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SIX Amphibian Medicine A. GENERAL COMMENTS Considering the wealth of information dealing with the physiology, bio- chemistry, genetics, developmental biology, and even behavior of amphib- ians, it is incredible that almost nothing of a practical nature is known about amphibian medicine. Entire books deal with the classification of amphibian infections and infestations, while therapy and prevention are usually dispensed with within a few pages. Moreover, those methods sug- gested, for the most part, have never been adequately evaluated. Yet, many amphibians brought to the laboratory do not survive for more than a few days under conditions effective for maintaining "healthy" wild-caught and laboratory~reared or laboratory-bred animals for years. No doubt our inability to define or measure amphibian disease in anything but the crudest terms has contributed to this situation. An amphibian can have a "good" appearance and yet be within hours of death as the result of massive destruction of internal tissues. On the other hand, we easily confuse mild inflammation of the skin with serious disease. Certainly, anyone using amphibians in their research should evaluate the potential impact of "disease" on their investigations and, insofar as possible, de" fine the state of health of the individuals being studied. While some diseases of amphibians interfere with laboratory experiments, other diseases may be exploited as animal models of human disease. Indeed, much of the interest in the renal adenocarcinoma of R. pipiens is not moti- vated by interest in the hazard of the tumor to frog populations but is re- lated to the similarity of the frog cancer to comparable tumors in humans (Duryee et al., 1960; Dawe, 1969~. It is beyond the scope of this document to review all the diseases of am- phibians and all of the suggested treatments (see Boterenbrood, 1966; Frazer, 115

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116 1966~. Since frog medicine is a relatively advanced specialty of amphibian medicine, it will serve as the best model and the following discussion will be restricted to several common frog diseases and how they may affect R. catesbeiana and R. pipiens in the laboratory. Additional background infor- mation and discussions regarding amphibian diseases may be found in E1- kan (1960), Gibbs (1963, 1973), Walton (1964, 1966, 1967), Inoue et al. (1965), Reichenbach-Klinke and Elkan (1965), Gibbs et al. (1966), Joiner and Abrams (1967), Abrams (1969), Crans (1969), Lom (1969), Mizell (1969), Rowlands (1969), Boyer et al. (1971), Mawdesley-Thomas (1972), Van der Steen et al. (1972), Cicmanec et al. ( 1973), and van der Waaij (in press). Serious students of amphibian diseases may also find the extensive collection of reprints at the Osborn Laboratories of Marine Sciences to be an invaluable source (Ross R. Nigrelli, Chief Pathologist, Osborn Labora- tories of Marine Sciences, New York, New York). B. BACTER IAL DISEASES 1. Pathogens Bacteria and viruses deserve special attention because they are responsible for the majority of deaths that occur in populations of laboratory frogs. Also, in the case of bacterial infections at least, there are methods that seem to be effective in preventing and treating the diseases. Although the list of bacteria identified from amphibian sources is lengthy (Walton, 1964, 1966, 1967; Reichenbach-Klinke and Elkan, 1965), only a few seem to be major pathogens. Thus, despite the complexity of the bacterial mil- lieux, only Aeromonas hydrophila (Ewing et al., 1961) has been repeatedly implicated so far in the large-scale mortality of leopard frogs since 1898 when Russet first isolated an organism that was probably A. hydrophila and that he called Bacillus hydrophilus fuscus (Russet, 1898~. A. hydro- phila has also been called Proteus hydrophilus and Pseudomonas hydro- philus (Gibbs et al., 1966; Reichenbach-Klinke and Elkan, 1965~. The disease produced by A. hydrophila has been "red-leg" but the symptom- atology is not sufficiently consistent or specific to warrant this title. Thus, Miles (1950) reported an outbreak of "red-leg" in tree frogs at the London Zoo but identified Bacterium alkaligenes as the causative agent. To avoid confusion, it would be better to define diseases on the basis of the bacteria responsible. ~ n essential step in the study of any disease is the isolation and classifi- cation of organisms from sick animals. In a major study of northern R. pipiens exhibiting a wide variety of symptoms, three organisms were found to be associated with serious illness (Gibbs et al., 1966~. This study is note

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117 worthy because it demonstrates the effectiveness of a systematic method- ology for studying pathogenic organisms and the treatment of the diseases they produce. Such methods have not been widely practiced in amphibian medicine. Blood samples for bacterial culture were obtained aseptically. Cultures were incubated at 37 C (98.6 F) and, in order not to overlook psychrophilic bacteria, at 25 C (77 F) for 1 week before any were as sumed to be negative. The samples were discarded if the animals from which they were obtained failed to survive for at least 8 hours. This pre- caution reduced the possibility of isolating nonpathogenic bacteria that might enter the blood and other tissues under conditions of shock and widespread collapse of tissue functions. A. hydrophila and types of Mimeae (Ballard et al., 1964) associated with A. hydrophila were found. The Mimeae are easily missed as they tend to grow relatively slowly and the Aeromonas overgrows them. These observations led to the conclusion that A. hydrophila and Mimeae were the major pathogens found in popu- lations of R. pipiens from the north central United States (Gibbs et al., 1966~. Although Staphylococcus epidermis was isolated repeatedly from purulent leg infections, such infections were not common and the causa- tive role of Staphylococcus remained unclear. Doubtless, many more organisms will eventually be identified as etiological agents of amphibian disease once techniques and observation become more refined. 2. Drug Selection and Administration The use of antibiotics should be guided by determining the antibiotic sensi- tivities of the organisms in question. This is readily done by using standard tests in which effective inhibition of growth serves as the indicator of sen- sitivity to the antibiotic. This method was applied to each of the three or- ganisms isolated in the study referred to above (Gibbs et al., 1966) and Tetracycline HC1 was selected as the drug of choice on the basis of sensi- tivity and wide tissue distribution observed in other animals. Other infections may require other drugs, few of which have been ade- quately tested on frogs. Since so little is known about the sensitivity of amphibians to drugs or about the fate of administered drugs, the potential complications of antibiotic therapy, both to the health of the animal and to the results of the experiments in which the animal is to be used, must be thoroughly researched before using a drug routinely. The Tetracycline treatment of R. pipiens provides a glimpse of possible complications (Gibbs, 1963; Gibbs, et al., 1966~. it was determined that placing Tetracycline in the tank water serves no useful purpose. It is not ingested in sufficient amounts or absorbed sufficiently to result in signifi- cant blood levels. Attempting to raise the water concentration of the drug

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118 only results in deaths due to skin damage. Intraperitoneal injection of Tetracycline is also contraindicated because of local irritation, although attempts to ameliorate this have been reported. The only route tested that has been generally effective in R. pipiens is the oral route. (Other routes may be possible with other drugs or other amphibians.) In addi- tion, Tetracycline must be administered by stomach tube since the drug is very bitter and stimulates a gag or vomiting response. Stomach tubes (Figure 28) are conveniently made from polyethylene uretal catheters or other small-gauge soft polyethylene tubing following the procedure described by Gibbs (1963~. The essential feature of the stomach tube is a small ball tip, which protects tissues from damage. The tube is open just behind the ball tip. Plastic disposable syringes with plastic plunger seals are recommended for use with the stomach tubes since the plungers do not slide while the stomach tube is being inserted. The stomach tube should be inserted gently into the frog's esophagus with light manual pres- sure so that the ball tip passes the pyloric sphyncter without unnecessary irritation and vomiting. Stomach tubes should be maintained in good re- pair, free of rough surfaces, to further minimize irritation. Smooth-tipped ,~ ~ _~;! 1 _~ _ :~ ~ (-I ~ it. ~_ _ .` - FIGURE 28 Use of a stomach tube on R. pip,ens. Note that the length of the tube allows gentle, fingertip manipulation while the syrinx rests on the base of the hand. The second blunt instrument seen below the stomach tube is inserted between the jaws of the frog to stimulate opening of the mouth and avoid injury by the larger stomach tube.

