Xenopus Care, Health & Disease: A Brief Overview

Masha Rand and Jennifer Kalishman, DVM, MS

Columbia University, New York, NY

 

The Kelley lab maintains colonies of several species of the pipid Xenopus: Xenopus laevis, Xenopus tropicalis, and a Gabonese Xenopus species.  The adult Xenopus laevis colony is derived from commercial breeders (Nasco, http://www.enasco.com/prod/Home and Xenopus One, http://www.xenopusone.com). The Xenopus (Silurana) tropicalis are derived from a Nigerian stock from the Grainger laboratory at The University of Virginia (http://faculty.virginia.edu/xtropicalis).  In addition, we are developing a colony of animals that Drs. Kelley and Tobias brought back from their 2001 field trip to Gabon, Africa.  These have been tentatively identified (Tobias, Evans, and Kelley, 2002) as a subspecies of Xenopus tropicalis.

 

Care:

Following is a broad description of each colony, maintenance, mating, and rearing.

 

Health & Disease:

These colonies have provided a rich case study of both common and rare diseases and infections affecting the different Xenopus species.  Here we describe the diseases encountered in our colonies, including symptoms, diagnosis, and treatment.


Xenopus laevis

Care:

The Xenopus laevis colony ordinarily consists of 10-50 animals.  These are housed at 18.3oC on a 12/12, light/dark cycle. 

We use carbon-filtered water (mixed bed deionizer, US Filter), to which we add Novaqua (0.5ml/gallon) and NaCl (13.3g/gallon).  The water is stored in large Nalgene carboys.  After the tank is filled, the water is allowed to stand for one day before use. Water in the animals' tank is changed completely twice per week via a static renewal system.

 

The frogs are kept in clear, polycarbonate tanks measuring 1'6"l x 9"w x 8"h.  The tank covers are made of stainless steel rims to which, neoprene mesh with 1/4" openings is attached.  The lids are secured to the tanks with wire and inspected frequently for holes.  Frogs dessicate rapidly out of water.  A maximum of five adult males or three adult females are housed per tank in 4L of water. 

The frogs are fed every other day with Nasco Frog Brittle (http://www.enasco.com/prod/Static?page=xen_brittle&seqid=4).  They are fed approximately 3-5 pellets per frog or until they are satisfied (as judged by a cessation of feeding).

 

Of all the colonies, the X. laevis seem the most susceptible to disease.  This is probably due to the fact that, with the high turnover rate, this colony most frequently contains recently shipped animals.  Shipping animals causes stress, which leads to significant immunosuppression and flora or fauna that are present ordinarily may become pathogenic as a result (Tinsley, 1996).  In addition, new animals could introduce novel pathogens into the colony.

 

Health & Disease:

Aeromonas hydrophila/caviae

Adult male X. laevis with snout lesions caused by Aeromonas infection.

 

For several months we witnessed the widespread occurrence of dermal ulcers.  Snout lesions, such as the one above, were accompanied by similar lesions on the digits of the forelimbs and less frequently of the hind limbs; the skin appeared to be eaten away, as did the upper phalanges.  In a few severe cases, the nasal septum was visible on the snout.  The symptoms were either present in a nascent form upon arrival from the breeder, or else developed soon thereafter.  Cultures of snout lesion tissue showed heavy growth of Aeromonas hydrophila/caviae. Necropsy of several symptomatic animals showed no obvious internal sign of infection. However, histological evidence indicated considerable liver damage, also due to Aeromonas infection. 

 

Antibiotic treatment was attempted, administering 4 courses of gentamicin, subcutaneously, at a dose of 2mg/kg.  However, the treated animals fared no better than a control group of equally symptomatic animals from the same frog shipment.  Significantly, all animals lived - the lesions healed, leaving a faint scar. We did not try other antibiotics; possible alternatives are tetracycline or doxycycline. 

 

Aeromonas hydrophila makes up a normal part of the fauna encountered by Xenopus in their environment and only under immunosuppressive conditions does it become pathogenic.  Aeromonas, along with a host of other gram-negative bacteria, is responsible for Red Leg, a commonly encountered disease plaguing stressed Xenopus colonies.  It is characterized by reddening on the abdomen and inner thighs, caused by vasodilation, and by internal hemorrhaging.  Red Leg has a very high mortality rate if left untreated.

