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Keeping Pathogens out of Your Zebrafish Facility
Christopher M. Whipps & Carolyn ChangState University of New York,College of Environmental Science and Forestry (SUNY-ESF)Syracuse, NY
cwhipps@esf.edu
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IntroductionMichael L. Kent and Zoltán Varga
Introduction: Use of Zebrafish in Research and Importance of Health and HusbandryHealth
Christopher M. Whipps, Christine Lieggi, and Robert Wagner Mycobacteriosis in Zebrafish Colonies
Justin L. Sanders, Virginia Watral, and Michael L. Kent Microsporidiosis in Zebrafish Research Facilities
Jan M. Spitsbergen, Donald R. Buhler, and Tracy S. Peterson Neoplasia and Neoplasm-Associated Lesions in Laboratory Colonies of Zebrafish Emphasizing Key Influences of Diet and Aquaculture System Design
Michael L. Kent, Claudia Harper, and Jeffrey C. Wolf Documented and Potential Research Impacts of Subclinical Diseases in Zebrafish
Marcus J. Crim and Lela K. Riley Viral Diseases in Zebrafish: What Is Known and Unknown
HusbandryStephen A. Watts, Mickie Powell, and Louis R. D'Abramo
Fundamental Approaches to the Study of Zebrafish NutritionAndrzej Nasiadka and Matthew D. Clark
Zebrafish Breeding in the Laboratory EnvironmentCarole Wilson
Aspects of Larval RearingChristian Lawrence and Timothy Mason
Zebrafish Housing Systems: A Review of Basic Operating Principles and Considerations for Design and FunctionalityMonte Matthews and Zoltán M. Varga
Anesthesia and Euthanasia in ZebrafishGeneral
George E. Sanders Zebrafish Housing, Husbandry, Health, and Care: IACUC Considerations
Stephen A. Smith Zebrafish Resources on the Internet
ILAR Journal Zebrafish Health and HusbandryVolume 53 Issue 2 June 2012
http://ilarjournal.oxfordjournals.org/content/53/2.toc
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0.0%
10.0%
20.0%
30.0%
40.0%
50.0%
60.0%
Pseudoloma neurophilia
Mycobacteriosis
Neoplasia
Hepatic megalocytosis
Myxozoans
Nephrocalcinosis
Gill epithelial hyperplasia and hypertrophy
Bacterial infection (non-acid fast)
Pseudocapillaria tomentosa
Other helminths
Pleistophora hyphessobryconis
Fungal infection
ZIRC Health Services Data (2006-2010)% facilities positive for a particular disease (5 yr. average)
ZIRC disease manual (https://zebrafish.org/health/diseaseManual)
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ZIRC Health Services Data (2006-2010)Just the infectious diseases
0.0%
10.0%
20.0%
30.0%
40.0%
50.0%
60.0%
Pseudoloma neurophilia
Mycobacteriosis
Myxozoans
Bacterial infection (non-acid fast)
Pseudocapillaria tomentosa
Other helminths
Pleistophora hyphessobryconis
Fungal infection
ZIRC disease manual (https://zebrafish.org/health/diseaseManual)
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Edwardsiella ictaluri• Enteric Septicemia of Catfish• Hole in the Head Disease
http://www.ag.auburn.edu/fish/mediagallery/2013/08/13/bacterial-diseases-in-warmwater-fishes-37/
CM Whipps
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Edwardsiella ictaluri
Challenges
• Virulent
Advantages
• Acute infections – likely ID in quarantine
• Avoid pet store/ commercial fish
• Not environmentally stable
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Pseudocapillaria tomentosa
http://zebrafish.org/health/Disease_images/CapillariaWetMounts4.jpg
• Nematode• Infects intestinal wall• Macroscopic (4-12 mm)
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Pseudocapillaria tomentosa
Challenges
• Environmentally stable eggs
• Direct transmission
Advantages
• Screen water for eggs
• Can be treated
Capillaria egghttp://www.cdc.gov/dpdx/intestinalCapillariasis/gallery.html#eggs
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• Associated with kidneys/urinary system• Nephritic ducts packed with plasmodia
Myxozoanosis – Myxidium streisingeri
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Myxozoanosis – Myxidium streisingeri
Image: Kent, M.L., and T.T. Poppe. 1998. Diseases of seawater netpen-reared salmonids. Pacific Biological Station Press, Nanaimo, British Columbia, Canada
• Typical myxozoan life cycle has 2 hosts
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Image: http://seedmagazine.com/place/place_labs-at-night.html#0
Myxozoanosis – Myxidium streisingeri
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Sump/Biofilter colonized by oligochaetes?
