Infectious diseases in amphibians

Amphibian populations are said to be declining on a world wide range. There now appears to be a growing body of public evidence to verify such declines, and probably even several extinction events in the last decades. Over the years, many hypotheses, such as habitat loss, the introduction of predators, global climate change, acid rain, and other effects of environmental pollution, have been proposed as explanations for these observations. However, until very recently infectious diseases have not been discussed in this context. One reason may be, that the introduction of foreign pathogens has not been generally accepted as an anthropogenic threat to the natural balance of an ecosystem.
Although wild amphibians have undoubtedly carried, suffered from, and died of unknown and unreported infectious diseases for as long as there have been wild amphibians (see picture, according to W. Busch, how draw a portrait of a convalescent frog more than 100 years ago), these infectious diseases are most probably the crucial factor within the scope of the recent worldwide decline of amphibian populations. Various demographic and nutritional changes in amphibian populations adapted to man combined with complacency about the role of opportunistic infectious diseases in general have brought about a resurgence in infectious diseases, especially waterborne ones, and with it challenges to the microbiology that are unprecedented in recent times. The amphibian population decline underscores the importance of understanding the interrelationship among stress, immune function, and susceptibility to disease. Especially the infectious diseases known as opportunistic diseases have been implicated as the proximal cause of death in several amphibian populations. Regarding macroclimate changes we have to state, that allmost all declining populations are located in more or less cold to moderate localities such as the mountain ranges in the Western United State. But, because montane populations have lived for generations in cold or changeable temperatures and climatic changes are not unusual at all, it is unlikely that a single climatic change is the causative factor in the widespread appearance of "new" infectious diseases. Cold itself, even slow adaption to cold, seems to immunocompromise anura as their normal physiological response, characterized by lymphopenia and low complement activities. Maybe, climate-induced immunosuppression may also act synergistically with some new, presumably man-made, environmental or mental stress. Thus, the final immunosuppression can be a consequence of a variety of noxious environmental factors. It especially affects the cellular immune response by changing the vitality of the cells involved. For amphibians, immunological parameters have been mainly investigated in Xenopus laevis. But, since Xenopus is the only cold-blooded vertebrate known to have three immunoglobuline isotypes (IgM, IgY, IgX), and it`s immune system is fundamentally similar to that of mammals (e.g. rearranging TCR and Ig genes, MHC class-I- and class-II-restricted T-cell recognition), it might not be an ideal model to study immunology in amphibians. For other species however, there is a deficiency of basic knowledge which is fundamentally necessary to assess the significance of immunosuppression in the genesis of infectious diseases in amphibians.
Unfortunately, our knowledge of the aetiology and pathogenesis of infectious diseases of amphibians is very poor, and knowledge of the effects of such diseases on population dynamics is poorer still. To confound matters, increasing doubt arises as to the scientific validity of traditional microbiological methods used and, more particularly, of the significance of the results reported for past investigations of infectious diseases caused by germs now known as opportunistic germs. The Microbiology Working Group of the DAPTF, therefore, gives emphasis to immunoregulatory mechanisms in native amphibians and captured exotic ones, will investigate the influence of captivity on the health status, and will carry out epidemiologic studies on amphibians on fungi, gut and blood parasites, mycobacteria and other opportunistic bacteria.

My special interest is focusing on a registration and balancing of mostly opportunistic behaving infectious germs associated with amphibian keeping. The microbiology and the hygienic problems of the interface man - exotic pet (amphibium) - feeder animal - terrarium is of a special interest of mine. The microbiological fauna of this interface is characterised by the appearance of a special type of infectious germs, so called "opportunistic germs". These ill-defined group of microorganisms do usually not infect or infest the host, but may cause even lethal disease in an immunologically debilitated animal or man, in rare cases even in amphibians and man mutually. There is a good characterisation of this type of germs by Karasawa et al, 2002 in the Brazil. Memorias Do Instituto Oswaldo Cruz 97(6): 779 concerning the Cryptosporidium (Apicomplexa, Cryptosporidiidae) in Crotalus durissus terrificus (Serpentes, Viperidae), which doesn`t make too much difference to the opportunistic germs in amphibians: The literature shows that elimination of oocysts in the feces is periodic (cit.), explaining the presence or absence of oocysts in samples from the same animal. The presence of Cryptosporidium sp. can be frequently associated with capture or recent import, suggesting that stress and altered immunological state can play an important role in the development of the infection (cit.). Cryptosporidiosis is selflimited in immunocompetent mammals and a life threat in immunosuppressed mammals, and this seems to also be true for reptiles. The elimination of environmental or nutritional problems and other diseases seems to be more effective than the use of anti-Cryptosporidium drugs to reduce the infection (cit.).
Some of the today acknowledged members of the group of opportunistic germs in amphibians are (text in red: taxa potentially pathogenic to man):

Iridoviruses (e.g. Bohle Iv),
Aeromonas hydrophila,
Pseudomonas sp.,
Weeksella virosa,

Salmonella enterica and S. arizonae,
Chlamydia psittaci,
Chlamydophila pneumonia,
Mycobacterium fortuitum biovar peregrinus,
Saprolegnia parasitica
Basidiobolus ranarum,
Blastocystis sp.,
Glugea sp.,
Plistophora sp.,
Myxosporida,
numerous intestinal protozoa, previously known as "Flagellata",
Opalina spp.,
Trichodina spp.
Cryptosporidium sp.,
Rhabdias sp. ? (R. bufonis),
Hannemania spp.

