PATHOLOGY IN THE AMPHIBIA

6

PATHOLOGY IN THE AMPHIBIA E. Elkan I. Introduction I I . Developmental Abnormalities I I I . Infectious Diseases A . Virus-Induced Conditions B. Bacterial Infections C. Tuberculosis I V . Protozoal Infestations V . Infestation b y Helminths V I . Leeches, Flies, and Crustaceans V I I . Fungal Infection VIII. Tumors A . Nonmalignant T u m o r s B. Malignant T u m o r s C. T h e N e w t " T e s t " D . Registry of T u m o r s E. Prerequisites for Histopathology I X . Conclusion References

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I. Introduction T h e science »of pathology often provides additional information about the physiology and immunological responses (see Chapter 5, this v o l u m e ) of any particular group of animals, and from this view point pathology has a justifiable position in a volume devoted to the physiology of these uniquely interesting vertebrates. It must be stated, however, that the pathology covering this group is still in its infancy, o n l y one systematic approach to this topic has so far been attempted ( R e i c h e n b a c h - K l i n k e and Elkan, 1965). Information to date is sparse, largely due to the fact that, in most cases, no easily observable s y m p t o m s are displayed b y the animals until the advanced stages of a disease have been reached. F u r thermore, in captivity they tend to remain in the regions of the cage which offer the greatest chance of concealment, particularly when they become ill. Thus., b y the time that such a diseased specimen is discovered, it is often t o o late to be of value for pathological analysis. 273

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In the wild, diseased specimens are rarely seen, since debilitated ani­ mals fall easy prey to predators, and are therefore rarely caught b y c o l ­ lectors. In so far as parasitic infestations are regarded as "diseases," h o w ­ ever, it must be born in mind that most amphibians, whether in their natural environment or in captivity, are hosts to one or several types of parasites. Chance observations on isolated cases are extensively d o c u ­ mented (see reviews b y Angel, 1947; Klingelhoifer, 1955; Cohrs et

al.,

1958; R e i c h e n b a c h - K l i n k e and Elkan, 1965, 1974; Harschbarger, 1968, 1969), but we are largely dependent on the data obtained from captive animals for our information about the types of pathological conditions that occur in the Amphibia, and only rarely do we find sequelae of i n ­ juries in wild-caught specimens which have not been sufficiently severe as to kill the animal, but extensive enough to produce permanent damage (Fig.l). It must be remembered t o o , that pathological conditions can be arti­ ficially

produced b y bad animal husbandry such as keeping animals in

unfurnished drawers without food or water. Amphibians are highly spe­ cialized animals and narrowly adapted to their natural environment. W e

Fig. 1. Skeleton of Ceratophrys ornata showing the spontaneous healing of several comminuted fractures of the right pelvis and the right elbow. T h e frog did not die from the trauma, and this was only accidentally discovered when the skeleton was prepared. From Elkan (1960a).

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275

sometimes hear of experiments, carried out "after the animals had become " a c c l i m a t i z e d . " Amphibians never acclimatize. Whether they die or surv i v e in the cages in which they are housed depends on whether they can tolerate the soil, the food, the humidity, and the temperature imposed upon them. If any of these factors transcends their natural tolerance and deviates t o o much from normal, the animal cannot feed and dies from starvation. T h e y cannot adjust themselves to uncongenial conditions in the w a y higher vertebrates d o . Thus, if experiments involving amphibians are t o have any meaning, care must be taken to ensure that the animals thrive in their artificial habitat, i.e., the habitat must be adjusted t o the animal, not vice versa. Unsuitable food, even if eaten without compulsion, m a y produce the most remarkable surgical catastrophies. It is not easy, for example, to see w h y a newt, normally a strict protein feeder, should fill its stomach with water plants. Y e t the author has seen a case where a c o m m o n newt (Triturus

vulgaris)

had its stomach distended like a b a l -

loon with decaying, fermenting vegetable matter, to such an extent that the overlying skin and muscle coat had ruptured. T h e sudden death of a whole collection of animals cannot always be blamed on Pseudomonas.

It m a y simply be due to electrocution

from

a faulty electric heater or the breakdown of the thermostat installed to regulate the temperature of the water. Artificially produced catastrophies m a y be of interest to the amateur—to the biologist they are of no interest at all.

II. Developmental Abnormalities T h e greater the fertility of a species the greater the likelihood of o c c a sional maldevelopment. Nowhere is this rule more evident than in the A m p h i b i a which, for this reason, are the favorites not only of the o n c o l o gists but of the geneticists as well. W h o l e books have been written about frogs and toads with supernumerary limbs, about melanotic and albinotic specimens, about the mechanisms of their development, and their ability or failure to regenerate lost parts (Rostand, 1934, 1955). Abnormal genes or abnormal combinations of genes appear in hitherto normal populations and can sometimes be transmitted to future generations. T h e y appear in the wild state as well as in the laboratory but normally specimens so affected are eliminated b y predators because their variations have no survival value. In the laboratory almost any kind of abnormality can be produced if insemination is carried out artificially or if premature or p o s t mature eggs are fertilized in water of an abnormal p H or at temperatures a b o v e or below the normal. T h e effects of such conditions on the induction

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of an intersexual state has already been discussed in Chapter 6, V o l u m e II. A m o n g the naturally occurring abnormalities the most interesting are perhaps the giant tadpoles which have, on several occasions, been r e ­ ported since they were first seen in 1905 (Angel, 1937; Witschi, 1952; R e i c h e n b a c h - K l i n k e and Elkan, 1965). These tadpoles grow to a size t w o to three times larger than normal and never metamorphose. Attempts to " c u r e " them b y the administration of a variety of hormonal extracts have failed. A t least three major endocrine glands, the thymus, the pitui­ tary, and the thyroid gland are histologically abnormal in these t a d ­ poles. This condition has been found both in Rana R. esculenta.

temporaria

and in

A so-called "Brassica f a c t o r " has been blamed for this m a l -

development which can, on occasion, affect all the tadpoles in a pond. This factor is thought to arise from the contamination of the pond b y drainage from adjacent fields in which cabbages are grown ( D o d d and Callan, 1955). In Xenopus,

which has been widely bred in captivity, one of the c o m ­

monest abnormalities is a hydropic condition of the subcutaneous lymph sacs which m a y occur before or after metamorphosis. A n example of such a condition in a y o u n g toad bred in the laboratory is shown in Fig. 2. T h e lymph sacs are normally drained b y several pairs of posterior and

Fig. 2. Severe subcutaneous hydrops in a young Xenopus F r o m Reichenbach-Klinke and Elkan (1965).

laevis, bred artificially.

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one pair of anterior lymph hearts (Elkan, 1957). T h e lymph hearts are present in the hydropic specimens, and histologically appear to be quite normal. T h e reason w h y they do not function is obscure but has been ascribed to a recessive gene (Uehlinger, 1965, 1969). Such hydropic froglets never reach maturity. Goiters can sometimes be seen in newts but the condition is extremely rare and a detailed study has not so far been undertaken. Like the p h e nomenon of the giant tadpole, this condition t o o , has sometimes been associated with a "Brassica f a c t o r / ' and an extract from cabbages has been found to be goitrogenous. H o w e v e r , it seems doubtful whether such a factor can account for every case of amphibian goiter. L a b o r a t o r y breeding can produce almost any abnormality. Figure 3,

Fig. 3. Female Xenopus laevis, bred in captivity. A cardiac hernia has developed through a deficiency in the pectoral girdle. F r o m Reichenbach-Klinke and Elkan (1965).

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for example, shows a Xenopus female which grew to maturity with a deficient area in the shoulder girdle through which the whole heart herni­ ated and could be seen beating under the skin.

Fig. 4. Dissection of an adult Xenopus right kidney From Elkan (1963).

laevis to show a nephroblastoma of the

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279

Perhaps the greatest rarities in the amphibian teratology are the genuine embryonic tumors, but only a few well-documented cases of n e p h roblastoma have been seen. These tumors m a y grow to a considerable size (Figs. 4 and 2 6 ) , compressing the residual normal renal tissue so that it m a y be reduced to a thin capsule surrounding the tumor. T h e condition is essentially benign, but causes the death of the affected animal b y the compression of the remaining normal renal tissue and b y the d i s placement of other vital organs.

III. Infectious Diseases Only rarely will infectious diseases play a dominant part in the life of amphibians in their natural habit, since, apart from the breeding season, they are solitary animals and, therefore, even when infected have little chance of spreading the disease. Conditions are totally different in laboratories or in the cages of dealers, where one m a y find animals being maintained in very crowded unhygenic conditions. Here, one sick animal will immediately infect others and the disease, if virulent, m a y wipe out the whole collection within a few days. Disasters of this kind are b y no means rare and it cannot be sufficiently emphasized that amphibians should never be kept in crowded conditions, particularly, during transportation over long distances where heat m a y add to the hazards. M u c h valuable material and data are lost due t o the lack of c o m m u n i cation between the pathologist and the keepers of laboratory amphibians and field collectors. T h i s is particularly evident in the realm of infectious diseases where material, if it is to be of any value, needs t o be quickly cooled for transportation, and blood smears have to be taken before the death of the animal. Under the latter circumstances, if aspiration from the heart or from a large b l o o d vessel is not practicable smears can s o m e times be obtained b y cutting the tail. A further point to emphasize is that where available it is of the greatest importance to collect simultaneously material from a normal healthy animal as well. W i t h o u t such controls it is often difficult to evaluate the pathological material since there are very few (if a n y ) books on the normal histology of the lower vertebrates, which can be consulted. A.

VIRUS-INDUCED

CONDITIONS

Our knowledge of the part played b y viral infection in the Amphibia would be even smaller than it is had it not been for the discovery of a neoplastic tumor in the leopard frog, Rana pipiens, b y L u c k e ( 1 9 3 4 ) . Since this discovery a great number of papers dealing with the p r o b a b l y viral nature of this tumor have been published (e.g., Balls, 1962; Balls

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ELKAN

and R u b e n , 1968; Barch et al, 1965; Freed and Rosenfeld, 1965; Lunger, 1966; M c K i n n e l l , 1965; M c K i n n e l l and M c K i n n e l l , 1968; Rafferty, 1964; R u b e n and Balls, 1967; Tweedell, 1967; Wilson et al., 1965; W o l f et

al,

1968). Herpetiform and several other viruses have been found in some cases, but in others no virus has been present. It has also been suggested that viruses m a y be involved in the lymphosarcoma described b y Balls and R u b i n ( 1 9 6 8 ) . T h e nature of these tumors is considered in greater detail in Section V I I I . B.

BACTERIAL

INFECTIONS

A m o n g the most virulent of all the infections encountered in the A m ­ phibia is that caused b y members of the Pseudomonaceae. These are Gram-negative, motile rods, the best known of which is Pseudomonas aeruginosa (Ambrus et al, 1951; Karasek, 1967). In human pathology it is known as Bacillus pyocyaneus. T h e number of different strains of pathogenic pseudomonads is very large, and over 100 have been described. T h e y frequently occur in c o m p a n y with B. alkaligenes faecalis, Proteus, Haementerococci and various Staphylococci, and reach their victims b y w a y of polluted water and infected food. T h e best known and most v i r u ­ lent disease they produce is that known as " r e d - l e g , " which takes its name from the typical red patches that appear on the ventral skin or on the extremities. B y the time these symptoms appear, the infection is already too advanced for any hope of successful treatment and whole collections m a y be destroyed within 24 hours. Isolation of the infected animal, thorough cleansing of the tank, and additions of small amounts of an antibiotic like K a n a m y c i n or Chloromycetin to the water, m a y stop the spread of the infection. Mercurochrome and Potassium permanganate are also sometimes added to the water. A u t o p s y of diseased frogs reveals that the infection is b y no means confined to the skin, and intense inflam­ mation m a y have spread to the lungs, the intestine, spleen, and the k i d ­ neys. T h e liver, heart, and brain often remain unaffected, even in acute cases, presumably because the animals die from toxemia before these organs show visible changes. Treatment of individual cases is hardly ever successful. W h e r e a pseudomonad epizootic appears, attention should be paid to prophylaxis rather than to therapy. F o r completely aquatic amphibia, such as Xenopus, the cheapest and most successful p r o p h y l a c ­ tic measure is that of raising the salt content of the water to just below frog saline (about 4 % ) . Terrestrial amphibians can be given baths in methylene blue or gentian violet solutions but it is easy to understand w h y these methods rarely meet with much success. T h e skin of the normal, healthy amphibian presents a most efficient barrier against infection but the smallest injury gives a foothold to b a c -

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AMPHIBIA

teria which are otherwise harmless commensals in the amphibian environment. In this w a y B. ranicidum

Ernst, and Diplobacterium

ranarum

Pet-

tenkofer and K a u f m a n n have been found to cause lethal epidemics in frog collections. Guarding against overcrowding and frequent cleaning of cages helps to avoid such catastrophies. C.

TUBERCULOSIS

It has been known since the beginning of the present century (Kuester, 1905, 1928) that mycobacteria can cause lesions analogous to human t u berculosis in poikilothermic animals, and there is an extensive literature on this subject (e.g., Cohrs et al,

1958; Darzins, 1950; Elkan, 1960b;

Ippen, 1964; R o w l a t t and R o e , 1966; Schlumberger, 1958). B o t h

hu-

mans and lower vertebrates live in an environment in which these b a c teria occur, and both, so long as they are otherwise healthy and not e x posed to massive infection, m a y never actually suffer from tuberculosis. T h e relationship between the host and the infecting organism, however, is not analogous in these two groups of vertebrates. Tuberculosis in the homoiothermic animals is caused b y a well-defined

invading

bacillus

which is not present in the healthy individual, whereas the cold-water mycobacteria associated with the amphibian infection are ubiquitous s a p rophytes quite c o m m o n l y present on the moist skin of these animals, which cause serious disease only in exceptional circumstances. T h e normal ecological temperature range of amphibians coincides with that of the mycobacteria, and strains of these organisms can be found in any tank or cage in which frogs or newts are kept. There are never any epizootic outbreaks of tuberculosis among laboratory populations, only

isolated

specimens m a y occasionally be found in an advanced stage of the disease, amidst others which remain healthy for a long time. Generally, tuberculous infections are much rarer events in these animals than pseudomonal epizootics. Nevertheless, they occur in laboratory populations and are o c casionally found even in freshly caught specimens. Suggestions for treatment cannot be offered; the disease, when discovered, is usually t o o far advanced for any intervention. A s to the mode of infection, injury is the crucial factor, and this can be illustrated b y Xenopus

which, if kept in t o o crowded a condition, bite

each other's toes at feeding time. It is at the toes or the finger tips that the first tuberculous granulomata are generally found, and an example of this is shown in Fig. 5. F r o m the foot the infection spreads, b y w a y of lymphatic channels, toward the pelvic organs and gradually to the other viscera (Figs. 6 - 8 ) , particularly the kidneys (Fig. 9 ) . W h e n about half the renal tissue is destroyed death supervenes. Occasionally captive frogs injure their mouth parts, and a resulting infection b y mycobacteria

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F i g . 5. Xenopus laevis with a primary tuberculous infection of the feet, probably the consequence of being bitten b y other toads when competing for f o o d .

gives rise to tuberculosis of the bronchi, lungs, and, in rare cases, of the pericardium. T h e disease m a y progress to a very advanced stage before the animal dies. T h e reason for this lies in the fact that the cold-water mycobacteria produce no exotoxin and are unable to pass through a ser­ ous membrane. A pulmonary lobe, for example, m a y be completely trans­ formed into one large granulomatous tuberculous mass (Fig. 10) while the pleural surface remains healthy and unaffected. M y c o b a c t e r i a l tuber­ culosis m a y therefore appear, after dissection, as a large isolated, sessile or pedunculated tumor. W i t h the naked eye the disease is indistinguish­ able from growths or malignant conditions. T h e clinical pattern of amphibian tuberculosis is that of a milder v a r i ­ ety of the disease seen in warm-blooded animals. T h e skin m a y be affected b y ulceration and the viscera b y miliary distribution, massive pneumonia, or subserous tumors, both single or multiple. In cases of this kind, the fat b o d y is usually found to be vestigial, the liver small, dark, and below its normal weight, and in females the o v a r y m a y be much reduced in size. Histologically, in the early-stage tubercles, the epithelioid cells are seen to be filled to capacity with slender acid-fast rods which are sometimes so short that they look more like cocci than bacilli. T h e tubercles have a core showing more or less advanced caseation. Calcifica­ tion does not occur, presumably because the animal is t o o short-lived

Fig.6 . A dissection of Bufo bufo with miliary tuberculosis of the liver. The photographs show the (A)

dorsal aspects of the liver.

ventral and

(B)

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for its development. W h e n the individual tubercles coalesce, large tumors m a y develop, y e t extensive caseation is rarely seen. Such tuberculous tumors, however large they m a y become, have no stroma and it is difficult to see how metabolism is still carried on at the center which has no visible blood supply. T h e bacteria involved do not seem to produce exotoxins since the animals are not killed b y toxaemia even in the case of tumors of large size. It is of importance to note that amphibian mycobacterial infection rarely produces giant cells. If a multitude of these are seen, the infection is p r o b a b l y not mycobacterial but m y c o t i c . W h e n examining cases of this kind one is again and again struck b y the degree to which even vital organs can be destroyed before the animal dies and, apart from a refusal to feed, it m a y , even minutes before its death, show no outward symptoms to warn us that more than half of its liver, its kidneys, or its lungs have been destroyed. W i d e l y disseminated dermal tuberculosis also occurs (Fig. 1 1 ) , p r o b ­ ably only where severly debilitated specimens are exposed to massive infection. Cerebral tuberculous granulomata, on the other hand, have not been seen in amphibians thus far.

IV. Protozoal Infestations T h e relationship of the Amphibia and the Protozoa which, quite n o r ­ mally, inhabit the same environment, is ambiguous. Amphibian species invariably show some degree of infestation, either externally or inter­ nally, b y some protozoan species which m a y cause death or severe d e b i l ­ ity if they manage to penetrate into vital organs, or if they become too abundant. M o r e often than not, however, the protozoan lives in w e l l balanced symbiosis with its amphibian host and m a y , b y virtue of its own digestive powers, even be an indispensable partner of the alliance. Catastrophies only occur when strange, unadapted species invade in n u m ­ bers which cannot be tolerated and against which there is no effective defense available. Such examples are found among the Flagellata ( M a s t i g o p h o r a ) , the Sarcodina ( R h i z o p o d a ) , the Sporozoa, and the Ciliophora. A massive infection with sporozoans such as Eimeria, Haemogregarina, Plasmodium, M y x o s p o r i d i a , and Microsporidia m a y , on occasions, wipe out whole populations of amphibians. A m o n g the Microsporidia, Plisto-

Figs. 7 - 1 0 . See color plate facing page 296.

6. PATHOLOGY IN THE AMPHIBIA

Fig. 1 1 . Xenopus Elkan (1965).

285

laevis with dermal tuberculosis. From Reichenbach-Klinke and

phora, usually a parasite of insects and fishes, has been responsible for a lethal epizootic among populations of the c o m m o n toad, Bujo bujo, in southern England (Canning et al., 1964). Infected toads showed m i c r o sporidial deposits which follow the direction of the fibers in the muscle sheets (Figs. 12 and 2 1 ) . T h e same pathogen was subsequently discovered in two specimens of the Tuatara (Sphenodon punctatus) which had been shipped to N e w Y o r k from N e w Zealand (Liu and W a y n e , 1971). Dermocystidium is another fish parasite found to infect amphibians where it produces dermal cysts in urodeles and anurans. Other offenders m a y be found among the Flagellates, R h i z o p o d s ( S a r c o d i n a ) , and Ciliates

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Fig. 1 2 . Bufo bufo infected with the microsporidian Plistophora myotrophica. Note that the arrangement of microsporidian deposits follows the direction of the muscle fibers.

