Brunner Lab

I am interested in getting a better sense of where and when ranavirus and Bd-related epidemics occur in the Northwest and Rockies. If you happen upon a die-off of larval or metamorphosed amphibians, I would definitely be interested in hearing about it. Pleae email me, making note of the following information:

• Where is or did this die-off occur? (Latitude longitude would be ideal, but driving directions or locations relative to fixed landmarks would suffice.)
• What sort of habitat is it? Is it a vernal pool permanent pond? Clearly artificial? Is it shallow or deep? Embedded in a forest or in agricultural lands? Full of emergent vegation or clear?
• What species were present in the pond? (I am interested in amphibian, fish, and any invertebrates you might note there.)
• Which species were dying or miribund? Did you see others that were apparently healthy?
• Roughly how many individuals of each species and life history stage were affected? How many were present?
• When did you notice the die-off? Had you been to this location before? If so, when?

I can potentially screen samples for ranavirus and Batrachochytrium dendrobatidis ("chytrid") infections. The samples need to be relatively fresh if they are to be usable. If the die-off happened weeks ago, or you have had the samples in your refridgerator for several weeks, they may not be usable. Contact me before hand to be sure. This also means that if you cannot get the samples to me (or mailed on ice) and cannot preserve them, then it would be best not to collect the samples.

There are a number of considerations in collecting samples and it is difficult to provide absolute guidlines. Therefore, I would recommend you contact me first (with answers to the above questions) in order to ensure that we get the most useful samples and no one wastes their time.

That said, there are a few general guidlines:

• Species. In general, it would be useful to collect a few individuals of all affected and unaffected amphibian species in the pond or pool. If there are fish in the pond, I would likely be interested in those as well. In most cases I will not be able to look at other vertebrates (e.g., birds, mammals), invertebrates, or plants.
• Whole animals or tissues? In general, I would like the whole animal. If this is not possible, I can use drink pouch and/or toe webbing to screen for Bd, and liver, spleen, renal tissues, gastrointestinal tissues, or even skin or tail clips for ranavirus detection (in that order of preference).
• Live or dead? I prefer live, apparently health animals as well as morbid (sick) and recently dead animals in about equal numbers. The animals need to be in reasonable shape; no piles of goop, please! Also, please use IACUC or veterinarian approved methods of euthanizia with the live animals. Get and keep all samples cold as soon as possible.
• Fresh, frozen, or preserved? In general, fresh samples are the best, provided that you can get the samples to me quickly. Plase keep them cool in a refrigerator or on ice for the duration. If you cannot, freezing them solid in a regular freezer is a very workable solution, but the usefulness of the samples will decrease the longer they are in your freezer. If you can preserve the animals in 70-95% ethanol, this would work, too. I would prefer the live and very recently dead animals be frozen to preserved, though, since this allows for the isolation of the pathogens, rather than just DNA-based detection.
• Sample size. Aim for about 5-10 individuals per species per location of the sick ones, and ~5 healthy individuals if possible. These are very rough numbers---even one individual can be useful if it is infected. Please do not kill the last few individuals of a population to get the sample size. Even after catastrophic die-offs in vernal pools, some tadpoles and salamander larvae somehow survive and metamorphose. Let's make sure they have that chance.
• Bag them separately! If at all possible, collect the samples individually and keep them in separate bags or containers. I like ziploc-style snack-sized or sandwich bags. All sorts of containers can work, so long as they seal. Make sure you label the bags either with a sharpie on the container or with a piece of paper and pencil inside the container. Label them with the location, date, and species of the sample. (You'd be amazed how unrecognizable a small tadpole can be after being in the cooler or freezer for a while! )

• Contact me! Email is the best means, but I do answer my office phone as well.
• Make sure the sample(s) are labelled individually with the location, date, and species.
• Make sure the bags or containers are sealed well.
• Place the sample containers inside a separate, sealable bag, and then in another one.
• Place the samples in their layers of bags in a cooler or styrofoam box with plenty of ice for the journey.