTGTTAAGTTCTCGGCCATT Present study

SRS-tnpA/R1 Rev AGCCTTCACAAATGTCAACAAGTGA Present study

elfP SRS-elfP/F Fwd GCCACKGCTAATTCAGCAA Present study

SRS-elfP/R Rev STGGAATGGTCAGCCACYT Present study

• Genetic characterization:

• Phylogeny of 16S rDNA-ITS

• Phylogeny using ten

concatenated housekeeping

genes (MLSA)

• Multilocus sequence typing

(MLST) method

Methods - Phenotyping

The Fish Disease Group / The Department of Biology

The phenotypic study:

- Growth medium experiment

- Optimization of growth medium

- Temperature experiment

- Antibiotic test

- Other test

- Indole test

- Gram-staining

- Oxidase test

- Catalase test

- Cefinase test

- Triple Sugar Iron Agar

- API ZYM kit

- Hydrogen sulfide strips

Results – Phylogeny of 16S rDNA-ITS

Ch15

Ch7

Ch14

Ch12

Ch8

Ch11

Ch16

Ch17

Ch18

Ch10

Ca20

Ca19

Ch3

Ch4

Ch5

Ch6

LF-89

Ch2

99

99

0.002

Clade G2

Clade G3

Clade G1

- Three clades: 2 Chilean - one Canadian

Results – Phylogeny of concatenated HK genes

0.02

Ch15

Ch4

Ch3

Ch12

Ch18

Ch14

Ch11

Ch6-Rt-L

Ch7

Ch8

Ch10

LF-89

Ch17

Ca19

Ch2

Ca20

Ch5

Ch16

100

100

100

100

100

Clade G1

Clade G3

Clade G2

- Isolates in clade 1 and 2 are better

differentiated

Results - MLST

Ch16

Ca19, ST2

Ch8

Ch7

Ca20, ST1

LF-89

Ch18, ST12

Ch15, ST10

Ch3, ST6

Ch2

Ch4

Ch11, ST7

Ch5

Ch14I

Ch10

Ch12, ST9

Ch17, ST11

Ch6, ST4

ST5

ST8

ST3

- Isolates from clade 1 and 2 are

even better differentiated into

several sequence types

• Chilean P. salmonis are differentiated into two groups, G1 and G2

• The Chilean isolates are genetically distinct from the Canadian isolates,

G3

• The three genetic methods used show the same grouping among the

18 isolates of P. salmonis analyzed.

• MLST gives the best separation

Conclusions

Results - Growth media test

Isolate SRS-BA CHAB CHAB w/Fe Austral-TSHem BA BA w/2 % NaCl

Ch11-G2-As-I +++ +++ +++ ++ ++ +++

Ch7-G2-As-L +++ +++ +++ ++ ++ ++

Ch14-G2-As-I +++ ++ ++ ++ ++ +++

Ch9-G2-As-na +++ +++ +++ ++ + +

Ch13-G2-As-I +++ ++ ++ ++ ++ +++

Ch17-G2-As-I ++ ++ + + ++ ++

Ch2-G1-As-I +++ ++ ++ + ++ ++

Ch12-G2-As-I +++ ++ ++ + + ++

Ch15-G2-As-I +++ + + W + +

Ch10-G2-As-I ++ ++ ++ + + ++

Ch8-G2-Rt-K +++ + + +

+ +

Ch18-G2-As-I +++ + + + - +

Ca19-G3-As-I +++ ++ ++ - + +

Ch16-G2-As-I ++ + + - + ++

LF-89 +++ ++ ++ - + +

Results - Optimal growth medium results The Fish Disease Group / The Department of Biology

Growth mediums:

1. SRS-BA

2. CHAB

3. CHAB w/ 0.2 mM Fe

4. Austral-TSHem

5. BA w/ 2% NaCl

6. BA

7. MA

8. FLPA

1 2 3 4

5 6 7 8

Results - Growth medium

• To improve the growth of P. salmonis on solid media, a new

optimized SRS blood agar (SRS-BA) was developed

• The composition of this agar was:

- 40g of TSA (BD, Difco)

- 20g of Red Sea Salt (RSS) (Red Sea, USA)

- 50 ml of defibrinated sheep blood (DSB) (Oxoid Limited, UK)

- 1g of L-cysteine (Sigma-Aldrich)

- 5g of D-glucose (Sigma-Aldrich)

- 50 ml of fetal bovine serum (FBS) (Thermo Scientific Hyclone, USA)

- 0.2 mM ferric nitrate (Sigma-Aldrich)

- Reverse osmosis water (RO) to a final volume of 1000 ml

Results - Temperature optimum

Isolate Optimum growing temp (°C)