(Cheng, 1967; Doflein and Reichenow, 1953; Elkan, 1960a). An example of a salamander, Gyrinophilits

porphynticus,

with such dermocystidial i n ­

festations is shown in Fig. 13. Infection with any of these parasites spreads very rapidly in crowded conditions, and speedy isolation of the first diseased specimen is of greatest importance.

Fig. 1 3 . A Dermocystidium.

salamander,

Gyrinophilus

porphynticus,

heavily

infested

with

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6. PATHOLOGY IN THE AMPHIBIA

Flagellated protozoa m a y be found in the blood or in other b o d y fluids. T h e borderline between those which live as commensals and those which cause definite damage and disease is not always easy to determine. V a r i ous species of Trypanosoma ples, and Hexamita,

m a y often be discovered in fresh blood s a m -

Retortomonas,

and Trichomonas,

are often

found

to inhabit the intestines of m a n y species. T h e best known and most u b i q uitous among the intestinal commensals in this group is undoubtedly Opalina,

a multiflagellate which occurs so regularly and in such numbers

that it has been suggested it might be a source of a vitamin necessary to the host. Amphibians raised under conditions of sterility, however, show no signs of any vitamin deficiency, indicating that this latter suggestion is unlikely. On the other hand, there is no positive proof that such flagellates cause any harm. A m o n g the R h i z o p o d a , Entamoeba

ranarum

is sometimes found in the

intestines or in the liver. T h e encysted form is indistinguishable

from

that of other Entamoebae. There remains the limited number of ciliates which c o m m o n l y share the same environment with amphibians and which, just as occurs in fishes, become dangerous to debilitated specimens only. T h e y m a y then suddenly appear in large numbers, and types like the circular or the ovoid Balantidium,

Trichodina

an inhabitant of the rectum, are sometimes

blamed for fatalities which they did not cause but only exploited. U s u ally, in such cases, the primary debilitating factor must be looked for elsewhere.

V· Infestation by Helminths Intestinal worms are c o m m o n l y found in the Amphibia. T h e y can, if they become t o o abundant, cause severe disability and death. Y a m a g u t i (1935a,b, 1961) lists 13 families of trematodes, 4 families of cestodes, and 20 families of nematodes (with 53 genera and 201 different species), that have been recorded as occurring in amphibians, and to this list m a y be added members of the N e m a t o m o r p h a (Gordian worms) and the P e n tastomida ( A r a c h n i d a ) . Thus, it can be appreciated that the threat of infestation b y helminths and other wormlike types is considerable ( B a y lis, 1930; Cheng, 1967; Cohrs et al, 1958; D a w e s , 1946; Fulleborn, 1928; Elkan and M u r r a y , 1952; Goin and Ogren, 1956; H y m a n , 1940; R e i c h e n b a c h - K l i n k e and Elkan, 1965; Schlumberger, 1958; Thurston, 1967; W a r d l e and M c L e o d , 1952). B o t h monogenetic and digenetic trematodes m a y be encountered. T h e

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commonest t y p e among the M o n o g e n e a , are species of Polystoma, ticularly P. integerrimum,

par­

which parasitizes m a n y kinds of frogs. This

w o r m m a y be up to 10 m m in length and preferably inhabits the urinary bladder. It can be recognized b y a crescent-shaped group of posterior sucking disks. In some parts of the world Polystoma

species show such a

degree of host specificity that m a n y of them have been named

after

their host. T h e variety of digenetic trematodes which infest amphibians is v e r y much larger than that of the M o n o g e n e a , and only the specialist can distinguish the m a n y types which occur and which m a y be very similar in appearance. Intermediate larval forms (Cercariae, Metacercariae) are frequently encountered in microscopical sections where they m a y be seen encysted in almost any organ (Fig. 1 4 ) , frequently in lymphatic spaces. It is remarkable to note in how m a n y instances the host tissue shows no detectable defensive reaction against such an invasion. I n the b l o o d , all that is usually seen as a result of such an invasion, is a relative eosinophilia. T h e surrounding tissues m a y make an attempt to wall off a cercarial cyst b y a second wall of host tissue which, in some instances, m a y contain melanophores

(Elkan and M u r r a y , 1952). A n example of this

is seen in Fig. 15. H o w e v e r , although metacercariae m a y thus be isolated b y a double wall, a heavy infestation kills the frog and the surrounding tissue shows no sign of defensive cellular infiltration. A complete survey of all the digenetic trematodes which infest Amphibia can be found in the larger textbooks (e.g., Cheng, 1967; H y m a n , 1 9 4 0 ) . On the continent of Europe, Opisthoglyphe

ranae

occurs as a c o m m o n intestinal parasite

of frogs. In the British Isles it is replaced b y Dolichosaccus while newts have their own parasites like Batrachocoelium T h e pulmonary trematode Haematoloechus

rastellus, salamandrae.

has a worldwide distribution.

T h e worms either share the host's food or live on desquamated epithelium, mucus, blood, or b o d y fluids. T h e hosts seem to have neither a humoral nor a cellular defense against them. Once the skin, the only line of defense, has been breached, walling off remains as the only further line of p r o t e c ­ tion. Inside the cyst the cercaria remains viable and will resume its life cycle when the host is eaten b y a larger predator. Here again the border line between commensalism, parasitism, and pathogenic invasion is diffi­ cult to draw. M e m b e r s of the Cestoda (tapeworms) infest some amphibians with great regularity, and are common in all parts of the world. Nematotaenia dispar, a slender worm, varying between 5 and 22 cm in length is c o m ­ monly found in the small intestine of m a n y European frogs and toads, and it is b y no means unusual t o see cases where this w o r m becomes so abundant as to cause intestinal obstruction, gangrene, and death (Fig.

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289

F i g . 1 4 . Xenopus laevis showing a cercarial infestation of all the neuromasts. N o r mally these are almost invisible to the naked eye, but are here easily seen due to a local melanosis stimulated b y the infestation. From Elkan (1960b).

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F i g . 1 5 . T h e underside of the skin of the specimen shown in Fig. 14. N o t e the congregation of melanocytes around the cercarial cysts. From Elkan (1960b).

1 6 ) . Chlamydocephalus namaquensis is a short intestinal cestode of the South African clawed toad, X. laevis (Fig. 1 7 ) . In the author's experience infested toads injected during pregnancy diagnostic testing with extracts containing bromphenol indicator, become cleared of their intestinal cestodes. In the United States several varieties of Diphyllobothrium infest both urodeles and anurans. While to the aesthetic eye all intestinal parasites l o o k unpleasant enough, the Acanthocephala have even more alarming features than the rest. Their proboscis, which m a y bury deeply into the intestinal wall,

6. PATHOLOGY IN THE AMPHIBIA

F i g . 1 6 . Dissection showing the duodenum of Bufo bufo obstructed b y enia dispar. T h e toad died from intestinal obstruction.

