HOW TO REDUCE THE RISKS OF YOU TRANSMITTING AN INFECTIOUS AGENT BETWEEN FROGS AND BETWEEN SITES

Rick Speare¹, Lee Berger¹ and Harry Hines ²

¹School of Public Health and Tropical Medicine, James Cook University, Townsville, Australia, 4811

² Department of Environment, Moggill, Queensland, Australia

1. PROBLEM

1. Infectious agents pathogenic to frogs are present in the environment eg. fungi, viruses.
2. We do not want agents transmitted by humans from an infected frog population to an uninfected frog population.
3. People working in infected streams need to ensure that they do not contribute to the declines by transmitting possible agents between frogs within a population.

This information sheet therefore aims to tell you what measures you can take to reduce the chances of a researcher working in an area with ill frogs transmitting organisms to a new area or increasing rates of transmission within a population.

2. BASIS FOR THE RECOMMENDATIONS

The recommendations are based on the following assumptions:

1. Micro-organisms are durable and can survive in a range of environments;
2. Micro-organisms are present in water as well as in infected frogs;
3. Procedures that reduce the infectivity of durable viruses such as parvoviruses will inactivate these agents.

Remember that with most infectious agents the number of infectious particles in the initial dose (inoculating dose) frequently determines the outcome in terms of disease. Low numbers of organisms may result in no disease or mild disease; high numbers of organisms may result in rapid onset of severe disease. So although the measures may not kill all particles of the agent or prevent frogs coming into contact with the agent, if the measures reduce the number of particles of the agent that enters a frog, outcomes may be much improved.

Remember also that the procedures followed in any disease control are done on a cost-benefit basis. For the cost of the control procedures what will be the benefit? For example, control procedures in an infected stream may have little impact and so expensive control procedures may not be justified. However, preventing an agent from getting from an infected to an uninfected stream, may be absolutely critical, and expensive and tedious control procedures may be justified. We recommend that this protocol be implemented for any studies on frog species considered endangered.

The following procedures do not guarantee against spread of disease, but greatly minimize the risk.

3. RECOMMENDATIONS

3.1 REDUCING RISK OF TRANSMISSION BETWEEN FROGS WITHIN INFECTED STREAMS

If infected frogs are in a stream, how could an agent be spread and would any activities be able to reduce this? We recommend that these procedures are used in all frog research programs, as it is often difficult to know whether infected frogs are present.

How Frogging Activities Could Potentially Affect Spread

In water: no effect

By handling infected frogs first and then handling uninfected frogs increase dose of agent.

Toe clipping infected frogs then clipping uninfected frog with agent inoculated into cut stumps of toes of uninfected frogs.

PIT tagging agent inoculated into body.

3.1.1 To decrease possibility of transmission from handling infected frogs

On arrival clean hands using hospital grade antiseptic solutions such as 4% chlorhexidine and rinse thoroughly. There are two methods of handling frogs that if used correctly will ensure agents are not transmitted between frogs.

Either:

• Use disposable vinyl gloves (e.g. Surgigloves), changing gloves after handling each animal, or

• Capture and handle frogs using new plastic bags for each animal.

In both cases the frogs do not come in contact with the frogger's skin or clothing. At times frogs prove difficult to catch and handle, or try to escape. In such instances where the frogger's skin or clothing comes in contact with the frog then these should be cleaned using an antiseptic solution before handling any other frogs.

Ensure that used gloves and bags do not come in contact with clean gloves or bags. When work is completed at a stream dispose of the bags or gloves and disinfect any clothing or equipment that came in contact with them. Clean hands with hospital grade disinfectant.

3.1.2 To decrease possibility of transmission from toe clipping or PIT tagging

Toe clipping is used to collect genetic material and/or to individually identify animals or cohorts in mark-recapture studies. Toe clipping has been associated with inflammation and local infection in the many studies and there is some evidence that it may decrease longevity. If individual identification is necessary then consider alternative means of identification e.g. pattern recognition..

If toe clipping is used, decrease the risk of transfer between frogs by ensuring every frog has a cutting instrument free of the agent, either:

1. Disposable sterile instruments,
2. Instruments sterilized previously and used once, or
3. Instruments resterilized in the field and reused.

A possible strategy for using disposable instruments is to use a 15G or 11G scalpel blade to cut off toes and a compound to stop blood flow. A scalpel blade will be difficult to use unless the toe is fixed against a reasonably solid surface. However, a small piece of card may be adequate for this, and could be disposed of after every frog.

All cutting instruments could be sterilized before field trips by autoclaving or other standard procedures. This could be done if the researchers know approximately how many frogs will be marked and they take sufficient sterilized instruments.

Resterilised instruments: if biologists had several sets of instruments, after use on one frog, the instruments could be cleaned, placed in a sterilising solution for the necessary period of time, and reused. Possible options:

1. Immersion in 70% methanol for 30 min, or
2. Dipped in 100% methanol and then flamed, or
3. Immersion in 1% glutaraldehyde for 15 minutes. or
4. Immersion in boiling water for 10 minutes.

Note that for options 1 and 3, the instruments should be rinsed well in water.

Most of the options are difficult to implement in the field. Take care not to let frogs contact the disinfectants. Do not contaminate streams.

PIT tags are supplied in sterile needles. However other instruments used in the implanting procedure (e.g. forceps) should be sterile prior to use on each animal (see above).

Assuming the water is contaminated, open wounds left by toe clipping or PIT tagging may increase risks of transmission by allowing easier access of the agent. Sealing the toe or PIT tag hole by rapid artificial means would decrease these risks. It is not feasible to hold frogs in sterile conditions until the wounds heal. However the use of a cyanoacrylate compound such will seal the wound until it heals naturally.

Christy (1996) reports using PIT tags after swabbing with 0.1% iodine and then sealing with medical grade cyanoacrylate. Captive Limnodynastes peronii and and free-ranging Litoria aurea showed no ill- effects.

3.2 REDUCING RISKS OF SPREAD TO NEW AREAS

Always go from uninfected streams to those suspected to be infected, not the reverse. It is much more important that these procedures are used for long distance movements by froggers than for short moves. Adjacent streams in the same major catchment are likely to have similar agents present, but the possibility of introducing new agents is greatly increased by moving between major catchments or over much longer distances

Spread on people: