Saturday 17th April 1993
Organised by the University of Bristol and the British Chelonia Group
THE ELKAN MEMORIAL LECTURE
The Elkan memorial Lecture was instigated by Professor John Cooper and this, the third Elkan Lecture, was given by Professor Peemel Zwart of the University of Utrecht, The Netherlands. In the absence of Professor Cooper in Africa, the lecture was introduced by Mrs. Margaret Cooper. An outline of the extraordinary life and work of Dr. Edward Elkan was presented in the first Elkan Lecture "The Guiding Hand - Foundations of lower Vertebrate Pathology" by John E. Cooper, published in Herpetopathology 1, 1-4 (1989). The second Elkan Lecture was given by Dr. Fredric Frye at the First World War Congress of Herpetology at the University of Kent in 1989.
Professor Zwart has lectured at B.C.G. Symposia on two occasions and the papers were published in Testudo. Biographical details were published in Testudo 3, (3), 11 (1991).
REPTILIAN DISEASES: A PATHOPHYSIOLOGICAL APPROACH
Utrecht University, DepL Veterinary Pathology,
Yalelaan 1, P.O. Box 80.158, 3508 TD UTRECHT, The Netherlands.
Presented at the British Chelonia Group Symposium, 17 April 1993 as the Elkan Memorial Lecture
This paper, is given in honour of the late Dr. Edward Elkan. Dr. Elkan died on July 4th 1983. It is a matter of good timing that this lecture, in honour of such an impressive man, is presented 10 years after his death.
I have tried to bring forward some ideas and views about disease and disease processes in which Dr. Elkan and I had a common interest. Of course Dr. Elkan had a different basis as a medical doctor. He had been directly involved in social life of man and in human interactions within families and populations, as was expressed most clearly by his involvement in family planning.
I came to know Dr. Elkan from relatively few contacts, mostly from 1964 - 1966. At that time, when Dr. Elkan was about 65 years of age, he was a very active man. If a letter was not immediately answered, he would remind you within a few days by writing a card starting with a phrase: "I know that the British post is no longer what it used to be, but have you received my letter dated ... and so on" I must confess, I only experienced this once. At that time we collaborated on the problem of Vitamin A deficiency in terrapins. Our first contact was initiated accidentally. In 1961 I presented a short paper on the histology of tissue structures of lesions in vitamin A deficiency in terrapins, which was presented at the Third International Symposium on Diseases of Zoo and Wild Animals in Cologne, Germany, (May 1961). It was Mr Fiennes, at that moment curator of the Zoological Gardens of the Zoological Society of London, who spoke to me and said "well do you know that Dr. Elkan has an almost identical manuscript?" I did not know. As a young scientist, I sent my text to Dr. Elkan to inform him about my activities and suggesting that, if my material was useful, we could perhaps consider a joint paper. And so our joint paper was released. The paper was entitled "The ocular disease of young terrapins caused by vitamin A deficiency" (Elkan and Zwart, 1967). At that moment it was the only ocular disease known in terrapins.
Later, we exchanged several items of common interest. Over the years we exchanged slides and transparencies of interesting cases. Dr. Elkan was always inclined to share new things he had discovered and had an open eye for and deep interest in things which were brought to his attention. I still remember that I sent him transparencies of a Nematode larva present in one of the mucous-glands of the skin of a frog. Dr. Elkan was very enthusiastic about this picture of a phenomenon he had never seen before. I may say now, that it is still surprising that Nematode larvae can be found in mucous glands of frogs, although later research, in which I was partially involved has demonstrated that the Nematode Oswaldocruzia invades the pond frog via the lachrymal or tear gland of the eyes.
A quite different aspect of the many facets of Dr. Elkan was his interest in humorous combinations of art and animal life. This came for vivid presentation at the first colloquium on pathology of reptiles and amphibians, 1983 in Angers, France. The paper had been prepared by Dr. Elkan but alas at the last minute he was too ill to attend the meeting. His paper was helpfully presented by John Cooper and his wife, and included transparencies from the slide collection of Dr. Elkan i.e. stuffed frogs, arranged as social activities of man such as a group of frogs imitating a chamber orchestra. 'The frogs were quite nicely arranged in characteristic positions of a violinist, one playing the piano etc.. Not only the positions were typical, but also the expression of the frog's faces were very individual and characteristic.