291

Nematota-

is armed with m a n y rows of barbed hooks (Fig. 18) and the w o r m , if it burrows t o o deeply, can cause intestinal perforation, peritonitis, general sepsis, and death. T h e most frequently seen species in frogs and newts, is Acanthocephalus ranae, a white w o r m , about 6 m m long which firmly attaches itself to the mucous lining of the stomach or intestine (Fig. 22) (Elkan 1960a,b). Just as in the parasitology of the higher vertebrates, the Nematodes play the largest part in the helminthology of the Amphibia. T h e y are round, thin, unsegmented, and have neither a proboscis nor sucking disks. Where they occur they are usually found in large numbers, free or encysted. T h e encysted worms m a y be found in the intestinal wall, under the peritoneum, in the skeletal muscle, in lymphatic channels, or in any of the parenchymatous organs (Fig. 1 9 ) . T h e y are harmless in small n u m -

292

Ε. ELKAN

F i g . 1 7 . Scolex of the small tapeworm, Chlamydocephalus namaquensis, to the intestinal mucosa. From Reichenbach-Klinke and Elkan (1965).

attached

bers but m a y seriously interfere with the host's metabolism if they b e ­ come too numerous. It m a y be taken for granted that all amphibians newly imported into the laboratory are carrying their burden of hel­ minths, and, in particular, Nematodes. Sometimes, for reasons which are little understood, specimens which carried this burden with impunity in their natural environment, die from it in captivity. Others again, live in apparent harmony with, for example, a severe Filaria infection for m a n y years, and then die suddenly for no obvious reason. E v e n more than in the higher vertebrates, the presence of helminths stimulates the host to produce an abnormal number of eosinophil leukocytes and these m a y be found either surrounding encysted worms like a barrier (Fig. 2 0 ) , or fill­ ing lymphatic sheaths which a c c o m p a n y blood vessels. Whether the leu­ kocytes become concentrated because they are attracted b y substances excreted b y the parasitic larvae is not known, but whatever the reason for their production, as a means of defense they are woefully inefficient. Nematodes m a y be found in the intestines, the blood vessels, the lymphatic channels, and the lungs. T h e y m a y , like Filaria, be of m i c r o ­ scopic size or they m a y , like the c o m m o n lung w o r m , Rhabdias, be visible to the naked eye. W h e n looking for nematodes it is advisable to dissect

Fig. 18. Acanthocephalus ranae with (A) the proboscis retracted and (B) extended.

6. PATHOLOGY IN THE AMPHIBIA

293

294

Ε. ELKAN

F i g . 1 9 . N e m a t o d e larvae encysted along the small intestine of Pipa pipa. Reichenbach-Klinke and Elkan (1965).

From

the suspected organ in Ringer saline. T h e worms will then float away and cannot get lost. T h e dissecting glass dish should be placed on a black surface. Attempts at ridding amphibians of their helminths are never made. A n y drug would p r o b a b l y kill the host before making any impres­ sion on the parasite.

VI. Leeches, Flies, and Crustaceans Leeches m a y occasionally attach themselves t o aquatic amphibians. T h e y probably present only a temporary hazard unless they manage t o perforate the skin and gain access to one of the subcutaneous lymph sacs whence, after a good meal, they m a y be unable to emerge. Specimens

6. PATHOLOGY IN THE AMPHIBIA

295

Fig. 2 0 . N e m a t o d e larvae encysting in the mesenterium of a metamorphosing tadpole of Rana esculenta.

of Batracobdella and of Philaemon ( M a n n and T y l e r , 1963).

have been found in this location

A m o n g the Crustaceans it is only Argulus, the well-known fish louse which m a y occasionally become v e r y numerous in ponds and attach itself to the skin of newts and frogs. Of much rarer occurrence are the tongue worms (Pentastomida) now classified with the Arachnids. W h e r e they occur in Amphibians they are found in the intestinal canal. Of much greater interest are the cases where frogs are found t o suffer, and are indeed killed, b y the depredations of flies. Several species of D i p tera, both in Europe, America, and Australia, lay their eggs selectively on the hind legs of their unwary victims. T h e larvae hatch, migrate t o the frog's head, gain entrance through the eyes or the nostrils and then proceed to devour the victim from within. T h e best known European " t o a d fly" is Bufolucilia bufonivora, but m a n y other D i p t e r a with the same behavior have been described (Elkan, 1965; Mertens, 1950; Zumpt, 1965).

VII. Fungal Infection W h e n examining tumors, and in particular when faced with amphibian dermal tumors of unknown origin, a search must always be made for

296

Ε. ELKAN

fungi which m a y have gained access through accidental wounds or insect bites. It is usually easy to demonstrate the presence of fungal mycelia in sections where they stain well b y the G r a m , the Grocott, or the P A S technique. On the other hand it is not easy, indeed it is often extremely difficult, to determine the actual fungal species, because once within the frog's tissues, the fungus does not produce fruiting bodies which are essen­ tial for exact identification. Only special laboratories have the facilities to culture such fungi and to do this they naturally require fresh, unfixed material. One of the best investigated amphibian fungi is ranarum,

Basidiobolus

c o m m o n in m a n y of the African states where it has also been

found to attack the human population (Lie K i a n Joe, 1956). Frog imports from South America require particularly stringent p r e ­ cautionary measures because some of the c o m m o n soil fungi occasionally infect the frogs. These infections are invariably fatal. T h e frogs

die

within a few weeks or months and the fungal epidemic spreads quickly through the collection. As in bacterial infections, the port of entry is usu­ ally to be found in those parts of the skin normally in contact with the ground or exposed to minor injuries, particularly the toes, the ventral skin, and the nasal region. (Fig. 2 7 ) . F r o m the periphery, the fungus spreads, not necessarily b y any prexisting channels but simply b y spread­ ing out in all directions, the intestine eventually becoming transformed into purely granulomatous tissue. Once some or all of the vital p a r e n c h y ­ matous organs have been transformed into fungal granulomata (Fig. 2 3 ) , death supervenes. T h e main South American fungal genera involved are, so far as we k n o w at present, strains of Phialophora

and

Cladosporium.

