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Appendix 7
PROTOCOL
This protocol is part of the project’s SOPs. Follow the
protocol to ensure the data we record in the Excel
worksheets are the result of consistent procedures and
are technically correct
Pre-purchase and pre-stocking testing of P. monodon PLs and juveniles
for viral pathogens
1. Post-larvae
Note that, for any batch of PLs, the tests below must be completed, with negative
results, before purchase of the batch can be confirmed and the PLs moved from the
hatchery.
Sampling procedure for PLs
If more than one nursery tank is to be used for stocking, collect and test PLs as
described below from each tank separately.
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Collect approximately 5,000 PLs by taking approximately 1,000 PLs from 5 different
places in the nursery tank, including the bottom.
Swirl the 5000 in a tub and select ≥ 110 weak PLs (i.e. a biased sample) from the
center of the tub. If not enough weak PLs, add normal PLs to make up the total.
Divide the 110 into:
o 10 PLs in ambient water for WSSV i-screen test; submit to laboratory, must
arrive live.
o 3 pools of 30 PLs each in 70% ethanol for WSSV PCR test; submit to
laboratory
o 10 PLs in modified Davidson’s fixative (used to give rapid fixation; see Flegel
course notes or CD for recipe) for microscopic examination of hepatopancreas
for MBV and HPV; submit to laboratory.
I-screen test for WSSV
Test the 10 PLs in one pool as described in the protocol. Since biased sample, and
assuming a test sensitivity of 100%, a negative result will give at least 95% confidence
that the PLs in the tank are infected at a prevalence of 26% or less.
If i-screen test result is positive, reject the tank for stocking; if the i-screen test
result is negative, submit the 3 x 30 pools to the laboratory for PCR testing.
Nested PCR test for WSSV
Test each pool of 30 PLs separately, using the procedure below.
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Macerate in 7.5 ml lysis buffer (after this the sample can be stored for over one year if
necessary)
Centrifuge and take 0.75 ml sample for DNA extraction
After DNA extraction, measure DNA to ensure that template does not exceed 300 ng.
Separately, follow PCR test procedures for WSSV according to standard method used
by the laboratory.
Record and report results
If the nested PCR test result on any pool is positive, reject the tank for stocking.
Accept the batch in relation to WSSV only if the nested PCR test result on all 3 pools is
negative.
Note that tests for HPV and MBV must also be negative before we accept the tank
for stocking.
Screening PLs for HPV and MBV using smears of hepatopancreas
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Separate the 10 PLs into 2 groups of 5.
For PLs in each group, use sterile scissors to cut through the cuticle over the
hepatopancreas and remove a portion of the hepatopancreas.
For each group, place the 5 portions in a drop of modified Davidson’s fixative on a
clean glass slide.
Using a sterile scalpel blade, finely divide and mix together the 5 hepatopancreas
portions.
Using the edge of a second glass slide, smear the mix onto the slide.
Air dry thoroughly.
Fix the smear by dipping the slide into 1% glacial acetic acid in distilled water.
Stain with H&E, mount and examine for the presence of MBV and HPV.
Retain the slide for future reference.
2. Juveniles
Sampling procedure for nursery-reared juveniles
Any juveniles to be stocked into BMP ponds must have negative WSSV test results at
the PL stage (see above) before being transferred to biosecure nursery tanks.
If more than one nursery tank is to be used for stocking into ponds, collect and test
juveniles as described below from each tank separately.
Collect ≥60 juveniles in ambient water for WSSV i-screen test; submit to laboratory,
must arrive live.
As described in the i-screen protocol, test juveniles in pools of 10, i.e. 6 tests each
involving 10 juveniles.
Stock into ponds only if a negative test result is obtained for all 6 pools. If any of the
pools tests positive, reject the batch.