Bearded Dragon Health: Parasites and Adenovirus

Worms

Pinworms
Bearded dragons have a number of parasites and commensals, one of the most common is pinworms or Oxyurids. These nematodes are debatable parasites, since they cause no real pathology unless they super infect a captive animal that is over exposed to the eggs. Most veterinarians consider them to be pests, but mostly commensals rather than parasites. They appear as small, short, white, tapered worms approximately 5-8mm long, though rarely some may be over 10mm. These worms reside in the colon and cloaca, rarely venturing outside to be seen by the owner. They lay eggs ranging in size around 95 microns long with a roughly triangular shape and recognizably sculptured, brown shell.
The life cycle is fairly straight forward. The worms reside in the colon where they lay eggs. Eggs are passed in the feces. In the environment they mature rapidly into infectious eggs and are ingested where they hatch in the bowel and attach to the colon to mature into adult worms.

Treatment
The treatment of choice is fenbendazole, though in recent years this has proven to be largely ineffective in treating mammal nematodes due to resistance, and may be ineffective in treating reptile nematodes as well. Working with several breeders, I have noticed a significant decline in the effectiveness of fenbendazole (sold commercially as panacur). One population I have worked with has shown little or no response to fenbendazole treatment, showing that resistance has occurred in at least one population to the point of virtual immunity.
Currently there is no real treatment that is decisively effective. Ivermectins have been used with some success in reptiles, but the margin of safety is so narrow that it is too risky for most people to try. Ivermectins are another class of drug that has treated nematodes very successfully in mammals, but reptiles are extremely sensitive to it and can easily overdose and die. For that reason, it is generally considered better to live with the pinworms than to give ivermectins.

Coccidia

Two species of coccidia are known to infect bearded dragons. The first was described by Cannon in the 1960's and is named Isospora amphiboluri. The second was described by Walden in 2009 and is named Eimeria pogonae.

Isospora amphiboluri
Isospora amphiboluri has been associated with mortality and poor doing in bearded dragons and the infections can be severe. The life cycle appears to be a direct one within the intestine and it moves through the intestine as infection progresses. The prepatent period can be up to 25 days. The prepatent period is the time between infection and detectable numbers of oocysts appearing in the feces. The best way to diagnose the presence of this parasite is with modified Sheather's solution fecal floatation. The parasite infects the host by fecal oral contact and reproduces and amplifies within the host. A single oocyst can give rise to thousands of oocysts that are shed in the feces. Sporulation largely takes place in the colon or cloaca, so oocysts are infectious when first deposited.
Eimeria pogonae
Eimeria pogonae is less well understood, and may be a gall bladder infecting species, but the site of infection is as yet undetermined, though it is shed in the feces and can be detected with modifed Sheather's solution fecal floatation. Presumably the life cycle is roughly similar to I. amphiboluri and other members of the Isospora and Eimeria genera and consists of an amplification phase followed by sexual phase and the formation of new infectious oocysts. Oocysts are largely passed sporulated and infectious.
To date there has been no established disease state associated with E. pogonae, and it may actually be better classified as a commensal.

Coccidia Life Cycle
The life cycle of the coccidia in the genera Isospora and Eimeria are similar. In gut limited species that do not form tissue cysts in other tissues of the body (like Toxoplasma for example), the life cycle begins with the ingestion of the oocyst, usually from fecal oral contact.

• Ingestion
• The oocyst passes unharmed through the stomach and into the intestine.
• Excystation - bile acids and enzymes trigger the oocyst wall to split at its seams and fall away exposing the sporocysts (2 sporocysts each containing 4 sporozites in Isospora and 4 sporocysts each containing 2 sporozites in Eimeria).
  
