Experimental Infections of a Ciliate Tetrahymena Pyriformis on Ornamental Fishes

FISHERIES SCIENCE 2000; 66: 1026–1031

Original Article

Experimental infections of a ciliate Tetrahymena pyriformis on ornamental ﬁshes

Aranya Ponpornpisit,1 Makoto Endo2,* AND Hisashi Murata3

1United Graduate School of Agricultural Sciences, Kagoshima University, Kagoshima, Kagoshima 890-0065, 2Laboratory of Fish Pathology and 3Laboratory of Aquaculture, Faculty of Agriculture, Miyazaki University, Miyazaki, Miyazaki 889-2192, Japan

SUMMARY: Experimental infection of Tetrahymena pyriformis was conducted on ornamental fishes with skin wounded by acetic acid treatment. Among fishes used in this experimental infection, guppy Poecilia reticulata, pristella Pristella maxillaris, neontetra Paracheirodon innesi and cherry barbs Puntius titteya proved to be sensitive to challenge, while medaka Oryzias latipes, dwarf gourami Colisa lalia, goldfish Carassius auratus, platy Xiphophorus maculatus and angelfish Pterophyllum scalare were resistant. In catfish Corydoras aeneus infection was unsuccessful. Exposure to Tetrahy- mena at ≥100 cells/mL at 25 to 30¡C and at pH of 6.0 to 8.0 resulted in the successful infection of some sensitive fish species. Histological and bacteriological observations suggest that deeply destroyed skin tissues from the acid-treated method are the primary factors in successful infection.

KEY WORDS: ciliate, experimental infection, ornamental ﬁsh, Tetrahymena pyriformis.

INTRODUCTION methodology and histopathology. No reports are yet available on the successful control of the Tetrahymena Ornamental fish farming is a worldwide growing branch infection in ornamental fishes. of aquaculture. Most of the ornamental fish traded in the This study describes the methodology and some world market come from South-East Asian countries.1 important conditions for the experimental infection of Because of the intensive farming system or ineffective Tetrahymena pyriformis in ornamental fishes, and its quarantine processes, many infectious diseases have been histopathology. causing serious damage to fish production and trade in recent years. Tetrahymena infection is one such disease.2–5 Tetrahymena is a free-living ciliate which consumes MATERIALS AND METHODS organic matter and bacteria in natural habitats. It becomes a facultative parasite in many groups of aquatic Fish animals including fish under some conditions where, for instance, they are maintained in a closed water system Ten species from seven families of ornamental fishes were or when they are wounded.3 For several decades, research used in this study: dwarf gourami Colisa lalia (3.5–4.0 cm) on Tetrahymena has focused mainly on its taxonomy, (Belontiidae), goldfish Carassius auratus (7.0–7.5 cm) physiology, genetics and ecology.6–9 Only a few studies (Cyprinidae), cherry barbs Puntius titteya (3.0–3.2 cm) have viewed it as a pathogenic agent and have paid little (Cyprinidae), pristella Pristella maxillaris (3.0–3.1 cm) attention to the pathological aspects of Tetrahymena (Characidae), neontetra Paracheirodon innesi (2.4– parasitism.5,10–13 Concerning the experimental infection 2.5 cm) (Characidae), angelfish Pterophyllum scalare of Tetrahymena, some researchers have tried to infect (2.9–3.1 cm) (Cichlidae), guppy Poecilia reticulata various kinds of animals including fish.10,14,15 However, (2.5–3.0 cm) (Poeciliidae), platy Xiphophorus maculatus these reports include no detailed descriptions about (3.2–3.5 cm) (Poeciliidae), medaka Oryzias latipes (3.3–3.5 cm) (Adrianichthyidae) and catfish Corydoras *Corresponding author: Tel: 0985-587223. Fax: 0985-582884. aeneus (3.0–3.1 cm) (Callichthyidae). All fishes were Email: [email protected] purchased from a commercial farm located in Miyazaki, Received 11 November 1999. Accepted 1 June 2000. southern Japan. Subsequently, 25 p.p.m of formalin Tetrahymena infection in ornamental fishes FISHERIES SCIENCE 1027

bathing for 24 h was employed to eradicate pathogenic subjected to routine light microscopy under a parafﬁn agents on these ﬁshes. They were then kept sepa- embedding technique, thinly sectioned and stained with rately according to their species for 1 week prior to hematoxylin-eosin. experimentation.

