External protozoan parasites in three trout species in the Eastern Black Sea region of the Turkey: intensity, seasonality, and their treatments

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1 Bull. Eur. Ass. Fish Pathol., 28(4) 2008, 157 External protozoan parasites in three trout species in the Eastern Black Sea region of the Turkey: intensity, seasonality, and their treatments F. Balta 1, S. Kayis 1 and I. Altinok 2 * 1 Rize University, Faculty of Fisheries Sciences, Department of Aquaculture, 53100, Rize, Turkey; 2 Karadeniz Technical University, Faculty of Marine Sciences, Surmene, Trabzon, Turkey. Abstract The seasonal fluctuation of the protozoan parasites (Ichthyophthirius multifiliis, Ichthyobodo necator and Trichodina spp.) on rainbow trout (Oncorhynchus mykiss), brook trout (Salvelinus fontinalis), and brown trout (Salmo trutta fario) in the Eastern Black Sea Region of Turkey was investigated monthly from April 2005 to September A total of 4260 fish was examined; the overall prevalence of the I. multifiliis, I. necator and Trichodina spp. infestation in the fish was 20.69, 37.93, and 41.38%, respectively. The most intensity and variety of parasites were observed in May and their infestation continued until October. None of the parasites was observed from October to April. Ichthyophthirius multifiliis, and Trichodina spp. are observed on three species of trout and I. necator was not observed on brook trout. After treating fish with different concentration of formaldehyde, trichlorfon, dichlorvos, amitraz, cypermethrin, KMnO 4, chloramin-t, salt, and vinegar, salt, formaldehyde, and vinegar were appeared to be the most effective chemicals to treat all parasitic infestation while the other chemicals were not effective or suitable for treatment of all infestation. Concentration of 0.2 ml/l formaldehyde was highly toxic to brown trout. Introduction Ichthyobodo necator, Trichodina spp. and Ichthyophthirius multifiliis are protozoan parasites that cause fish mortality (Durborow, 2003). One of the chief causes of mortality along intensively farmed fish is infections with ectoparasites such as the flagellate I. necator that causes ichthyobodosis and attacks the skin and gills of fish is probably the most frequent external flagellate parasitosis among farmed fish, including rainbow trout (Tojo & Santamarina, 1998; Durborow, 2003). Trichodina is a very common organism, occurring in freshwater, marine and euryhaline environments. About 70 species were identified in marine fishes (Kinne, 1984) and more than 112 from freshwater fish worldwide (Lom & Dykova, 1992). Ichthyophthirius multifiliis is a ciliated protozoan, which causes white spot disease. The ciliate I. multifiliis is an important pathogen of freshwater teleosts occurring in both temperate and tropical regions throughout the world (Matthews, 2005). It is exclusively a parasite of fish and frogs (Lasee, 1995). Since external protozoan parasites are the most common parasites encountered in freshwater fish hatcheries (Pillay, 1995), it is essential to determine seasonality of these parasites and to determine suitable chemical agent to treat their infections. *Corresponding author s ialtinok@ktu.edu.tr

2 Bull. Eur. Ass. Fish Pathol., 28(4) 2008, 158 Chemotherapeutic Dose Time Toxic to fish Effective for parasites Rt Brt Brwt Icht Ich Tri Formaldehyde (37% Merck, Germany ) Trichlorfon (Neguvon, Bayer, Germany) Dichlorvos (DDVP, Bayer) Amitraz (Atabay, Istanbul, Turkey) Cypermethrin (Topkim, Istanbul, Turkey) KMnO4 (Merck) Chloramines-T (Merck) Vinegar (4% acetic acid) 0.1 ml/l 60 No No No Yes Yes Yes 0.15 ml/l 60 No No No Yes Yes Yes 0.2 ml/l 60 No No Yes Yes Yes Yes 0.5 g/l 90 No No No No NA NA 1 g/l 20 Yes Yes Yes No NA NA 2 mg/l 60 No No No No NA NA 4 mg/l 60 No No No No NA NA 0.5 ml/l 60 No No No No NA NA 0.6 ml/l 5 Yes Yes Yes No NA NA 0.01 g/l 60 No No No No NA NA 0.02 g/l 60 No No No No NA NA 0.07 g/l 10 Yes Yes Yes No NA NA 10 mg/l 30 Yes Yes Yes Yes Yes Yes 20 mg/l 30 Yes Yes Yes Yes Yes Yes 5 mg/l 60 No No No No No No 10 mg/l 60 No No No No No No 15 mg/l 60 No No No No No No 10 ml/l 3 No No No Yes Yes Yes 10 g/l 20 No No No No No No Salt 15 g/l 20 No No No No No No 20 g/l 20 No No No Yes Yes Yes NA; Not applied Table 1. Various chemotherapeutic drugs were tested by bath administration for in vivo activity against a natural infestation of rainbow trout (Rt), brook trout (Brt), and brown trout (Brwt) by I. multifiliis (Ich), I. necator (Icht) and Trichodina spp. (Tri). Toxicity of chemotherapeutics to fish and effectivity of chemotherapeutics to parasites were tested. Control of fish parasites is generally achieved by application of chemotherapeutic agents to infested fishes. A wide range of chemicals can be used in aquaculture industry (Tonguthai, 1997). Copper sulphate (CuSO 4 ), potassium permanganate (KMnO 4 ), formaldehyde, chloramines-t, malachite green and salt are commonly used chemicals to treat parasitic infestations (Lasee, 1995; Durborow, 2003). Use of copper sulphate and malachite green as therapeutic is restricted because of accumulation of CuSO 4 in the treated fish tissue and carcinogen affects of malachite green (Daramola & Oladimeji, 1989; Srivastava et al., 2004).

3 Bull. Eur. Ass. Fish Pathol., 28(4) 2008, 159 Material and methods A total of 4260 rainbow trout (Oncorhynchus mykiss), brook trout (Salvelinus fontinalis) and brown trout (Salmo trutta fario) infested with protozoan parasites, ranging from fry (1.0±0.2 g) to juvenile (40±0.7 g) was sampled monthly from April 2005 to September 2006 in 16 fish farms located in the Eastern Black Sea Region of Turkey. Each month 9 or 10 fish farms were sampled for parasites. Fish were examined for external parasites (AFS FHS, 2003) and parasites if present were identified according to method described by Joyon & Lom (1969). Temperature was measured and recorded at the time of sampling. Various chemotherapeutic drugs were tested by bath administration for in vivo activity against a natural infestation of fish by I. multifiliis, I. necator and Trichodina spp. Fish were also monitored for signs of drug toxicity. Nine groups of ten fish each in four static aquaria were treated with one of the different concentration of following chemotherapeutics: formaldehyde, trichlorfon, dichlorvos, amitraz, cypermethrin, KMnO 4, chloramin-t, salt, and vinegar (Table 1). Trichlorfon, dichlorvos, amitraz, and cypermethrin were not used to treat I. multifiliis and Trichodina spp. infestation. Before adding chemotherapeutics, water was aerated with air stones. Mortality and fish behaviours were recorded during the exposure periods. After the treatment, fish were examined for parasites under the microscope at 40X magnification. During the chemical treatment, water characteristic [(mean ± SD) temperature 14 ± 0,5 C, ph 6,8 ± 0,7, dissolved oxygen 12,3 ± 0,7 mg/l and total hardness 28 ± 1,3 mg/l as CaCO 3 ] were measured. Spectroquant Nova 30 (Merck, Darmstadt, Germany) was used to measure total hardness. Water temperature, dissolved oxygen and ph were determined with thermometer, polarographic oxygen meter, and ph meter. Results Three species of protozoan parasites (I. multifiliis, I. necator and Trichodina spp.) were detected from fish between April and September except I. multifiliis and Trichodina spp. that was not observed in April and I. necator that was not observed in August. Although I. multifiliis and Trichodina spp. were observed from gills and skin of rainbow trout, brown trout and brook trout while none of the I. necator was found in brook trout. However, in the laboratory, brook trout was infested with I. necator. Presentation of the monthly prevalence of parasites I. necator infestations was first observed in April and its infestations reached the highest levels in May. Overall prevalence of the I. multifiliis, I. necator and Trichodina spp. infection in the fish was 20.69, 37.93, and 41.38%, respectively. Ichthyophthirius multifiliis and Trichodina spp. infestations were observed in May and their infestations reached the highest levels in August (Figure 1). Although I. necator infestations were observed among the fingerlings, I. multifiliis and Trichodina spp. infestations were observed in both fingerlings and yearlings. Hyperventilation and excessive mucus and then mortality were observed during the treatments of highest concentration of cypermethrin, amitraz, trichlorfon and KMnO 4 used.