• The sporocysts then split along tiny seams or, in those species with a Steida body, the Steida body swells and then literally pops out followed by the sub Steidal body (in those species in which a sub Steidal body is present).
• The freed sporozoites then look for host cells and penetrate the cells using organelles in their anterior end (specifically rhopteries, dense granules and micronemes). The sporozoite uses the host cell's own membrane to make a protective bubble around itself called a parasitiphorous vacuole as it pushes into the host cell's cytoplasm and takes over machinery causing the host cell to feed the parasite.
• The sporozoite then becomes a meront (sometimes called a schizont - pronounced skizont). The meront undergoes merogony (schizogony), which is asexual reproduction, and forms numerous merozoites.
• Merozoites burst from the host cell and infect more host cells. The merozoites repeat the same process as the sporozoite and a new round of merogony begins in each new infected cell, continuing the amplification phase of the infection.
• The number of rounds of merogony is species dependent, but most Isospora average around 3, though some species of Isospora and Eimeria can go through 5 or more.
• The last round of merogony results in infected cells where the merozoites differentiate into female or male and form gamonts.
• Female gamonts (macrogamonts) are a single cell. Male gamonts (microgamonts) form many cells analogous to sperm and called microgametes. These burst out of the host cell and fertilize the macrogamonts to form zygotes.
• Zygotes mature into oocysts which are released into the lumen of the gut and defecated into the environment. The oocysts of some species form new sporocysts and sporozoites (a process called sporulation) quickly and are infectious when they first are deposited. However, some species must take some time to sporulate and are not infectious for a certain period of time ranging from hours to days.

Coccidia Treatment
Treatment for coccidia has classically been sulfadimethoxine (commercially called Albon). Studies by Walden (2009) have shown that sulfadimethoxine was effective in treating coccidia, but even after 21 consecutive days of treatment, some animals were still positive. Studies conducted by Walden also evaluated the probiotic Pediococcus and oregano oil (both of which have had some success in controlling coccidia in poultry) and found that these treatments had no significant effect. Ponazuril (commercially marketed as Marquis) compounded as a 90mg/ml solution and given at 30-45mg/kg was very effective in treating coccidia.

Reference: Michael R. Walden, MS, DVM, PhD; Characterizing the Epidemiology of Isopora amphiboluri in Captive Bearded Dragons (Pogona vitticeps). PDF available at http://etd.lsu.edu/docs/available/etd-05292009-214931/
You can also check out my amazon.com reading list if you are interested in herpetoculture, herpetology or herp medicine by clicking the "Selected Herpetology References" link below.
Selected Herpetology References - New Category (1)

• Selected texts for the herpetologist, hobbyist or veterinarian.

Parasite Fecal Floatation Pictures

Oxyurid ovum with coccidia oocyst of Isospora amphiboluri. Bar = 100 microns. 200x. Used with permission.

Isospora amphiboluri oocyst. Bar = 20 microns. 600x. Used with permission.

Eimeria pogonae oocyst. Bar = 20 microns. 600x Used with permission

Adenovirus

Adenoviruses are a widespread group of viruses that infect many species including amphibians, reptiles and mammals. In recent years a poorly characterized adenovirus has been observed killing bearded dragons.
The adenovirus kills dragons by caused necrosis (death) of numerous cells, particularly the enterocytes (gut lining cells) and hepatocytes (liver cells). Recently Louisiana State University developed a PCR test to test for adenovirus in bearded dragons. The test was conducted by the Dr. Alma Roy of the diagnostic lab along with Dr. Shawn Zimmerman and Dr. Michael Walden. The standard test to date had been electron microscopy of the feces to see the viruses, and it had been assumed that the EM was sufficiently diagnostic. The study places doubt on that.
What does adnovirus do?

1. Adenovirus causes interference with cellular machinery and ultimately ruptures and kills the host cell.
2. Its cellular specificity is not understood, but it is known to use enterocytes, hepatocytes and renal cells as hosts.
3. Fatality usually results from hepatitis in severe cases or starvation in milder, more protracted cases.
4. Gross lesions are not present externally, making diagnosis difficult for the clinician.
5. Some animals have pale livers resembling glycogeniasis, lipidiasis or lipidosis grossly.
6. At least 2 outbreaks have been reported in the U.S. recently.

EM has certain pitfalls.
Threshold – is around 1,000,000 (106) particles per gram of unprocessed feces.
Time consuming – very time consuming.Preparation of samples – hours.
Grid preparation – hours.
Staining and sample to grid – minutes.
Grid examination – hours.
Expensive

PCR in a nutshell
 Disadvantages of PCR
Currently not as widely available as EM

Advantages of PCR
Quicker than EM
More sensitive than EM
Probably as specific in reality as EM
Chance for false negatives is lower than EM
Chance for false positives is higher than EM – Huh?