 

Finally, complete isolation of infected animals was successful in controlling the spread of infection.  New animals were quarantined for a minimum of two weeks: they were housed in separate tanks and kept only on the bottom shelves to prevent water from one tank splashing into tanks with uninfected animals.  When animals were transferred from shipping containers, none of the water or plant material in which they were shipped was transferred with them.  Animals already in the colony are removed to quarantine tanks immediately upon detection of infection. 

 

Chromomycosis

The first sign of infection appeared immediately following a change of water: a female in a tank of two began expelling blood from her mouth.  Upon closer inspection, we noticed that the extension of the dorso-lateral surface due to the underlying, inflated lung, was missing from the right side and the frog was tilted towards that side.  Necropsy revealed a fully deflated, right lung filled with black/pustulous fluid.  The bottom 3/4 of the left lung was normally air-filled, but it was similarly necrotic at the apex.  The only other sign of infection was a copious amount of recently clotted blood between the surface skin and the muscle layers.  Histological examination of the lungs revealed a chromomycosis infection.  Chromomycosis is an opportunistic fungal infection caused by a variety of pigmented fungi.

Lymphosarcoma

The first symptoms of lymphosarcoma appeared as unusual antero-lateral swellings.  These swellings increased in size.  After approximately a month swellings were also present caudally in the dorsum as discrete lumps.  All swellings were symmetrical, as is typical of lymphosarcoma (the lymph vessels are symmetrically arranged).  Histological evidence supported the anatomical evidence, showing tissue from the lymphatic bumps, as well as from most of the internal organs (ovaries, fat bodies, liver, etc.) to be overrun by lymphocytes of a uniform size and shape. 

 

Above: dorsal view of lymphatic tumors.  Below: ventral view of same.

 

In addition, tumorous growths were evident on the hind limbs, both dorsally and ventrally.  The entire lymphatic system was affected, in essence creating a perfect anatomical map of the otherwise difficult to visualize system.

 

Lymphosarcoma occurs very rarely in Xenopus laevis.  There is some suggestion that it is related to mycobacterial infectious granuloma (Asfari, 1988). However, it is not clear whether the presence of Mycobacterium marinum in such cases is causative or a secondary infection due to weakening of the immune system (Clothier and Balls, 1973). In our case, no Mycobacterium marinum grew in culture.  However, a negative result is not conclusive since mycobacteria are notoriously hard to culture. 

 

Xenopus tropicalis

Care:

The X. tropicalis colony is housed in the same 1'5"x9"x8" polycarbonate tanks and mesh covers as the X. laevis, in a 26oC warm room with a 12/12 hr light/dark cycle.  Each tank contains a maximum of 12 animals.  They are fed Zeigler's Salmon Starter Pellets, size #3, twice a week (http://www.zeiglerfeed.com/finfish.asp).  Their water is made up of carbon-filtered water with Novaqua (0.5ml Novaqua/1L water) allowed to stand for 24 hours to release gases and to to warm to warm-room temperature.  The water is changed twice a week, completely, several hours after feeding to allow frogs time to digest. 

 

The colony consists of 40-70 animals, originally from the Grainger lab http://faculty.virginia.edu/xtropicalis as well as approximately 50 lab bred adults and juveniles. So far, the minimum period from fertilization to sexual maturity has been three months. 

Xenopus tropicalis cap esteria

Care:

This colony was founded with frogs collected by Drs. Kelley and Tobias in Cap Esterias, Gabon in August of 2001. Preliminary analyses indicate a chromosome number of 20 and a call distinct from the X. tropicalis Nigerian colony; 16S ribosomal sequence analysis places this population in the Silurana clade (with the X. tropicalis population from Nigeria).

X. tropicalis cap esterias housing at the Kelley lab.

 

The colony currently contains approximately fifty wild-collected animals and approximately 300 lab-bred (both in vitro (link to in vitro protocol) and natural) tadpoles and juveniles.  The animals are housed at 26oC on a 12/12hr, light/dark cycle in 5L or 7L Rubber MaidÔ containers with perforated snap-shut lids; maximum 4 animals per container. 

 

The X. tropicalis cap esterias are cared for almost exactly like the X. tropicalis nigeria. The water is changed twice a week (carbon-filtered water with 0.5mL/1L Novaqua) using valves (Nalgene 1/2” Barbed Bulkhead Fittings) at the bottom of the containers for drainage in order to minimize disturbance.  They are fed Zeigler #3 Finfish Starter pellets thrice a week.


