Summary and Tissue Checklist
Tissues for Histopathology (Samples can all be placed in a single container of 10% buffered formalin)
____ skin of ventral pelvic region ("drink or pelvic patch”)
____ skin of ventral hindlimb
____ skin of dorsal body
____ cross section of foot at the level of the metatarsals
____ heart ____ esophagus
____ larynx/trachea ____ stomach
____ lung ____ small intestine (at least 2 sections)
____ liver and gallbladder ____ large intestine
____ spleen ____ pancreas
____ kidney ____ bone/bone marrow
____ ovary ____ brain
____ testicle and Bidder’s organ ____ eye
____ urinary bladder ____ skeletal muscle
____ tongue ____ peripheral nerve
____ lymph heart
____ any other organs/tissues with significant abnormalities
Send formalin fixed tissues to pathologist used by your institution and send a copy of the final histopathology report to the SSP pathology advisor at the address below
Special Tissue Request:
___ frozen liver for vitamin A analysis (if applicable, see written protocol item # 4); call prior to shipment to SSP pathology advisor at the address below
____ routine aerobic bacterial culture (heart blood or liver, see written protocol item # 2)
____ other (coelomic fluid or other grossly identified lesions)
Tadpoles (see written protocol):
____ whole (opened) formalin-fixed carcasses
____ whole frozen (-70) carcasses
Questions regarding this protocol can be directed to:
Allan P. Pessier, DVM
Department of Pathology
Zoological Society of San Diego
1354 Old Globe Way
San Diego, CA 92101-1635
619-231-1515, Ext. 4510#
SSP Necropsy Protocol for the Wyoming Toad (Bufo baxteri)
General guidelines for amphibian necropsy have recently been published and may be useful (Nichols DK: Necropsy, in Wright KM, Whitaker BR (eds): Amphibian Medicine and Captive Husbandry. Malabar, FL, Krieger Publishing, 2001, pp 331-334
1. Deaths of all advanced subadult (yearlings) and adult animals should be investigated by gross necropsy with submission of samples for histopathology and ancillary testing as detailed below. Exceptions might include animals in advanced stages of autolysis, however, limited histopathology (such as examination of the skin for chytridiomycosis) could still provide valuable information about group/colony health.
2. While necropsy and histopathology of all small recently metamorphosed animals may not be feasible, examination of a subset of these animals from each institution is strongly recommended.
3. For freshly dead to mildly autolyzed animals routine aerobic bacterial culture is recommended. Suggested samples include aseptically collected heart blood from adult animals or alternatively, liver from subadult to adult animals. Coelomic fluid or other grossly visible lesions, if present, are also a desirable sample.
4. Freezing at –70 or 80 of portions of gross lesions (examples include visceral nodules, cutaneous or mucosal ulcers, organs with petechiae, among many others) is highly recommended. If grossly observed lesions are too small to accurately section and divide for multiple purposes (histopathology, microbiology, freezing ect), samples for histopathology should be given preference.
5. For continued investigation of suspected hypovitaminosis A/short tongue syndrome in captive toads, frozen liver is requested from freshly dead animals. After collection of a small section of liver and the gallbladder for histopathology, freeze the remainder of the liver for later shipment to the SSP pathologist. Freezing at –70 or 80 and shipment on dry ice is preferable.
6. All major organs, including the brain, should be sampled for histopathology. A tissue checklist is provided with this protocol. Complete dissection of individual organs and submission/fixation of representative samples is encouraged, particularly for adult animals. Important tissues that are frequently overlooked in Wyoming toad necropsies include tongue, urinary bladder and multiple skin sections (as indicated on tissue checklist). For animals with evidence of subcutaneous edema, the lymph hearts should be sampled. The posterior lymph hearts are most easily located and appear as very delicate pale white-yellow tissue in the subcutis adjacent to the caudal aspect of the urostyle.
7. Following standard pathology practice, all samples should be fixed in 10 times their volume of 10% neutral buffered formalin.
8. Small animals (less than 10-15 grams) can be submitted whole after opening of the coelomic cavity to allow penetration of the fixative. Large full-thickness incisions into the coelomic cavity work best; separation of the head from the body allows more optimal fixation of the brain. Under limited circumstances, larger (adult animals) can be submitted whole as for smaller animals, however, this results in varying degrees of sample degradation and is suboptimal.
1. In tadpole mortality events, euthanized sick animals or animals known to be very recently dead are the preferred samples. Whole representative animals should be selected for histopathology and others frozen at –70 or 80.
2. Samples for aerobic bacterial culture can be collected from animals designated for histopathology. The coelomic cavity is opened as aseptically as possible and sterile swabs of coelomic contents/fluid are obtained. Alternatively, a small section of liver (usually very large in tadpoles) can be aseptically collected for culture. The remainder of the opened carcass can be immersed in 10% neutral buffered formalin as described below.
3. In animals for histopathology, the coelomic cavity should be opened as described for small toads and the carcass immersed in 10% neutral buffered formalin.