I am grateful to Prof. John E. Cooper and the British Chelonia Group to have this opportunity to present this lecture in honour of Dr. Elkan. I personally feel happy that I have at least to some extent worked together with Dr. Elkan and have experienced the warm personality and broad interests and the capability of this honourable scientist, who has initiated and contributed, so much in the area of veterinary science with regard to amphibia and reptilians.
I think it is a marvellous initiative of Prof. John E. and Mrs. M. Cooper to commemorate his works and to maintain, protect and extend the Edward Elkan collection in such a way that future generations of scientists have access to the material and can derive new ideas and new spirit from them.
It is with this historic background, and the present stage of knowledge of reptilian diseases that I proposed as a subject for my Elkan memorial lecture the pathophysiology of reptilian diseases. Up till now, the majority of the work done on reptilian diseases was the recognition and description of diseases and problems.
This title of my talk is perhaps daunting. So let us have a look at it.
Most people in this audience will have some ideas and thoughts about disease in reptiles. As disease is unavoidably connected with life, many of you will have been confronted with diseases in your captive animals. However, in this talk I will try to use reptilian diseases in a broader sense than just the individual patient. It is my intention to talk about the mechanisms of some problems which can be met in reptiles. At this very moment the knowledge of diseases of reptiles is still fairly restricted. It can be expected that in the years to come this area of knowledge will increase more and more. For the scientist, reptilian diseases are of interest because this group of animals was the first of the vertebrates to reproduce on land, and thus were superior to all vertebrates existing at that period. In the process of evolution onward from fishes and amphibian the ways animals react to diseases have also evolved. But reptiles still did not reach the development and specialisation of defensive mechanisms known in mammals and birds. However, this would perhaps be subject of a separate paper.
This word may need some explanation. It is a combination of two words, Pathology and Physiology.
Pathology is the science of the study of disease. It investigates the essential nature of disease (Thomson 1984).
Pathology is subdivided in two areas:
- the study of morphological changes, which means changes in the form and structure of organs, tissues and cells. It is this part of pathology where lesions are studied with the naked eye, with a magnifying glass, with the microscope and, in some cases with the electron-microscope. This type of study in general gives a clear view of the extension and severity of the lesions in an individual. However the application of such studies to a live, sick animal is restricted compared with the study of dead (post mortem) specimens. In most cases, it implies that samples of tissue arc taken for study. In the live patient this may be very complicated. For instance, if we want to take a biopsy (living tissue sample) to look at the severity of changes in a kidney of a patient in which a probable diagnosis of nephritis is to be made, morphological pathology is not the most appropriate way of making a diagnosis. The technique is very invasive and often puts a heavy burden on the patient. It requires that the patient is under narcosis, undergoes surgery, and that the organs to be examined can be localised with absolute certainty.
- A second area of pathology is the study of the changes in the function of a diseased organ or system. In many cases it is technique to assess what is left of the normal function and how seriously the function of an organ is affected. This area uses a wide variety of techniques. An example may be that in a case of nephritis (kidney disease). Mammals suffering from nephritis are to a large extent unable to concentrate urine in the kidneys, so the amount of urine produced and excreted is more than normal. To compensate for the greater loss of fluid, the patient has to drink more than normal. These simple observations which help diagnosis can be made by the owner, who notes that his beloved pet drinks more than normal and produces a larger amount of urine. If the nephritis is very extensive, it may occur that waste products are no longer excreted sufficiently. It is in this stage that man or animals become poisoned by their own waste products which continue to circulate in the blood. In man, this stage can for longer periods be compensated by removing the urea from the blood at regular intervals, using a technique known as dialysis (kidney machine used in medical hospitals). In the end a final solution might be to replace an organ, which in man is nowadays a fairly reliable technique, but in animal patients let alone in reptiles, is far from realistic.
In the case of nephritis the regular check of the amount of urine produced, its specific weight and the presence of inflammatory cells, bacteria and/or (abnormal) proteins are of importance in assessing the character and the severity of the process. The blood can also be tested for specific changes or to measure an increase of waste products such as uric acid in the reptile.
Now let us say something about reptiles in particular. In reptiles the resorption of water from the urine mainly takes place in the cloaca and not in the kidneys themselves. Most reptiles produce mainly urates (a white deposit) as the end-product of their protein metabolism.
In the veterinary clinic, the vet combines the two approaches of morphological changes and functional or physiological changes to make a diagnosis. A simple example is a broken leg, where you can see structural changes like a swelling by bleeding and accumulation of fluid (edema) in the tissues, as well as a kink caused by displacement of the fractured bones and functional changes in that the animal is unable to stand or walk on his leg.
