The Growth of Spironucleus Vortens, an Intestinal Parasite of the Freshwater Angelfish

DISEASES OF AQUATIC ORGANISMS Vol. 38: 47-52,1999 1 Published October I I l Dis Aquat Org

Efficacy of various chemotherapeutic agents on the growth of Spironucleus vortens, an intestinal parasite of the freshwater angelfish

Somboon Sangmaneedet, Stephen A. Smith*

Department of Biomedical Sciences and Pathobiology. Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University. Blacksburg, Virginia 24061, USA

ABSTRACT: Seven chemotherapeutic agents (dimetridazole, metronidazole, pyrimethamine, albendazole, fenbendazole, mebendazole and magnesium sulfate) were examined for growth inhibition on the cultivation of Spironucleus vortens. Dimetndazole and metronidazole were effective in inhibiting the parasite's growth. At concentrations of 1 pg ml-' or higher, both dramatically decreased numbers of parasites. At 24 h exposure, 33 % of parasites were inhibited when exposed to dimetridazole or metronidazole at concentrations of 2 and 4 pg ml-l, respectively. Dimetridazole at 4 pg ml-l or higher concentrations decreased the number of organisms to 50":, or less after 48 h exposure. During the same period of time, the numbers of parasites decreased to 50% or less when exposed to metronidazole at 6 pg ml-' or higher. Pyrimethamine at concentrations of 1 to 10 pg ml-' was not effective in inhibiting the parasite's growth. Albendazole and fenbendazole at concentrations of 0.1 and 0.5 pg ml-' were similar in inhibiting the growth of the organism. Both compounds suppressed parasite growth at concentrations of 1.0 1-19 ml-' or higher after 24 h exposure. Mebendazole inhibited the parasite's growth at concentrations of 0.5 pg rn' or higher. At 72 h exposure. 45 to 50'',, of the parasites were inhibited when exposed to mebendazole at concentrations higher than 0.5 pg ml-' Magnesium sulfate at concentrations of 70 mg ml-' or higher also suppressed the growth of parasites after 24 h exposure. These results indicate that dimetridazole, metronidazole and mebendazole are the most effective chemotherapeutic agents in vitro at inhibiting the growth of S vortens.

KEY MrORDS. Diplomonad Spironucleus . Treatment. Chemotherapy . Angelfish

INTRODUCTION mental tropical aquarium fish (Sommerville 1981, Gratzek 1988, Woo & Poynton 1995). Thus, treatment Hexamita and Spironucleus are diplomonad flagel- and control of these organisms are important in both lates frequently found in the digestive tract of both the foodfish and tropical fish industry. Most studies of freshwater and saltwater fish (Kulda & Lom 1964, Fer- chenlotherapeutic agents for this group of parasites guson 1979, MO et al. 1990).These motile flagellates have been evaluated using Hexamita sp., a parasite of can cause cellular damage in the intestinal tract of salmonids (Yasutake et al. 1961, McElwain 1968, Tojo infected fishes and heavy infections may interfere with & Santamarina 1998). Although there is a report of normal growth of the host (Yasutake et al. 1961). They chemotherapeutic activity on Spironucleus rnuris, a often cause disease when the host has a low resistance parasite of the mouse intestine (Oxberry et al. 1994), or is adversely affected by some predisposing factors there are no published reports of chemotherapeutic such as inadequate nutrition, low oxygen content, poor efficacy on S. vortens of fish. sanitation or overcrowding (Lom & Dykova 1992). Metronidazole, l-(2-hydroxy ethyl)-2-methyl-5-nitro- They have been reported as pathogens causing enteri- imidazole, is the active compound of nitroimidazole tis and mortalities in salmonids, cyprinids and orna- used in the treatment of infections caused by anaerobic bacteria and protozoa (Amon et al. 1978, Baines 1978, 'Addressee for correspondence. E-mail: [email protected] McLean et al. 1984). The drug is rapidly absorbed from

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the gastrointestinal tract and is effective against obli- Unlike the previously discussed chemotherapeutic gate anaerobic organisms having an active hydro- agents, magnesium sulfate (MgSO4,Epsom salt) is fre- genase (Church et al. 1996). It has been applied quently used as purgative. It has been used for the frequently in the treatment of flagellate infections treatment of parasitic diseases, especially lumina1 par- causing urogenital trichomoniasis and giardiasis in asite infestations. When the magnesium salts reach the humans (Catchpool 1980). In addition, metronidazole intestine, magnesium ions cause the release of chole- (FlagylB)and metronidazole-related compounds (Ipro- cystokinin, which increases peristaltic activity of the pran" and Emtrylm)have been used in the treatment of intestine (Jenkins 1988). Increased movement then hexamitiasis in trout, ornamental fish and several helps the host to expel or decrease the number of par- species of cichlids (Gratzek 1983). Therefore, metron- asites in the intestine. Free flagellates of diplomonads idazole and dimetridazole, a metronidazole-related are the most commonly encountered stage found in the compound, may be effective in the treatment of Spiro- fish's intestine (Woo & Poynton 1995) and are capable nucleus vortens, an aerotolerance anaerobe in angel- of disseminating to other tissues during stressful con- fish. ditions of the host (Molnar 1974). Therefore, it was The benzimidazole anthelmintics is another group of hypothesized that magnesium sulfate may also be chemotherapeutics that has been shown to be effective helpful in reducing the number of parasites in the against intestinal protozoa such as Giardia lamblia intestine of the fish. (Edlind et al. 1990, Bell et al. 1993), G. duodenalis, Tn- In the present study, 7 different chemotherapeutic chomonas vaginalis and Spironucleus muris (Oxberry compounds were examined at various concentrations et al. 1994). Being less absorbable, albendazole a.nd for any effect on the ability of the parasite Spironucleus mebendazole were 30 to 50 times more active than vortens to survive and reproduce in an in vitro culture metronidazole (Edlind et al. 1990). Albendazole has system. also been reported to be successful in the treatment of Hexamifa salrnonis infection in rainbow trout (Tojo & Santamarina 1998). The target of benzimldazoles in MATERIALS AND METHODS Giardia sp. is a structure called P-tubulin (Edlind et al. 1990). The internal structures of S. vortens such as Parasite cultivation. Spironucleus vortens were cul- recurrent flagella, peripheral ridges and body (be- tivated in TYI-S-33 medium (ATCC, Cat. No. 350-X)at neath the cell surface) are also supported by micro- pH 6.8. The antibiotics, penicillin (2000 U ml-') and tubules (Poynton et al. 1995). Therefore, benzimida- gentamicm (50 pg ml-l), were added to the medium zoles may be effective in the treatment of S. vortens. during routine culture, but were omitted during exper- Pyrimethamine (2,4-diamino-5-p-chlorophenyl-6-imental treatments. The organisms were cultivated in ethylpyrimidine) is an antiprotozoal drug commonly a sterile flask and placed in an incubator at 25OC with- used for antimalarial activity and the treatment of tox- out light. oplasmosis (Pratt & Fekety 1986, Rosenblatt 1992, Parasite cell counting. The concentration (average Bradyley & Marciano-Cabral 1995). Pyrimethamine in cell number ml-l) of Spironucleus vortens in the cul- combination with sulfadiazine has also been used in ture medium was determined daily by automatic cell the successful treatment of chronic intestinal coccidio- counter (cAsY"~; model TTC, Scharfe System. GmbH, sis in man (Trier et al. 1974). The drug is chemically Germany). The parasite suspension was gently mixed, related to trimethoprim and is slowly absorbed from aliquots of either 5 or 10 p1 were added into counter the gastrointestinal tract (Catchpool 1980). Parasitici- containers with 10 m1 PBS, and the resulting cell mix- dal efficacy of pyrimethamine is due to inhibition of the ture analyzed. synthesis of dihydrofolate reductase which catalyzes Growth inhibition assay. All chemicals, except mag- the conversion of dihydrofolate into tetrahydrofolate in nesium sulfate, were prepared at different concentra- organisms (Pratt & Fekety 1986, Bradyley 81 Marciano- tions by dissolving in dimethyl sulfoxide (DMSO, Cabral 1995). The folate coenzyme, tetrahydrofolate, is Sigma Chemical Co., St. Louis, MO, USA). Concentra- necessary in the reactions involved in purine, pyrimi- tions of Epsom salt (MgSO,) were prepared by dissolv- dine and amino acid synthesis (Pratt & Fekety 1986, ing in fresh culture medium. Chemotherapeutic agents Bradyley & Marciano-Cabral 1995). Thus, organisms were divided into 3 separate testing groups. Chemicals exposed to pyr~methamine are killed due to a defi- in the first group were dimetridazole, metronidazole ciency of the folate coenzyme that results in the in- and pyrimethamine; the second group were albenda- hibition of cell division (Catchpool 1980). With the zole, fenbendazole and mebendazole; and the last energetic self-division of Spironucleus vortens, pyri- group was magnesium sulfate. Control groups were methamine may be effective in the treatment of spiro- established for each chemotherapeutic testing group, nucleosis. and an additional group was established to determine Sangrnaneedet & Smith: Growth of Spironucleus vortens 4 9

