Designing ranavirus surveillance studies: careful considerations

1. Designing
Ranavirus Surveillance Studies
University of Tennessee
Center for Wildlife Health
Department of Forestry, Wildlife and Fisheries
Matthew J. Gray

2. Outline
III.Amphibian Surveillance Example
• Importance of “Aseptic” Sampling
I. Uses and Biases of Surveillance Data
• What are the limitations?
I. Sample Design and Required Sample Size
IV. Disinfecting Procedures
Part One
Part Two
Part Three

3. Goal of Surveillance
To detect a pathogen/disease or obtain an
unbiased estimate of pathogen/disease
prevalence (or incidence) in a population
Pathogen Prevalence
An estimate of the proportion of individuals in
a population that are infected with a
pathogen
Infection Disease
Challenge
Studies:
Background
Levels
Incidence

4. Detection Biases
Dead vs. Morbid vs. Health
Vegetation
Water Depth
Seasonality
Sample Frequency?

5. Uses of Surveillance Data
Occurrence and Distribution
Evidence of Emergence
Pathogen or disease that is increasing in
distribution, prevalence, or host range
Baseline
http://www.bd-maps.net/

6. Uses of Surveillance Data
Evidence of Hotspots
Identification of Mechanisms of Emergence
Elevated?
$$
Population
Effects?

7. Uses of Surveillance Data
Disease Intervention Strategies
1. Transmission: DD
2. Distribution expansion
3. Stressors
Interrupt Host-Pathogen Cycle
Reduce Stressors
OIE: Routes of Entry
4. Novel
(Introduced)
Isolate

8. Surveillance Designs
Random Sampling
All individuals or surveillance
locations have an equal
probability of being sampled
Random Numbers Table or
Programs
Collecting Unbiased, Representative Sample
1) Sample all captured individuals
2) Sample up to n captured individuals
3) Randomly select individuals from sample of n
captured individuals
Avoid Systematic or Haphazard Sampling
Objective?

9. Detect a Pathogen
Estimating Required Sample Size
•Assumed Pathogen Prevalence Level (APPL)
•Estimated Host Population Size
•Confidence in detection (95%)
Information Needed
50
100
250
500
2000
>100,000
20
23
25
26
27
30
35
45
50
55
60
60
50
75
110
130
145
150
Population Size 10% APPL 5% APPL 2% APPL
(Amos 1985,
Thoesen 1994)
epiR (epi.detectsize)

10. Precise Estimate of Prevalence
Estimating Required Sample Size
n p p
d
= −





( )
.
1
1 9 6
2
Prevalence from a
previous study
d = error in estimation
Zα/2 =1.96
(95% confidence)
p =
“Error in Estimation” is the amount of error you are
willing to tolerate in your estimate of prevalence
p = 85%
Error = 5%
n =





 ≈( . ) ( . )
( . )
.
0 8 5 0 1 5
1 9 6
0 0 5
1 9 6
2
p = 85%
Error = 10%
p = unknown
Error = 10%
n =





 ≈( . ) ( . )
( . )
.
0 8 5 0 1 5
1 9 6
0 1 0
4 9
2
n =





 ≈( . )
( . )
.
0 2 5
1 9 6
0 1 0
9 6
2
What happens if estimation error increases?
What happens if prevalence is near 0.5?
0.01< P(1-p) < 0.25
Two
Proportions

11. Wildlife Surveillance:
An Amphibian (Ranavirus) Example
University of Tennessee
1
Center for Wildlife Health
2
CVM Department of Pathobiology
Matthew J. Gray1
and Debra L. Miller1,2

12. Enclosure (Pipe) Sampling
Count Number of Dips
Dip until No Larvae
Captured after 10 dips
Is probability of
transmission
affected?

13. Co-housing Animals
Mean =
0.8 – 0.9 contacts/min for 40 tadpoles/m2
(10 tadpoles per 5-gal [19 L] bucket)
Uniform Contact = 9 Minutes
10, 20, 40%
X
15, 30, 60 min
What about
gloves?
Ranavirus Example

14. Holding Containers
One Individual per Container
Plastic Bags
1-L or 2-L
Plastic Tubs
Mason Jars

15. Area Searches
Search under Cover Objects
Record Time and Observers: CPU
Return to Approximate Capture Location

16. Aseptic Processing Station
People that
collect do
NOT
process!!

17. Aseptic Processing Station
Station 1
ID Rinsed & Labeled

18. Weigh SVL
Aseptic Processing Station
Station 2

19. Swabs
Aseptic Processing Station
Station 3

20. Tail Clip
Data Recording
Aseptic Processing Station
Stations 4 and 5

21. Aseptic Processing Station
Sample Storage
90% EtOH or Dry Ice

22. Aseptic Processing Station
Station 6
Releasing or Collecting
Individuals

23. Collecting Additional Data
Water
Quality
Vegetation
Apparent
Stressors

24. Take the Opportunity to Instruct

25. Questions??
Gray: mgray11@utk.edu
Miller: dmille42@utk.edu

26. Matt Gray, Debra Miller, and Amanda Duffus
Diseases, Pathogens and Parasites Task Team
Biosecurity Precautions:
Disinfecting Procedures

27. Wear Disposable Gloves
Gloves Rinsed with Distilled H2O
Greer et al. (2009)
Latex Vinyl
Nitrile
$12/box of 100

28. Disinfecting Equipment
Scrape Mud and Scrub

29. 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

30. 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)

31. Questions??
Gray: mgray11@utk.edu
Miller: dmille42@utk.edu
Duffus: aduffus@gordonstate.edu