Experimental infection under various conditions Tetrahymena culture Effects of pH, temperature and Tetrahymena cell density The Tetrahymena used in this study was isolated from on the infection rate were examined by using guppies as lesions of naturally infected guppies, cultured and main- experimental ﬁsh. In test waters with a temperature of tained in a Tetrahymena culture medium (ATCC culture 25°C and 100 cells/mL of Tetrahymena cell density, pH medium 357; 10801 University Boulevard, Manassas, levels were adjusted to 5.0, 6.0, 7.0 and 8.0, buffered by

VA) at 25°C. This Tetrahymena was classified as T. pyri- 1 mM K2HPO4 and 1 M HCl. In test waters with a pH of formis as discussed later. It was subcultured every 3 weeks 7.0, and 100 cells/mL of Tetrahymena cell densities, tem- by adding 1 mL of the old culture medium to 10 mL of peratures were kept at 20, 25, 28 and 30°C. In waters fresh medium. One day before the experiments started, with a 25°C temperature and a pH of 7.0, Tetrahymena 10 mL of the Tetrahymena culture medium was trans- cell densities were adjusted to 5, 100, 200 and ferred to 500 mL of fresh medium. Immediately before 400 cells/mL. Experimental infection was tried in each the start of experiments, the densities of stationary phase test using eight to 10 acid-treated guppies, according to cells were adjusted as required with filtered water. the methodology noted above. Number of Tetrahymena cells, infection rates and mortality rates were determined at 2, 4, 6 and 24 h after Tetrahymena challenge. Infection Methodology of experimental infection and its trial was confirmed as previously described. This experiment on various fishes was repeated twice.

The duplicated experiments were composed of a negative control test and two challenge tests. Eight to 12 Bacteriological investigation in experimental infection individuals from each species of experimental fishes were allocated to a test. In the negative control test and one Bacteriological investigation was performed to clarify the of the challenge tests, all fishes were anesthetized with involvement of bacteria in the experimental infection. FA100 (Tanabe Pharmaceutical Co. Ltd., Osaka, Japan) Eight wounded guppies were subjected to the experi- and the base of the tail fin was covered with a 10% acetic mental infection as mentioned above. During the test acid soaked cotton strip (5 mm in width) for 3 min. This process, 25 p.p.m. of oxytetracycline was added to the process was referred to as acid-treated method. In the test water to eradicate bacterial contamination. One day other challenge test, fish were similarly anesthetized, but later, the skin mucus was scraped off from the damaged the acid treatment was not conducted. The negative skin and streaked onto tryptic soy agar and on tryptone- control fish were kept in clean water free from Tetrahy- yeast extract-agar plates. The Tetrahymena infection rates mena cells for 1 day. Fishes allocated to the challenge and bacterial examination results were recorded. tests (one challenge test without acetic acid treatment) were exposed to 100 cells/mL of Tetrahymena cells for 1 day. All tests were performed with a water temperature of 25°C and a pH of 7.0. Five hundred mL beakers RESULTS with 300 mL of aerated test water were used for guppy, pristella, neontetra, cherry barbs, medaka and catfish. In Experimental infection the case of dwarf gourami, goldfish, angelfish and platy, 1000 mL beakers with 600 mL of the test water were The isolated Tetrahymena cells from naturally infected used. During the exposure, Tetrahymena cell density, fish guppies which varied from 50 to 80 mm in long-axis mortality and infection rates were recorded at 2, 4, 6 and length were pyriform in shape and had 20 to 30 ciliary 24 h. After the exposure, the fish mortality and infection meridians with two postoral meridians without caudal rates were scored every 24 h over the next 2 days. Infec- cilium. They contained a microstome and an apparatus tion was confirmed under a binocular microscope by the consisting of a paroral membrane and three membranells appearing of Tetrahymena cells underneath the skin of (Fig. 1), they multiplied by binary fission and did not the tail fin. Two of the live fishes were sampled and form a cyst. fixed in Bouin’s fluid for 24 h and changed to 70% Tetrahymena cells in water showed independent fast alcohol until histopathological processing was done. movement with a single macronucleus and few translu- All samples for histopathological observations were cent food vacuoles in their cytoplasm. After infection, 1028 FISHERIES SCIENCE A Ponpornpisit et al.