the effect of DMSO (solvent) on the parasite's growth. All cultures were established in triplicate in sterile 4 m1 screw-capped glass tubes which were placed in a thermo-regulated incubator at 25OC. The average number of parasites (cells ml-l) was determined every 24 h until no parasites were observed alive. Dimetridazole, metronidazole and pyrimethamine were tested at concentrations of 1, 2, 4, 6, 8 and 10 pg ml-l. Log-phase Spironucleus vortens concentration of 5.44 X 106 cells ml-l was established on the first day of cultivation with 2 ml of culture medium per vial and incubated at 2S°C without light. Albendazole, fenbendazole and mebendazole were examined at concentra- 0 2 4 4 8 7 2 9 6 tions of 0.1, 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 pg ml-l. Log- hour phase S. vortens concentration of 4.23X 106 cells ml-' was established on the first day of cultivation with 2 m1 Fig. 2. Spironucleus vortens. Growth in different concentra- of culture medium per vial and incubated as described. tions (pg ml-') of pyrimethamine Magnesium sulfate was prepared at concentrations of 5, 10, 20,30,40,50, 60,70, 80,90 and l00 mg ml-l. Log- Compared to the growth of organisms in cultures with phase parasites were established in 2 m1 of culture DMSO, pyrimethamine at concentrations of 1 to 10 pg medium per vial at 3.78 X 10"ells ml-' on the first day ml-' also did not inhibit the growth of the parasites of cultivation. The cultures were incubated as de- (Fig. 2). Under microscopic observation, the conditions scribed. DMSO (solvent) was examined for its effect on (morphology and movement) of organisms in cultures the parasite's growth. Cultures with different concen- with DMSO or pyrimethamine were not different from trations of DMSO (0.025, 0.05, 0.10, 0.15, 0.20 and those of the control (medium only) cultures. 0.25 %) were established and examined along with the Dimetridazole weakly inhibited the organism's experiment of dimetridazole, metronidazole and pyri- growth at a low concentration (1 pg ml-l) (Fig. 3). methamine. However after 24 h incubation with 2 pg ml-' dimetridazole, 33.46% of the parasites displayed inhibited growth. The percentages of parasites which were RESULTS inhibited from growing were not significantly different from those exposed to dimetridazole at 4 1-19 ml-' Growth of Spironucleus vortens at different concen- or higher concentrations. More than 50% of the trations of DMSO are shown in Fig. 1. DMSO dissolved organisms were killed after 48 h exposure to dimetri- in the artificial medium at concentrations of 0.025 to dazole at a concentration of 4 pg ml-' or higher. 0.25% minimally affected the growth of S. vortens. Under microscopic observation, a large number of

0 24 48 7 2 9 6 0 2 4 48 72 9 6 hour hour

+no DMSO *0.025 *O.OS Jt0.l+O 15 U02+O2S +conlrol -*I --62 Jt4 +6 -0-8 +l0

Fig. 1. Spironucleus vortens. Growth in different concentra- Fig. 3. Spironucleus vortens. Growth in different concentrations (%) of dimethyl sulfoxide (DMSO) tions (pg ml-l) of dimetridazole 50 Dis Aquat Org 38: 47-52, 1999

0 0 0 2 4 48 72 9 8 0 24 48 72 96 hour hour +control +0.1 eO.5 +I +!.S *2 +2.5 -3

