SAMPLING SIZE IN SCREENING OF DISEASES IN SHRIMP
From: robinliew [mailto:robinliew@myjaring.net]
To: shrimp@yahoogroups.com
Sent: 24 February 2003
QUESTION:
For general screening of diseases in shrimp, what is the acceptable sample
size needed, say for a population > 100,000 PL's to be
statistically acceptable.
Do we collect sample size that will give 95% confidence level
(if disease is present) ? In this case, 150 specimen or
60 specimen with assumption of 2 % or 5% infection prevalence. Some
hatcheries claim their PL's were screened for WSV but not able to provide
any statistically acceptable results.
Would appreciate if members can provide some possible standards or method
that will be statistically acceptable.
e-mail:
robinliew@myjaring.net
***************
comments 1 :
The international standard supported by the Office
International des
Epizooties (World Organisation for Animal Health) is demonstrating that the
pathogen (WSV) is present at less than 2% prevalence with 95% confidence.
Having said that, the actual sampling regime is harder to specify.
With a "perfect test", this would require the testing of 150
individuals and obtaining a negative result with each. Unfortunately,
the perfect test has not yet been produced and the specificity (propensity
to yield false positive results) and sensitivity (propensity to yield false
negative results) of most tests are not known. From the few tests that
have been validated, it appears that the sensitivity and specificity of a
"typical" PCR test is between 95-98%. This does not include
any loss of sensitivity or specificity due to lack of experience with the
operators.
The point that I would like to make is that the much discussed figures of 60
and 150 are not accurate because the diagnostic tests are not perfect.
The misuse of these figures is so widespread and concerning that the OIE has
totally rewritten the chapters on surveillance in the 2003 edition of their
"Diagnostic Manual" to remove these figures.
If one accepts that a WSV PCR test had similar performance characteristics
to other PCR tests ie Sensitivity = 95% and Specificity = 95% then including
these figures, the population size (100,000) and the expected prevalence
(2%) in a computer program called FREECALC (see www.ausvet.com.au
), the sample size required is 1854
samples with no more than 108 positive results observed. This is often
a difficult concept to grasp that because the test is not perfect, the
results will include results apparently positive for WSV even in the absence
of the actual virus in the test population. This happens because the test is
not perfect and a number of false positives are observed. In a recent survey
of crustaceans in Australia, we observed a rate of approximately 3% false
positive results which were all subsequently checked for accuracy by a
second laboratory.
Clearly, for screening for commercial purposes, testing 1800 individuals is
not realistic from an economic stand point, even if the samples are pooled
in groups of 10 and tested as batches.
An alternative to consider depends on your geographic location and your
farm's WSV disease status. If you are in a WSV endemic area, then
management of the virus rather than complete avoidance may be appropriate.
Studies in Thailand and Taiwan have shown that PLs that are negative for WSV
after the first step of a nested PCR test generally survive the grow-out
period without a disease event even if they are positive after the second
step of the nested PCR. In contrast, the PLs which are positive after
the first step of the PCR usually have disease problems during growout.
There are several scientific publications discussing this available in the
literature. There are of course no guarantees and results are
dependant upon water quality, stocking rates and a range of other
environmental factors.
Iain East
Aquatic Animal Health
Office of the Chief Veterinary Officer
Agriculture, Fisheries and Forestry - Australia
GPO Box 858
CANBERRA ACT 2601, Australia
Phone: 61-2-6272-3106
FAX: 61-2-6272-3150
e-mail:
iain.east@affa.gov.au
***************
comments 2 :
The alternative solution to produce "clean
seed" for shrimp farming is to start with certified pathogen free (SPF)
broodstock in a biosecure hatchery.
Jim Wyban PhD
High Health Aquaculture Inc.
tel/fax: 808.982.9163
email: wyban@gte.net
www.hihealthshrimp.com
***************
comments 3 :
What would then constitute a reasonable sampling size
to certify "clean seed" and frequency of screening ?
Robin
e-mail:
robinliew@myjaring.net
***************
commeNts 4 :
Jim Wyban is right that SPF broodstock is the
ultimate solution to this
deadly problem. Further success is assured if the farm also operates in a
biosecure manner. Even if your larvae are negative to WSSV, cross
contamination could spoil your day.
Chandrasekar
e-mail:
aqua@omanfisheries.com
***************
comments 5 :
As an addendum to the sample size posting, one of
Australia's leading epidemiologists suggested that I add the following to
the posting on the use of imperfect tests.
In terms of the expected number of positives in a free population evaluated
with an imperfect test, what would be expected for
a quantitative assay (eg an antibody ELISA for FMD in cattle) is a
smattering of low titre positives with no particular pattern obvious.
However, if these (the positive results) were geographically clustered, then
it would be a reason to do some additional investigation.
The major point is not to always takes tests at face value but to look for
problems. The paragraph above would equally apply to a PCR test and
several strong positive results or clustering of positive results would
certainly warrant further investigation.
Iain
e-mail:
iain.east@affa.gov.au
***************
comments 6 :
We are planning to use a template only WSSV PCR Kit,
single tube and single step reaction (unlike nested, that requires two
step). Only extract the DNA material and transfer into reaction tube and run
PCR. The results will be qualitative - positive or negative only.
Your info regarding PL's that are -ve (first step nested PCR) and +ve
(second step of the nested PCR) and vice versa is really interesting.
Our main concern is the PL's status from hatcheries since not all
farms have their own hatcheries (as such, unable to ascertain all
procedures and steps are adhered to unless, we inspect them regularly).
