Facts on upper respiratory tract disease in tortoises and turtles are revealed in this exclusive interview with the world renowned scientist, Professor Mary B. Brown, Ph.D.  She was part of a team that worked to identify Mycoplasmaas the organism causing URTD in 1992. Professor Brown has continued to address new and difficult questions about the effects of this disease on tortoises and she contributes highly valued expertise to many organizations working to save tortoises. The interview took place in Palmdale on January 25, 2003 when Dr. Brown was visiting California and about to be honored by the Desert Tortoise Preserve Committee with the Golden Tortoise Award.

Stephanie Pappas (SP): Dr. Brown if we know that healthy looking tortoises can test positive for M. agassiziishould tortoise collectors and hobbyist mix a healthy looking tortoise with another healthy looking tortoise without testing for Mycoplasma? And if you have a healthy looking tortoise that has tested positive but never shown a clinical sign of infection can that tortoise transmit the disease to another tortoise?

Mary Brown (MB): Boy, we don't definitively know the answer to that question. Common sense is most important. If the animal has never shown clinical signs, that means shedding of the organism is not occurring in very high numbers. However, say you put two males together and they start fighting and causing stress then perhaps they could spread the disease. We just don't know. Therefore, it is up to the individual owners discretion, but they should take into account how crowded the area is; how long they have had that tortoise under observation; and how comfortable they feel as an owner risking the health and safety of their animal. If you have another species of tortoise that is of great value I would not mix the animals because what if that tortoise had another disease like a virus that could be transmitted and fatal. Whenever you start mixing species you risk transmitting other diseases besides Mycoplasma.

SP: So would you advise our members to limit their populations of turtles and tortoises within a given area and to provide the most space possible for each species?

MB: Yes, use common sense. If you think about it crowding animals increases the chance for contact of infections and mixing species increases contact. I will give you a great example of why you should worry about mixing species. We also have Mycoplasma infections in the America Alligators. In Alligators and Caimans, Mycoplasma is rapidly lethal. By rapidly lethal I mean it kills the animal within two weeks of contact, from sepsis and joint infections. In Crocodiles, however, it causes minor problems with their tonsils. This lethal strain of Mycoplasmain the American Alligator and Caimans was originally spotted in a large area with many different species of animals. So why lethal in Alligator and Caimans? Keep in mind that bacterial or viral infections can be benign in one animal and jump to another animal and become a severe lethal disease. Classic cases of that are observed with Ebola, HIV, or West Nile Virus. So with that in mind it is always better to be cautious rather than having to deal with the die off of your collection of tortoises and turtles.

SP: Dr. Brown, we have talked in great detail about the transmission of the disease, so now I would like to focus on the diagnosis of the disease. Some people believe that the enzyme-linked immunosorbant assay (ELISA) test used to diagnosis M. agassizii is not reliable. Can you explain what an ELISA test is and how you diagnose whether your tortoise has Mycoplasma agassizii

MB: The ELISA test is a way to determine how many antibodies are in the blood of the tortoise that recognize specifically the surface antigens of Mycoplasma. When an animal is exposed to a foreign substance (a bacteria or virus) it makes an immune response. What it actually makes is a type of protein called an antibody. Think of it as Mycoplasma having blond hair and blue eyes with a striped coat. When the host (the tortoise) sees the Mycoplasma it recognizes the blond hair with the striped coat. The ELISA test, tests all the proteins (the antibodies) that recognize the Mycoplasma surface. The test itself is done in a plastic plate with a solid surface and 96 wells in it. In each well we put the Mycoplasma antigen and allow it to absorb or coat the plate. Than we add the tortoise serum (blood). There will be many antibodies that do not have anything to do with recognizing M. agassizii, but if any are there they will specifically recognize the Mycoplasma and bind tightly to the antigens that they recognize, the blonde hair and stripped coat. After binding occurs we wash off the material that did not bind to the antigen. We now have a sandwich, plastic with Mycoplasma antigen with specific antibody attached. Now we have to detect the tortoise antibody, so we prepare a special protein that recognizes specifically tortoise antibody. This antibody was developed at the University of Florida. We also grow the antigen Mycoplasma, which is the hardest part. The tortoise antibody is referred to as a monoclonal antibody and it has an enzyme tag on it which is a detector molecule and referred to as a conjugate. It binds and recognizes the tortoise immunoglobulin. Than you wash that off and add the substrate that comes in contact with the enzyme and turns from clear to yellow.  Once all the steps are complete, you measure how intense the yellow coloring is in the plate using an instrument called a plate reader. The more yellow the more antibody present recognizing Mycoplasma and indicating that the tortoise has tested positive.