Trichodina xenopodus, a Ciliated Protozoan, in a Laboratory-Maintained Xenopus laevis

Abstract

A postmortem evaluation of a domestically bred, adult, female Xenopus laevis revealed the presence of a urinary bladder protozoan consistent with Trichodina xenopodus. T. xenopodus is considered an incidental finding, as its presence in the urinary bladder in frogs has not been correlated with disease or with urinary bladder epithelial lesions. Trichodina spp. are ciliated protozoa known to colonize many species of amphibians and fish. These protozoa frequently inhabit the skin and gills, but may also be present in the urinary bladder of infected animals. Their presence on the skin and gills in low numbers is not related to disease; however, large numbers may indicate poor water quality and overcrowding.

Trichodina spp. are ciliated protozoa (diameter, 50 to 100 µm) considered to be nonpathogenic ectocommensals of amphibians, fish, and invertebrates.^7,12 These protozoa are part of Trichodinae, which is a diverse family comprising more than 200 species in 10 genera.¹² They have a direct life cycle, and they reproduce asexually by binary fission and sexually by conjugation.⁷ Trichodina spp. can colonize the skin, gills, gastrointestinal tract, oviduct, and urinary bladder of their host.^8-11 Although they have a sucking disc which they use to attach to their host, they do not feed on the host directly but on suspended organic material and bacteria. These protozoa are transmitted through several different mechanisms, the primary method being direct contact with another infected animal or contaminated water.^2,4 In aquatic housing systems, they can be introduced by fomites such as live plants and by aerosol droplet transmission between aquaria.¹⁰ Trichodina xenopodus, the urinary bladder ciliate of Xenopus laevis, can survive for a short time in water outside the host and is thought to be transmitted during amplexus.⁵

The characteristic feature of Trichodina spp. is its skeletal ring with radially arranged denticles that are readily apparent when viewed dorsoventrally (Figure 1).^4,7 These organisms have a saucer-to-bell shaped body that is about 84.5 ± 8.2 µm in diameter, numerous (48 to 64) denticulate rings, a highly developed basal adhesive disc, and an adoral zone of cilia arranged in a spiral and are motile.^4,6,7 The number, arrangement, and shape of the teeth on the denticle have been used to identify Trichodina spp. at the species level by using silver-staining methods.⁵

Figure 1.

Photomicrograph of T. xenopodus, demonstrating the denticulate rings (arrow). Hematoxylin and eosin stain; magnification, 40×.

Case Report

An adult female X. laevis presented with skin wounds and a decreased appetite during daily health rounds. The frog was group-housed by sex in accordance with standards promulgated in the Guide for the Care and Use of Laboratory Animals³ and in an AAALAC-accredited facility. The frog was fed daily with commercial frog pellets (Adult Xenopus Diet, Zeigler Brothers, Gardners, PA) and maintained in a 50-L tank on a recirculating custom aquatic housing system (Aquaneering, San Diego, CA) at a water temperature of 64 °F (17.8 °C), 7.4 pH, conductivity of 1500 microsiemens, and general hardness of 100 to 200 ppm. Experimental use of this frog was approved by Weill Cornell Medical College's IACUC. The frog had undergone dorsal lymph sac injections of 500 U human chorionic gonadotropin once every 4 mo to stimulate egg laying and oocyte harvest via gentle massage. The frog was estimated to be 7 y old and had been purchased from a closed colony (Nasco, Fort Atkinson, WI) 5 y previously.

On examination, the frog was thin and had an area (diameter, 0.2 cm) of ulceration on the proximal aspect of the right forelimb; the underlying tissue was exposed, and the lesion was surrounded by a thin rim of white tissue. There was a 0.1-cm-diameter focus of hemorrhage within the subcutis of the left axillary region and an area (1.5 × 1.0 cm) of healing ulceration, with a red rim on its caudal edge, on the proximal aspect of the left hindlimb. The frog was isolated from the other frogs in a flow-through tank on the same housing system to monitor appetite, decrease the risk for potential food-related aggression and injury, and decrease the potential risk of disease transmission. However, shortly after isolation, the frog was found dead.

At necropsy, the frog had muscle wasting and no visible coelomic fat stores. A few linear hemorrhages were present in the subjacent skeletal muscle. The gallbladder was markedly enlarged with a thick yellow content that was difficult to express. The ovary had no apparent active folliculogenesis. Aeromonas hydrophila was cultured from blood obtained via cardiocentesis at necropsy.

Microscopic examination of the kidney, heart, lung, liver, urinary bladder, gall bladder, stomach, intestines, pancreas, ovary, oviduct, skin, fore foot, hind foot, and coronal sections of the head, vertebrae, leg, and stifle region revealed subacute, moderate, focally extensive necroulcerative dermatitis of the skin with short gram-negative rod-shaped bacteria adhered to the ulcerated surface, with mild, multifocal, myofiber degeneration of the subjacent skeletal muscle; minimal multifocal renal tubular mineralization; and diffuse, marked zymogen depletion of the pancreas. In addition, the urinary bladder contained luminal ciliates consistent with T. xenopodus but was otherwise normal (Figure 2). In this case, the protozoan contained approximately 50 denticulate rings and was approximately 80 µm in diameter.

Figure 2.