1 3

Fig. 1 A free-living form of Tetrahymena pyriformis with 15 ciliary meridians on a lateral surface. The microstome (MT) contains paroral membrane and three membranells and there are translucent food vacuoles in the cytoplasm (arrow). Silver impregnation stain. Magnification ¥781. Fig. 2 Parasitic Tetrahymena pyriformis cells underneath the skin of the host fish. Food vacuoles are filled with melanin pigments (arrow). Fresh tissue preparation. Magnification ¥400. Fig. 3 Acetic acid treatment causing scale lifting and necrosis of dermal and muscular tissues in guppy Poecilia reticulata skin (sensitive species). The epidermis is exfoliated and disappeared in this figure. An arrow indicates an infecting Tetrahymena cell. H&E stain. Magnification ¥126. Fig. 4 In platy Xiphophorus maculatus skin (resistant species) injured by the acid treatment. Necrosis is limited within the epidermal and inter-scale connective tissues. H&E stain. Magnification ¥147. Fig. 5 Injured skin of catfish Corydoras aeneus (uninfected species) by the acid treatment. It has mild damage only on the skin surface. H&E stain. Magnification ¥257. Tetrahymena infection in ornamental fishes FISHERIES SCIENCE 1029

they invaded under the skin of the host fish with slow In the histopathological observations on negative movement, ingested the host pigment cell, and became control and challenge test fishes, the acid-treated larger in body size. Therefore, when the invasion under- method caused marked skin damage on the sensitive neath the host’s skin and the increased food vacuoles species. Pathological features consisted of necrosis in the filled with the pigment cell debris were confirmed, we epithelial cells, complete loss of mucous cells, and dis- judged that the infection had succeeded, and the Tetrahy- figured dermal and subcutaneous tissue with edema and mena cell to be in a parasitic form (Fig. 2). hemorrhage (Fig. 3). The necrotic appearance extended Experimental infections were successful with higher to the muscular tissues. However, the effects of the acid- rates in sensitive species of guppy, pristella, neontetra treated method on the resistant species were localized and cherry barbs than in resistant species of medaka, only in the epidermal and connective tissues adjacent to dwarf gourami, goldfish, platy, and angelfish. The guppy the scales (Fig. 4), because the tightly laminated thick showed the highest mortality rate. The parasitic Tetrahy- scales prevented damage from spreading to the under- mena cells were observed in the injured sites, caused lying tissues. The Tetrahymena cells were distributed by the acid-treated method, under stereomicroscopic between spaces in the damaged tissue of all the infected and histological observations. In catfish, however, the fishes. However, there was almost no inflammatory experimental infection was unsuccessful. There was response to the Tetrahymena cells. In catfish (uninfected neither success in infection nor mortality in the chal- species), the structure of the club cell layer, dermis and lenge test group without acid treatment. Skin damages muscular tissue stayed intact after acid treatment, in negative control caused mortality rates lower than although there was mild damage to the skin surface 10% in some species (Table1). caused by the acid-treated method (Fig. 5).

Table1 Mortality and infection rates of ornamental fishes in Tetrahymena challenge tests Fish species Control Challenge test Challenge test with acid treatment with acid treatment without acid treatment Mortality Infection Mortality Infection (Dead/total) Average (Infected/total) Average (Dead/total) Average (Infected/total) Average Trial 1 Trial 2 (%) Trial 1 Trial 2 (%) Trial 1 Trial 2 (%) Trial 1 Trial 2 (%) Poeciliidae Guppy Poecilia 0/8 0/8 0 0/8 0/8 0 2/8 2/8 25 8/8 8/8 100 reticulata Platy Xiphophorus 0/10 0/10 0 0/10 0/10 0 1/10 1/10 10 3/10 3/10 30 maculatus Characidae Pristella Pristella 0/8 0/8 0 0/8 0/8 0 0/8 0/8 0 8/8 8/8 100 maxillaris Neontetra 2/12 0/12 8.3 0/12 0/12 0 0/12 0/12 0 8/12 10/12 75 Paracheirodon innesi Cyprinidae Goldfish Carassius 0/8 0/8 0 0/8 0/8 0 0/8 0/8 0 2/8 2/8 25 auratus Cherry barbs 1/12 1/12 8.3 0/12 0/12 0 0/12 0/12 0 6/12 6/12 50 Puntius titteya Cichlidae Angelfish 0/10 0/10 0 0/10 0/10 0 0/10 0/10 0 3/10 5/10 40 Pterophyllum scalare Belontiidae Dwarf gourami 0/8 0/8 0 0/8 0/8 0 0/8 0/8 0 1/8 3/8 25 Colisa lalia Adrianichthyidae Medaka Oryzias 0/10 0/10 0 0/10 0/10 0 1/10 1/10 10 1/10 1/10 10 latipes Callichthyidae Catfish Corydoras 0/8 0/8 0 0/8 0/8 0 0/8 0/8 0 0/8 0/8 0 aeneus 1030 FISHERIES SCIENCE A Ponpornpisit et al.