Health & Disease:

Dermal Infection


 


 

 

 

 

 

We have encountered a dermal infection, occurring only in the X. tropicalis cap esterias colony.  The symptoms are rust colored, diffuse patches occurring dorsally on the torso and legs. White, fuzzy tufts localized to the discolored areas can be distinguished in profile. Histological evidence indicates a parasitic infection.  Slides of naturally shed skin with areas of discoloration show lengthy, winding tunnels, occurring in localized areas and containing numerous brown eggs inside.  This matches Tinsley’s description of the nematode Pseudocapillaroides xenopodis of which he says they “create tunnels in the epidermis within which embryonated eggs and all developmental stages occur” (Tinsley, 1996).    


Oval shaped eggs in tunnel (indicated by arrow) surrounded by epithelial cells. 
Slide made from skin shed by X. tropicalis cap esterias.

Treatment with a 5% Potassium Permanganate (KMnO4) solution and with MarOxy, a commercially available freshwater fungal and bacterial medication, were not successful in eliminating the skin lesions.  The infection does not to affect the general well being of the animal: level of activity and food intake remain normal in affected frogs. Finally, the symptoms remit and reappear spontaneously.

References

Articles

Care:

Able, D.J.  1988.  An economical, balanced diet for Xenopus.  Institute of Laboratory Animal Resources (I.L.A.R.) News 3: 20-21.

 

Brown, L.E. and R.R. Rosati.  1997.  Effects of three different diets on survival and growth of larvae of the African clawed frog Xenopus laevis.  Progressive Fish-Culturist 59(1): 54-58.

 

Davys, J.S.  1986.  The breeding of Xenopus laevis on a large scale in the laboratory.  Animal Technology: Journal of the Institute of Animal Technology 37(3): 217-223.

 

Dawson, D., T.W. Schultz and E.C. Shroeder.  1992.  Laboratory care and breeding of the African clawed frog.  Lab Animal 21(4): 31-36.

 

Deuchar, Elizabeth Marion.  1975.  Xenopus: The South African Clawed Frog.  Wiley; London, New York.

 

Etheridge, Albert L. and Stephen M.A. Richter.  1978.  Xenopus laevis: Rearing and Breeding the African Clawed Frog.  NASCO, Publishing Agencies, Fort Atkinson, WI.

 

sHilken, G., F. Iglauer, H.-P. Richter, Kathleen M. Cromwell, J. Dimigen and H. Kahler.

Der Krallenfrosch Xenopus laevis als Labortier: Biologie, Haltung, Zucht und experimentelle Nutzung (The Clawed Frog Xenopus laevis as a Laboratory Animal).  F. Enke, Publishing Agencies; Stuttgart, Germany.

 

Hilken, G., J. Dimigen and F. Iglauer.  1995.  Growth of Xenopus laevis under different laboratory rearing conditions.  Laboratory Animals 29: 152-162.

 

Hoogstraten-Miller, S. and D. Dunham.  1997.  Practical Identification Methods for African Clawed Frogs (Xenopus laevis).  Lab Animal 26(7): 36-38.

 

Kaplan, M.L.  1993.  An Enriched Environment for the African Clawed Frog (Xenopus laevis).  Lab Animal 22(5): 25-27.

 

Major, N. and R.J. Wassersug.  1998.  Survey of current techniques in the care and maintenance of the African clawed frog (Xenopus laevis).  Contemporary Topics in Laboratory Animal Science 37(5): 57-60.

 

McBride, Gordon.  1978.  South African Clawed Frog, Xenopus laevis: Rearing & Breeding Manual.  Ann Arbor Biological Center, Publishing Agencies; Ann Arbor, MI.

 

Mrozek, M., R. Fischer, M. Trendelenburg and U. Zillman.  1995.  Microchip Implant System Used for Animal Identification in Laboratory Rabbits, Guineapigs, Woodchucks and in Amphibians.  Laboratory Animals 29(3): 339-344

 

Phillips, R.J.  1979.  The Care and Induced Breeding of Xenopus laevis Laboratory Animals.  Institute of Animal Technicians 30(1): 11-16.

 

Rizzo, A.M., R. Gornati, F. Rossi, G. Bernardini and B. Berra.  1999.  Effect of Maternal Diet on the Distribution of Phospholipids and their Fatty Acid Composition in Xenopus laevis Embryos.  The Journal of Nutritional Biochemistry 10(1): 44-48.