In the study of diseases the scientist tries to understand the disease in terms of alterations which occur in organs and tissues and what types of effects the changes or lesions have on the organism. Disease is a very complicated process.
Disease is the result of many processes, processing with time, until in the end the compensatory processes are no longer able to cope with all changes which have occurred and disease becomes manifest with recognisable symptoms.
To give an example:
If we consider the process of invasion of an organism with a low level of pathogenicity. Let us say an Oxyurid (a roundworm or Nematode) in the large intestine of a tortoise. Before a worm's egg is capable of producing an infection, a fairly complicated process has to take place. In the egg, a larva develops. The larva is only infectious if it enters the new host at the right moment in its life history, which in general means that is has sloughed or moulted up to three times. Only at this stage can the larvae successfully invade the host. If one or more eggs at the infectious stage are ingested, some larvae hatch and begin feeding on the predigested vegetable contents present in the large intestine. Although infected, the patient will show no sign whatsoever of disease. It can stand this very slight infection and there is enough food, both for the tortoise and for the few worms, even if these worms mature. If however the number of worms increases, they will start to consume a considerable part of the food in the gut, in addition they will start damaging the epithelium or gut lining by compressing it. Many epithelial cells degenerate and are no longer able to absorb food from the intestinal tract. In the end, as well as being deficient in its digestive and absorptive function, the degenerated epithelium will no longer protect the underlying tissue. The exposure of the unprotected tissue of the tortoise to direct contact with the contents of the large intestine has considerable consequences. Bacteria and occasionally fungi or other organisms are likely to invade the patient. Abnormal products from the intestinal tract pass the damaged epithelium and enter into the body. Many of these abnormal products are toxic and intoxicate the patient or will at least put a heavy burden upon the detoxifying capacities of the body and especially on the liver.
Not only do toxins enter the body, but the process will also proceed in the opposite direction: several products leave the body via the damaged epithelium.
An example is the lymph-fluid. In the healthy animal this is produced by an absolutely normal process of leakage of fluids and some proteins from the blood-capillaries into the surrounding tissues.This is a part of the fluids normally present in the tissues between the cells. The fluid is collected in lymph-vessels - from this moment it is called lymph - and transported towards the heart, to be added again to the blood. In reptiles, the circulation of lymph is even more important than in mammals or birds. Reptiles have lymph-hearts, acting in much the same way as the normal heart which makes the blood circulate and these consist of an atrium and one ventricle or chamber, separated by valves. If you visit The National History Museum in London, there is an impressive skeleton of one of the prehistoric ancestor reptiles. If you look at its tail, you'll find that over a certain distance, the lateral processes are flattened at their very end. This situation exists over a distance of about 80 cm. These were the sites where the 2 lymph-hearts (one on either side) were located. It has been calculated that these lymph-hearts, with each contraction, forced 20 litres (!) of lymph-fluid in the direction of the heart. It must be stressed that a normal functioning lymph-system is essential for the health of a reptile. It is surprising that the number of cases in which the lymph-vessels were diseased, is very low in reptiles. In fact in all those years I have seen only one case of a true inflammation of lymph-vessels in a boa constrictor. In the same period two cases of distinct pathological dilatation, and some cases of local overfilling of lymph-vessels have been noticed, and there were 2 or 3 cases of gout in which the lymph-vessels were involved. There is not one chelonian case.
If we try to consider the functioning of a diseased reptile, it seemed necessary to obtain an impression of the importance of the vast streams of fluid whirling through the bodies of our animals.
So let us return to the events on the border between the intestinal contents and the tissues of the host. If the epithelium covering the surface of the intestine is damaged, or even worse, completely lost, then fluids produced near these sites, are likely to be drained in the direction of the intestinal lumen and lost. Not only is fluid lost, but also proteins and electrolytes such as calcium, phosphorus, potassium and sodium. Due to the loss of this valuable fluid, the body will desiccate, and also lose mineral substances which are essential to maintain normal osmotic pressures (or water balance) in the tissues. The situation is even worse, due to loss of phosphorus and potassium. Calcium will be precipitated in renal tissues, so the function of the kidney will also be affected. Shortage in potassium in the body fluids may lead to muscle fatigue and difficulties with all muscular functions. Even the muscles of the intestinal tract may be affected; the motility of the intestine stops and constipation may develop. The functioning of the heart-muscle is impaired which may be rccognised by an abnormal electrocardiogram.
