Archie Carr Center for Sea Turtle Research

• Lawrence H. Herbst, D.V.M., Ph.D., Dipl. A.C.L.A.M.
  Institute for Animal Studies
  Albert Einstein College of Medicine
  Bronx, New York 10461 USA
• Elliott R. Jacobson, D.V.M., Ph.D., Dipl. A.C.Z.M.
  College of Veterinary Medicine
  University of Florida
  Gainesville, Florida 32610 USA

The following specific recommendations are designed for capture-and-release field studies. A similar approach should be used for animals in captivity. The primary objective of these recommendations is to minimize the risk of either introducing a new pathogen into a population or amplifying the rate of transmission from animal to animal of an endemic pathogen when handling, tagging, and collecting samples from animals. Depending on field conditions, some of the recommendations may seem costly, impractical, or impossible to follow without compromising the ability to complete the study in a timely manner. However, these are ideals that one should strive toward while maintaining a reasonable perspective. For example, if capture methods result in multiple captures at once (trapping or netting turtles), or if field conditions are such that numerous animals are already in close contact (mating aggregation, nesting beach arribada, hatchlings in a nest, snake hibernaculum) then it becomes easier to rationalize not using strict individual to individual barrier practices such as separate holding containers and hand cleaning between animals. Once investigators become aware of the potential problem of pathogen transmission between animals either directly or indirectly on instruments and contaminated surfaces, they will be better prepared to develop specific protocols that apply to their specific situation.

Recommendations

1. Use separate holding (primary) containers for each individual. Avoid commingling animals if possible. For example, use separate sanitized bags for each snake or lizard, separate buckets or holding boxes for each turtle. If commingling is unavoidable, try to minimize holding times and group sizes.
   Note: A special consideration involves large reptiles such as marine turtles brought into boats. It is impractical to thoroughly clean each boat between the boarding of each turtle. It may be possible to use plastic sheets on the bottom of the boat where animals are held until released and then the sheets changed between each capture. We realize, however, that this may not be practical. Another possibility is to spray down the working surface of the hold with an environmentally safe disinfectant, such as dilute sodium hypochlorite (bleach; see below under “Disinfectants” for dilution). The most important thing to do is to inspect each animal thoroughly at the time it is boarded to note any lesion or disease. Then it is imperative that an ill animal be segregated from other apparently healthy animals already on board and that the next animal boarded should not come in contact with any secretions or other material from the ill animal.
2. Do not commingle species. Use separate holding (primary) containers for each species. Again, complications arise when boats are being used to hold marine turtles until they are released.
3. Containers and surfaces that contact the animal should be sanitized or disinfected between animals. Minimally, this should be done before and after groups and between species. The least expensive and most widely used disinfectant for surfaces is sodium hypochlorite (bleach). Certain disinfectants such as those containing phenolic compounds are toxic to reptiles and are to be avoided.
4. Wear exam gloves and discard or disinfect them between animals. This also protects the handler from zoonotic agents.
5. Sanitize hands before handling animals, sanitize them between animals, and after handling them. Use soap and water or a skin compatible disinfectant. If you cut your skin with a contaminated instrument be sure to wash the wound several times, preferably with a disinfectant soap such as betadine (povidone iodine) scrub. Wash for at least 3 minutes. A first aid kit should always be available.Note: The need for hand decontamination can be debated. However, one should be aware that feces, urine, and other body secretions may carry potential pathogens that can invade across intact epithelial surfaces or colonize epithelial breaks too small to be visibly detectable.Note: A special consideration for additional personal protective gear (face masks) involves procedures that may generate fine particulates or aerosols such as when a biopsy is taken from the shell. Power tools such as Striker saws and Dremmel tools, can be used to collect shell biopsies and to enter the coelomic cavity surgically through the shell. The keratin and bone that is aerosolized can be irritating to the respiratory tract and may cause foreign body reactions. These aerosols may also contain zoonotic pathogens. Remember that the respiratory tract is an excellent avenue for pathogens to invade your body. So avoid inhaling this material. Surgeons’ masks and particle filter masks provide the wearer with some level of protection although they are not specifically designed to fully protect the wearer from airborne pathogens or allergens.
6. When animals with obvious clinical disease are captured, they should be segregated from the unaffected animals and handled last.
7. Disinfect the skin or surgical site whenever any invasive procedures will be employed no matter how trivial (injection). This minimizes the risk that surface contaminants will be carried into the wound. Minimally this would involve wiping the surface with a disinfectant soaked gauze prior to injection.
8. Use only sterile single-use disposable instruments (needles, scalpel blades) for invasive procedures. Implantable transponders (PIT) tags should be sterile; punch tags should be sterile or disinfected.
9. Instruments and equipment that are used repeatedly for invasive procedures must be sterilized or disinfected between animals. This includes tagging devices and laparoscopic equipment. Realize that even the strongest cold sterilization products, such as the various aldehydes, do not kill all pathogens immediately on contact. Dipping instruments in these chemicals will reduce the load of pathogens on the surface but may not kill everything. If, during an invasive procedure, the instrument comes in contact with a lesion, such as a granuloma or an abscess, this instrument should not be used again until it is sterilized. Several sets of instruments should be available in case one set becomes contaminated by a lesion that may harbor infectious agents.
10. Wound treatment. Although it probably does no harm to treat minimally invasive wounds on animals that will be immediately released, the benefits of such treatment are questionable. Certainly there is little benefit in treating a wound that will be immediately contaminated after release. For many wounds fresh air and sunlight make the best bandages (see note below). For wounds resulting from an invasive procedure that enters the body cavity, such as laparoscopy, premedication with an injectable antibiotic and closure with a surgical glue should be considered. For body cavity penetrations in aquatic and semi-aquatic species, it is especially important to try to make the wound closure watertight.Note: Although it seems intuitively obvious that a smaller wound will be less likely to be a problem for the animal, this is not always the case. Generally, if wound edges are apposed, a 2-cm skin laceration will heal as rapidly as 2-mm one of the same depth. The tenfold difference in wound size will have some effect on the risk of contamination but not much. Puncture wounds, no matter how small the surface defect created, may result in fatal infection if the wound is not sterile. Deep narrow puncture wounds are likely to favor anaerobic conditions and the germination of anaerobic bacterial spores such as Clostridium spp. Larger defects that are open to the air will be less likely to have problems. A good example for discussion concerns the use of PIT tags versus standard punch-through metallic (flipper) tags in sea turtles. The larger punch through tag wound does present a larger surface for contamination and may take a bit longer to heal. However, this wound is open to air and actually heals with little problem. The implanted materials placed subcutaneously through a puncture wound must be sterile and the skin must be surgically prepared to avoid infection.