 

Rogers, W.P., T.W. Simpson, L.M. Jones and D.M. Renquist.  1992.  An Innovative Aquatic Non-Recirculating System for Use in Housing African Clawed Frogs (Xenopus laevis).  Contemporary Topics in Laboratory Animal Science 36(6): 72-74.

 

Sackin, N. and Sackin, H.  1991.  A new method for feeding captive frogs (Xenopus laevis).  Lab Animal 20(8): 44-46.

 

Smith, J.M. and K.C. Stump.  2000.  Isoflurane anesthesia in the African clawed frog (Xenopus laevis).  Contemporary Topics in Animal Science 39(6): 39-42.

 

Health & Disease:

 

Asfari, Maryam.  1988.  Mycobacterium-induced infectious Granuloma in Xenopus: histopathology and transmissibility.  Cancer Research 48: 958-963.

 

Ber, Artur and Zieleniewski.  Schozenia Zab Xenopus Laevis Daudin Obserwowane W Hodowli Zakladu Endokrynologii Am W Lodzi (Diseases of the Frogs Xenopus laevis daudin Observed in the Colony at Endocrinological Department of the Medical Academy in Lodze). (link to polish article) 

 

Bercovier, H. & V. Vincent.  2001.  Mycobacterial infections in domestic and wild animals.  Revue scientifique et techinique (International Office of Epizootics) 20(1): 265-90. 

 

Clothier, R.H. & M. Balls.  1973.  Mycobacteria and lymphoreticular tumors in Xenopus laevis, the South African clawed toad.  Oncology 28: 458-480.

 

Elsner, H.A., H.H. Honck, F. William, H.J. Kreienkamp & F. Iglauer.  2000.  Poor quality of oocytes for Xenopus laevis used in laboratory experiments: prevention by use of antiseptic surgical techniques and antibiotic supplementation.  Comparative medicine 50(2): 206-211.

 

Green, S.L., D.M. Bouley, R.J. Tolwani, K.S. Waggie, B.D. Lifland, G.M. Otto & J.E. Ferrell, Jr.  1999.  Identification and management of an outbreak of Flavobacterium and meningosepticum infection in a colony of South African clawed frogs.  Journal of the American Veterinary Medical Association 214(12): 1833-1838.

 

Howeth, E.W.  1984.  Pathology of naturally occurring chlamydiosis in African clawed frogs (Xenopus laevis) [Chlamydia].  Veterinary pathology 21(1): 28-32.

 

Hubbard, G.B.  1981.  Aeromonas hydrophila infection in Xenopus laevis water borne bacillus.  Laboratory animal  science 31(3): 297-300.

 

Iglauer, F., F. Willmann, G. Hilken, E. Huisinga & J. Dimigen.  1997.  Anthelmintic treatment to eradicate cutaneous capillariasis in a colony of South African clawed frogs (Xenopus laevis).  Laboratory animal science 47(5): 477-482.

 

Miller, J.C. & R. Landesman.  1977.  Magnesium deficiency in embryos of Xenopus laevis.  J. Embryol. Exp. Morpholo. 39, 97-113.

 

Parker, J.M., I. Mikaelian, N. Hahn, & H.E. Diggs.  2002.  Clinical diagnosis and treatment of Epidermal Chytridiomycosis in African clawed frogs (Xenopus tropicalis). Comp Med  52(3): 265-8.

 

Pearson, M.  1996.  Studies on the Role of Aeromonas spp. in bacterial septicaemias of cultured frogs.  Aquaculture News 21(24).

 

Tinsley, R.C.  1996. Parasites in Xenopus. Pp. 233-261 in R.C. Tinsely and H.R. Kobel, (eds.), The Biology of Xenopus.  Oxford University Press, New York.


Websites

IACUC Learning Module, includes section on health and disease http://www.ahsc.arizona.edu/uac/iacuc/xenopus/xenopus.shtml

 

Xenopus laevis Frog Colony Care, includes section on Health & Disease http://www.xlaevis.com

 

Xenopus Express husbandry and disease sites

http://www.xenopus.com/husbandry.htm

http://www.xenopus.com/disease.htm

 

Bibliography of amphibian diseases http://www.jcu.edu.au/school/phtm/PHTM/frogs/bibliog.htm

 

African Clawed Frog links

http://members.aol.com/sirchin/froglink.htm

http://www.xenopus.com/links.htm