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119 eye droppers have been used successfully with R. catesbeiana, and fabric cased stomach tubes for small animal work are available commercially. Soluble Tetracycline HCl should be made up at known concentrations of about 25 mg/ml in distilled water so that the injection volume can be kept to approximately 0.2 ml for R. pipiens, as larger quantities are easily regurgitated. Approximately 1 ml may be given to R. catesbeiana without a serious risk of regurgitation. A number of small aliquots of the solution should be prepared to minimize the chance for contamination, which may cause the Tetracycline to precipitate. The stomach tube and syringe should be rinsed with distilled water after each use and periodically with dilute hydrochloric acid in order to prevent precipitation within these parts. During treatment, the frogs should be kept in sloping tanks, half filled with water. Care should be taken to keep the water clean since, as mentioned above, the Tetracycline can damage the frog's skin and much of the dosage is excreted or passed unchanged. Food is not well digested and therefore not offered during treatment, but it is very important that the frogs be maintained in an optimum environment with optimum tem- peratures (see Chapter VI) as their immunity systems are very tempera- ture dependent (Volpe, 1971~. Twice daily administration of a dosage of 5 mg Tetracycline HCl/30 g body weight produces effective blood levels and is well tolerated. Al- though apparently unnecessary, the dosage can be doubled without ill effects. The effectiveness of the dosage was tested in vivo by injecting a control and a treatment group of frogs with a live culture of Aeromonas hydrophila (Gibbs, 1963~. The treatment group was placed immediately on a 1-week course of Tetracycline. Of the untreated group all died within 9 days and of the treated group only one died on the first day and none thereafter. Similar experiments with varying periods of treatment have demonstrated that a 5- to 7-day course of therapy is necessary in most cases. The usefulness of the Tetracycline treatment has been confirmed in many laboratories (Papermaster and Gralla, 1973~. Once the frogs have completed a 5- to 7-day course of treatment, they should be offered live food and an optimal environment (see Chapter VI) for a recovery period of at least 3 weeks. This period allows diseased or damaged tissues to heal and most residual Tetracycline to be excreted. Note, however, that some Tetracycline will be bound to tissues contain- ing calcium and may be retained for long periods of time. Although wide spectrum light is recommended for most amphibians (see Chapter V, Sec- tion B.2.c), frogs treated with Tetracycline should not be exposed to ultra- violet light for about 2 weeks after the termination of medication since this drug may produce photosensitivity. The remarkable recovery demonstrated by treated frogs attests to the

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120 effectiveness of their immunity system since Tetracycline is not bacteri- cidal under the conditions of the treatment. This fact has important impli- cations regarding the contagion of bacterial disease and the need for isola- tion procedures and sterilization of tanks. Tank sterilization, as distinct from cleanliness, is not necessary; A. hydrophila can be readily isolated from the feces of healthy, treated frogs. Consequently, if septecemia at- tributable to A. hydrophila develops, it must represent a response to some factor that triggers a decrease in the frog's bacterial defenses. This factor might be viral, nutritional, other as yet unidentified bacteria, or stress. At present, the relative significance of these possible factors cannot be evalu- ated, but the user is cautioned to be sensitive to their possible role in the disease process. From a practical standpoint, Tetracycline-treated frogs have been held for years without recurrence of serious bacterial infec- tions (Gibbs et al., 1966~. The major cause of death in such a treated population is likely to be the Lucke renal adenocarcinoma. 3. Identifying Diseased Frogs Perhaps the most common mistake made by those working with frogs is to attempt to distinguish between healthy and sick animals purely on the ba- sis of appearance. Nearly half of the frogs heavily infected with A. hydro- phila wd1 exhibit only mild somatic signs, such as being thin or lacking brilliance of the skin coloration. Their behavior may lack purpose and they may be either hyperactive or sluggish. Since these signs overlap with those of uncomplicated malnutrition, fright, cold torpor, or the irritability associated with overheating, it is quite impossible to identify healthy frogs with certainty. Shortly before death, frogs, including some which may even have looked "healthy,' usually vomit blood and convulse. When present, the more striking and traditionally recognized symptoms include slumped posture (palms turned outward), disinclination to move when prodded, tense abdomen, cutaneous hemorrhages, eroded toes and feet with bare bones exposed, eroded jaws, perforations of the skin on the dorsal surfaces (particularly the nose), rough and bleeding nictitating membranes, hemor- rhaging within the eyes, and numerous neurological signs. The course of illness may extend over several months. Spontaneous recoveries are rare, although several authors have reported them (Russet, 1898; Emerson and Norris, 1905~. The presence of Mimeae in conjunction with A. hydrophila does not result in specific symptoms or special complications differing from those already described (Gibbs et al., 1966~.