Hatcheries would have screened their PL's. We want to screen to
confirm before stocking. The farm may be WSV free previously but problems
occur when supplied PL's are not WSV free. Hatcheries may (not all)
mix or take PL's from a few different tanks to supply to a farm. Same
problem with antibiotic tainted feed. Supplier say everything OK since
assurance was given by the upstream supply chains.
How to minimise the risk ? Is there a proper "auditing"
system that has been developed for farms.
Robin
e-mail:
robinliew@myjaring.net
***************
comments 7 :
Your request raises the issue of what is practical
and what actually happens in a commercial environment. I cannot speak
about the reputation and practices of hatcheries in your area because I am
not familiar with them and in fact, I don't even know what country your farm
is located in.
There are some steps that you can take that will increase the chances of
obtaining quality PLs. I can only suggest that you consider these.
1. Only deal with a reputable hatchery that has no history of WSV (or other
diseases)
2. Establish exactly which batches of PLs that you would like to purchase
and agree with the hatchery on how they will be stored/maintained until you
have completed the testing (they should be kept away from other batches and
should not share water circulation)
3. Have your PCR conducted by experienced personnel that have recent
experience with the exact test that you are using. PCR is not an easy
technique to use without experience
4. If your PCR shows a positive test, don't buy the PLs - go to
another batch or another hatchery
5. If the hatchery claims that they have tested the PLs and that they are
free of WSV, insist on a health certificate that states that the PLs are
free of WSV (this may give you some legal redress if a problem occurs).
On top of this, you should have completed all the standard management
practices of a complete dry-out of your ponds, removal of any dead stock
from the bottom of the pond, liming of the pond, screening of inlet water
etc.
As I said in my previous post, nothing will guarantee that disease won't
occur, you can only take reasonable precautions and adopt best management
practices to reduce the risk.
Iain
e-mail:
iain.east@affa.gov.au
***************
comments 8 :
Do you take PLs from the hatchery directly or through
some sort of supply chain like nurseries? what I understand from your mail
is you are taking your PLs from intermediate sources. Sorry if I understood
wrongly.
The point is to obtain your PLs regularly from a trusted hatchery directly.
In general the cross contamination in a hatchery is an unusual phenomenon
compared to nursery operators. Once you select your PLs in a hatchery tank
and screened for any viral infections, insist on taking your PLs from the
same tank. If a hatchery regularly screens their brood stock and larvae,
then the chances of infection in other tanks are less likely. In case you
are the one insisting on screening of larvae and you are doing so yourself,
then you need to be more careful by insisting the hatchery staff to avoid
cross contamination. As I mentioned earlier, cross contamination in any
stage could spoil your hard work.
Chandrasekar
e-mail:
aqua@omanfisheries.com
***************
comments 9 :
I don't have a precise number to answer your question
about how many PLs you need to sample to demonstrate the absence of specific
viruses. But I want to pick up Jim Wyban's thread about the advantages of
the SPF approach to providing 'clean' seed. The approach that is going to
give the most reliable results in the long run is not the testing of
individual batches of PLs as they roll off the production line but the
inspection of the hatchery that produces the PLs and checking the systems
that are in place to maintain biosecurity.
Some places, such as Texas, are very strict about the PLs they let in but
they do not in fact require that every batch be screened by PCR. What they
do require is that the hatchery involved routinely submits samples for
testing and that a disease-free track record is built up. One problem with
testing the PLs (typically PL 5-15) that leave the hatchery is that at this
stage they are not very susceptible to viral attacks. So to increase the
chance of detecting virus, samples of more susceptible PL 30 - 40 (from
earlier batches) must also be submitted. What's more these larger PLs must
be grown in unpurified water taken directly from a maturation tank, be fed
with the remains of any freshly dead broodstock, and ideally they should be
of a genetic strain that has not been selected for viral resistance. These
measures are designed to maximize the chances of sending contaminated PLs
for testing, and to show that the whole hatchery is 'clean' rather than just
its output.
PCR screening, particularly for WSSV, is plagued by the problem of false
positive results. Statistical theory (as explained in detail by Iain East)
suggests that a certain frequency of false positive results is normal in
'clean' seed. But this undermines the whole screening process if a few
positive results are ignored and not used to reject the PLs. One way that
this problem can be contained is to submit samples for histological analysis
along with samples for PCR analysis and to retain parallel samples. Thus if
cellular changes consistent with a viral infection are observed then a true
positive PCR result can be confirmed. If the cellular changes are not
observable however, this is not enough to confirm that a positive PCR result
is false. The procedure in such cases usually involves re-testing the
suspect material with a different set of PCR primers, possibly sending it
off to an independent lab for re-testing, and submission of additional
samples of PLs from the same batch for further testing. In most cases these
measures will determine if a positive PCR result is true or false. (If it is
confirmed as true then the hatchery's SPF status is lost and a major
clean-up operation is called for.) Backing up the testing of PLs is the
screening of broodstock by taking pleopod samples, and the exclusive use of
SPF broodstock from a facility that also has a long track record of being
virus-free. All samples need independent 3rd party confirmation that they
are genuine and are not interfered with between collection and testing.
This information may be of limited use in your specific and immediate
situation. If you want to know further details and numbers of PLs that are
tested each month from this hatchery please contact me off list. But my main
point is that deciding on a precise number is not the key issue in providing
SPF PLs.
Daniel Lee
Hatchery Manager
High Health Shrimp / INA S.A.
Dominican Republic
danlee@codetel.net.do