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Unpublished  Figured  Deleted  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Unpublished  Figured  Deleted  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?