I t is interesting to note that the former is equally responsible for the human disease of Chromoblastomycosis, endemic in some parts of South America. Reports on outbreaks of

fungal epidemics in collections

amphibians m a y be found in papers b y Scott

( 1 9 2 6 ) , Dhaliwal

of and

Griffiths ( 1 9 6 4 ) , and Elkan ( 1 9 6 0 b ) . Ichthyosporidium

(Fig. 28) of which several species are known, is a

fungus which migrated from the sea where it infected fish of fresh waters. It is n o w occasionally found to occur in and on the skin of amphibians, particularly Urodeles where it can do considerable damage. Attempts to cure it with fungicidal lotions are rarely successful. Fig. 7. Xenopus laevis with tuberculosis of the rectum. The rectum has b e c o m e the seat of a large obstructing tuberculoma, but there are no signs of a tuberculous peritonitis. The toad died from intestinal obstruction. Fig. 8. Bufo bufo with tuberculosis of the liver. N o t e the typical tubercles. F i g . 9. Tuberculous kidneys of B. bufo. Fig. 1 0 . Lobar, tuberculous pneumonia in Xenopus laevis. One lung is transformed into a solid tuberculous granuloma.

Figs. 7 - 1 0 .

Figs. 2 1 - 2 6 .

297

6. PATHOLOGY I N T H E A M P H I B I A

Fig. 2 7 . Bufo

bufo with a heavy fungal infestation of the head.

Some particularly interesting papers on fungal infections in amphibians have come from W . Frank and his collaborators (Fig. 29A and B ) . In 1970 Frank and Roester described cases of Hormiscium drum)

infection in American toads (Bufo alvarius),

(Hormoden-

a fungus which they

also hold responsible for the cases of chromoblastomycosis which occur in South American agricultural workers. T h e paper should be read in conjunction with Elkan's (1973) which described the destruction of a whole collection of South American frogs. Only, this time the pathogens found were Phialophora

and Cladosporium,

the former of which is also

held responsible for the same skin disease in South America. T h e identity of these soil fungi and the degree in which they are responsible for c h r o m o mycosis needs further investigation, preferably b y laboratories in their countries of origin. M e a n w h i l e , importers of South American frogs and

Fig. 2 1 . Deposits of Plistophora myotrophica in striated muscle of Bufo bufo. Fig. 2 2 . Several specimens of Acanthocephalus ranae attached to the gastric mucosa of Rana temporaria b y their hook-lined proboscis. Fig. 2 3 . Intestinal fungal infection in Hyla caerulea. N o t e the discoloration of the liver and the numerous adhesions due to almost total destruction of the parenchyma b y this yeastlike fungus (Cladosporium). Fig. 2 4 . A facial epithelioma in Xenopus laevis. Frogs occasionally develop cancerous ulcerations of the rostral or the orbital region which behave very much like an "ulcus rodens." T h e y gradually invade the underlying structures. Fig. 2 5 . A gastric carcinoma in Xenopus laevis. Spontaneous intestinal carcinomata are extremely rare in amphibians. T h e tumor shown here involved the stomach, the pancreas and in the form of metastases, both kidneys and the urinary bladder. Fig. 2 6 . A rare nephroblastoma in Xenopus laevis. T h e tumor arises from a developmental error and destroys the kidney b y compression.

298

F i g . 2 8 . Ichthyosporidium, in Amphibians.

Ε. ELKAN

& fungus which usually infests fish, has also been found

toads should be alert to the danger. It is not very great for humans but very great indeed for frog collections which m a y be wiped out within a few weeks b y any of these fungi.

VIII. Tumors A guide, assessing the risk to life and limb in the A m p h i b i a would have to rank predators first, debility through starvation and ecological disasters next, then infestations and infections of all kinds, and, lastly, spontaneous tumors, both benign or malignant. T h e time m a y come when we shall have to assign the tumors to the infection-induced group. A t present we k n o w only of a few tumors where this regrouping might be justified, and these have already been discussed in Section Ι Ι Ι , Α . T h e relative rarity of tumors in amphibians should not induce herpetologists to neglect them, since, of all the pathological phenomena observed in lower vertebrates, the tumors are scientifically the most interesting. A t the outset, however, we must distinguish between true and false t u ­ mors, and discard from this chapter all those growths whose center is found t o contain more or less well-preserved remnants of parasites, fungi, or bacteria. This determination between true and false tumors is not a l ­ ways easy to achieve, since there m a y even be borderline cases where, due t o cellular reaction against the pathogens, they can no longer be d e m -

6. PATHOLOGY IN THE AMPHIBIA

299

Fig. 2 9 . ( A ) Liver of a frog infected with mucor, a c o m m o n mold. ( B ) Smear from rawcor-infected tissue from a frog. Showing developmental stages in the form of "spherules." (Courtesy W . Frank et al, 1974).

300

Ε. ELKAN

onstrated because they have been destroyed b y

cellular defenses.

In

everyday laboratory routine examinations, dermal tumors are most likely caused b y fungi or parasites, while internal ones m a y be caused b y m y c o ­ bacteria. Other tumors are rare, but fairly extensively documented (e.g., Arifmann, 1963; Balls, 1962; Balls and R u b e n , 1968; Barch et al,

1965;

Brunst, 1967; D i Berardino, 1965; D u r y e e , 1965; Elkan, 1963, 1968; Freed and Rosenfeld, 1965; Granoff et al, and Schlumberger,

1949; M a t e y k o

1965; L u c k e , 1934; L u c k e

and K o p a c ,

1965; M u r r a y ,

Rafferty, 1964; Schlumberger and Lucke, 1948; Stewart et al,

1908; 1959;

Stolk, 1957-1959; Willis, 1960). A.

NONMALIGNANT TUMORS

A few words only need to be said about the benign tumors. Just as in human pathology they arise mainly from the soft tissues. Epitheliomata (see

Fig.

24),

papillomata,

lipomata,

fibromata,

and

chondromata

have on occasion been seen. T h e y c o p y the tissue from which they arise, rarely grow to a large size, and do not usually present a direct threat to life. Several authors have described groups of granulomata, 2 - 5 m m in diameter, on the underside of the skin of frogs and newts, bulging into the lymphatic sacs. A n example of such a tumor in Xenopus

is seen

in Fig. 30. Searches for causative organisms have so far been fruitless;

F i g . 3 0 . Adult Xenopus laevis with a granuloma on the inner surface of the dorsal skin. From Elkan (1960b).

6. PATHOLOGY IN THE AMPHIBIA

301

the tumors are harmless. Nonmalignant visceral tumors are exceedingly rare. If a thorough search is made, such tumors will mostly be found to contain remnants of parasites. As a word of caution it might here be mentioned that excessive or particularly widespread melanosis of any v i s cera, does not necessarily represent a sign of disease. In Fig. 31, for e x a m ple, an intestinal loop of Bufo

bufo is shown in which there is a h e a v y

deposition of pigmentation around the capillary network of the intestinal wall. Apart from this melanosis, however, the tissue was perfectly healthy and normal. All amphibians store melanin apparently in amounts in e x cess of their needs, and deposits m a y be found not only in the liver, k i d ey, lungs, and anywhere in the connective tissue, but m a y also appear in the form of large dendritic deposits in the peritoneal lining of the intestine. This melanosis is entirely normal, but where, in the course of fungal or tuberculous destruction, large parts of the liver are transformed into granulomata, melanin granules become mobilized, enter the circulation and are then reabsorbed selectively b y cells of the renal tubuli. In these tubuli some cells m a y be seen crammed with melanin while others i m mediately adjacent are free of pigment (Fig. 3 2 ) . This cellular selectivity remains, so far, unexplained.