Chance for false positives is higher than EM. Why is that an advantage?
Production situations would rather have false positives than false negatives when introducing new stock.
False negatives introduced into a true negative population can cause severe economic consequences.
In most cases culling 100 new introductions to maintain a negative breeding population is more cost effective in the long run than having a costly outbreak in a population of several thousand.

Comparison of Results Between EM and PCR
EM
5 of 132 samples were positive for Adenovirus (3.79%).
8 of 132 samples were positive for Parvoviral like particles consistent with Dependovirus (6.06%).
PCR
117 of 132 samples were positive for Adenovirus (88.64).
So PCR is far better for detecting adenovirus in bearded dragons. So if you think you have adenovirus in your population, contact LSU.

Treatment
Unfortunately there is no treatment and positive animals should be culled from the population.

Source
Association of Reptilian & Amphibian Veterinarians - proceedings from 2007 in New Orleans.

Adenovirus EM

Electron microscopy colorized photo showing Adenovirus (red) and associated dependovirus (yellow). Copyright Dr. Michael Walden, 2009, all rights reserved.

Be Careful of Hobbyist and Birding Book Terms

Concerning Birding Sites and the Ornithology Books
You have to be careful about many of the birding books and sites. Most PhD's in ornithology know very little about chromatophore biology and they throw words around carelessly. To be fair, ornithologists are not physiologists, cell biologists or pathologists and have most of their education in behavior, ecology and taxonomy. So they can perhaps be forgiven for not really understanding the finer points of pathological conditions and the need for consistency within the scientific community that deals with these issues. Hobbyists are really bad about using terms incorrectly and that comes from much misinformation on the net and from reading books by those who are not really qualified to make a diagnosis of albinism or leucism (leukism). There are several sites with really weird definitions out there that have no pathological basis. One site that has been brought to my attention:

http://listserv.arizona.edu/cgi-bin/wa?A2=ind0011c&L=birdband&T=0&F=&S=&P=778

This one does a fairly good job at pointing out the inconsistencies in the birding literature regarding leukism.

Another problem with the bird literature is the superficial examination of the animals themselves with no real scientific scrutiny. The tendency to throw the word leucism (leukism) around is rather frightening. Any animal with a messed up patch of feathers that have gone white is called leucism by these people. Just look at the Cornell folks:

http://www.birds.cornell.edu/pfw/AboutBirdsandFeeding/Albinism_Leucism.htm

SCARY for anyone concerned with accuracy and precision, the way scientists are supposed to be.

The problem with this "bird brained" approach to the question is they are examining feathers. These are structures composed of keratin and they have no idea whether or not there is a lack of pigment, reduction of pigment or if there is an impairment of pigment transfer to the keratin. Transfer impairment would constitute a whole new pigment disorder not leukism, which has already been established as a defect involving the survival or migration from the neural crest of chromatophores. To date this has not been examined. What is more, is that many birds called leukistic are not really leukistic. They have pigment on most of their feathers, they just have patches of white. That is not really classical leukism. Pattern mutations, piebaldism, or some other disorder should be considered before the diagnosis of leukism is applied. To make a diagnosis of a disorder without understanding the basic pathogenesis is at the least VERY BAD FORM, and MALPRACTICE at worst!
Furthermore all you have to do is look at a calico cat as a good example. Due to the X chromosomes having different color genes you get two colors, right? No, you get three. One color on calicos is white. Tuxedo cats also have black and white. This is not leucism. It is a pattern mutation. The abnormal patchy distribution of white in the feathers of birds may be something similar, and this must be ruled out before they can be called leukisitc.

Regarding pigments, other sites which are not so good, I will not mention. But several of you have asked about another birding term, that really has no pathological authority. Xanthochroism is an odious term to be frank about it. It really has no real biological basis. It often is seen in psittacines and in aquarium fish. The cause is generally a lack of the melanins and other pigments that cause the yellow pigments to be all that is left, so yellow predominates. There is a problem with this term. First the term means yellow skin. Not a good term for birds, since we are talking about the feathers not the skin itself. Also it is a lack of pigments that causes the problem, not excess of yellow. To understand the pathogenesis, it is necessary to understand the proper name to give the condition. By the way, xanthism is also used for this and that is a really poor choice.
Here is a list of color abnormality definitions so you can see why this is a problematic condition to name.

