WHAT TO DO WITH DEAD
OR ILL FROGS

2. ASSUMPTIONS

3. BASIC INFORMATION REQUIRED ON ALL SPECIMENS

4. PRESERVATION TECHNIQUES

5. DEAD FROGS

6. ILL FROGS

7. TRANSPORTATION OF SPECIMENS

8. AMPHIBIAN SUBMISSION FORM

9. HOW TO MAKE UP GOOD QUALITY FORMALIN

1. INTRODUCTION

At our present stage of knowledge about diseases of Australian frogs, we need all the additional specimens that we can get. Only by thorough studies of more dead and ill frogs will we be able to piece together the overall disease picture. For that reason every ill or dead frog is valuable, and efforts to get the maximum amount of information from each specimen are well worth while.

Deciding what to do with an ill or dead frog is a complex process. At the current time, if you find a frog that is ill, do not allow it to die in the wild; collect it and allow it to be used in the effort to understand what is happening to Australia's frogs. This document will assist you in deciding what to do with a particular ill or dead frog given the particular circumstances associated with it.

1.1 Amphibian Disease Group

The main members of the Australian Amphibian Disease Group are Lee Berger, Rick Speare, Alex Hyatt and Rick Speare. Lee, Rick and Diana are veterinarians, while Alex is a scientist with much experience in searching for and identifying unknown pathogens. However, there is a growing number of scientists involved in frog diseases research through their peripheral activities on pathogens. A list of frog disease people is available at this site.

Diana Mendez and Rick Speare at School of Public Health and Tropical Medicine, James Cook University, Townsville are the initial contact point for any ill or dead amphibians. Alex Hyatt is a Senior Principal Researcher at the CSIRO Australian Animal Health Laboratory and will also accept ill or dead amphibians.

The JCU group is funded by grants from the National Heritage Trust via Environment Australia with assistance from Queensland Department of Environment and Heritage.

2. ASSUMPTIONS

1. More information will be obtained from a postmortem done by an experienced pathologist than from one done by a person with little experience in collecting specimens from frogs.

2. Less information will be obtained from a dead frog than from a frog that is processed in the terminal state. Therefore, if a collector has dissecting skills and equipment, postmorteming a terminal frog will provide more information than shipping the frog and having it die in transit.

3. Processing of specimens will depend on:
   • whether the frogs are ill or dead
   • if ill, whether they are terminal or non-terminal
   • time from collection to processing
   • level of expertise of finder / dispatcher
   • facilities available

4. Any stage (eggs, tadpoles, metamorphs, juveniles and adults) should be sent if dead, ill or abnormal.

5. Dead or ill cane toads should be treated in the same way as frogs.

3. BASIC INFORMATION REQUIRED ON ALL SPECIMENS

To make the most of every specimen, please record and pass on the following information:

1. Species of frog.
2. Date and time.
3. Where found - be very detailed. Use map if helpful.
4. Brief description of area where specimen was found.
5. Why you think it is abnormal. In this include details on appearance and behaviour.
6. Whether other frogs were ill.
7. What proportion of population was affected.
8. Is finding ill or dead frogs a common occurrence? If "yes", please provide details on locations, dates, signs, species.

The submission form at the end of this document can be used to record this information.

4. PRESERVATION TECHNIQUES

Once a frog dies, its tissues start to degenerate (postmortem degeneration). The rate of degeneration is dependent on ambient temperature. If a frog is dead, action has to be swift to get the maximum amount of information possible from the specimen. Postmortem changes decreases the amount of histological information as well as the chances of isolating an infectious agent. Postmortem degeneration can be stopped by:

1. chemicals called fixatives (alcohol, formalin, or glutaraldehyde), or
2. freezing.

Preserving agents, while protecting the tissues from postmortem degeneration, also kill infectious agents. So if viruses or bacteria are present, once the tissue is preserved the infectious agent cannot be grown. This is the major disadvantage of preserving specimens. Infectious agents in tissues are best protected by freezing the specimen.

4.1 Ideal Processing Technique

The ideal solution to get the maximum amount of data is to:

1. necropsy the frog using an aseptic technique
2. cut organs into pieces
3. preserve parts of organs in 10% buffered neutral formalin for histology (please remember that fresh well buffered formalin is best).
4. preserve small parts of organs in 2.5% glutaraldehyde for transmission electron microscopy; this enhances the possibility of identifying intracellular pathogens, and is very important when looking for a new infectious agent).
5. freeze the remainder of organs at -70øC for isolation of infectious agents. Freezing at -20øC is better than leaving in the fridge at 4øC.

Obviously this option requires skill, knowledge and access to the correct chemicals.

