Biosecurity and Disease

Biosecurity Protocol

Protective Wear & Equipment Disinfecting & Sanitizing Methods nonpermeable boots or waders rinse in bleach solution immediately after leaving each study site3 (fig. 46) vinyl gloves1 dispose of gloves after each handling incident nets rinse in bleach solution immediately after leaving each study site plastic bags (for holding specimens)2 properly dispose after each use needles & syringes (for blood extraction) properly dispose after each use scalpel blades, PIT tag cannula, forceps, etc. immerse in sterilizing solution

¹Only vinyl gloves should be used when handling amphibians. Some people are allergic to latex gloves,
and latex gloves are toxic to amphibians (Gutleb and others, 2001).
²Use one bag per specimen.
³Premixed bleach solutions can be carried in containers large enough to step into and immerse boots, nets,
and equipment. If this is not possible, bleach solutions can be carried in a spray backpack firefighting pump.

Solution Formulas

bleach one (1) capful per gallon water sanitizing solution (for instruments) 70% methanol for 30 minutes, then flamed; or, 1% glutaraldehyde for 15 minutes; or, boiling water for 10 minutes

Additional Precautions

• Avoid contact between used and unused protective wear and equipment.
• Separately house specimens.
• Avoid contact between gloved hands and face, especially the area of the nose.
• Do not urinate in or near ponds and streams.
• Wash hands thoroughly with soap and water, or use a sanitary wipe, after urinating.
• Wash hands thoroughly with soap and water, or use a sanitary wipe, after handling specimens known or suspected of being diseased or contaminated.
• Wash hands thoroughly with soap and water, or use a sanitary wipe, after leaving each site.
• Do not use insect repellent on hands when handling amphibians.

Disease Protocols

Figure 46. Biosecurity. Washing boots and stump ripper in bleach solution.

       The following information is taken from the U.S. Geological Survey’s STANDARD OPERATING PROCEDURE (Kathryn Converse and D. Earl Green; ARMI SOP No. 105; revised March 2, 2001) entitled “Collection, Preservation & Mailing of Amphibians for Diagnostic Examinations.” It was developed by the National Wildlife Health Center, Madison, Wisconsin (http://www.nwhc.usgs.gov/research/amph_dc/sop_mailing.html).

       The best diagnostic specimen is the live, sick amphibian. Live amphibians are necessary to obtain meaningful bacterial cultures and most types of fungus cultures. In addition, blood for various blood tests can be obtained only from live amphibians. Dead amphibians have limited usefulness because aquatic animals decompose much more rapidly than terrestrial animals which means amphibian carcasses 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, but dead amphibians may still have usefulness for virus cultures, histology and toxicological tests, if promptly and properly preserved.

       If the amphibians will be captured and euthanized as part of other studies, then first observe and record their behavior. Blood should be collected and saved prior to euthanasia. If the euthanized amphibians will be preserved in a fixative, then collect swabs for bacterial, viral and fungus cultures from the mouth, vent, skin, and any skin abnormalities (lesions) prior to emersion of the animal in the fixative.

       At a casualty site, the priority specimens for diagnostic examinations are live, sick amphibians. Divide dead amphibians into two groups: promptly preserve about half the carcasses (preferably the most recently dead amphibians) in 10 percent formalin (or 70-75 percent ethanol); promptly freeze the other dead amphibians (for virus cultures and possible poison tests). In 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 lethal disease may affect a population simultaneously, so submission of multiple animals is always encouraged. Collect specimens that represent the species that are affected and the geographic areas. 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 or ethanol).

       If possible, submission of invading (alien or introduced) amphibians from the casualty site is desirable, even if they appear healthy or unaffected, because invasive species can be the vectors of infectious diseases. If any other endemic amphibians, fish, or reptiles are present at the casualty site, these animals also may need to be examined as part of a wider epizootiologic investigation into the cause of the casualties.

       Many amphibian die-offs are fleeting. This means the casualties must be collected the hour and day they are found. Returning to the casualty site the next day to collect sick amphibians and carcasses invariably fails because of the highly efficient activity of scavengers during the night and rapid autolysis of carcasses.

Methods