Fig. 4. Spironucleus vortens. Growth in different concentra- Fig. 6. Spironucleus vortens. Growth in different concentrations (pg m]'') of rnetronidazole tions (pg ml-l) of fenbendazole

trophozoites of Spironucleus vortens exposed to Growth inhibitions of Spironucleus vortens by alben- dimetridazole were not in their normal pyriform dazole and fenbendazole are shown in Figs. 5 & 6, re- shape, but were multinucleated trophozoites that spectively. Albendazole and fenbendazole failed to in- were larger in size with numerous flagella. hibit the parasite's growth at concentrations less than Growth inhibition of Spironucleus vortens by rnetro- 0.5 1-19 ml-l. At higher concentrations (1 to 3 pg ml-'), nidazole was similar to that observed with dirnetrida- both albendazole and fenbendazole minimally inhib- zole (Fig. 4). At 24 h exposure, 33.09% of parasites ited the growth of the organism. Compared to albenda- were dead after incubation with 4 pg ml-l. Metronida- zole and fenbendazole, mebendazole (Fig. 7) was more zole at concentrations higher than 4 1-19 ml-l decreased effective in inhibiting the parasite's growth. Mebenda- the number of parasites by more than 50% at 48 h zole inhibited the parasite's growth at concentrations of exposure. Compared to dimetridazole, metronidazole 0.5 pg ml-' or higher. The numbers of parasites gradu- was less effective in decreasing the number of para- ally decreased throughout the cultivation period when sites at the same concentration and exposure period. exposed to an increasing concentration of mebenda- Metronidazole (2 pg ml-l) killed only 17.46% of the zole, with the greatest period of growth inhibition seen parasites, whereas 33.46% of the organisms were after 24 h exposure. After 72 h of exposure, approxi- killed with dimetridazole at the same concentration mately 45 to 50% of the parasites were killed when in- and same exposure period (24 h). cubated with mebendazole (1.0 to 3.0 pg ml-').

0 0 2 4 48 72 9 6 0 2 4 4 8 72 9 6 hour hour +control -U- 0.1 0.5 +l +control +O. l eO.5 JtI + +I 5 -0-2 +2.5 -3 +1.5 -0-2 + 2.5 - 3 Fig. 5. Spironucleus vortens. Growth in different concentra- Fig. 7. Spironucleus vortens. Growth in different concentrations (pg ml-'1 of albendazole tions (pg ml-l) of mebendazole Sangrnaneedet & Smith: Growth of Spironucleus vortens 5 1

intestinal flagellates in fish. The present study showed that trophozoites of S. vortens were inhibited and misshapen in the cultures exposed to dimetridazole or metronidazole. This may have been caused by the toxicity of the intermediates accumulated in the cells and the reducing of cytoplasmic materials. It has been pro- posed that the toxicity of the intermediates is due to their interaction with DNA, decreasing the stability and integrity of DNA, and possibly with other macro- molecules (Edwards 1980, Miiller 1983). These radical anions generated within intact hydrogenosomes also may damage the radical-generating system and the 0 0 24 48 72 96 120 hydrogenosome membrane, and are capable of efflux hour from isolated organelles (Chapman et al. 1985). The -4-0 +S +l0 Jt20 +30 -0-40 +50 U60 -70 -0-60 -*g0 6-100 results of misshapen or damaged cell organelles may also lead to a failure of cell division. Thus, the large rig. 8. Spironucleus vortens. Growth in different concentra- number of irregular, multinucleated trophozoites tions (pg ml-')of MgSO, found in cultures exposed to dimetndazole and metronidazole may have been a result of cell division failure. Growth of Spironucleus vortens in different concen- Trophozoites of Spironucleus vortens cultivated in trations of magnesium sulfate are shown in Fig. 8. medium with the benzimidazoles were also mis- Epsom salt at all concentrations (5 to 100 mg ml-') shapen. However, the number of malformed tropho- affected the growth of S. vortens. The average num- zoites and multinucleated trophozoites were lower bers of parasites at concentrations of 5 to 60 mg ml-' than those incubated with the nitroimidazoles. At the gradually increased with exposure bme, but gradually same concentration, mebendazole was more effective decreased at 70 mg ml-' or higher of MgSO, after 24 h than albendazole and fenbendazole in inhibiting the exposure. growth of S. vortens. The effect of benzimidazoles on parasites is different from nitroimidazole in that the target of benzimidazole is a structure called P-tubulin DISCUSSION (Edlind et al. 1990). Mebendazole has been shown to damage p-tubulin by distorting cell structures and Spironucleus vortens, an aerotolerance anaerobe, rapidly disrupting the ventral disk and flagella of Giar- are highly energetic flagellate protozoa. They are nor- dia lamblia (Edlind et al. 1990), while albendazole mally pyriform in shape and they actively move in a binding to the cytoskeletal proteins causes tropho- forward direction in the culture medium. In the present zoites of G. duodenalis to become swollen, misshapen study S. vortens were cultivated and examined in an with cytoplasmic dark granule materials and finally die artificial medium with different types and concentra- (Oxberry et al. 1994). Albendazole also increased gly- tions of chemotherapeutic agents. Nitroimidazole, par- cogen granules and cytoplasmic vesicles and caused ticularly metronidazole, is the most widely prescribed thickening of the parasite's cell coat with many invagi- drug in the treatment of anaerobic protozoa (Amon et nations (Oxberry et al. 1994). However, albendazole al. 1978, Edwards 1980, Church et al. 1996). Com- had no effect on the cytoskeleton or any other gross pounds in this group enter cells and are accumulated morphological characteristics of S. muris (Oxberry et inside the cells by passive diffusion without an active al. 1994). transport process (Miiller 1983, Chapman et al. 1985). Pyrimethamine is most widely used clinically as an Their intermediate products, the reduced nitro-group antimalarial chemotherapeutic agent (Pratt & Fekety forms, function as a short-lived cytotoxic effect, and 1986). The drug has a parasiticidal effect by inhibiting the accumulation of these intermediates enhances the the process of cell division. In the present study, rate of entry of the chemical into the susceptible cells pyrimethamine was examined for its efficacy on the (Edwards 1980). Metronidazole also reduced the growth of an energetic flagellated diplomonad, Spiro- homogeneous material within the cytoplasm and nucleus vortens. The results indicated that pyrimeth- caused misshapen trophozoites of S, muns, but did not amine did not affect the cell division of trophozoites. It affect'l the cytoskeleton (Oxberry et al. 1994). Metron- is suggested that the parasites may not need the coen- idazole at 5 ppm in water (Bassleer 1983, Gratzek zyme, tetrahydrofolate, in the cell division process or 1983, Jenkins 1988) or at 10 mg in 1.0 g of food the parasites may have alternative pathways that help (Stoskopf 1988) is recommended for the treatment of in purine, pyrimidine and amino acid synthesis. 52 Dis Aquat Org 38.47-52, 1999