4.2 Specimens to Collect

Specimens for disease investigation are mainly collected for 4 reasons:

1. histopathology - to observe the pattern of changes in cells in tissues and visualisation of some infectious organisms. Organs affected by disease can be identified. For histopathology, tissues have to be preserved in formalin or alcohol based liquids. Please try to use quality fixatives, e.g. fresh well buffered formalin, this enables follow-up electron microscopy to be used.
2. identification of agent - if disease is caused by an infectious agent, fresh or frozen specimens may allow the micro-organism to be isolated, grown in-vitro, identified using antigen detection techniques, or identified using molecular biological techniques.
3. transmission electron microscopy - if an infectious agent is present, it may be identified using TEM even if the micro-organism cannot be isolated. To get the best result, tissues are preserved in glutaraldehyde (if glutaraldehyde is not available, use formalin).
4. toxicology - frozen tissues are necessary. Liver, kidney, fat and stomach contents are considered likely to have concentrated levels of toxins.

For histology, collect the following in 10% formalin:

1. Any abnormal changes or lesions
2. Skin
3. Skeletal muscle
4. Liver
5. Kidney
6. Spleen
7. Lung
8. Heart
9. Stomach
10. Intestine
11. Urinary bladder
12. Eye
13. Brain
14. Bone of femur
15. Gonads

For isolation of infectious agents or toxicology, collect the following organs and freeze:

1. Any lesions
2. Liver
3. Kidney
4. Lung
5. Skeletal muscle
6. Brain
7. Stomach contents
8. Fat bodies
9. Remainder of carcase.

To detect chytrids on the skin by histopathology,

1. cut strips of skin to include any lesions or surface roughening,
2. cut strips of skin from inguinal regions
3. cut off toes or feet

A protocol for collecting skin samples in surveys for amphibian chytrids is available at this site.

To detect chytrids on the skin by direct microscopy or culture:

1. gently scrape the superficial layer of skin
2. place scrapings into 0.8% saline in a sterile tube
3. refrigerate and send to laboratory.

5. DEAD FROGS

Dead frogs can be handled in three ways:

1. Frozen whole
2. Preserved whole in formalin or alcohol after opening the belly
3. Necropsied.

5.1 Problems with Dead Frogs

The main problem is that postmortem degeneration in dead frogs is very rapid; the higher the temperature, the greater the rate of degeneration. The aim is to get the maximum amount of information from the frog before it decays any further.

5.2 Processing of Dead Frogs

If a postmortem can be done immediately by someone with experience and the right chemicals, then do a postmortem, divide organs if possible into two, put specimens for histology in 10% buffered neutral formalin, freeze the other half of the organs, freeze the carcase.

6. ILL FROGS

The key question here is mainly for people that have found a terminal (almost dead) frog, and are trying to decide whether:

1. to forward the frog to the laboratory, and run the risk of it dying in transit, and have the lab get a dead frog; or
2. to kill the frog on site, and process it for subsequent examination by the laboratory.

6.1 Severity of Illness

An "ill" frog for our purposes has at least one of the following:

1. behaves in an abnormal fashion;
2. is very thin;
3. has abnormalities of eyes or skin;
4. is deformed

Ill frogs should be classified into two groups: terminal and non-terminal.

Terminal = a frog which will probably die within 24 hours.
Signs: not able to move; moves very slowly; very thin; can be turned onto back and does not attempt to right itself.

Non-terminal = a frog that is ill, but will probably not die in 24 hours.
Signs: attempts to escape; moves actively.

6.2 Problems with Ill Frogs

An ill frog may become a dead frog during transit. Terminally ill frogs and toads usually die in transit. Once an amphibian dies, degeneration occurs rapidly. If a frog is terminal, it is better to process it prior to dispatch, or to dispatch it in a cooled container.

6.3 Benefits of Collector Doing Processing

Since more comprehensive information can be obtained from live amphibians, it is better for us to receive live, ill frogs than dead frogs. So the main aim should be to get a live, ill frog to the Amphibian Disease Group. However, after considering the various factors below, the best option for the collector may be to process the frog, and then to send the specimens to us.

6.4 Processing of Ill Frogs

6.4.1 Terminal Frogs

Collector Has Minimal Dissection Experience

If you have minimal scientific facilities and skill:

1. Put frog in plastic bag in freezer to kill.
2. If there are many ill frogs, some can be fixed and the rest frozen. 10% formalin is preferred, although 70% alcohol can also be used. After euthanasing, cut through the skin and muscle on the belly before placing in fixative.
3. Notify Diana Mendez at JCU.

Collector has Dissection Experience

If you have appropriate facilities and skill:

1. Weigh frog.
2. Measure snout-vent length.
3. Anaesthetise with chloroform, or barbiturate.
4. Open ventrally along abdomen.
5. Collect blood from heart using needle, capillary tube or incision.
   1. make smears (dry at room temperature)
   2. collect into plain tube (store in fridge at 4øC)
   3. collect 0.3 ml into EDTA or sequestrene tube for haematology (store in fridge at 4øC).
6. Examine internal organs for abnormalities.
7. Divide organs into 2, one part for histology (placed in 10% formalin), and other into sterile tube (for culture of micro-organisms - freeze in fridge at - 20øC or -80øC).
8. If possible, a small section of the organs and lesions (~1mm thick) should be placed in 2.5% glutaraldehyde at 4øC for electron microscopy. 25% glutaraldehyde is usually obtained which needs to be diluted in 0.1M phosphate buffered saline shortly before use. After 24 hours the samples should be transferred to phosphate buffered saline.
9. Fresh tissue specimens should be frozen at -20øC.
10. Notify Diana Mendez at JCU.

