Tortuga Gazette 39(7): 8-9, July 2003
MYCOPLASMA LADY
Professor Mary Brown
Interviewed by Stephanie Pappas
PART 3
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 Mycoplasma as 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
Dr. Brown if we know that healthy looking tortoises can test positive for M. agassizii should 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
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.
Stephanie Pappas
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?
Mary Brown
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 Mycoplasma in 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.
Stephanie Pappas
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?
Mary Brown
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 stripped coat. When the host (the tortoise) sees the Mycoplasma it
recognizes the blond hair with the stripped 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.
Stephanie Pappas
What is the reliability of the ELISA test and can you explain what is meant by a positive, negative and suspect result?
Mary Brown
ELISA test is the best screening tool available for identifying M. agassizii. We have a quality control procedure
and for every assay we run we have a positive and negative control with a dilution of known serum so we know the final
titer end point. Than we do a statistical analysis and generate a standard curve. We plot a straight line and determine
what titer values are positive or negative. There are ways to determine sensitivity and specificity of the test, and we
have published the details. A Positive value is one that is over 128 in the titer system (this indicates there is no
question M. agassizii is recognized). A Negative is below this and indicates no M. agassizii are present.
However, you could get a negative result if it is the first 6 to 8 weeks of exposure and if M. agassizii is not
the organism causing URTD. Remember, the current ELISA test uses antigen to M. agassizii only and therefore may
not detect infections by other mycoplasmal species that may be involved in URTD. If M. cheloniae is present the
ELISA test could still be Negative because M. cheloniae may not cross react. A Suspect or Gray Zone result means
you may want to repeat the test. Only 5% of all the animals tested have shown up in the gray zone so the statistics are
very low. Even in a clinical test animal, when you know what you are going to see, you can get blips-negative, suspect,
positive.
Stephanie Pappas
Have you ever retested a positive tortoise and found a negative result?
Mary Brown
You would never see a high positive drop to negative. The only exception would be in a hatchling where you have maternal
antibody. Antibody is transmitted through the yoke sac, so hatchlings can have maternal antibody that is declining over
time. We do not know how long maternal antibody can remain in a hatchling before they will lose all of it.
Stephanie Pappas
Dr. Brown, this has certainly been a very informative interview, but before I let you go can you tell us how you
actually got started studying this bacteria (Mycoplasma)?
Mary Brown
When I left Birmingham I commented to the people in the lab that I really did not care what host I worked with as long
as I could keep working with Mycoplasma. This is a host that I truly care about. I have worked with many species
affected by Mycoplasma; humans, rats, mice, goats, chickens, tortoises and others animals. I have become attached
to this host! I was sitting in my office one day and Elliott Jacobson walked across the hall with an electron microgram
and said, "Mary do you know what this is?" I said, "Cool, Mycoplasma, where did you get it?" He said, "Do you
think you can grow it?" I said, "Sure", but little did I know that that Mycoplasma would be the most difficult
organism I would ever grow in my life. That is how I first became involved with Mycoplasma and how we discovered
M. agassizii is the host that causes URTD. After that we had wonderful support from all these people involved in
working to save the desert tortoises: Kristin Berry, Ken Nagy, Dave Morafka, Mary Christopher, and all the great
students and veterinarians we have had the pleasure to work with. These individuals educated me on the ecology and the
desert tortoise and we all worked together as a team. When I think of my career, having the opportunity to determine
what causes a disease in an animal as important to the ecosystem as the Desert tortoise has been one of the most
rewarding things in my life.
Stephanie Pappas
In conclusion, Dr. Brown what makes the Desert tortoise so valuable to the ecosystem and what one piece of advice could
you give to our CTTC members that they could do to help save the Desert tortoise?
Mary Brown
The Desert tortoise is a keystone species. Their burrows provide refuge for many other species living in the desert. The
tortoise is very active in terms of speed dispersal and an early warning system that something is going wrong in their
habitat, because when a tortoise dies you see a shell but when another amphibian dies there is no evidence of death. The
tortoises are like a canary telling us to wake up and do something to save the habitat. URTD is only one very small
component of what is happening to both the Gopher and the California Desert tortoise. More critical than the disease is
the destruction of the habitat for human population expansion, as we continue to develop areas in the desert that
affects not only the tortoise survival but many other species of plants and animals living there. If we cure the disease
what good will it be if there is no land for these animals to survive? If people truly want to help the Desert tortoise
they should get involved with the Desert Tortoise Preserve Committee. These individuals are dedicated and truly working
hard to buy the land needed to save the animals. Volunteer, become educated, and start letting others know how important
it is to preserve the desert - the environment and all the species living in it.. The desert is a fragile bio-diverse
system, and if you pull one or two pieces out the whole system can collapse.