An example of the impact of an infection of a frog with such an opportunistic bacterium, Weeksella virosa, is shown to the right (fig. 2): These aerobic, gram negative, Pseudomonas-related rods are ubiquitous soil and water inhabiting bacteria which are known to be opportunistically infectious to immunosuppressed or traumatised vertebrates in which they lead to sepsis. Weeksella is apparently able to colonise cysts and, probably, to form necrotic foci in the epithelium of otherwise apparently healthy anurans. (cit. from Hassl A, Url A, Rebel-Bauder B., 2001). The intention of the research work is the creation of recommendations on hygiene for manipulations of amphibians in special regard to the prevention of man-made infectious diseases in amphibians.

Infectious Diseases and Defense Mechanisms

Diseases and Immunology

Like other creatures, amphibians are integrated into their environment, interacting with each other and with the environment. As a result of internal factors such as aging or external influences such as infections, individual body cells, tissues or organs may fail or mutate; regulatory functions may collapse, tissue may die or be attacked by foreign organisms such as protozoa or bacteria that use it as a substrate. As vertebrates, amphibians are in principle exposed to the same pathogenic mechanisms as humans or domestic animals, although some peculiarities exist due to their mode of life and phylogeny.

Diseases in Amphibians Not much is known about systematic diseases, infectious diseases, immunology and disease defense in all amphibians with the exception of the clawed frog (Xenopus laevis), which is often used as a laboratory specimen, as few case histories have been recorded in each field. Above all, the relevance of individual factors which are expected to be pathogenic by themselves or to modulate the immune system - for example, increased UV radiation or the resorption of industrial toxins - in disease manifestation is unknown altogether. This is why it is currently impossible to predict the survival of amphibian populations or even the worldwide well-being of this class in the context of the global amphibian decline. However, precise studies in pathology and immunology in special laboratories can reveal the cause of disease or death in an individual.

Before attempting to organize amphibian diseases into logical and systematic categories, we should take a closer look at amphibians' living conditions. Veterinary studies on amphibians in their natural environment yield findings that are considerably different from those obtained from terrarium specimens. In nature creatures that are weakened by disease are the preferred prey of predators. This means that, in comparison to specimens kept in captivity, chronic infections, diseases that result from aging processes and benign tumors are less frequently diagnosed. Furthermore, the range of diagnosed infectious or viral diseases is considerably different.

In captivity the vectors necessary to the life cycle of some germs are almost always absent, but unnaturally high population densities create epidemic conditions that are specific to captivity. In nature, however, limited germ colonization is normal and sometimes even conducive to the health of the host. This relates to a broad spectrum of usually hardy or non-pathogenic germs that are slowly propagating; among them are many opportunists. Only when considerable stress occurs - for example, during an increase of toxin exposition or constant uneasiness with the connected immunosuppression - do these germs multiply in an uncontrollable way and begin to develop pathogenic characteristics.

The colonization of free-living amphibians by opportunistic germs is clinically barely noticeable and it is thus rarely documented; nevertheless, it is the main factor for a lasting regulation of population density. In captivity, however, we see a preponderance of infections caused by a small number of usually highly pathogenic species of germs that multiply rapidly and have a tendency to autoinfection. Diseases in amphibians can be classified into the following groups:

Cancer (Tumors): Benign tumors are difficuit to recognize because there is an even transition to encystation of foreign tissue. Malign tumors in amphibians are almost invariably epithelial in origin. They may be striking because they can reach unbelievable dimensions. The transplantable, renal adenocancer of Rana pipiens (Lucke tumor) is a long-known and relatively well-researched cancer whose herpes virus-induced genesis is, however, still in question.

Metabolic Diseases and Regulatory Malfunctions: This category includes ontogenetic abnormalities and malformations that are unusually frequent among amphibians and are caused by abnormal genes or abnormal gene combinations. Abnormalities are well researched because they are easy to induce in a laboratory setting and are well documented in the genetic literature. Amphibian larvae with thyroid hypofunction do not metamorphose, but continue to grow and sometimes even reach sexual maturity.

Deficiency Diseases manifest themselves clinically almost exclusively as a result of poor nutrition in captivity. Skeletal damage has been described for the clawed frog as a result of insufficient calcium intake. The standard values of required vitamin and mineral intake for individual amphibian species have not been determined until now.

Infectious Diseases are caused by transmitable germs that harm the host by robbing it of its energy, but which are not lethal to it, at least at the initial contact. Amphibians are susceptible to viruses, bacteria, fungi, protozoa and metazoa. Although infectious diseases may threaten the life of the host, they are the driving force behind the immunological and - to a smaller degree - the biological evolution.