• Erythrism (Erythristic) - excessive production and deposition, or distribution of red pigments (orange possibly).
  
• Anerythrism/Anerythristic - lack of production of pigments in the darker orange to red range.
• Hypoerythrism/Hypoerythristic - reduction in the amount of darker orange to red pigments so that the appearance of this color is largely absent except for traces or appears "washed out."
  
• Xanthism (Xanthic) - excessive production and deposition, or distribution of yellow pigments (orange possibly).
• Axanthism/Axanthic - lack of yellow and lighter orange pigments, depending on the point in the pigment cascade, this mutation can also cause corresponding anerythrism since erythric pigments (drosopterins) appear to come from the more yellow pteridines biochemically.
  
• Hypoxanthism/Hypoxanthic - reduction int he amount of yellow or lighter orange pigments so that the appearance of this color is only found in trace amounts or appears "washed out." This may also result in hypoerythrism since the red pigments appear to be made from the yellow pteridines.
  
• Melanism (Melanistic) - excessive producution and deposition, or distribution of melanin pigments (may be orange if pheomelanin to black if eumelanin).
• Amelanism/Amelanistic - lack of melanin production. At least three basic forms are possible, though whether all forms have been observed is questionable. 1) amelanism where the chemical cascade is defected before eumelanin and pheomelanins take separate biochemical routes, resulting in a complete lack of melanin production. 2) aeumelanism - where only eumelanin production is blocked. 3) apheomelanism where only production of pheomelanins is blocked.
  
• Hypomelanism/Hypomelanistic - condition resulting in the reduced production of melanins. At least three types are possible by restriction of production at the initial stages of melanin production, at the eumalnin production cascade or at the pheomelanin cascade.
• Iridism (Iridistic) - excessive production and deposition, or distribution of iridophore platelets (this is, as yet, only a theoretical condition).
• Aniridism/Aniridistic - (again theoretical - I have not heard this reported) lack of the formation of refractile platelets in iridophores.
• Hypoiridism/Hypoiridistic - (theoretical) reduction in the number of refractile platelets formed in iridophores.
  

So if the condition in psittacines is caused by reduced melanin for example it is really a hypomelanism or amelanism not xanthism or xanthocroism. Furthermore, many birds have blue in the feathers as a structural color, that means it is not caused by pigments but by structural design of the feather causing the reflection of blue light back toward your eye. If yellow pigment and blue structures are combined then you will perceive green. In this case if the feathers turn yellow it is a structural change in the feather and that is another defect entirely. That may be a good condition to use xanthocroism for, or perhaps another name (I am open to suggestions here).
A Word on Blue
Blue is generally a structural color and is the result of the interaction of iridophores and other chromatophores. There should be a note here that some species possess other forms of chromatophores. Leukophores (leucophores) are described in fish, but they are really iridophores that have platelets reflecting white back at the observer's eye. Being a bit of a lumper, I really do not consider leukophores a separate cell line, but some people do. I must also admit to and point out a bit of hypocrisy here, since I tend to talk about xanthophores and erythrophores, but lump iridophores and leukophores.
Among the other cells out there are some that have been called cyanophores. No they do not produce lethal cyanide. Cyan = blue. "Bluephores" then are cells that would contain blue pigments. This has thus far been unusual. Most animals do not have blue pigments, but use iridophores and other chromatophores to produce blue. However, that does not mean that cyanophores do not exist. They have been demonstrated in fish, and I strongly suspect they are present in cephalopods and would be surprised if they are not. Are they present in amphibians and reptiles? No, not so far as I can find, but that does not mean they are not present in some species and just have not been described. I do not look to find them in any known reptile species (though I would be pleased if I was surprised), but hope to find a paper one day where they have found them is some rain forest amphibian.