Myxozoanosis – Myxidium streisingeri
Image c/o Mike Kent
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Many Remaining Questions
Myxozoanosis – Myxidium streisingeri
Challenges
• Unknown life cycle
• Impact on host??? (kidney function studies?)
Advantages
• Likely requires oligochaete host
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Signs of Disease
Skinny fishBent spine
Pseudoloma neurophilia
(Matthews et al. 2001 J. Eukaryot. Microbiol., 48(2), 227–233)
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Pseudoloma neurophilia
Figure from:Sanders et al., 2013 - Verification of Intraovum Transmission of a Microsporidium of Vertebrates: Pseudoloma neurophilia Infecting the Zebrafish, Danio rerio. PLoS One
Microsporidian spore
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Pseudoloma neurophilia
Challenges
• Environmentally stable/resistant spores
• Fish may not have clinical signs (chronic infection)
• Spores intra-ovum
• Spores resistant to disinfectants (more to come…)
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Mycobacteriosis
• Swollen belly
• Emaciation
• Skin ulcers
• General malaise
• Exopthalmia
Signs of mycobacteriosis
None at all?
• Granuloma
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Mycobacteriosis
Caused by Mycobacterium spp.
• Over 100 species in the genus
• Infect wide range of animals
• Acute to chronic infections
• Notoriously difficult to treat
• Fastidious
• Environmentally derived
• Form biofilms
http://www.futura-sciences.com/magazines/sante/infos/actu/d/vie-afrique-situation-urgence-tuberculose-6992/
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Mycobacteriosis
Mycobacterium spp. of zebrafish
Zebrafish• M. chelonae
• M. fortuitum
• M. marinum
• M. haemophilum
• M. peregrinum
• M. abscessus
• M. gordonae
• M. fortuitum-like
• M. marinum-like
• Mycobacterium spp....
Culture and PCR of infected tissues
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Mycobacteriosis
Many species of Mycobacterium
•Some common, others less so
•M. marinum and M. haemophilum more serious pathogens
Variation within hosts
•TU fish more susceptible
•Other lines of fish with some genetic resistance?
Different bugs + Different fish = Different Impacts
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Mycobacteriosis
Direct cause of mortality
Contributing cause of mortality
Morbidity
Source of non-protocol variation
Safety of employees
Mycobacterium marinum zoonotic
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Transmission
• Sharing of fish between facilities
• Vertical transmission?
http://www.epa.gov/region5/superfund/ecology/images/fishcartoon.gif
• Feeding on infected tissues•Important to remove “sick” fish
• ‘Ubiquitous’ in aquatic systems• Role of Biofilms in tsm
http://unsolvedmysteries.oregonstate.edu/microarray_02
Mycobacteriosis
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Zambrano (2005) Cell, 123(5): 762
http://paulorwin.blogspot.com/
Biofilms harbor and protect mycobacteria
Biofilms
Tank swabs
Water sump
Food (-ve)
Sampling at a Zebrafish facility
Mycobacteriosis
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Biofilm transmission... (?)-When tanks are cleaned, does the suspension of biofilm in water
increase transmission of mycobacteria??
Scrubbed+ debris
siphoned laterEntire tank replaced
Low risk? Higher risk?
Scrubbed+ increased
flow to flush debris
Low risk?