In recent years certain germs have received a lot of attention for different reasons: virus identification and detection continue to present a diagnostic and technical challenge. Specific viruses (for example, iridovirus-like particles) can now be identified quite easily and efficiently with the help of electron microscopy, so they are now frequently detected in amphibians. The clinically conspicuous and epidemic-causing “red-leg” disease in frogs has excited interest across the world; the germ Aeromonas hydrophila is a ubiquitous, mobile, gram-negative bacterium regularly found in the intestine of amphibians. The cold-water mycobacteria, which cause tuberculosis in amphibians, are similarly ubiquitous opportunists; they colonize the skin of the host. Protozoa from the phylum Microsporida create completely different scenarios: In recent years they led to catastrophic mortality among non-adapted hosts (Bufo bufo) in southern England. This was caused by intensified commercial fish breeding and the introduction of fishes foreign to the local fauna.

ImmunologyImmunology is a basic and vital function of any union of cells. In all higher animals it maintains individual integrity or, put very simply, maintains health. The key function of immunology is to distinguish reliably between the body's own working cells and foreign and/or mutated cells. In amphibians, self-recognition is realized with the help of complex and multi-structured Protein molecules at the cell surface (MHC complex). Protein structures that do not carry these recognition signals are captured by means of a complicated mechanism by phagocytic cells that are part of the body (macrophages); divided through digestion, the parts are then kept at the macrophages surface and are utilized by other immune cells as a starting point to control their own activity. This “presentation” sets in motion a cascade of several reactions that ultimately lead to a complete destruction of the foreign structure. Unique characteristics of the amphibian immune system are a strong seasonal activity cycle and a partial functioning at temperatures that would lead to paralysis of the immune functions in other poikilothermic animals. Another immune function is the production of proteins (antibodies) floating in bodily fluids that attach to foreign proteins according to the “keyhole” principle. Its task is to clot and thus immobilize the foreign structure and to mark the surface for the purpose of targeted attack by phagocytes. These antibodies are divided into several classes according to their specialized tasks. In Anura we find the so-called IgX (dimension: probably 170 kD), during an infection early arising IgM (900 kD) and IgY (170 kD); in the phylogenetic older Urodela only IgM and in some species IgY are present. Antibodies are produced during the active stage of an infection and are still present in small amounts months later. Hence, identifying and documenting antibodies that target a specific germ provides conclusive proof of an infection without having to isolate the germ itself. Such immunological-diagnostic procedures are currently becoming rapidly more significant in amphibian research.

Amphibians as Vectors

When thinking about a possible link between human health and amphibians, cases in which people were harmed by poison excreted through these animals' skin, by touching or coming into contact with a wound, immediately come to mind. There is, however, another frequently overlooked aspect of amphibian impact on the health of human beings: certain taxa of this group of animals can serve as intermediate hosts for pathogenic germs, and can transport these germs or cause contact between man and infectious foci.

Amphibians as Intermediate Hosts

The most severe and most dangerous illness that humans can contract from a germ transmitted by amphibians is an infection caused by the larvae of the tapeworm genus Spirometra. These parasites are located in the muscles of frogs as outwardly undifferentiated worm larvae. They can actively move into human tissue if direct contact occurs, stay there permanently, perhaps even metastasize, move, and increase in size to several cubic inches or centimeters. Especially in South East Asia, where direct contact with frog meat is quite common, since compresses containing amphibian meat are used as an antibiotic remedy and placed on patients' eyes or on festering wounds. Particularly serious cases of these sparganoses [infestations with plerocercoids of the genus Spirometra] may lead to the loss of one's eyes; in minor cases, surgery is required to remove the worm. The development of a mature tapeworm in human beings is impossible.

Amphibians as Carriers of Potentially Pathogenic Germs

Several potentially pathogenic bacteria have such a low host specificity and are so widespread that they can be found in sick amphibians as well as in sick humans. These include Aeromonas hydrophila (red leg, sepsis), Pseuolomonas sp., and Salmonella (diarrhea, sepsis). Contrary to the situation with reptiles, an actual transmission of these germs from amphibians to humans has not been proven until now. Nevertheless, caution is advised when dealing with excrement or in treating wounds.

Amphibian Breeding as Source of Human Infections

In cases where human beings keep amphibians, it is inevitable that the caretakers come into contact with a special flora of germs, specific to aquaterrariums. The human pathogenic germs of this flora include Mycobacterium fortuitum (but not M. ranae and M. xenopi!), free-living, potentially pathogenic amoeba Acanthamoeba spp. Naegleria fowleri; a germ that produces brain abcesses) and the pathogen that causes a subcutaenous mycosis in Africa, Basidiobolus ranarum.
So far, there are no basic scientific studies in existence concerning the health risk to humans in handling amphibians. Also, the risk to the animals during their first contact with humans, when germ exchange and "germ adaptation" occurs, has not been examined, although this phenomenon has a significant impact on physiological studies and on their chances of survival in captivity.

Infectious diseases in amphibians

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