Fig. 3 1 . H e a v y pigmentation along the intestinal capillaries of Bufo bufo, without any accompanying pathological process.

302

Ε. ELKAN

Fig. 3 2 . A section of the kidney of Xenopus laevis which had died of visceral tuberculosis. M a n y of the cells lining the renal tubules have taken up the melanin mobilized b y the disease.

B . MALIGNANT

TUMORS

T h e malignant tumors found in amphibians, though numerically insig­ nificant, are of great interest to the pathologist. W i t h the exception of the lymphomata they are nearly always of epithelial origin (Figs. 33a and b ) . D e r m a l , pulmonary or visceral growths m a y become malignant, and, a few true teratomata of embryological origin like nephroblastoma (see Figs. 25 and 26) have also been described. T h e epithelial growths m a y remain local for a long time before they metastasize. L o c a l l y , they sometimes grow to such a size that their mere presence becomes incompatible with the life of the animal (Figs. 34a and b ) . W h e n ever an amphibian dies under such circumstances it is surprising t o see to what extent vital organs m a y be compressed or destroyed before death supervenes (Fig. 3 5 ) . Fortunately, since the discovery b y Lucke (1934) of the renal a d e n o ­ carcinoma in some populations of North American Rana pipiens, there has been ample material of this malignant amphibian tumor for observa­ tion and experimentation (Zambernard and M c K i n n e l l , 1969). C o n s e ­ quently a considerable bibliography has accumulated about this growth. T h e tumors arise in the kidneys of both sexes in some populations, but

6. PATHOLOGY IN THE AMPHIBIA

Fig. 3 3 . Section of a malignant epithelioma of Rana temporaria region, and ( B ) infiltrating the cornea. From Elkan (1963).

303

in ( A ) the loreal

not in others. It has the appearance of a typical renal adenocarcinoma (Fig. 3 6 A ) where the abnormal carcinomatous tubuli are seen lined b y abnormal, basophil cells (Figs. 36B and C ) showing m a n y mitoses. T h e tumor is infiltrating and no capsule develops. A t later stages of its growth metastases develop in the lungs and the liver. A greater number of t u m o r bearing frogs are found in the spring than at other times of the year, and female frogs show a higher incidence ( 4 . 8 % ) than males ( 2 . 8 % ) ( M c K i n n e l l , 1965). Frogs m a y develop the tumor while held captive in the laboratory even if they are well fed and the cages suitably heated. Repeated transplantation experiments have shown that the speed of growth of this tumor rises with increased environmental temperature. This is not surprising since all metabolic processes are dependent on the temperature of the environment in poikilothermic animals. It is to be expected that in such a tumor, which is easily transplanted to the anterior chamber of the eye and can thus be grown under visual control, every effort has been made to find the causative agent, and since its discovery m a n y thousands of frogs have been screened for such tumors, and the tumors subjected to every k n o w n method of experimentation. Y e t , even now, it is not definitely known whether a viral or nonviral etiolo g y should be accepted. As has already been mentioned (Section I I I ) , herpeslike and other viruses have been found in some cases, but not in

304

Ε. ELKAN

Fig. 3 4 . Xenopus laevis with ( A ) an orbital adenocarcinoma causing an extreme dislocation of the eyeball. ( B ) Shows a section of the papillary adenocarcinoma aris­ ing from the ophthalmic glands.

every ease, and genetic, chemical, and physical factors have all, in turn, been suggested as causal agents. It m a y eventually be found that a c o m ­ bination of several of these factors is needed to produce the tumor. There remains one other t y p e of malignant tumor which has been r e ­ ported as occurring spontaneously in specimens of Xenopus laevis, and which has been described as a lymphosarcoma (Balls, 1962; Balls and

6. PATHOLOGY

IN THE

AMPHIBIA

305

Fig. 3 5 . Dissection of Xenopus laevis showing a large central necrotic area in the liver caused b y a biliary adenoma. F r o m Reichenbach-Klinke and Elkan (1965).

R u b e n , 1968). Similar tumors have also been seen in a few urodeles. T h e y are extremely rare and their interest, like that of the L u c k e tumor, lies in the fact that they too are transmissible, particularly if y o u n g specimens of the same or a closely allied species are being used as recipients. Here again the causative agent is as y e t n o t k n o w n with certainty, but the experimental evidence points in the direction of an infective agent. W h a t e v e r the agent is, it is of low virulence and, like so m a n y others, only seems to be able to attack otherwise debilitated animals. T h e power to resist any of these attacks lies apparently entirely in the epithelium. Where this barrier has been broken, either accidentally or experimentally, the animals show themselves to be devoid of any resistance to the t u m o r inducing agent. C. T H E NEWT

"TEST"

A n attempt has been made b y German pathologists (Arffmann, 1963; K r a c h t and W a i b l e , 1966) to use the newt as a test animal for cancerous disease, but the results produced have shown that the cellular infiltration

306

Ε. ELKAN

F i g . 3 6 . ( A ) Section of the kidney of Rana pipiens with Lucke's adenocarcinoma ( B ) T h e tumor at a higher magnification to show the numerous eosinophilic intra­ nuclear inclusions in the tumor tissue (lower end of p h o t o ) , but not in the regior of normal tubules (top right of p h o t o ) . ( C ) Enlargement of the boxed area of ( B ) Nearly all nuclei have prominent Cowdry T y p e A inclusions, i.e., large eosinophilic masses with marginated chromatin.

6.

PATHOLOGY

IN T H E

AMPHIBIA

307

or hyperplasia seen in the newt's tail after the injection of extracts suspected of carrying oncogenic substances is not specific, and can be i n duced equally well b y the injection of m a n y organic and inorganic agents. T h e test, described as the " M o l c h T e s t , " produces cellular reactions in the tail, but these m a y equally well be elicited b y the base in which the test substance is dissolved, and, in any case, they eventually regress. D.

R E G I S T R Y OF

TUMORS

A Registry of T u m o r s in L o w e r Animals (both vertebrates and invertebrates) has been established b y a collaboration between the American National Cancer Institute and the Smithsonian Institution in Washington D . C . Specimens are accepted alive, fixed, embedded in paraffin or s e c tioned, and assistance is given with their diagnosis and bibliographic references provided. Annual reports of the activities of the Registry can be obtained on application. It is hoped that all those interested in o n c o l o g y will cooperate with the Registry so as to make their collection as representative as possible. Meanwhile, since it is often difficult to define what constitutes a " t u m o r " and what does not, the Registry is slowly developing into a center for lower animal diseases generally, a d e v e l o p ment which can only be sincerely applauded. (Harshbarger 1968-1975.) E.