Murray et al. (2011 Comp Med)
Fish density also important (crowding stress)
Mycobacteriosis
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Biofilms
Fish
‘DNA fingerprinting’Whipps et al. (2008)
DAO 82, 45-54
Several other biofilm mycobacteria
are not found in fish
(not shown)
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Mycobacteriosis
Challenges
• Several species
• Acute to chronic infections
• Form surface biofilms (no need for fish)
• Waxy hydrophobic cell wall (tough to kill)
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Prevent Introductions
• Source History
• Quarantine
• Disinfected Eggs Only
• Specific Pathogen-Free Fish
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Prevent Introductions – SPF fishSpecific Pathogen Free fish
•Artificial Diet•Controlled personnel access/procedures•Monitoring/screening population and morts•‘Retiring’ fish at 1yr •Sentinel screening
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Prevent Introductions – SPF fish
Pseudoloma-free fish availableMycobacteria difficult to eliminate
Table from Kent et al. (2011)
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Prevent Introductions - Quarantine
• Separate System
• 2 weeks – 3 months (depending on pathogen of concern or history of fish)
• Screening recommended for:• Morts• subsample
• Regular cleaning between housing incoming fish
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Prevent Introductions - Quarantine
• Weeks • Likely good for acute infections• i.e., Edwardsiella ictaluri
• 6 wks - months• Mycobacterium haemophilum• Mycobacterium marinum
• May or may not see signs of:• Other Mycobacterium spp.• Pseudocapillaria tomentosa• Pseudoloma neurophilia• Myxidium streisingeri
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Prevent Introductions – Eggs Only
Quarantine
Surface disinfect eggs /embryos
Main Facility
Shipment from lab X
Nursery
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Prevent Introductions – Eggs Only
Focus testing on mycobacteria•Very common•Same methods likely effective on other bacteria
Notes on other pathogens•Pseudocapillaria
•Eggs very resistant•Screening in quarantine should be effective
•Myxidium•Not known to be associated with eggs•Eggs-only should avoid
•Pseudoloma•Spores very resistant•Intra-ovum a possibility•Screening parents important
Sanders et al., 2013 -PLoS One
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What do you do when mycobacteria detected?
Depopulate
Disinfect system1000ppm bleach >1hr
Re-establish via ‘eggs only’ policy
Disinfection - Mycobacteria
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Follow up
Mycobacteria re-establish in biofilms within weeks after bleaching (and fish added)
A few cases of infected fish since (M. chelonae)
No further cases of M. haemophilum (as of Feb 2015)
Disinfection - Mycobacteria
ILAR Journal 2012, 53(2) 95-105
Described outbreak of M. haemophilumDepopulation, re-establishment
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Prevent Introductions – Eggs Only
Some treatments:•Bleach (25-50ppm, 5-10min)•Bleach (150ppm, 5-10min)•PVP-Iodine (100ppm, 2min) + bleach (30ppm, 10min)
Rinse or inactivate bleach with water/sodium thio
Surface disinfect
General Method of Surface Disinfection
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Chlorine Bleach Hydrogen Peroxide Povidone Iodine
Current
Usage
• Zebrafish
• 5-10 min @ 25-150
ppm
• Catfish
• 450-900 ppm in a bath
or flow-through
• Salmonids &
Zebrafish
• 2-5 min @ 100 ppm
Our
Study
• Clorox®
• Unbuffered &
Buffered
• CDFB
• 50, 100 & 150 ppm
• 5 and 10 minutes
• Drugstore brand
• Unbuffered
• 15 000 & 30 000 ppm
• 5 minutes
• Drugstore brand
• Unbuffered
• 12.5-100 ppm
• 5 minutes
Mycobacteria - Disinfectant Choice
Prevent Introductions – Eggs Only
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Not all disinfectants are equalPercent survival differs significantly (p<0.05) between treatments.
Mycobacterium chelonae
• Less than 1% survival for:
• Buffered Bleach
• PVP-I
Bleach (CDFB)
Bleach (MILQ)
Hydrogen Peroxide
PVP Iodine
Disinfection - Mycobacteria
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Not all species are equally susceptiblePercent survival differs significantly (p<0.05) between species.
Mycobacterium chelonaeMycobacterium abscessus
Mycobacterium peregrinumMycobacterium gordonae
Disinfection - Mycobacteria
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One week post plating differences in colony growth are observed in representative
(A) M. abscessus, (B) M. chelonae, (C) M. gordonae, and (D) M. peregrinum
control plates and (A’, B’, C’, D’) 25 ppm iodine treatment plates.
