Zebrafish Health Monitoring: Recommendations from the ...

Zebrafish Health Monitoring:

Recommendations from the

FELASA-AALAS Working GroupDr J-P Mocho MRCVS MVDr DVM [email protected]

C. Collymore, S. Farmer, E. Leguay, K. Murray, N. Pereira

The Working Group

Federation of European

Laboratory Animal Science

Associations (FELASA)

Emmanuel Leguay

J-P Mocho (convenor)

Nuno Pereira

American Association for

Laboratory Animal Science

(AALAS)

Chereen Collymore

Susan Farmer

Katy Murray

The Context

Follow steps of similar rodent working groups

development of a harmonized health monitoring program

Become the reference

FELASA-AALAS Working Group on Health Monitoring for Fish in Research

Focus first on zebrafish to set a framework

The Mission

Identify and describe most relevant pathogens

and pathology

Recommend a screening pattern

Create a reporting template

Describe applications to transfer of animals

between facilities

Figure 2: P. tomentosa egg detected during sludge analysis

by microscopy. Magnification used was 400X. Arrows

indicate the bipolar plugs. Scale bar = 50 μm.

Mocho, J.P., Martin, D.J., Millington, M.E., Saavedra Torres, Y.

Environmental Screening of Aeromonas hydrophila,

Mycobacterium spp., and Pseudocapillaria tomentosa in

Zebrafish Systems. J. Vis. Exp. (130), e55306, doi:10.3791/55306

(2017).

What have we achieved so far?

Survey

Diseases and pathogens references

Screening pattern

Reporting

Scenarios

COMPLETE

FINAL REVIEW

FINAL REVIEW

80% COMPLETE

WORK IN PROGRESS

Survey

Survey entries

145 entries

Europe: 111

North America: 24

Other: 10

Wide range of fish speciesFigure 3: Breeding device submerged in a fish holding tank to collect

sludge for analysis. This tank is set on a bench for the purpose of the

picture; it is set otherwise in the recirculating system.

Mocho, J.P., Martin, D.J., Millington, M.E., Saavedra Torres, Y.

Environmental Screening of Aeromonas hydrophila, Mycobacterium

spp., and Pseudocapillaria tomentosa in Zebrafish Systems. J. Vis. Exp.

(130), e55306, doi:10.3791/55306 (2017).

Survey outcome

Health Monitoring

Consensus on

Main pathogens to monitor

Wide range of non-infectious

disease

Majority have a health monitoring

system in place using

Colony fish

PCR and histopathology

Adult and old fish only

No gender selection

Biosecurity

Common practice are:

Import of fish

Compulsory PPE

Basic biosecurity measures

>15% without quarantine

Very few with bio-secured

quarantine

Pathogens and diseases

Identifying Relevant Pathogens and Diseases

Relevant Pathogens and Diseases

Most prevalent

And/or

Reported/potential impact on

fish welfare or science

Prevalence

ZIRC data

Disease Vs pathogens

HISTOPATHOLOGY IS REQUIRED

Pathogens' prevalence in ZIRC submissions 2006-2016

Pseudoloma neurophilia

14%

Mycobacterium spp. 5%

Pseudocapillaria tomentosa

1.5%

Edwardsiella ictaluri 0.1%

https://zebrafish.org/wiki/_media/health/submission/submissions_s

ummary_2006-2016.pdf

Prevalence and virulence of pathogens

SMOP

Screen More Often Pathogens

VIP - Very Important Pathogens

Mycobacterium spp.

Pseudoloma neurophilia

Pseudocapillaria tomentosa

SLOMScreen Less Often Microorganisms

Edwardsiella ictaluri

Flavobacterium columnare

Ichthyophthirius multifilis

Myxidium streisinger

Picornavirus

Piscinoodinium pillulare

Pleistophora hyphessobryconis

Screening pattern

Screening pattern – Epidemiological rules

Sample number depends on:

Prevalence

Test sensitivity and specificity

Define epidemiological unit

ZIRC’s records on prevalence is an

indication but intra-facility prevalence

varies widely.

http://epitools.ausvet.com.au/content.ph

p?page=FreedomSS

Specificity set for100%

95% confidence

Run several scenarios with different

sensitivity

Sampling to achieve SPF status will be in

addition to the routine health monitoringSample size for varying prevalence and population size to

confirm absence of pathogen (sensitivity 100%)

Prevalence

Population size 1% 5% 10% 20%

N = 500 226 57 30 15

N = 1 000 259 59 30 15

N = 5 000 291 60 30 15

N = 10 000 296 60 30 15

N = 100 000 300 60 30 15

Screening pattern – Epidemiological bias

Bias detection

Once a year, set-up

Pre-filtration sentinel tank

50 mixed gender wild types

> 180 dpf

Samples GAA colony fish too

Variety of age categories

Add environmental samples

Figure 1: Pre-filtration sentinel tank out of the recirculating system. An 8 L

tank is filled up with water and bio-media from the sumps of the systems

to screen. The two white ceramic bio-media beads sit at the bottom of

the tank (in the middle of the picture). 12 fish are selected

according to their age, strain, and gender, and they are added to the

sentinel tank.

Mocho, J.P., Martin, D.J., Millington, M.E., Saavedra Torres, Y.

Environmental Screening of Aeromonas hydrophila, Mycobacterium spp.,

and Pseudocapillaria tomentosa in Zebrafish Systems. J. Vis. Exp. (130),

e55306, doi:10.3791/55306 (2017).