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121 4. The Need for Treatment Since bacterial disease can seriously affect experimental results, treatment or prevention is essential. This is emphasized by the extensive histological studies of animals infected by Aeromonas, which date from Russet (1898~. The work of Russet and later investigators (Emerson and Norris, l90S; Kulp and Borden, 1942; Rose, 1946; Gibbs et al., 1966) has shown that virtually every organ and tissue of the frog is affected by this organism. As one illustration of the physiological impact of such infection, a ret cent study of the sartorius muscle of infected R. pipiens is instructive. The muscles of newly arrived untreated and treated and recovered frogs were examined histologically and physiologically. The untreated frogs were carefully selected to exclude certain diseases but to include Aeromonas and Mimeae infections and malnutrition, which could not be studied sepa- rately. The muscles from diseased animals showed a combination of de- pleted metabolic stores, edema, necrosis, and hemorrhage that would introduce serious errors in biochemical determinations made on such muscles. Function, as measured electrophysiologically, was also seriously impaired in the muscles of the untreated animals (Gibbs, 1973~. Un- questionably, use of the frog as a model of physiological processes suffers the same limitations of all animal model research. If the frog is not healthy, the value of the investigation must be held in serious doubt. C. Vl RAL DISEASES 1. General Comments As a practical matter, it is virtually impossible to obtain virus-free frogs, yet very little can be done to treat virus infections (Lunger, 1966; Mizell, 1969; Granoff, 1969, 1972~. Consequently, the investigator must study animals infected with viruses that are, for the most part, unidentified, of unknown effect on the host, and of equally unknown effect on the experi- ment (Whipple, 1965~. Since no effective, specific treatments are available for any viruses, the only protection for an amphibian colony is good ani- mal husbandry. As already mentioned, the Lucke renal adenocarcinoma virus or Lucke tumor herpesvirus (LTHV) is the major cause of death in populations of laboratory frogs that have been treated for their bacterial infections and maintained under optimal conditions. In the experience of one laboratory, it appears that, provided the treated frogs are held long enough, almost all of them will eventually succumb as the result of kidney tumors. Appar

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122 entry, the virus or its oncogenic effects can remain latent for years. Frogs held at warm temperatures do not shed the Lucke virus, but refrigerated frogs develop the virus quickly in the kidneys. Tadpole edema virus may also be responsible for the loss of adult laboratory frogs. It is felt that occasional epidemics of bloating, eventually leading to death, may be re- lated to this virus, which is highly lethal in tadpole populations. For the most part, however, adult frogs appear to be immune to the virus (Wolf et al., 1969~. Kidney tumors do not have an obvious effect on the health of frogs until the tumors destroy essentially all useful kidney tissue or until they metastasize to other more critical tissues, such as the lungs or liver. 2. Lucke Tumor Herpesvirus ~ LTHV) The virus of the Lucke renal adenocarcinoma is believed to be a herpes" virus; it is icosahedral with a capsid consisting of 162 capsomeres (Lunger, 1964) and has a DNA core (Zambernard and Vatter, 1966; Wagner et al., 1970~. A substantial body of evidence has emerged to suggest a causal re- lationship between LTHV and the Lucke renal tumor (McKinnell, 1973~. Tadpoles injected with virus-containing extracts develop tumors (Tweedell, 1967, 1972; Mizell et al., 1969), and LTHV is omnipresent in all cold weather renal tumors (McKinnell and Ellis, 1972~. The viral genome is present in summer-phase "virus-free" tumors (Collard et al., 1973~. Tumor prevalence varies from 6-9 percent for grossly observable neo- plasms (McKinnell, 1965) to almost 100 percent for tumors detectable by microscopic examination (Marrow and Mizell, 1972~. Frogs with this herpesvirus-associated tumor will ultimately succumb to the disease in the laboratory. Another herpesvirus, known as frog virus 4 (FV-~), was isolated origi- nally from the urine of a tumor-bearing frog (Rafferty, 1965~. The DNA of FV-4 differs from that of LTHV (Gravel!, 1971), and the virus does not produce tumors when injected into tadpoles. Its effect on the health of the frog is unknown. 3. Amphibian Polyhedral Cytoplasmic Deoxyribovirus {PCDV) This virus-also known as frog virus 3 (FV-~) (Lunger, 1966) and tadpole edema virus (TEV) (Wolf et al., 1968)-was isolated from normal and tu- morous R. pipiens (Granoff et al., 1965) and from R. catesbeiana larvae of a number of localities in the eastern United States (Wolf et al., 1969~. Injections of FV -3 are lethal to embryos and larvae of R. pipiens (Tweedell and Granoff, 1968) and will cause death of young R. catesbeiana when

