zebrafish health monitoring: recommendations from the
TRANSCRIPT
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