Screening pattern – detection assays

PCR, culture, microscopy

Restricted range of pathogens

Histopathology:

Wider range of pathogens

Impact of infection

Non-infectious disease

Husbandry

Mortality, morbidity Figure 3. Pseudocapillaria tomentosa egg next to artemia egg as

seen during microscopic screening of sludge. green arrow =

P.tomentosa egg white arrow = artemia egg. 200x.

Mocho, J.P. Three-Dimensional Screen: A Comprehensive Approach

to the Health Monitoring of Zebrafish. Zebrafish. 2016 Jul;13 Suppl

1:S132-7. doi: 10.1089/zeb.2015.1200.

Screening pattern – 6 month period

Routine Health Monitoring

Origin of sample Months 1 & 4 Months 2 & 5 Months 3 & 6Histopathology and/or

PCR

Colony fish Collect carcasses of fish with clinical signsScreen 5 fish

< 6 months oldMonth 3: SMOP

Month 6: SMOP+SLOM*Pre-filtration sentinels Sample 10 fish

Sludge analysis 1 sample for PCRMycobacterium spp.,

P. tomentosa

Sump surface swab 1 sample for PCR Mycobacterium spp.

Feed PCR Mycobacterium spp.

Quarantine Screen imports (fish, sludge and/or clutch) or quarterly SMOP+SLOM

Mortality Monitor and increase number of fish sample if mortality increases N/A

*: At least 15 fish are tested quarterly by PCR or histopathology for routine health monitoring. Found dead fish are screened by PCR only. Speciation of Mycobacterium spp positive samples is required to differentiate between commensal and species reported as pathogenic for zebrafish.

Reporting

Aims of reporting

Historical data collection

Internal use to follow

Trends

Prevalence

Money spent wisely…

External use to ease transfer of lines

Check health status

Easy highlight of prevalent

pathogens

Facility description for

external use

Be aware of past

outbreaks and disinfection

Assess import risk by

understanding biosecurity

and husbandry of exporter

Scenarios

Screening pattern to achieve SPF status

Samples to achieve SPF status are in addition to the routine health

monitoring ones (SPF=Specific Pathogen Free)

As long as biosecurity measures are in place to prevent contamination,

6 quarterly sets of negative samples as described would qualify for the SPF

status

SPF screening pattern defined for

M. haemophilum and marinum

P. tomentosa

P. neurophilia

Example for SPF status – main room

Zebrafish only facility

1 main room with 1 recirculating system

Only bleached embryos from quarantine go to main system

RO water

10 000 litres

25 000 fish

pH 7.5

Conductivity 0.5 mS

GH 3, KH 2

27 ºC

14h light – 10h dark

10% daily water renewal

Live feed: rotifer and artemia

Dry feed

Example for SPF status - quarantine

Busy quarantine in separated and

dedicated room

All imports go to quarantine

Imports are triaged (refused if

suspicious)

Imports are screened and triaged

Treated, euthanized or kept

Flow through quarantine system

Fully barriered quarantine:

Dedicated full PPE:

Tyvek

Overshoes

Dedicated gloves

One way flow

Last animal duty of staff

Items are double bagged out

Tanks are hand cleaned,

autoclaved and then

machine washed

Example for SPF status – SPF screening pattern

additional quarterly samples required

P. neurophilia M. haemophilum M. marinum P. tomentosa

Colony fishPCR or

histopathology on 85 fish

PCR or histopathology on

30 fish

PCR or histopathology on

30 fish

PCR or histopathology on

15 fish

Sludge analysis1 sample per

system for PCR1 sample per

system for PCR1 sample per

system for PCR

Sump surface swab

1 sample per system for PCR

1 sample per system for PCR

Feed1 sample per

relevant feed for PCR

1 sample per relevant feed for

PCR

Mortality Increase number of samples if mortality increases

Routine & SPF screening pattern for

M. marinum, M. haemophilum and P. tomentosa

Origin of sample Months 1 & 4 Months 2 & 5 Months 3 & 6Histopathology and/or

PCR

Colony fish Collect carcasses of fish with clinical signsScreen >35 fishincl >5 that are< 6 months old Month 3: SMOP

Month 6: SMOP+SLOM*

Pre-filtration sentinels Sample 10 fish

Sludge analysis 2 samples for PCRMycobacterium spp.,

P. tomentosa

Sump surface swab 2 samples for PCR Mycobacterium spp.

Feed2 rotifer samples for

PCRMycobacterium spp.

Quarantine Screen imports (fish, sludge and/or clutch) SMOP+SLOM

Mortality Monitor and increase number of fish sample if mortality increases N/A

• More on zebrafish at the FELASA 2019 congress

• Fish health monitoring

• Euthanasia, anaesthesia, analgesia

• Husbandry and management of multi-species fish facilities

• International harmonization of zebrafish shipping regulations (Zoltan Varga)

• Severity classification for zebrafish and their immature form

• Refinements in disease modelling of zebrafish (Claire Allen, sponsored by ZHA)

• And plenty more including Cephalopods, Killifish, cave fish (D. Baumann)

• http://www.felasa2019.eu/resource/felasa2019/files/FELASA-Preliminary-Programme.pdf

THANK YOU

Chereen Collymore

Susan Farmer

Emmanuel Leguay

J-P Mocho (convenor)

Katy Murray

Nuno Pereira

[email protected]