Urinary bladder. Several ciliated protozoa consistent with T. xenopodus (arrow) are present within the lumen. Hematoxylin and eosin stain; bar, 100 µm.

Discussion

The ulcerative skin lesion was presumably traumatic and became colonized by A. hydrophila, a ubiquitous bacterium in aquatic systems, leading to sepsis, anorexia, and death. The zymogen depletion in the pancreas, lack of coelomic fat stores, and enlarged gallbladder suggest inanition may have contributed to the frog's demise. Tubular renal mineralization is a common finding in frogs subject to acute or chronic stress.⁸ Luminal ciliates confined to the urinary bladder were consistent with T. xenopodus and are an incidental finding. To our knowledge, this case represents the first report of T. xenopodus infection described in laboratory Xenopus laevis.

Trichodinads are found normally in low numbers on infested animals and do not cause pathology.^4,7,8 However, these organisms can become pathogenic if they are present in large populations, which on the skin or gills usually indicates poor nutrition, overcrowding, poor water quality, or another disease process.^4,7,10 Trichodinads increase in association with an increase in organic material, on which they feed.¹⁰ Mechanical tissue damage can result, because their adhesive discs can cause epithelial cell sloughing, which can subsequently be ingested by the ciliate.⁴ Associated clinical signs include ulcerative skin lesions, skin discoloration, dermatitis, excessive cutaneous mucus production giving a white to gray appearance to the affected animals, and reddened gills.^4,7,9,10 In addition, frogs with skin infestations may be observed rubbing against solid objects in an attempt to dislodge the parasites.⁷ Larval amphibians may be dyspneic if their gills are infected.⁷

T. xenopodus, a bladder parasite of Xenopus laevis, was initially described in 1924 and again in 1958 after the use of a silver impregnation technique.⁵ The parasite has been found adhered to the bladder lumen of wild-caught X. laevis worldwide and has not been described to occur in other species. T. xenopodus measures 84.5 ± 8.2 µm in diameter and has a characteristically high number of denticles (48 to 64).⁵ Interspecies transmission has not been studied. The parasite is able to survive off the host in water at least 30 min. It is spread during amplexus, in which the frogs remain attached to one another for a few hours to 2 d.⁵ The stimulus resulting in parasite migration from the urinary bladder is unknown; however, some authors suspect it may result from hormonal changes.⁵ The parasite has not been identified in urine samples collected from infected frogs.

Several other trichodinads including T. urinicola, T. pediculus and T. fultoni have been shown to colonize amphibians.^2,5-7,11 T. urinicola has been identified in the urinary bladder of some species of frogs, toads, and newts worldwide.⁷ In addition, frog tadpoles have been reported to have skin infestations by T. pediculus, and T. fultoni has been isolated from the gills of mudpuppies.⁷ Although all of these trichodinads have similar morphology, T. xenopodus is unique due to the high number of denticles it has.^5,6 T. urinicola has an average of 28 to 36 denticles.⁶ Both T. xenopodus and T. urinicola have short survival times in water outside of the host.^5,6 T. fultoni and other trichodinads are exposed to the host's environment due to their location on the gills and skin.⁶

There are several ways to diagnose Trichodina infection. Skin infections can be diagnosed by lesion skin scrapes.^2,4,7,9 Gill infections can be diagnosed via wet mount of gill tissue or a gill biopsy.^4,7,8 The organisms are easily identifiable due to their structure and characteristic movement, ^4,7 in which they appear as spinning discs in the water.² Their motion has also been described as rotating, scooting, and erratic.¹⁰ The presence of a circular ciliate with a prominent denticulate ring is diagnostic.¹⁰ When attached to host tissue, trichodinads can have a domed appearance. They can be diagnosed by using histopathology,^4,10 as described in the current case.

Few treatments for this infestation in amphibians have been reported. The most common treatment for skin and gill infestation is immersion of the animal in a saltwater bath.^2,4,7 For frogs, immersion in a sodium chloride bath (10 to 25 g/L for 5 to 30 min or 4 to 6 g/L for 24 h) has been reported to kill the parasite successfully.^7,9 In addition, immersion in distilled water for 2 to 3 h may be effective.⁷ Other reported treatments include immersion in malachite green, potassium permanganate, copper sulfate, formalin, sodium chlorite, and Steinberg solution or modified Holtfreter solution.⁹ However, some of the treatments, such as formalin, are contraindicated in animals with skin ulceration.⁹ Increasing the number of water changes and correcting water-quality issues will help to eliminate the species that colonize external structures.⁸ There are no known treatments for the parasite that colonizes the urinary bladder.

Prevention of disease can be achieved by maintaining appropriate husbandry and water quality.^4,8 Quarantine or immersion of frogs in a saltwater solution during quarantine may prevent the entry of these parasites into an established colony. The parasite likely is not transmissible between tanks with a correctly designed and functioning water filtration and UV sanitization system.

Conclusion

Trichodina spp. typically are a commensal of amphibians, fish, and invertebrates in the wild, rarely causing clinical disease. The presence of high numbers of these protozoa on the skin and gills may reflect poor water quality, overcrowding, or immune suppression; therefore husbandry methods and housing conditions should be evaluated. Presence in the urinary bladder is generally incidental and has not been associated with disease or water-quality issues.