4 Bull. Eur. Ass. Fish Pathol., 28(4) 2008, 160 Figure 1. Monthly prevalence of parasites and water temperature. No parasites were observed between October and April. One-hour formaldehyde treatment at 0.1 and 0.15 ml/l concentration was very effective to treat all parasites and safe for fish species; however, 0.2 ml/l concentration of formaldehyde had toxic effect on brown trout. None of the trichlorfon, dichlorvos, amitraz, cypermethrin chloramines-t doses was effective to treat I. necator. Trichlorfon, amitraz, and cypermethrin were toxic to fish when concentrations increased to 1 g/l, 0.6 ml/l, and 0.07 g/l, respectively (Table 1). Although potassium permanganate (10-20 mg/l) treatment was effective on the parasites, it was toxic to fish. On the other hand, vinegar (4% acetic acid; 10 ml/l), formaldehyde (0.15 ml/l), and salt (20 g/l) were not toxic to fish and they were very effective to treat I. necator, I. multifiliis, and Trichodina spp. infestations. Fish had no abnormal behaviour during and after the vinegar, chloramines-t, salt and dichlorvos treatments. Discussion There are positive relationships between occurrence of protozoan parasites and water temperature (Antonia & Hedrick, 1995; Buchmann et al., 2001). Present study indicated that most of the infestation occurred in May when temperatures were between 8 and 15 o C. Similarly, Nilsen (1995) reported that Trichodina hippoglossi caused heavy infections when a temperature rise from 12 to 18 C and Rintamaki-Kinnunen and Valtonens (1997) reported that I. multifiliis and I. necator infestations in salmon usually occurred at temperatures between 10 and 20 o C. On the other hand, I. necator infestation occurred in a wide temperature range from 3.5 to 38 o C (Robertson, 1979). In the present study, I. necator, Trichodina spp. and I. multifiliis were not detected above 20 o C. Similar to Rintamaki-Kinnunen & Valtonens (1997) findings, we found that the prevalence of parasites in fish in winter ceased.

5 Bull. Eur. Ass. Fish Pathol., 28(4) 2008, 161 Protozoan parasites are commonly observed on fingerling and yearling fish in freshwater systems (Rintamaki-Kinnunen & Valtonens, 1997). Similarly, I. necator infestations were observed more frequently on fingerlings than yearlings in the study. However, I. multifiliis and Trichodina spp. infestations were observed in both fingerlings and yearlings of fish. Ichthyobodo necator infestations were most commonly found on brown trout fingerlings (Rintamaki-Kinnunen & Valtonens, 1997). Besides I. necator, I. multifiliis and Trichodina spp. infestations were also observed on tree species of trout in the present study. Although fish fry are reported to be highly susceptible to both I. necator and I. multifiliis infections (Robertson, 1979; Urawa, 1992), like in the present study, mortality among older fish has also been reported (Valtonen & Keranen, 1981). Trichodinids are a widely dispersed group of ectoparasites (Van As & Basson, 1989). In the present study, species of Trichodina was not identified because many species of Trichodina are morphologically variable and show low host specificity, which make their determination difficult (Lom & Dykova, 1992). Fish disease is result of complex interactions between the fish, disease agents and the environment. Understanding these interactions is very important to successful diseases prevention, diagnosis and treatment (Warren, 1991). Although there are many treatment methods for protozoan infections in fish (Floyd, 1995; Roberts & Stepherd, 1997; Shao, 2001; Durborow, 2003; Kayis et al., 2005), we suggest that salt and vinegar (4% acetic acid) are the most effective and harmless chemicals to treat freshwater fish protozoan parasites. Although acetic acid is not generally used today for treatment of protozoan parasites in salmonids, it has been used in the past (Richardson, 1938; Fish and Burrows, 1940). Further more, formaldehyde is also effective to treat the parasites, however, formaldehyde was highly toxic to brown trout. Chloramines-T and other chemicals are not effective chemicals for treatment of parasites. The best treatment or protection of fish from parasites is good health management practices. Acknowledgements This project was funded by State Planning Organization (Devlet Planlama Teskilati; Project no: 2003K ). References AFS-FHS (American Fisheries Society Fish Health Section) (2003). Suggested procedures for the detection and identification of certain finfish and shellfish pathogens, 5th edition. Fish Health Section, American Fisheries Society, Bethesda, Maryland, USA. Antonio DB & Hedrick RP (1995). Effect of water temperature on infections with the microsporidian Enterocytozoon salmonis in chinook salmon. Diseases of Aquatic Organisms 22, Buchmann K, Sigh J, Nielsen CV & Dalgaard M (2001). Host responses against the fish parasitizing ciliate Ichthyophthirius multifiliis. Veterinary Parasitology 100, Daramola JA & Oladimeji AA (1989). Accumulation of copper in Clarias anguillaris and Oreochromis niloticus. Water, Air, and Soil Pollution 48, Durborow RM (2003). Protozoan parasites, SRAC Publication, No: Floyd RF (1995). The use of salt in aquaculture, University of Florida Cooperative Extension Service Institute of Food and Agricultural Sciences, Fact Sheet VM 86.

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