Via the damaged intestinal wall, organisms such as bacteria and also toxins may enter the body while valuable products are also drained from the body. At the same time, the functioning of vital organs is affected. The animal will get intoxicated and lose its capacity to combat infection.
This kind of knowledge is basic for a therapeutical approach.
It is logical that in an attempt to cure a patient, a causative organism is combated. But it is also essential to compensate for the loss of fluid, proteins and electrolytes, by dosing these either orally or, preferably, by injection under the skin or in the abdomen.
Let us return again to our tortoise with its helminth or worrn infection. The animal has been ill for several weeks. The owner notices that the animal stops growing and does not eat. Under these circumstances the reserves of the body are exhausted. This concerns especially the vitamins of the B -complex group. As you will know, the B -vitamins are not stored in the body, thus there are no extensive reserves for periods the animal is in need. Within some weeks or months, depending on the intensity of the metabolism, the vitamins present are exhausted.
In herbivorous animals, the B-vitamins are produced in by the intestinal bacteria, in the intestinal canal. Thus such herbivores are unlikely to develop a deficiency in vitamin-B. However, omnivorous reptiles, can to a large extent be compared with mammals and birds such as the fowl. It may be expected that some degree of hypovitaminosis B2 or B6 deficiency may develop in omnivorous reptiles.
Within the order of the reptiles, little has been known about the signs of hypovitaminosis B. The group of B-vitamins, comprises about 6 vitamins of which Vitamins B1, B2, Panthotenic acid and B6 are the most important It is only vitamin B1 of which clinical signs of hypovitaminosis are known. Hypovitaminosis B1 occurs under very specific circumstances. A distinct and clinically impressive situation develops, when fish containing a specific antivitamin called thiaminase, is given to reptiles. This antivitamin destroys the vitamin B1, and lesions develop. This is especially the case when fish - eating snakes such as garter snakes, are fed raw cod-fish as their sole food. After some weeks or months these animals develop neurological signs, caused by a degeneration of the nerves which innervate specific groups of muscles. The muscles (especially the muscles along the dorsal side of the neck) can get into a spastic state. This is seen as a backward movement of the head. Here again we meet a specific pathobiological process, in which the functional changes are distinct.
The most illustrative example of pathobiological processes is in the deviations of calcium metabolism.
Before we enter into the pathobiology of bones, I would like to give you a short impression of the development of the carapace of testudines.
In testudines which have just hatched, the carapace and plastron are built of protein fibres of connective tissue. The fibres are haphazardly arranged. In this connective tissue, the ribs can be recognised.
In the process of ossification, when the carapace becomes the specific bony structure, the ribs broaden by outgrowth of the bony cortex. In addition in the areas of connective tissue, islets of bone formation make their appearance. Thus the formation of the bony shield is a complicated process of at least two activities: broadening of the ribs and ossification of the connective tissue (the latter type of bone is called dermal bone - it differs from the bone in the legs and others in that there is no earlier stage of cartilage).
Even in very young animals the normal process of calcification may be disturbed. We have seen such cases in animals of 4-6 months.
In these cases, several processes can be recognised. We have had a good example of an inflammation of the inner lining of the bowel (enteritis) present in a group of about 6 months old captive bred Testudo hermanni. The enteritis was caused by a combined action of Blastocystis sp. and Balantidium sp..
Here again some explanation about the physiological processes and the pathophysiology seems appropriate.
First a statement about the normal animal has to be made, which is essential for the understanding of these processes: In the normal animal, growth is normal if a complete and adequate diet is provided. In respect of calcium and phosphorus this means that the ratio between calcium and phosphorus is 1.25 : 1 at a level of ± 1% calcium. Under these circumstances absorption of calcium is optimal. In the process of calcium absorption, metabolites of vitamin D, play a role. When the ratio between calcium and phosphorus it optimal, only minimal amounts of vitamin D3 are needed. If however, the ratio between calcium and phosphorus is less optimal, larger amounts of vitamin D3 are needed in the process of absorption. If the ratio is very poor, even larger amounts of vitamin D3 are unable to compensate the resorption, and a calcium deficiency develops.
In our young tortoises suffering from enteritis, there are several factors to be considered:
- Loss of calcium through the damaged intestinal epithelium.
As in mammals, the combined symptoms of a) insufficient calcification of the skeleton and b) enteritis may indicate that calcium can be lost through the damaged epithelium.
