Collection, Preservation and Mailing of Amphibians for Diagnostic Examinations

The following guidelines pertain to collection of healthy, sick or dead amphibians for diagnostic examinations.

The best diagnostic specimen is the live sick amphibian; dead amphibians have limited usefulness because aquatic animals decompose much more rapidly than terrestrial animals; this means dead amphibians nearly always will have large numbers of decompositional bacteria and fungi throughout their bodies. This rapid decomposition (autolysis) makes it very difficult to obtain meaningful or useful bacterial and fungal cultures. Therefore, live amphibians are necessary to obtain meaningful bacterial and fungus cultures; in addition, blood for various "blood tests" can be obtained only from live amphibians. If the amphibians must be sampled and euthanized as part of other studies, then first observe and record their behavior. If no sick amphibians are available, collect freshly dead carcasses and divide them into two groups: place about half the carcasses (preferably the most recently dead amphibians) into 10% formalin after first opening the body cavity with a straight incision through the ventral midline of the body wall (sternum to pubis bone); immediately freeze the other dead amphibians (for virus cultures and possible poison tests). In some cases, especially cases involving less well-known species, submission of live healthy amphibians as "control" or "baseline" specimens will be necessary to assist in the interpretation of findings in the sick or dead animals. More than one disease may be affecting a population simultaneously, so submission of multiple animals is always encouraged. Collect and ship specimens that represent the species that are affected and the geographic areas. If possible, submission of invading (alien or introduced) amphibians from the casualty site is desirable, even if they appear healthy or unaffected. If any other endemic amphibians (except threatened or endangered species), fish or reptiles are present at the casualty site, these animals also could be submitted as part of a wider epizootiologic investigation into the cause of the casualties. Wherever possible, do not place live and dead animals in the same container, and do not put multiple species in the same container (except, it is acceptable to put dead animals of multiple species in one container of formalin).

When you go into field to collect amphibians, take along a cooler containing ice to immediately chill (assuming warm weather) the amphibians. Freezing and thawing makes isolation of some pathogens difficult and usually damages tissues for microscopic examination; therefore, guidance on when or if to freeze samples will be provided on a case by case basis. However, if you are in the field and cannot call or ship within 24-36 hours, freeze and formalin-fix equal numbers of dead amphibians, and keep live amphibians chilled.

Place live aquatic amphibians (hellbenders, mudpuppies, neotenes, tadpoles and larvae) in a solid container with lid (Glad or Ziploc storage containers are good - not bags, which tend to open during shipping and do not protect specimens from being squashed by the blue ice blocks) to hold them prior to shipment. It is very important to assure enough oxygen is present in each container; containers that allow a larger surface area of water to air are preferred. There should be at least as much air space as the volume of water in each container. Place terrestrial amphibians in a plastic container or bottle with holes in the lid. To this container add a small amount of moist substrate (not dripping wet), such as aquatic or emergent vegetation, leaves, or moss from the site, or moistened unbleached (brown) paper towels. Do not use sponges--many are toxic (poisonous) to aquatic and terrestrial amphibians. Plastic containers or bags must be labeled, preferably with an indelible ink felt-tip pen ("Sharpie") or grease pencil that can be erased and the container reused.

• species
• location (state/county/town)
• collector (name/address/phone)
• date collected
• found dead or euthanized
• additional history on back of tag

To prepare for shipment, select a hard-sided cooler or a Styrofoam cooler placed in a cardboard box. Line the cooler with a large plastic bag. Pack individual bags or containers with amphibians next to frozen blue ice to keep carcasses cold. (If you had to use bags for the specimens, they must be placed within a hard sided container to keep the bag from shifting around or opening, or the specimens getting squashed.) Blue ice is preferred to wet ice to avoid leaking during shipment. If amphibians are alive, pack containers to allow for air circulation around the lid. Place crumpled newspaper or similar absorbent material in the cooler with the bagged carcasses to prevent shifting, keep ice in contact with carcasses, provide insulation, and absorb any liquids.

Tape the cooler or box securely and label: National Wildlife Health Center, 6006 Schroeder Road, Madison, WI. 53711. Note that in addition to the NWHC address, you need to add DIAGNOSTIC SPECIMENS -WILDLIFE. This label will direct coolers with specimens to our necropsy entrance. Contact NWHC (608-270-2400) (FAX 608-270-2415) prior to shipping animals by 1-day (overnight) service and after shipment to confirm the estimated time of arrival.

Biosecurity must be considered at any site with dead and dying animals. Biosecurity involves three equally important aspects: safety of the humans and scientists in the area, decontamination/disinfection of field equipment (especially boots and nets) to prevent spread of the possible infectious agent to other sites and other populations of animals, and careful quarantining (isolation) of live sick animals from all other populations in the field and in laboratory animal colonies. First, note whether there are sick and dead animals of more than one vertebrate class and phyla (i.e., dead birds, frogs, fish, snails, insects, etc); if so, then there is a much greater chance the animal deaths are due to a toxin (poison), and use utmost caution in entering the area, because mammals (i.e., the field scientists) could also be poisoned. Secondly, if only amphibians (or only amphibians and fish) are affected, there is a greater likelihood the deaths are due to an infectious disease; it would then be important to wash and disinfect all field equipment that came into contact with animals and surface waters (i.e., boots, rubber gloves, nets, minnow traps, tripods, water quality instruments, etc) which may also include tires and wheel wells of vehicles that drove through surface waters at the site. Disinfection is easily accomplished by putting 4 ounces of bleach in 1 gallon of clean water and using the solution to rinse off all field equipment before going to another site. For this disinfection process, a long handled brush and 5-10 gallon bucket should be considered standard equipment for field investigators and when visiting casualty sites. Third, amphibians (dead or alive) from a casualty site should be considered contagious specimens; such live sick animals and carcasses should never be released or discarded at other sites and should not be taken into laboratory settings with other amphibians, fish or reptiles. Finally, simple sanitary precautions should be taken by field scientists when handling sick or dead amphibians (and fish and reptiles): rubber gloves or disposal gloves should be worn when handling sick and dead amphibians. If you do not have gloves, then insert your hand into an inside-out specimen bag to collect an animal and then close the bag around the animal.

PLEASE DO NOT ADDRESS SPECIMENS TO AN INDIVIDUAL. LABEL THE PACKAGE "DIAGNOSTIC SPECIMENS"