Magnesium sulfate (Epsom salt) minimally affected the microscopical observations on Hexamita salmonis (Moore, parasite's growth at low concentrations but inhibited 1992) related to mortalities in ra~nbowtrout fry Salmo growth at concentrations higher than 60 mg ml-l. It is gairdneri Richardson. J Fish Dis 2:57-67 Gratzek JB (1983) Control and therapy of fish diseases. Adv suggested that magnesium sulfate at high concentra- Vet Sci Comp Med 27:297-324 tion may interfere with homeostasis of ions between Gratzek JB (1988)Tropical fish medicine. Parasites associated the inside and the outside of the cell. Further informa- with ornamental fish. Vet Clin North Am Small Anim Pract tion about the mechanism of action of pyrimethamine 18:375-399 Jenkins WL (1988)Drugs affecting gastrointestinal functions. and magnesium sulfate on diplomonads is needed. In: Broth NH, Mcdonald LE (eds) Veterinary pharmacol- Of the 7 chemotherapeutic agents investigated in ogy and therapeutics. Iowa State University Press, Ames, this study, dimetridazole, metronidazole and meben- p 657-671 dazole were the most effective compounds in vitro at Kulda J, Lom J (1964) Remarks on the diplomastigine flagellates from the intestine of fishes. Parasitology 54:753-762 inhibiting the growth of Spironucleus vortens. How- Lom J, Dykova I (1992) Developments in aquaculture and ever, the success of individual treatments was depen- fisheries science, Vol 26. Protozoan parasites of fishes. dent on both drug concentration and time of exposure. Elsevier Sclence Publishers RV,New York, p 65-68 McElwain IB (1968) Efficacy of cyzine for trout hexamitiasis Prog Fish-Cult 30:84-91 Acknowledgements. The authors wish to express thanks to Dr McLean AJ, Ioannides-Demos LL, Spicer WJ (1984) Current David S. Lindsay for providing advice and some of the chem- clinical applications and dose regimens of metronidazole icals used in this research, and to Dr Sarah L. Poynton for and related nitroimidazoles. 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Editorial responsibility: Wolfgang Kortlng, Submitted: Apnl23, 1999; Accepted. June 4, 1999 Hannover, Germany Proofs received from author(s): September 13, 1999