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LIVE AND SICK AMPHIBIANS
Eggs __ Place eggs in heavy mil plastic bag or plastic container. Equal volumes of air and water should be present in the bag or container to assure adequate oxygen exchange. Do NOT fill bags or containers completely with water. If bottled oxygen is available, it may be placed into the air cell in the bag or container, but this is optional. If possible, place plastic bags in a solid container for support and to avoid crushing specimens or puncture of the bag.
Tadpoles, Larvae, and Neotenes __ Same as for eggs. For small amphibians (<2 grams each), multiple live animals may be placed in one container, but avoid mixing species. For larger aquatic larvae and neotenes, one animal per bag or container is recommended. Enough air must be present in each container; containers that have a large surface area of water to air are preferred; hence, flat food storage-type plastic boxes with lids (available at nearly any grocery store) are preferred to tall narrow plastic bottles. If bottled oxygen is available, oxygen may be placed into the air cell in the bag or container, but this is optional.
Adult Amphibians (Terrestrial Amphibians) __ Plastic boxes or bottles with wide lids may be used for mailing. Sick amphibians should be mailed in separate containers. Two or more live adult amphibians of the same species may be placed in one container, but avoid crowding. Note: if an infectious disease is the cause of the casualties, the disease may be transmitted between amphibians in the container if more than one animal is placed in each container. Wet unbleached (brown) paper towels or wet local vegetation should be added to the container to prevent dehydration of the animal; do not use sponges, because many contain chemicals that are toxic to amphibians. Three or more small holes should be made in the lid of each container. Plastic bags are not recommended for terrestrial amphibians.
About half the dead amphibians should be immediately placed into 10 percent buffered neutral formalin or 75 percent ethanol for histologic examinations. When possible, the freshest carcasses (those with the least amount of decomposition) should be selected for fixation. Prior to immersing the carcass in the fixative, slit open the body cavity along the ventral midline to assure rapid fixation of internal organs. For the first 3-4 days of fixation, the volume of fixative to volume of carcasses should be 10:1. After 3-4 days of fixation, the carcasses may be transferred to a minimal amount of fresh fixative that prevents drying of the specimen.
Freezing __ About half the carcasses should be promptly frozen. Preferred freezing temperature is -40 degrees, but any freezing temperature is preferable to a chilled carcass. Do NOT freeze amphibians in water. Frozen carcasses can be used for virus cultures, toxicological examinations, and molecular (DNA) tests. Frozen and preserved carcasses are not suitable for bacterial and fungus cultures; generally, bacterial and fungus cultures will be attempted only on amphibians that are submitted live.
Decomposed Carcasses __ Clearly decomposed carcasses may have some diagnostic usefulness for molecular testing and toxicological analyses. Very decomposed carcasses with fluffy growths of fungus on the skin; maggots in the mouth, vent, and body cavity; or carcasses of only skin and bones should be frozen and saved if fresher carcasses are not available.
Each container must be labeled. Paper labels written in pencil are preferred, especially if there is ethanol in any containers. Most ink will dissolve in ethanol or become streaked during freezing and thawing. Each label should have the following information:
Shipping Container __ Use a picnic cooler or styrofoam-lined cardboard box.
Ice __ Ice packs (blue ice) is preferred to wet ice to avoid leaking during shipment. Most amphibians from temperate climatic zones should be mailed with ice packs. Ice packs should be wrapped with about 5 layers of newspaper before being placed at the side of containers of amphibians. For live amphibians, position ice packs on the side of the shipping container, not under the specimens, as this allows live amphibians to move away from cold zones.
Frozen Specimens __ Frozen samples should be mailed with dry ice. Ice packs are an alternative, especially if the ice packs were frozen in an ultra-low freezer (-40 or lower). Avoid mailing frozen specimens in the same shipping container as live animals or specimens in formalin. If frozen samples and live amphibians (or specimens in formalin) must be mailed in the same shipping container, never put dry ice in the shipping container. If frozen samples and live amphibians (or specimens in formalin) must be mailed in the same shipping container, separate the shipping container into two compartments with styrofoam panels and place the ice packs at one end of the container next to the frozen samples.
Preserved Specimens __ Once specimens have fixed in a large volume of formalin or ethanol for 3-4 days, the preserved samples may be mailed in a minimal amount of preservative that prevents drying. It is not necessary to mail large volumes of liquid fixative. Preserved carcasses may be wrapped in gauze or a paper towel that is moistened with the fixative. If preserved specimens are transferred to plastic bags, always double-bag the specimen and pack it into the shipping box to avoid crushing the sample during transport.
Packing the Shipping Container __ Plastic boxes and bags containing live amphibians may be stacked, but keep air holes clear; some plastic boxes will stack tightly on each other and may seal air holes of lower containers. Do not place live amphibians directly on top of ice packs, because this may cause water in the animal's container to freeze. After placing ice packs and specimen containers in the shipping box, add crumpled newspaper, plastic peanuts, or other filler around the containers to minimize shifting of contents during mailing and crushing the plastic-bag samples. If a styrofoam-lined cardboard box is being used for mailing, then line the box with a heavy mil plastic bag and place all ice packs and specimens into the bag to minimize leaks and moisture condensation into the cardboard box.
Double Bagging __ Frozen samples and specimens in formalin (or ethanol) should be double bagged. This is especially important to avoid fixative leakage. If glass vials or jars must be mailed, these too should be placed into a plastic bag.
Taping __ Tape should be wrapped completely across the lid, sides, and bottom of each plastic cooler in at least two places to prevent accidental opening of the container during mailing. Nylon-reinforced tape is recommended, but 2-inch-wide clear tape also may be used.
Overnight Couriers __ should be used for sick, live, and frozen amphibians.
Dates for Mailing __ Only mail boxes of specimens by overnight couriers on Mondays, Tuesdays, and Wednesdays. Most diagnostic laboratories are not open on weekends, so specimens mailed on Fridays may be held in hot or freezing delivery vans over the weekend. A significant percentage of packages mailed by overnight courier on Thursdays, do not arrive in 24 hrs, and these can suffer the same fate.
Mailing __ Overnight courier service should be used. Securely tape the cooler or box and mail to: National Wildlife Health Center, 6006 Schroeder Road, Madison, WI 53711. Note: in addition to the NWHC address, add DIAGNOSTIC SPECIMENS_WILDLIFE to the outside of the box. This label will direct coolers with specimens to our necropsy entrance. Do not label the container with statements like, “Live Animals,” as this could interrupt or prohibit shipment because of courier policy. 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.
QUARANTINE OF AMPHIBIANS
Amphibians (dead or alive) from a casualty site should be considered contagious specimens. Live, sick animals and carcasses should never be released or discarded at other sites and should not be taken into laboratory settings with other live amphibians, fish, or reptiles. Release of sick amphibians or discarding carcasses at other sites may result in the spread of infectious diseases.
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