Disinfection - Mycobacteria
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Disinfection - Mycobacteria
Chlorine bleach•Variable by species, 100ppm+ for 10 min largely effective•Buffered (to pH7) chlorine very effective at 50ppm+
Peroxide•Moderately effective at 3% •This level likely lethal to fish
Iodine•25ppm+ effective
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Impacts of Germicides on Fish - Chlorine
•Impacts of chlorine (0, 6.25, 12.5, 25, 50, 100 ppm)•5 vs. 10 minutes•Fish at 6 and 24 hours post fertilization•Buffered and unbuffered chlorine
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Kent et al. (2014) DAO 107; 235-240
Mortality (buffered chlorine)
Mortality (unbufferedchlorine)
Malform(buffered chlorine)
Malform(unbufferedchlorine)
6 hpf – 5min exposure 6 hpf – 10min exposure
24 hpf – 5min exposure 24 hpf – 10min exposure
Impacts of Germicides on Fish - Chlorine
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Impacts of Germicides on Fish - Chlorine
Kent et al. (2014) recommend 100ppm chlorine (unbufferred):•10min for fish 6 hpf•5 min for fish 24 hpf
Mycobacterium peregrinumMycobacterium gordonae
Our data on Mycobacterium spp.
52
Viability of embryo disinfection with povidone-iodine tested:
2 Developmental Stages• 6 and 24 hours post-fertilization (hpf)
3 Treatment Concentrations• 12.5, 25, and 50 ppm
2 Treatment Times• 2 and 5 min
2 Brands of PVP-I• Drugstore and Ovadine (Western Chemical)
Impacts of Germicides on Fish - Iodine
Modeled on chlorine protocol of Kent et al. (2014)
Chang, Amack, Whipps (In Prep)
Incubate in 96 well plate at 28.5°C
until 5 dpf
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RO H2O
RO H2O
RO H2O
RO H2O
PVPISol’n
E2 Medium
6 hpf
24 hpf
Embryos at 2 Developmental Stages
Impacts of Germicides on Fish - Iodine
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Impacts of Germicides on Fish - Iodine
Concentration and duration of treatment solutions has an effect on zebrafish health at 6 hpf exposures.
Percent mortality differs significantly (p<0.05) for 50 ppm 5 min treatments.
NS mortality between buffersfor 25 ppm at 6 hpf
Drugstore Brand (Unbuffered) Ovadine (Buffered)
55
Impacts of Germicides on Fish - IodineConcentration and duration of treatment solutions has an effect on
zebrafish health at 24 hpf exposures.Percent mortality differs significantly (p<0.05) for 50 ppm 5 min treatments.
Ovadine (Buffered)
NS mortality between buffersfor 12.5 & 25 ppm at 24 hpf
NS between treatments
Drugstore Brand (Unbuffered)
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Rinsing alone is not enough to remove mycobacteria
RO H2O
RO H2O
RO H2O
RO H2O
PVPISol’n
E2 Medium
RO H2ORO H2O RO
H2ORO H2O
25 ppm 5 min PVPI
E2 Medium
105 cfu/ml
Incubate:Overnight
28.5°C50 rpm
Average518 571 cfu/mL
Average56 666 cfu/mL
No Growth
Average2611 cfu/mL
No Growth No Growth
Average2383 cfu/mL
Control
Impacts of Germicides on Fish - Iodine
Rinse only
Iodine & rinse
57
Impacts of Germicides on Fish - IodineFresh Preparation of Iodine is criticalAvoid light exposure
Test solutions after 24 hrs
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• Treatment with PVPI not harmful to embryos except 50 ppmfor 5 min
• 6 hpf embryos were generally more resilient to treatments compared to 24 hpf
• No difference in embryo mortality between unbuffered and buffered iodine at 12.5 and 25 ppm
• Rinsing embryos is not enough to eradicate mycobacteria
Recommendations for Egg Disinfection- Iodine
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• PVPI may be a better alternative to chlorine bleach for preventing mycobacterial spread between facilities
• Facilities should incorporate a embryo disinfection protocol to suit their needs– Take into account:
• Source
• Strain
• Age
• Generally recommend disinfecting embryos at 25 ppm PVPI for 5 min– Modified as necessary
Recommendations for Egg Disinfection- Iodine
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