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123 added to aquarium water (Wolf et al., 1969~. Morphology as revealed by electron microscopy and biochemical studies suggests that amphibian poly- hedral cytoplasmic viruses isolated from leopard frogs, bullfrogs, and newts areindistinguishable(Clarket al., 1969~. 4. Lymphosarcoma Virus of Xenopus The South African clawed frog, Xenopus Levis, is susceptible to lympho- sarcomas (Xenopus L-1 and L-2) thought to be caused by viruses (Balls and Ruben, 1967, 1968) that have thus far not been detected with the electron microscope (Hadji-Azimi and Fischberg, 1972~. D. PARASITIC DISEASES That frogs and other amphibians can be heavily infested with a seemingly endless number of metazoan parasites is well known (Reichenbach-Klinke and Elkan, 1965; Walton, 1964, 1966, 1967), and the list of even the most commonly encountered parasites is too long to recount. In spite of their ubiquity, the effect of parasites on the frog's health is far from clear. Para- sites have been blamed as the direct cause of death and indirectly as vec- tors of virus and bacterial disease. However, parasite infestations tend to decrease in wild-caught frogs as they are held in optimal conditions (see Chapters V and VI) free of intermediate hosts; for all practical purposes, parasites do not appear to seriously affect the health of the laboratory frog (Gibbs et al., 1966~. This is not to say that the presence of parasites will not affect the frog's health to some degree, for they can most cer- tainly cause local destruction and irritation of tissues. Parasitic infestation of frogs, however, must have important conse- quences for the investigator. The modified physiology resulting from the infestation certainly has a potential for modifying the results of many types of physiological investigations. Biochemical investigations may also be effected by the presence of parasites. It is because of the implications of parasitic infestations to the investi- gator that the definitions of experimental animals for the laboratory (Chap- ter III, Section B) sharply distinguish between those animals directly or indirectly exposed to intermediate hosts and those protected from such exposure. In this regard, the selection of food items is restricted by the fact that living or unprocessed foods may introduce parasites into the laboratory environment unless these foods are, themselves, isolated from the life-cycle sequences of potential parasites. This is illustrated by R. catesbeiana deaths experienced at the Louisiana State University amphib- ian facility when heavy infestations of a nematode, Eustrongylides sp.,