Experimental infection under various conditions and few numbers of translucent food vacuoles in their cyto- bacteriological survey plasm. In the host tissues after infection, they showed slow movement, their food vacuoles contained the host High Tetrahymena infection rates were established under pigment cell debris, and the cells were enlarged. These water temperatures between 25 and 30°C, pH levels morphological shifts of the Tetrahymena cells in para- ranging from 6.0 to 8.0 and more than 100 cells/mL sitism are consistent with the reports of Edgerton et al.13 of the Tetrahymena cell densities (Table2). In most and Seaman et al.15 experiments, the infection was first confirmed at 4-h Experimental infection of Tetrahymena in fish was dif- post-exposure, when the number of free-living Tetrahy- ficult despite its high pathogenicity to the fish.5 In this mena cells in the test water increased from 100 to more study on the experimental infection of Tetrahymena pyri- than 800 cells/mL (data not shown). With an additional formis in fish, however, the four following conditions 2 days after the Tetrahymena exposure, the infection did seemed to be reasons for high infection rates: (i) a viru- not occur in the uninfected fish. The addition of oxyte- lent strain of T. pyriformis used; (ii) skin injured by acid- tracycline to the test water did not influence the infec- treated method; (iii) exposure to more than 100 cells/mL; tion rate of 100%. In this antibiotic-addition test, any and (iv) suitable water conditions at a 25 to 30°C tem- bacteria could not be isolated from the experimentally perature and a 6.0 to 8.0 pH level. Although in the pre- infected guppies. liminary tests procedures such as toothbrush scratching, blade cutting, or scale removing were used to injure the fish skin they were not useful for successful duplication DISCUSSION because of their inconstant injury levels. Concerning the water temperature and pH, their ranges were similar The isolated ciliates used in this study from naturally to the optimal ones for T. pyriformis cell replication.16 infected guppy were classified as Tetrahymena pyriformis Thus, the number of Tetrahymena cells in the test water according to the descriptions by Elliott16 and Corliss.17 increased, although it could survive in a wider range of They differ from other species in the same genus because temperature and pH levels. of their typical pyriform shape, size and microstome So far, some researchers have succeeded in carrying structure, although they seem to have more ciliary out experimental infection of Tetrahymena with methods meridians than in the descriptions of Elliott16 and of cohabitation with, or inoculation on invertebrate and Corliss.17 The lack of caudal cilium and the cyst forma- vertebrate animals.14,15 In fish, Thompson10 has reported tion distinguish them from the species belonging to experimental infection using a wounded guppy. These the T. rostrata complex. They were different from the reports revealed susceptible hosts, target organs of a host T. patula complex, which are all only free-living, greater and physiological and morphological transformations of than 100 mm in length and form distinct microstome and the Tetrahymena cells. However, these reports have no macrostome.16,17 description about key factors which would affect the In culture medium, free-living Tetrahymena seemed to experimental infection. A histopathological survey in exhibit fast movement with a single macronucleus and this study revealed that the skin of all the sensitive

Table2 Infection rates of guppy Poecilia reticulata challenged under various conditions Water pH Water temp. (°C) Tetrahymena cell density (cells/mL) Infection (Infected/total) Average Trial 1 Trial 2 (%) 5.0 25 100 2/10 2/10 20 6.0 25 100 10/10 10/10 100 7.0 25 100 10/10 6/10 80 8.0 25 100 8/10 8/10 80 7.0 20 100 5/8 5/8 62.5 7.0 25 100 7/8 8/8 93.8 7.0 28 100 8/8 8/8 100 7.0 30 100 8/8 6/8 87.5 7.0 25 5 0/8 0/8 0 7.0 25 100 6/8 8/8 87.5 7.0 25 200 8/8 8/8 100 7.0 25 400 8/8 8/8 100 7.0* 25 100 8/8 8/8 100

* Oxytetracycline added in challenge water. Tetrahymena infection in ornamental ﬁshes FISHERIES SCIENCE 1031

species to T. pyriformis suffered from serious damage by REFERENCES the acid-treated method. Considerable damage extended to the dermal, subcutaneous, and sometimes muscular 1. Biffar M. The worldwide trade in ornamental fish: Current status, tissues. However, in both the resistant and uninfected trends and problems. Bull. Eur. Ass. Fish Pathol. 1997; 17: species the acid-treated method resulted in shallow 201–204. 2. Speide CC. The occurrence of amicronucleate Tetrahymena as damage, limited only within the inter-scale dermal layer facultative parasites in embryos of the catfish Ameiurus. Biol. Bull. or the epidermal surface layer. Deep dermal and muscu- 1954; 115: 366. lar injuries in the sensitive species did not result from the 3. Nigrelli RF, Jakowska S, Padnos M. Tetrahymena as pathogenic Tetrahymena infection because the negative control fish epibiont in fishes and urodeles. J. Protozool. 1956; 3 (Suppl.): 10. had also suffered identical damage to their skin and mus- 4. Stolk A. 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