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Sample collection and shipping: what's important and why


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Delivered 11 August 2015 by Dr. Deb Miller at a Southern Appalachia Bsal meeting in Asheville, NC, USA.

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Sample collection and shipping: what's important and why

  1. 1. Sample Collection and Shipping What’s important ____and why___ Deb Miller1,2 and Matt Gray1 1Center for Wildlife Health 2College of Veterinary Medicine
  2. 2. Collecting Animals
  3. 3. Enclosure (Pipe) Sampling Count Number of Dips Dip until No Larvae Captured after 10 dips Is probability of transmission affected?
  4. 4. 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
  5. 5. Methods: Cohousing 450 Total Animals Cohouse in buckets for 15, 30, or 60 minutes; 10%, 20%, or 40% infected; 50 animals per treatment
  6. 6. Results:  Cohousing Unpublished  Figured  Deleted  
  7. 7. 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  
  8. 8. Methods: Glove Change Swab individual
  9. 9. Results:  Glove  Changing Unpublished  Figured  Deleted  
  10. 10. Smokies Example Searching and Numbered Bags
  11. 11. Isolating Animals
  12. 12. Holding Containers One Individual per Container Plastic Bags 1-L or 2-L Plastic Tubs Mason Jars
  13. 13. Processing Station
  14. 14. Aseptic Processing Station People that collect do NOT process!!
  15. 15. ID and Morphometrics: Stations 1 and 2
  16. 16. Aseptic Processing Station Station 1 ID Rinsed & Labeled
  17. 17. Weigh SVL Aseptic Processing Station Station 2
  18. 18. Swabbing: Station 3
  19. 19. 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
  20. 20. 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
  21. 21. 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.
  22. 22. Photo courtesy Dale McGinnity , Nashville zoo What if you see a lesion?
  23. 23. watch?v=a5CtPrGOK8c
  24. 24. 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
  25. 25. Clipping: station 4
  26. 26. Tail Clip Aseptic Processing Station Station 4
  27. 27. Sample Storage Individual tubes LABEL!! Use marker that will not come off with Ethanol 90% EtOH or Dry Ice
  28. 28. Data Recording!!!: Station 5
  29. 29. Data Recording Aseptic Processing Station Station 5
  30. 30. Release: Station 6
  31. 31. Aseptic Processing Station Station 6 Releasing or Collecting Individuals
  32. 32. Whole animals
  33. 33. Biosecurity
  34. 34. Matt Gray, Debra Miller, and Amanda Duffus Diseases, Pathogens and Parasites Task Team Biosecurity Precautions: Disinfecting Procedures
  35. 35. Wear Disposable Gloves Gloves Rinsed with Distilled H2O Greer et al. (2009) Latex Vinyl Nitrile $12/box of 100
  36. 36. Disinfecting Equipment Scrape Mud and Scrub
  37. 37. 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
  38. 38. Amphibian Biosecurity References 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)
  39. 39. Shipping
  40. 40. Debra Miller and Matt Gray Diseases, Pathogens and Parasites Task Team Shipping Amphibians for Diagnostic Testing
  41. 41. Transporting Amphibians Most Diagnostic Labs Prefer Fresh Specimens if Possible Tent Design No Direct Contact with Dry Ice or Ice Packs
  42. 42. Preserving Animals 95% EtOH 10% neutral buffered formalin Separate Containers for Each Specimen! DO NOT USE GLASS FOR SHIPPING 50 mL
  43. 43. Shipping Animals (1) Call the Diagnostic Lab for Specific Instructions (2) Follow Courier Guidelines
  44. 44. Shipping Specimens Triple Packaging First Layer Label Each Layer!
  45. 45. Shipping Specimens Triple Packaging Second Layer Do not use Biohazard Bags (unless known to be infected with a BSL-2 agent) Absorbent Paper Towel
  46. 46. Shipping Specimens Triple Packaging Third Layer ThermoSafe* Polar Pack Place Cooler in Cardboard Box Only Use Dry Ice for Frozen Samples
  47. 47. 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
  48. 48. 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
  49. 49. Excellent Example
  50. 50. 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.
  51. 51. Tadpole liver Necropsied approximately 12 hours after death Tadpole liver Necropsied immediately after death
  52. 52. 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
  53. 53. Tadpole liver Collected fresh and shipped overnight with plenty of cold packs Tadpole liver Collected fresh and shipped overnight with too few cold packs
  54. 54. Questions?