6.4.2 Non-Terminal Frogs

If the frog is ill, but it seems likely that it will not die within 24 hours, do the following:

1. Notify Diana Mendez at JCU.
2. Place frog in a bag made of cloth
3. Wet bag
4. Dispatch so air supply is not restricted (i.e., don't put into airtight bottle or plastic container).

6.5 Sending Specimens

6.5.1 Terminal Frogs

Full postmortem

1. To be useful, blood specimens should arrive within 2 days of collection. If delivery is impossible within 2 days, the blood tubes should be frozen as for fresh tissue samples.
2. Dispatch frozen samples with dry ice or if not possible, with ice brick.
3. Dispatch fixed samples in 10% formalin.
4. Before dispatch notify Diana Mendez at JCU.

Frozen Specimens

1. Notify Diana Mendez at JCU.
2. Forward with dry ice or, if not possible, with ice brick.

6.5.2 Non-Terminal Frogs

1. Notify Diana Mendez at JCU.
2. Forward as livestock.

7. TRANSPORTATION OF SPECIMENS

If sending specimens by air, the fresh tissues are regarded as diagnostic specimens and are not classified as dangerous goods. They have to be packed and dispatched according to IATA Regulation 650. Labelled "Diagnostic specimen" on consignment note.

If dry ice is included, the package is treated as a Dangerous Good. Label "Diagnostic specimen and dry ice" on consignment note, but no shipping bill required. If 10% formalin is included, since formalin is a dangerous good, the package has to be packed in accordance with Dangerous Goods Regulations. Shipping bill required.

If you have specimens to send to us, please contact either Diana Mendez or Alex Hyatt to discuss how the specimen should be processed and transported to either JCU or AAHL respectively.

Contact numbers are:

Initial:	Diana Mendez or Assoc Prof Rick Speare	Ph. 07-47-225777 Fax. 07-47-225788 email: diana.mendez@jcu.edu.au richard.speare@jcu.edu.au
Other:	Dr Alex Hyatt	Ph. 052-275000 Fax. 052-275555 email: alex.hyatt@csiro.au

Diana Mendez or Rick Speare School of Public Health and Tropical Medicine James Cook University Townsville 4811 Ph: -61-(0)7-47-225777 Fax: -61-(0)7-47-225788 Email: diana.mendez@jcu.edu.au

Alex Hyatt Australian Animal Health Laboratory Ryrie St, Geelong, Victoria 3220 Ph: -61-052-275000 Fax: 61-052-275555 email: alex.hyatt@csiro.au

SENDER'S NAME AND ADDRESS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
TELEPHONE............. FAX........... EMAIL.....................
SENDERS ID NUMBER....................................
SPECIES..................................... STAGE..................... AGE.................... SEX ............
LENGTH Snout-vent............... WEIGHT..............................
DATE & TIME COLLECTED.............................DATE SUBMITTED............................
LOCATION FOUND............................................................ ...................................
TYPE OF ENVIRONMENT...................................................... ................................
ARE PESTICIDES etc USED?............................................................ .......................
ABNORMAL BEHAVIOUR........................................................ .............................. ................................................................. .........................................................
EFFECT OF HANDLING......................................................... ...............................
ABNORMAL APPEARANCE: Skin............................................................. ................................................................. ....
Eyes...............................................Orifices...... ...............................Other............................. .
............................................................. ................................................................. ....
ARE OTHER FROGS ILL?............................................................. ...........................
PROPORTION OF FROGS AFFECTED......................................................... ..............
IS FINDING ILL/DEAD FROGS A COMMON OCCURRENCE?...................................... ................................................................. ..........................................................
EUTHANASED?......................... HOW?............................................................. ...
FIXATIVE USED (if any)?............................................................ ............................
FROZEN?....................WHAT TEMPERATURE? ......................................................
TIME FROM DEATH TO AUTOPSY/FREEZING................................................. ........
IF CAPTIVE, HAS ANY MEDICATION/DISINFECTANT BEEN USED?...........................
COMMENTS......................................................... ............................................... ................................................................. ........................................................ ................................................................. ................................................................. .............. ................................................................. ................................................................. .............. ................................................................. ................................................................. .............. ................................................................. ................................................................. .............. ................................................................. ................................................................. .............. ................................................................. ................................................................. .............. ................................................................. .......................................................

9. HOW TO MAKE UP GOOD QUALITY FORMALIN

10% Neutral Buffered Formalin

The quality of the histology depends on using good quality fixatives. The pH of the fixative is critical. The best histology is obtained using 10% formalin with a pH about neutral. The following is a formula that will produce 10% formalin of neutral pH.

Chemicals needed

Di-sodium orthophosphate (Na2HPO4) Potassium dihydrogen orthophosphate (KH2P04) 40% formaldehyde solution Distilled water

6.5 gm 4.0 gm 100 ml 900 ml

Procedure

1. Dissolve salts in small part of the water with heating.
2. Add remaining water, then formaldehyde.
3. Add 1-2 ml of methylene blue solution as a colour indicator.