PREREQUISITES FOR

HISTOPATHOLOGY

T h e pathologist's demands with respect to tissue preservation have to be exacting. A semi-decayed or shriveled specimen, which m a y y e t be of some use for taxonomic purposes is useless to the bacteriologist or the histologist. W h e n collecting amphibian material, an ample supply of 5 to 1 0 % formaldehyde should be injected into the peritoneal cavity, the specimen wrapped in cotton wool soaked in the same solution. I t then should be placed, into a well-closed plastic bag.. In this w a y it will keep indefinitely. Bacteriological, virological, and some parasitological investigations can only be carried out if and when live material is sent to the laboratory. Cooled material is acceptable. H a r d freezing produces intracellular ice crystals and entirely destroys the histological pattern (Fig. 3 7 ) . B l o o d smears should preferably be made while the animal is still alive. C o l o r photographs should be taken showing the diseased parts in situ. Pictures of organs removed from the b o d y are rarely v e r y i n f o r m a tive. T h e intestinal canal should be opened in Ringer saline if possible and the sites and number of free parasites noted. W o r m s attached to the intestinal wall should not be removed since they would be mutilated in the attempt. Specimens m a y be killed b y immersion in a solution of 5 % urethane or in 6 % of Sandoz M S 222 ( M a h o n e y , 1966). Injections o f soluble b a r -

308

Ε. ELKAN

Fig. 3 7 . Hard freezing completely destroys the histological structure of any paren­ chymatous organ like the spleen shown in this picture.

biturates in suitable concentrations are equally effective. Methods which irritate the skin and produce overproduction of mucus should not be used.

IX. Conclusion This short chapter on the pathology of the Amphibia can only serve as an introduction to a discipline which is, as yet, very much in the early stages of its development. Much of the progress that has already been made can be attributed to the temporary popularity of Xenopus as a test object for the diagnosis of pregnancy, and it is to be hoped that further progress will be made on a wider variety of amphibian species. Two bullfrogs have already been reported to have died on the altar of space exploration. How much did those who sacrificed these animals know about the physiological tolerance of the Amphibia?

References Ambrus, J. L., Ambrus, C. M., and Harrison, J. Y . F. (1951). Prevention of Proteus hydrophilus infections (red leg disease) in frog colonies. Amer. J. Pharm. 123, 129. Angel, F. (1937). Sur deux tetards geants de Rana Natur. Paris 9, 54-55.

esculenta

L. Bull. Mus.

Angel, F. (1947). " V i e et moeurs des Amphibiens.' Payot, Paris. Arffmann, E. (1963). Studies on the Newt Test for carcinogenicity. Acta Microbiol. Scand. 57, 375-384.

Hist.

,

Pathol.

6. PATHOLOGY IN THE AMPHIBIA

309

Balls, M . (1962). Spontaneous neoplasms in Amphibia. A review and description of six new cases. Cancer Res. 2 2 , 1142-1154. Balls, M., and Ruben, L. N . (1968). L y m p h o i d tumours in Amphibia. A review. Progr. Exp. Tumour Res. 10, 238-260. Barch, S. H., Shaver, J. R., and Wilson, G. B. (1965). Some aspects of the ultrastructure of cells of the Lucke renal adenocarcinoma. Ann. N.Y. Acad. Sci. 126, 188-203. Baylis, H . A . (1930). T h e parasite worms of British reptiles and amphibians. In " T h e British Amphibians and Reptiles" ( M . Smith e d . ) , p p . 267-284. Collins, L o n d o n . Brunst, V . V . (1967). Tumours in Amphibians I. Histology of a neuroepithelioma in Siredon mexicanum. J. Nat. Cancer Inst. 38, 193-304. Canning, E. U., Elkan, E., and Trigg, P. I. (1964). Plistophora myotrophica spec, n o v . causing high mortality in the C o m m o n T o a d Bufo bufo L. with notes on the maintenance of Bufo and Xenopus in the laboratory. J. Protozool. 11, 157-166. Cheng, T . C. (1967). " T h e B i o l o g y of Animal Parasites." Saunders, Philadelphia, Pennsylvania. Cohrs, P., Jaffe, R., and Meesen, H . (1958). "Pathologie der Laboratoriumstiere." Springer, Berlin. Darzins, E. (1950). Tuberculose das Gias. Sep. Arch. Inst. Brazil Invest. Tubercul. 9, 1. Dawes, B. (1946). " T h e Trematoda." Cambridge Univ. Press, L o n d o n . Dhaliwal, S. S., and Griffiths, D . A . (1964). Fungal disease of Malayan toads (Bufo melanostictus). Sabouraudia 3, 279-287. Di Berardino, M . A. (1965). Renal adenocarcinoma promoted b y crowded conditions in laboratory frogs. Cancer Res. 25, 1910-1912. D o d d , J. M., and Callan, H . G. (1955). N e o t e n y with goiter in Triturus helveticus. Quart. J. microsc. Sci. 96, 121-128. Doflein, F., and Reichenow, E. (1953). "Lehrbuch der Protozoenkunde." Fischer, Jena. Duryee, W . R . (1965). Factors influencing development of tumours in frogs. Ann. Ν. Y. Acad. Sci. 126, 59-84. Elkan, E. (1957). Observations on the lymphatic system of the South African clawfooted toad (Xenopus laevis D a u d i n ) . Brit. J. Herp. 2, 37-53. Elkan, E. (1960a). T h e c o m m o n toad (Bufo bufo L.) in the laboratory. Brit. J. Herp. 2, 177-182. Elkan, E. (1960b). Some interesting pathological cases in amphibians. Proc. Zool. Soc. (London) 134, 375-396. Elkan, E. (1963). Three different types of tumours in Salientia. Cancer Res. 2 3 , 1641-1645. Elkan, E. (1965). Myiasis in Australian frogs. Ann. Trop. Med. Parasitol. 59, 51-54. Elkan, E. (1968). T w o cases of epithelial malignancy in Salientia. J. Pathol. Bacteriol. 96, 496-499. Elkan, E., and Murray, R . W . (1952). A larval trematode infection of the lateral line system of the toad Xenopus laevis. Proc. Zool. Soc. (London) 122, 121-126. Elkan, E. (1973). Mycotic infections in frogs due to a Phialophora-like fungus. Sa­ bouraudia 11, 99-105. Elkan, E., and Reichenbach-Klinke, H . (1974). " C o l o r Atlas of the Diseases of Fishes, Amphibians and Reptiles." Tropical Fish Hobbyist, Neptune, N e w Jersey.

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