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Dear All, I was just looking through the OIE manual again, based on Greg's suggestion, and was struck by the lack of validated PCR, especially qPCR assays in it. From my experience (and I believe that of others, though I won't speak for them), quantitative real time PCR is much more (analytically) sensitive than standard PCR (e.g., with the MCP4/5 primers in Mao et al. 1997) and more sensitive even than virus isolation in cell culture. We are consistently detecting the equivalent of <1 plaque forming unit in asymptomatic animals from experiments and from the wild. Jaramillo et al. have a validated assay for ranavirus in fish that seems to support the assertion that qPCR is more sensitive: "The assay had 100-fold greater analytical sensitivity compared to virus isolation in Bluegill fry (BF-2) cells, detected all viruses in a panel of 20 ranaviruses from Australia, America, Europe and the United Kingdom, did not detect two disputed members of the genus Ranavirus (doctorfish virus and guppy virus 6) and did not detect a megalocytivirus or two cyprinid herpesviruses." (Note: I do understand that analytic sensitivity is not the same as diagnostic sensitivity, but if virus isolation is our gold standard then qPCR cannot, by definition, have greater diagnostic sensitivity!) So I guess my question is, if someone is interested in ensuring that their animals are ranavirus free, shouldn't we be pointing them to these more sensitive assays? What are your thoughts? Would it be worth "stressing out" some subset of the animals to see if animals carrying inapparent infections might recrudesce? The question of whether they can develop full blown, transmissible infections seems very relevant in these cases. I would also like to point out that the protocols in the OIE manual state, in section 6: "Statistically valid sampling practices need to be used but these cannot presently be defined for amphibians." I am not sure why amphibians would present a special case in epidemiology, but I was not at these meetings, so perhaps someone could clear up my confusion. Barring that, I might suggest using the standard formulas for calculating the sample sizes required to be, say, 95% confidence that the prevalence of infection is at or below some level (e.g., 10%) given a population of N (e.g., 10^6)… here n = 30. I'm sure there are other references for the formula, but this is the one I have: Cannon, R.M., and R.T. Roe. 1982. Livestock Disease Surveys: A Field Manual For Veterinarieans. Canberra: Australian Bureau of Animal Health I've attached a spreadsheet I created a while back to help me see the relationships between confidence-level, population sizes, the number of disease animals, and the sample size. It is rather busy, but some playing with the numbers in different colors should clear up which graph is associated with which equation. Or I'm happy to answer questions about it. In any case, if there is a better way of establishing statistically valid sample sizes, I would love to hear them. It would be great for the members of this list to be on the same page, more or less, about how best to sample. Best wishes, Jesse Jesse Brunner School of Biological Sciences Washington State University PO Box 644236 Pullman, WA 99164 USA [log in to unmask] 509-335-3702 http://www.oie.int/international-standard-setting/aquatic-manual/access-online/ Jaramillo D, Tweedie A, Becker JA, Hyatt A, Crameri S et al. (2012) A validated quantitative polymerase chain reaction assay for the detection of ranaviruses (Family Iridoviridae) in fish tissue and cell cultures, using EHNV as a model. Aquaculture On Jun 18, 2012, at 7:09 AM, Victor Gregory Chinchar wrote: > The fellow to ask is Alex Hyatt as he has worked up the sampling protocols for amphibian ranaviruses. Protocols are in the OIE Manual, but I’d drop Alex an email. His email address is (and I suspect he is part of the GRC): [log in to unmask] > > > > Greg > > > > > > From: Global Ranavirus Consortium [mailto:[log in to unmask]] On Behalf Of marja kik > Sent: Monday, June 18, 2012 5:13 AM > To: [log in to unmask] > Subject: question from the netherlands > > > Dear all, > > In the Netherlands different institutions raise toads from the common spade foot toad or Garlic Toad (Pelobates fuscus) for reintroduction purposes. The eggs were collected in many different parts of the country. At this moment the larvae are supposed to be released in nature. > But Ravon (reptile, amphibian and fish research Netherlands) wants the animals tested on chytrid an ranavirus before releasing them. > Swabs are being examined for chytrid infection by PCR. > But the main challenge now is how to detect in a reliable way whether ranavirus is present within these groups of animals. The following questions came from the involved veterinarian. > How many animals should be tested to have a statistical reliability that they are really free of ranavirus infection. The animals are housed in separated tanks. In some of them approximately 100 are housed, in others a thousand. > Further what would you use as test samples? Is a piece of tail? The whole animal? > > I’m aware if the work of Greer and Collins and the risk of underestimating the true prevalence of infection, as PCR does for early-stage infections. > But please could you advise us how to proceed. > > All the best > Marja > > > > Individuals who have received this information in error or are not authorized to receive it must promptly return or dispose of the information and notify the sender. Those individuals are hereby notified that they are strictly prohibited from reviewing, forwarding, printing, copying, distributing or using this information in any way.