This is one way by which there is a shortage in calcium in the body.
- Insufficient uptake of calcium from the intestine:
There are several different processes which function in reptiles in much the same way as in mammals and birds.
(i) A poor ratio of calcium and phosphorus in the food.
As said, an optimal ratio of calcium and phosphorus is about 1.25 : 1.0 at a calcium content of about 1 % in the food. If the ratio between calcium and phosphorus is somewhere around 1: 3 or less, like in meat where it is about 1: 20, and the calcium level is low, the body is unable to resorb sufficient calcium from the intestinal canal. We have noticed before that the presence of vitamin D3 is important in the absorption of calcium, and in the case of a severe imbalance between calcium and phosphorus, even the dosing of larger quantities of vitamin D3 cannot compensate for the lack of calcium.
Insufficient uptake of calcium prevents the normal calcification of the skeleton and in the young animal rickets will develop, while in the older animal osteodystrophia fibrosa will develop. Osteodystrophia fibrosa is a disease in which bone is broken down. The organism tries to compensate for the weakening of the skeleton by producing larger amounts of connective tissue.
In the treatment of poorly calcified tortoises it is essential to check the food given to the animals and rectify the diet, this is especially the case in carnivorous species.
(ii) Deficiency in vitamin D3
A deficiency in vitamin D3 leads to a complicated cascade of events.
In the normal animal vitamin D3 is resorbed from the intestine and stored in the liver of reptiles in the specific form of 25-hydroxy-cholecalciferol (Dacke, 1979)
In the process of metabolism, the amount of Vitamin D3 needed for further metabolic activities in the body, is mobilised from the liver and transported to the kidneys. In the kidneys the vitamin D3 (25-hydroxy-cholecalciferol) binds a second hydroxyl group to produce 1,25-dihydroxy-cholecalciferol. It is this last form which is active. Research has shown that this metabolic path is also present in testudines. The curious thing about the active form of Vitamin D3 (1,25-dihydroxy-cholecalciferol) is that it acts more or less like a hormone.
The active hormone-like 1,25-dihydroxy-cholecalciferol is active especially in several ways
- It helps in the resorption of calcium from the intestinal canal.
- It is active in the process of calcification of the early stage of formation of bone, i.e. the osteoid.
- It is active during remodelling of the bone, for instance during growth.
Knowing these processes of absorption, storage, mobilisation, transformation of the Vitamin D3 into the active hormone-like substance, one can understand several possibilities where the process may be disturbed.
The resorption of vitamin D3 from the intestine can be disturbed, for instance if there is a long standing enteritis.
This leads to a deficiency in vitamin D3 and contributes to the insufficient uptake of calcium. On the other hand the calcification of bone cannot take place or is severely disturbed.
In case of nephritis, the transformation of vitamin D3 can be insufficient Here again the resorption of calcium and the mineralisation of the skeleton is affected. As nephritis occurs predominantly in older reptiles, this last case will occur especially in elderly animals.
Separate from the influence on the transformation of vitamin D3 nephritis also leads to loss of calcium and phosphorus via the kidneys. The loss of calcium especially has serious consequences. The animal tries to maintain a normal calcium level in the blood.
It therefore starts to produce extra of a specific hormone which acts in the process of resorption of calcium from the bone. This is the parathormone, which is produced in the parathyroid glands. If the number of cells normally present in the parathyroid glands are no longer able to produce sufficient amounts of parathormone, the number of cells start to increase (a process known as proliferation). This is macroscopically seen as an enlargement of the glands.
We have had several of these cases in young captive Testudo hermanni, which illustrate these possibilities.
In one case there was distinct evidence of hypervitaminosis A due to an overdose of vitamin A.
These intoxicated animals developed an enteritis, caused by the protozoan Ciliate Balantidium sp. It thus is likely that the animals were unable to absorb sufficient vitamin D3 and calcium. Even after more than half a year the skeleton was not calcified. The same occurred in animals suffering from a very severe infestation with the protozoan Blastocystis sp.(which up till now has not been recognised as a pathogen for reptiles - this is a new possibility, presented here for the first time). Inspection of the carapace in these cases reveals a slight elevation of the rims of the horny scutes, in combination with a weak, somewhat flattened carapace.
In the carapace there was only a minimal indication of dermal bone formation.
Allow me to make a step away from the tortoises to give another example, which elucidates the time involved in these processes.