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124 were traced to the use of live fish from outdoor ponds in the diet for the frogs. Since very little is known about the treatment of the parasitic diseases of amphibians, the only protection available to the amphibians and to the integrity of the experimental results is to maintain the animals in an op- timal environment, free of intermediate hosts, or to use animals that meet the requirements defined for the laboratory-bred classification (see Chap- ter III, Section B). Lists of parasites that infest amphibians are not given because no recom- mendations for specific treatments can be made. Those interested in the identification of amphibian parasites are referred to the authors cited above. E. MYCOTIC DISEASES Only a relatively few amphibian fungal infections have been identified. Saprolegnia is reported to be the most common fungus afflicting amphibia (Reichenbach-Klinke and Elkan, 1965; Walton, 1964, 1966, 1967~. It often attacks nonviable amphibian eggs in an egg mass and may pose a threat to the other eggs. Basidiobolus ranarum occasionally infects amphibians. A1- though Fonsecaea pedrosoi has been identified in B. marinas, R. catesbeiana, and R. pipiens, transmission studies using B. marinas resulted in infestation and death in only the stressed group; the unstressed group remained free of the disease (Cicmanec et al., 1973~. Thus, while fungi can be the direct cause of disease and death in some cases, as a rule, their infestations are secondary to other infections or nutritional disorders. As in the case of parasites, fungi and algae do not appear to represent a primary threat of serious disease in populations of well maintained laboratory amphibians. Some authors have recommended increased salinity, dips of potassium permanganate or formaldehyde, or topically applied tincture of iodine as treatment for fungal disease. However, in view of the highly sensitive and vital nature of amphibian skin, such practices must remain open to ques- tion, since they may only serve to produce additional insult and the ef- ficacy of such treatments has not been clearly established under controlled conditions. F. EUTHANASIA AND ANESTHESIA Considerations of humane treatment and the research requirement that animals in both acute and chronic protocols be subject to a minimum of stress introduce the need to apply the best available procedures for eutha- nasia and anesthesia. Kaplan (1969) recommends an excessive dose of

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125 ether or pentobarbital for euthanasia of all amphibians. The action of these agents is not instantaneous, however, and the level of stress pro- duced is unknown. The least cumbersome and least stress-producing procedure may still be brain and spinal pithing, an instantaneous tech- nique. A sharp needle of a diameter appropriate to the size of the animal is quickly inserted in a cranial direction through the foremen magnum and is rotated in a manner to crusts the brain bilaterally. The needle is then inserted in a caudal direction at the same point of entry and rotated to destroy the spinal cord. The location of the foremen magnum may be readily identified by a slight depression in the skin on the midline just posterior to the eyes when the animal is held between the thumb and last three fingers and when the index finger, placed on the head, is used to press the head in a ventral direction. If not immediately evident vi- sually, the tip of the needle may be placed on the skull and slid caudally along the midline. The depression marking the location of the foremen magnum is readily detected as the needle slides over the junction between the skull and first vertebra. For procedures that require that the brain and spinal cord remain intact, we recommend the initial induction of deep anesthesia. The most useful but most expensive anesthetic reagent is tricaine meth- anesulfonate (MS-222~. Larvae may be fully immobilized within 80 s at room temperature by immersion in a 1: 2,000 or 1: 3,000 solution of this reagent. Recovery occurs within 4-14 min upon return to normal medium, depending on the stage of development and length of exposure to the re- agent. Exposure to MS-222 for as long as 1 h does not result in abnormal development (Kaplan, 1969~. Adults may be anesthetized by immersion in the same preparation. This is slow, however, and more certain control of the depth of anesthesia is possible by injecting the reagent into either the dorsal lymph sac or intraperitoneally. Good results are obtained in R. pipiens by intraperitoneal injection of 0.1 ml of 1 percent MS-222/10 g of frog. If a frog is not completely anesthetized in 5 min. inject half the origi- nal dose. The frog will be in deep anesthesia appropriate for surgical manip- ulation for about half an hour (Nace and Richards, 1972b). Repeated doses can be used for longer periods, and recovery is good when the animals are set aside on a moist towel in a vegetable crisper. Note that the animal will drown if placed in deep water. Kaplan (1969) discusses other procedures and also notes that, although there is no literature on the errors introduced into experiments because of the lack of or misuse of preanesthetics, analgesics, or anesthetics, post- anesthetic care of amphibians has no problems and requires no special procedures. Among useful procedures Kaplan (1969) describes the use of hypothermia and chloretone (immersion in 0.2 percent solution) and

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126 discusses the disadvantages of several other anesthetics that have been used. The investigator who expects to make extensive use of anesthesia in amphibians should consult this paper. It must be cautioned that anesthesia must be used with care in certain physiological studies. Farrar (1972) demonstrated that repeated treatment of R. pipiens with Finquel (MS-222) causes increased blood glucose and lactate and discussed the mechanism of this response.