Sterilization and Disinfection of Instruments and Equipment Used for Minimally Invasive Field Procedures

Sterilization kills all life forms, including resistant spores. Disinfection kills almost all vegetative microbial life forms, but resistant spores are usually not killed. Therefore, disinfected materials are not sterile. There are a variety of methods available for sterilization and disinfection, and use depends on the sensitivity of the instruments or equipment to be sterilized and on field conditions. Equipment that will be used repeatedly in invasive procedures should at least be sterile at the start of a session and be disinfected between animals.

• Heat sterilization:
  • Steam autoclaving (5 minutes at 135 C or 15 minutes at 121 C under 15 psi) is excellent for metallic instruments and tools that will not lose their temper during repeated heating and for some non-plastic materials (paper, cloth).
  • Dry heat (170 C for 1-2 hours) is effective, but cannot be used for any flammable or meltable material, or metallic materials that can lose temper.
  • Flaming instruments by dipping in alcohol and burning off the fluid is an extremely effective field method, but it is an extreme fire hazard. Note: Soaking instruments in boiling water for 20-30 minutes will disinfect but will not sterilize them.
• Cold sterilization: Chemical (cold) sterilants must be used for electronic devices, plastic, rubber, etc., that cannot be sterilized by heat. One should always check with the manufacturer of a device before selecting a method to sterilize it, as some agents will damage the materials. Whether an agent is a sterilant or a disinfectant often will depend on its concentration and the amount of time it is in contact with the material.
  • Aldehydes such as glutaraldehyde 2% or formaldehyde (vapor or 10% solution) are highly effective sterilants but are highly toxic. Instruments must be thoroughly rinsed before coming into contact with living tissues. These agents are environmental hazards, so their use should be carefully managed, and they should not be carried into the field. For 2% glutaraldehyde, a minimum contact time (soaking) of 5 minutes for disinfection and a minimum contact time of 20 minutes for sterilization (sporocidal activity) are recommended.
  • Chlorine dioxide (Alcide, Cidex) is a useful sterilant as well as disinfectant. It is more stable than bleach. The disadvantages are similar to those for bleach (below).
• Disinfectants:
  • Bleach (Sodium hypochlorite) – a 1:10 dilution of household bleach (5.25% sodium hypochlorite) is an inexpensive, readily available, quick acting disinfectant. A 30-second treatment with bleach should be extremely effective. Standard practice is a spray followed by a wipe down (< 5 seconds). It is also quite effective to allow the spray to dry on the surface. Disadvantages are that bleach solutions bleach surfaces and clothing, corrode many metallic materials, irritate tissues, and are easily inactivated by ultraviolet light and organic matter. Rapid inactivation, however, is also an advantage from an environmental perspective. Bleach is not suitable for skin disinfection. Materials that will contact living tissue should be thoroughly rinsed.
  • Chlorine dioxide (as above).
  • Povidone iodine (10%) (Betadine) is bacteriocidal, virucidal, fungicidal, and protozoacidal. It is stable, highly effective, and nonirritating to mucous membranes. Therefore it can be used on living skin as surgical prep or surface wound treatment. Standard practice is a spray followed by a wipe down (< 5 seconds). It is also quite effective to allow the spray to dry on the surface. The disadvantages are that it is corrosive to some metals and may stain surfaces (wood, paper).
  • Chlorhexidine diacetate 2% (Nolvasan) is bacteriocidal and virucidal. Diluted (3 oz per gallon) it can be used for cold disinfection of instruments and diluted further (1 oz per gallon) can be used for general surface and premises disinfection. Standard practice is a spray followed by a wipe down (< 5 seconds). It is also quite effective to allow the spray to dry on the surface. It is not highly effective against certain Gram positive bacteria at these dilutions, however, and although specifically labeled for premises disinfection, open dumping is prohibited.<
  • Alcohols (ethanol, isopropanol) are inexpensive and readily available but are not very effective by themselves. The amount of time required for adequate disinfection–many minutes–is impractical. However, they can be effectively used with other disinfectants, for flaming instruments, or for clean-up and rinsing materials.