LF-89 16 - 19

Ch2-As-I 16 - 19

Ch3-Rt-L 16 - 19

Ch4-Rt-L 16 - 19

Ch5-As-I 16 - 19

Ch6-Rt-L 16 - 19

Ca20-As-K 16 - 19

Ch7-As-L 19 - 22

Ch8-Rt-K 19 - 22

Ch9-As-na 19 - 22

Ch10-As-I 19 - 22

Ch11-As-I 19 - 22

Ch12-As-I 19 - 22

Ch13-As-I 19 - 22

Ch14-As-I 19 - 22

Ch15-As-I 19 - 22

Ch16-As-I 19 - 22

Ch17-As-I 19 – 22

Ch18-As-I 19 – 22

Ca19-As-K 19 – 22

Results - Antibiotic sensitivity test

• Most isolates

sensitive to

Florfenicol and

Oxytetracycline

• One isolate with low

sensitivity for

Florfenicol

• Two isolates show

low sensitivity to

Oxytetracycline

• Most isolates has low

sensitivity to

Streptomycin

Isolate Streptomycin Oxytetracycline Penicilin Ceftazidime Ampicilin Florfenicol

LF-89 5 23 12 15,5 19,5 24

Ch2-As-I 0,5 0,5 1,5 10 3 14,5

Ch3-Rt-L 3,5 8,5 0,5 0,5 2,5 4

Ch4-Rt-L 2 18 6,5 17,5 14 19

Ch5-As-I 1 17 12 16,5 12,5 19,5

Ch6-Rt-L 0 16,5 13 15,5 13 18

Ch7-As-L 0 10 10 13,5 10,5 14,5

Ch8-Rt-K 1 21 2 13,5 13,5 24,5

Ch9-As-na 2 25 7 16 9,5 24

Ch10-As-I 32,5 27,5 1,5 18,5 22 23,5

Ch11-As-I 1 21 4 5 7,5 19

Ch12-As-I 0,5 17 9,5 12,5 1,5 22

Ch13-As-I 0,5 22 2,5 10 10 25

Ch14-As-I 0 22 9,5 11,5 11,5 24,5

Ch15-As-I 1 20 6 12 9 20,5

Ch16-As-I 0 3,5 6,5 12,5 8 17,5

Ch17-As-I 0,5 24,5 5,5 11 14,5 23

Ch18-As-I 2,5 20 0 10 1,5 15,5

Ca19-As-I 2 17 21,5 22 25 24,5

Ca20-As-I 2,5 24 13 13,5 15 27,5

Summery – Phenotypic characterization

• Based in growth the isolates were defined as ‘less fastidious’ and

‘fastidious’ which correlates with clade 1 and 2

• The fastidious had 16 ºC – 19ºC as optimum growth temperature

and less fastidious had 19ºC – 22ºC which correlates with clade 1

and 2

• Growth (speed, medium and numbers) and temperature optimum

correlates with clades 1 and 2

• Most isolates were susceptible to Florfenicol and Oxytetraclycline

• No significant differentiation in the other biochemical tests

P. Salmonis challenge experiment and treatment with Florfenicol

Background

• In Chile we can experience rapid reinfections of SRS resulting

in several antibiotic treatments

• We wanted to investigate if the fish was reinfected by itself

(No clearance) or from the environment.

• We also wanted to identify minimal inhibitory concentrations

for 13 of our isolates and look at treatment regimes.

Challenge and treatment of SRS

• Objective

- Identify if a Florfenicol and Flumequin treatment clears the P. salmonis

infection

• Secondary objectives

- Identify MIC values for Cermaq Chile P. salmonis isolates.

- See if the does 10mg pr. kg for 10 or 20 days outperform or is equal to

the florfenicol treatment dose used in Chile

MIC testing

• 13 isolates of P. salmonis collected in 2012 from Cermaq

Chile sites were tested using 8 different doses for Florfenicol

(a bacteriostatic) and Flumequin (Bactericidal)

• An inoculum of Mcfarland 4 was used and 100 ul was plated

out on SRS-BA in triplicate, negative and positive controls

were included

• Plates were read after 7 and 14 days to document growth

0

1

2

3

4

5

<0,25 0,25 0,5 1 2 4 8 16 >16

Nr.

of

iso

late

s

MIC ug/ml

The MIC for 13 isolates of P.salmonis, 13 from Cermaq Chile

Florfenicol

0

1

2

3

4

5

<0,25 0,25 0,5 1 2 4 8 16 >16

Nr.

of

iso

late

s

MIC ug/ml

The MIC for 13 isolates of P.salmonis, 13 from Cermaq Chile

Flumequine

Summary

• There were considerable variation in MIC for different isolates

for both Florfenicol and Flumequine

• Different sensitivity phenotypes of P. salmonis exist within the

same outbreak of SRS

• 3 of 4 isolates with quinolone resistance were isolated from

rainbow trout

• We recommend an surveillance of the MIC in the future

Challenge trial setup

• 10 ip infected shedders

• 50 kohabitants of 65 gr. at start of treatment - 15 fish sampled during trial

• We used three treatment regimes: - 10 mg/kg for 10 days

(suggested by the supplier)

- 10 mg/kg for 20 days

- 25 mg/kg for 18 days

• Skretting 3mm pellet standard or with 1 or 2 gram floraqpharma

Sampling

• Kidney for CFU and qPCR - Kidney of five fish is quantified and homogenized before plating in

triplicates on SRS-BA for CFU counting

- Kidney is quantified for qPCR quantifiaction (NA)

• Sampling was performed the day before treatment start and the day after treatment end

• Plasma and muscle tissue is sampled for Florfenicol quantification

• Gill and heart for detection of viral pathogens

• Water samples to monitor changes in infection pressure

Summary challenge

• Shedders mortality 11 dpi, develops different in the tanks

- 3 weeks post challenge 9 of 70 shedders are alive

- Positive control Cohab mortality starts 29 dpi

- Total accumulative mortality 91%

• Treatment effects:

- No cohab mortality in treated groups

- No treated cohabs positive for P. salmonis by cultivation on agar

- No differentiation between treatment regimes

Conclusions

• Chilean Piscirickettsia isolates in this study can be divided

into two genogroups, 1 and 2

• Canadian isolates in this study are genetically distinct from

Chilean isolates

• Growth speed, media preference and temperature optimum

correlates with genogroups

• MIC values varies among the tested isolates in Chile and it is

recommended to do MIC testing going forward

Conclusions 2

• Florfenicol seems to be able to clear P. salmonis under

experimental conditions

piscirickettsia salmonis workshop - bc cahs · ch10-as-i chile chiloé sur (x) ... results -...

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