Sample collection and shipping: what's important and why
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Transcript of Sample collection and shipping: what's important and why
Sample Collection and
Shipping
What’s important ____and why___
Deb Miller1,2 and Matt Gray1
1Center for Wildlife Health 2College of Veterinary Medicine
Collecting Animals
Enclosure (Pipe) Sampling
Count Number of Dips
Dip until No Larvae Captured after 10 dips
Is probability of transmission
affected?
Co-housing Animals
Mean = 0.8 – 0.9 contacts/min for 40 tadpoles/m2
(10 tadpoles per 5-gal [19 L] bucket)
All should contact each other in 9 Minutes
10, 20, 40% X
15, 30, 60 min What about
gloves?
Ranavirus Example
Methods: Cohousing 450 Total Animals
Cohouse in buckets for 15, 30, or 60 minutes; 10%, 20%, or 40% infected; 50 animals per treatment
Results: Cohousing
Unpublished Figured Deleted
Methods: Glove Change (600 total animals)
• Change gloves or don’t change gloves • Vary density of infected individuals
5%
40%
10%
20%
100 animals per treatment for 14 days 600 animals total
Methods: Glove Change
Swab individual
Results: Glove Changing
Unpublished Figured Deleted
Smokies Example Searching and Numbered Bags
Isolating Animals
Holding Containers One Individual per Container
Plastic Bags
1-L or 2-L Plastic Tubs
Mason Jars
Processing Station
Aseptic Processing Station
People that collect do
NOT process!!
ID and Morphometrics: Stations 1 and 2
Aseptic Processing Station Station 1
ID Rinsed & Labeled
Weigh SVL
Aseptic Processing Station Station 2
Swabbing: Station 3
Type of swab: 1. Individually packaged. 2. Wire or plastic shaft (plastic often is designed with breaking point so no need for additional instrument to cut the shaft when placing in tube). NOTE: wood shafts may interfere with molecular testing 3. Microtip is easier for smaller species
Swabbing Anurans Bd and Bsal Surveillance
Non-lethal Techniques: Brem et al. (2007)
Swabbing Preferred
Swab 5 times in 5 locations
A. Cressler, USGS
A. Cressler, USGS
• Ventral feet
• Inner thighs
• Ventral Abdomen
Larvae: Swab Oral Cavity 5 times
Adults: if needed, lightly rinse with sterile water to remove any dirt/debris
Store:
1. Dry swab (refrigerate or freeze)
2. 70% EtOH if you cannot dry the swab or keep it cool
Swabbing Salamanders Bd and Bsal Surveillance
Ventral surfaces (all feet, belly, tail); 5 times each, often they will grab the swab and you can twirl it while they hold onto it.
Photo courtesy Dale McGinnity , Nashville zoo
What if you see a lesion?
https://www.youtube.com/watch?v=a5CtPrGOK8c
Sample Storage Individual tubes (if shipping, USE SCREW TOP TUBES)
LABEL!! Use marker that will not come off with ethanol/alcohol
90% EtOH or keep cool
Clipping: station 4
Tail Clip
Aseptic Processing Station Station 4
Sample Storage Individual tubes
LABEL!! Use marker that will not come off with Ethanol
90% EtOH or Dry Ice
Data Recording!!!: Station 5
Data Recording
Aseptic Processing Station Station 5
Release: Station 6
Aseptic Processing Station Station 6
Releasing or Collecting Individuals
Whole animals
Biosecurity
Matt Gray, Debra Miller, and Amanda Duffus Diseases, Pathogens and Parasites Task Team
Biosecurity Precautions: Disinfecting Procedures
Wear Disposable Gloves
Gloves Rinsed with Distilled H2O Greer et al. (2009)
Latex Vinyl
Nitrile
$12/box of 100
Disinfecting Equipment Scrape Mud and Scrub
Disinfecting Equipment Spray Bottle or Immersion
• Bleach >4% • EtOH >70% • Virkon >1%
• Nolvasan >0.75%
Johnson et al. (2003), Bryan et al. (2009), Gold et al. (2013)
$50/ bottle
Amphibian Biosecurity References
www.separc.org
Dodd, C. K., editor. 2009. Amphibian Ecology and Conservation: A handbook of techniques. Oxford University Press, UK.
ISBN 9780199541188
Pessier, A.P. and J.R. Mendelson (eds.). 2010.
Miller et al. (2015)
Shipping
Debra Miller and Matt Gray Diseases, Pathogens and Parasites Task Team
Shipping Amphibians for Diagnostic Testing
Transporting Amphibians
Most Diagnostic Labs Prefer Fresh Specimens if Possible
Tent Design
No Direct Contact with Dry Ice or Ice
Packs
Preserving Animals
95% EtOH 10% neutral buffered formalin
Separate Containers for Each Specimen! DO NOT USE GLASS FOR SHIPPING
50 mL
Shipping Animals
(1) Call the Diagnostic Lab for Specific Instructions
(2) Follow Courier Guidelines
Shipping Specimens Triple Packaging
First Layer
Label Each Layer!
Shipping Specimens Triple Packaging
Second Layer Do not use Biohazard Bags
(unless known to be infected with a BSL-2 agent)
Absorbent Paper Towel
Shipping Specimens Triple Packaging
Third Layer
ThermoSafe* Polar Pack
Place Cooler in Cardboard Box
Only Use Dry Ice for Frozen Samples
List of Contents & MSDS if Needed
• Detailed list of all contents • Description and location of die-off
• Requested services
• Contact information of the shipper ▪ General Pathological Screening ▪ Specific Pathogen Testing
MSDS Required
▪ EtOH or Formalin
Labeling University of Tennessee (Matthew J. Gray, Ph.D.) Department of Forestry, Wildlife and Fisheries Institute of Agriculture 274 Ellington Plant Sciences Building Knoxville, TN 37996-4563 USA
Debra L. Miller, D.V.M., Ph.D. Veterinary Diagnostic Laboratory The University of Georgia
43 Brighton Road
Tifton, GA 31793-1389
Contents: Exempt Animal Specimen (Refrigerate upon Arrival)
Phone: 229-386-3340
(5 lbs) (>1 L or 33.8 oz) (<500 mL, <30 mL Container)
Dangerous Goods Excepted Quantity Dangerous Goods in
Hazardous Quantity
No statement required for
environmental samples
Excellent Example
Why? � Culture for pathogen (virus isolation or fungal
culture or if looking for the causative bacteria): often is not possible from autolysed tissues because of growth/contamination of postmortem organisms.
� PCR: DNA becomes compromised and can be harder to extract and detect with autolysis, contamination (bacterial overgrowth).
� Histology!! Huge difference in what can be seen under the microscope.
Tadpole liver Necropsied approximately 12 hours after death
Tadpole liver Necropsied immediately after death
Adult salamander liver Dead for greater than a day before fixing in alcohol & shipping
Adult salamander liver Necropsied immediately after death
Adult liver Fixed in ethanol shortly after death and then shipped
Tadpole liver Collected fresh and shipped overnight with plenty of cold packs
Tadpole liver Collected fresh and shipped overnight with too few cold packs
Questions?