In a group of chameleons, caught in Madagascar, 14 days before arrival in the Netherlands, some animals were ill, they died within 4-6 weeks. These chameleons revealed an enteritis, caused by an adenovirus infection (in one animal combined with a Cryptosporidium infection) The adenovirus infection is known in chameleons (Jacobson and Gardiner, 1990,) as is the Cryptosporidium-infection (Dillehay et a]., 1986).
Histological examination of the skeleton revealed a larger number of cells, known as osteoclasts, which are capable of removing bone, and which are always involved in processes of breakdown of bone as for instance in osteodystrophia fibrosa. In the case of these chameleons, the process was just in an early stage, and fibrous tissue had not yet developed. These cases illustrate that within about 4 weeks demineralisation of the skeleton may start.
We have been looking at the delayed ossification in some young Testudo hermanni. However, also animals of some years of age may reveal insufficient ossification of the skeleton. This was noticed in a 3 year old Testudo hermanni. This animal had a distinctly flattened carapace and again slightly elevated rims of the horny shields. In addition it had a weak carapace. At histology, only minimal indications of ossification of the carapace were observed. This was distinctly a carapace which had never been ossified before, as could be derived from the pathomorphology. In the connective tissue no blood-vessels surrounded by pigmented cells, the so called melanophores were present.
Here again this needs some explanation. The normal carapace of a tortoise contains a network of holes, in which blood-vessels are present These blood-vessels are surrounded by a rim of melanophores. In the case of osteodystrophia fibrosa, the bone is broken down and compensating connective tissue is produced. However, the original anatomical structure can still be recognised from the presence of blood-vessels surrounded by melanophores. In addition the structure of the newly produced connective tissue is much more dense while the arrangement of the connective fibres also differs from the situation in the newborn animal.
To return to the 3 year old Testudo hermanni, in this animal nephrosis was diagnosed, thus there is a fair possibility that there was a loss of calcium via the kidneys. This may have hampered the ossification and calcification of the skeleton.
This brings me to a problem of which there is no pathomorphological basis, and thus all ideas about the pathophysiology are highly speculative. I am referring to specimens of especially Testudo hermanni, which grow excessively quickly. Such animals develop a thick though somewhat flexible carapace with slightly bulging horny scales. It is known that, if such animals are damaged, the bleeding from the carapace is markedly more that in specimens which have grown normally and developed a normal ossification of the carapace.
There is a distinct lack of information about the processes in such abnormal animals. I am not aware of any radiological examination of such specimens. It is tempting to speculate that such animals have more or less juvenile structure of the carapace.
There would have been much more to say about pathological changes in bones, specially in cases of osteodystrophia fibrosa or remodelling after an injury.
I want to finish with a remarkable phenomenon which recently came to my attention. I was confronted with a Testudo hermanni, suffering from a flattened carapace, due to a severe dietary decalcification. Under the influence of an optimal diet, over a period of many months, the carapace rose slowly to regain its normal shape.
It is a fascinating assumption that the pathophysiological background of this process might be to a combined action of an optimal dosing of calcium and vitamin D3 as additives to the food. It can be speculated that the action of muscles, and physical processes lead to a remodelling by a breakdown of bone which is superfluous under the new situation and the production of new bone where needed. This remodelling even counteracts the influence of gravitation.
This lecture was based on a rather vague idea formulated two or three years ago, without knowing exactly what the end stage would be. For me preparing this lecture in honour of late Dr. E. Elkan, was something like a voyage of discovery.
One of the conclusions of this intellectual exercise is, that by close cooperation, we as an internationally orientated group of fanciers of tortoises, are able to discover a wide spectrum of fascinating aspects concerning the incredible chelonians.
Dacke, C.G. (1979) Calcium regulation in sub-mammalian vertebrates. Academic Press New York and London.
Dillehay, D.L., Boosinger, T.R. and MacKenzie, S. (1986). Gastric cryptosporidiosis in a chameleon. Journal of the American Veterinary Medical Association 189 (9), 1139-1140.
Elkan, E. and Zwart, P. (1967) The ocular disease of young terrapins caused by vitamin A deficiency. Pathologica Veterinaria 4, 201-222.
Jacobson, E.R. and Gardiner, C.H. (1990) Adeno-like virus in esophageal and tracheal mucosa of a Jackson's chameleon (Chameleo jacksoni). Veterinary Pathology 27, 210-212.
Thomson, R.G. General Veterinary Pathology. Saunders Cie Philadelphia 1984.
Testudo Volume Three Number Five 1993