Chromatophores

I have had several questions of late regarding pigmentation and chromatophores.  There is a lot of information out on the misinformation super highway about chromatophores, but it is highly confusing.  Part of the reason for this is many people take information from studies done in mammals and think that can be lumped into one big pot with studies done in reptiles.  I would even argue that lumping mainstem reptile studies with studies in archosaurs might be a mistake.  The fact is they do not function in the same way, they do not go through the same development and they do not even have the same cells.  Mammals lack xanthophores (and the subclass erythrophores) and iridophores.  Mammals also lack dermal melanophores.  Mammals (some argue) do not even have melanophores, but instead have melanocytes.  The point is that the misinformation super highway (MiSH - not to confuse with MSH which is melanophore (melanocyte) stimulating hormone) is full of people that do not do the proper research and do not fully understand the subject they are writing about.  Some in the misinformation super highway's drunk lane (abbreviated 'wikipedia') do much more than confuse the issues, they actually write things that are incorrect and when it is corrected, change it back the the incorrect information (see the wikipedia article on leucism that a colleague of mine at another college tried to correct and wound up getting his stuff changed back to the incorrect information and told that he did not offer credible citations when he used research papers, peer reviewed literature and expert's text books as references).

The result is that there is a mass of confusion and it stems from the MiSH and wikipedia.  For an entry level of understanding about reptile and amphibian chromatophores you should start with the following three resources:

Reptile and amphibian variants - Bechtel, 1995 (book).

Dermal Chromatophores - Taylor and Bagnara, Am. Zoologist, 122:43-62(1972)

The Dermal Chromatophore Unit - Bagnara, Taylor and Hadley, The J of Cell Bio, 38:67-79(1968)

I will have a more thorough discussion on this topic later in the semester.

In brief, mammalian melanocytes do not appear to be the same as the melanophores in reptiles and amphibians.  Indeed, they do not appear to be the same as the chromatophores of invertebrates or fish either.  The chromatophore is a neural crest cell in its typical origin, though chromatophores not from neural crest develop in the eye.  They start out as a protochromatophore or chromatoblast.  They then differentiate into one of three, or four, types.

Chromatophore Subtypes - xanthophores, iridophores and melanophores contain all elements of all the chromatophore types.  Thus, melanophores contain pterinosomes and the iridophore plates (called reflecting platelets), but what makes them distinctly one type or another is the degree to which they contain the other structures.  Melanophores are melanophores because they contain around 99.9% melanosomes and only a small percentage of the other structures.  This is important to note, because this fact is what gave rise to the single progenitor theory for chromatophres.

Melanophores - contain mostly melanosomes and are capable of two forms of pigment production.  Eumelanin is brown to black and pheomelanin is orange to rust or rusty brown.  Melanophores, unlike melanocytes in mammals, generally do not inject their melanosomes into keratinocytes.  They are also usually able to move their melanosomes into their dendrites or into the perikaryon depending on neurohormonal stimulation.  The melanins are contained within the melanosomes.

Xanthopores - contain two major pigment bodies the pterinosomes containing pteridines and vesicles that contain fats with stored carotenoids.  Another class of organelle may exist in which the pteridines are converted to drosopterins and some people have suggested the name drosopterinosome.  However, since drosopterins are made from pteridines, this may be a bit of a splitter attitude, and really may not be valid.  But it cannot be denied that yellow pteridine rich cells occur within microns of orange or red drosopterin rich cells, so there may be something to the separation.  At any rate, xanthophores can be divided into at least two subtypes.
          Yellow xanthophores - pterinosome and pterinidine rich.  Since they are yellow to yellow orange and the term xanthophore can apply to the red xanthophores as well, there is a good argument to refer to this subtype as luteophores, but that term has yet to catch on. 
          Red xanthophores (erythrophores) - pterinosomes (drosopterinosomes) are rich in drosopterins which range from orange to red and even violet.

 Iridophores -  while possessing all the organelles of the other chromatophores, the iridophores primarily use refractile platelets formed by crystals of the uric acid based DNA components called purines.  Specifically the purines hypoxanthine, guanine and possibly adenine.  Basically theses platelets act as prisms and refract light to form certain colors and interact with different pigment bearing chromatophores to vary the colors.