Pathogenicity of Diplostomum cercariae in rainbow trout, and alternative measures to prevent diplostomosis in fish farms

The pathogenicity of Diplostomum cercariae shed from two different snail species (Lymnaea. stagnalis and Radix auricularia) to rainbow trout fry (5-6 cm body length) was investigated. Thus, 1000 cercariae per fish elicited 100 % mortality within 24 h. Cercariae shed from L. stagnalis (Diplostomum pseudospathaceum) elicited higher mortality in rainbow trout compared to cercariae (Diplostomum paracaudum) from R. auricularia. Furthermore, cercariae from R. auricularia were more successful in terms of migrating and establishing as metacercariae in the lenses of rainbow trout. Alternative measures to prevent diplostomosis in fish farms were subsequently studied. Diplostomum cercariae could be eliminated by mechanical filtration methods. Filters with 32 micrometer mesh size eliminated 99 % while 160 and 200 micrometer meshes removed approximately 50 % of the cercariae. Treatment of water with sodium percarbonate (20 mg/L or higher) killed all infective larvae. Therefore, filtration and sodium percarbonate treatment of fishpond water are suggested as sustainable methods for control of eye-fluke populations in fish farms. Introduction Diplostomid metacercariae (eye-flukes) are frequently found in the eye lenses of fish in fresh and brackish waters (Chappell et al., 1994). These parasites use fish as their second intermediate host, and have been reported from more than 125 species of freshwater fish world-wide (Skrjabin, 1964; Sweeting, 1974). Diplostomum spp. cercariae are released from many different snail species (the first intermediate host), but usually host specificity is high and a particular diplostomid limited to one or two related snail species. D. spathaceum exploits Radix balthica (syn. Lymnaea ovata and L. peregra (Glöer, 2002)), and R. auricularia (syn. Lymnaea auricularia (Glöer, 2002)) as their first intermediate hosts (Niewiadomska and Kiseliene, 1994), whereas D. pseudospathaceum develops in L. s t a g n a l i s a n d S . p a l u s t r i s . F i n a l l y, D . paracaudum use R. balthica, R. auricularia, and in rare cases S. palustris, as their first intermediate host (Niewiadomska and Kiseline, 1994). Fish become infected when cercariae invade through the skin and migrate to the eyes where they often elicit parasitic cataract (Betterton, 1974; Shariff et al., 1980). Heavily infected fish become easy prey for birds, and growth is reduced even when food is abundant (Crowden and Broom, 1980; Owen et al., 1993; Buchmann and Uldal, 1994). Thus, chronic infections with * Corresponding author’s email: [email protected] Bull. Eur. Ass. Fish Pathol., 25(1) 2005, 21 diplostomids are of wide economic importance to freshwater fish producers (Whyte et al., 1988). However, information on the pathogenic effect of cercarial penetration into fish has been limited. Therefore, the present experiments have been conducted in order to elucidate the pathogenicity of the penetrating cercariae. In addition, possible alternative ways for the fish farmers to prevent outbreaks of diplostomosis were evaluated. Previous studies have shown that treatment with praziquantel supplemented feed or in water baths affects the metacercariae (Bylund and Sumari, 1981; Székely and Molnár, 1991). In addition, increase of water current velocity will reduce infections (Field and Irwin, 1994). The present study deals with the mechanical removal (filtration) of Diplostomum cercariae, and treatment of cercaria contaminated water with formalin and sodium percarbonate. Methods and materials Snails and cercariae Two infection experiments were carried out with Diplostomum cercariae shed from two different snail species L. stagnalis and R. auricularia, respectively. Due to limitation of infected snails the Diplostomum cercariae used in the filtration experiment were taken from L. stagnalis whereas studies on the effect of sodium percarbonate and formalin on Diplostomum cercariae were conducted with cercariae shed from R. auricularia . The L. stagnalis snails were collected at a fish farm in the Northwestern part of Denmark in August 2003, and the R. auricularia snails were collected at Lake Furesø on Zealand, Denmark, in September 2003. The snails were brought to the laboratory, placed in separate beakers with de-chlorinated water and monitored for cercarial shedding at room temperature as described by Lyholt and Buchmann (1996). For identification purposes, a sample of the cercariae was fixed in buffered neutral formalin (4%) or glutar-aldehyde (2.5 %). The cercariae fixed in formaldehyde were later stained using Alcian Blue and Meyer ’s haematoxylin, and mounted on slides with glycerol gelatine. The cercariae were characterised by using light microscopy and SEM (Scanning Electron Microscopy). Measurements in μm of five cercariae were taken from each snail species using a compound microscope (Olympus CH-2), and means were calculated. For SEM cercariae were post-fixed in tannic acid, critical point dried, sputtered with gold/palladium and examined in a JEOL JSM840 SEM. Fish Rainbow trout Oncorhynchus mykiss (Walbaum, 1792) were obtained from a pathogen free re-circulated fish culture system. The mean body length of the rainbow trout exposed to Diplostomum cercariae from L. stagnalis was 52.83 mm (SD 4.86 mm), and the mean body weight was 1.434 g (SD 0.513 g). The mean body length of the rainbow trout exposed to Diplostomum cercariae from R. auricularia was 57.92 mm (SD 5.94 mm), and the mean body weight was 1.981 g (SD 0.760 g). Filters Cylindrical filters (diameter 100 mm, height 40 mm) were delivered from Retsch GmbH and Co. KG (Haan, Germany) with the following mesh sizes (in micrometers): 32, 80, 112, 160, 200, 300 and 500. Bull. Eur. Ass. Fish Pathol., 25(1) 2005, 22 Infection procedure Two infection experiments comprising a total of 50 fish were performed. Four groups of rainbow trout with five fish in each group were exposed to cercariae from L. stagnalis and correspondingly four groups were exposed to cercariae from R. auricularia. Individual fish in each of the four groups were either placed in a suspension of 100, 300, 600, and 1000 cercariae in 250 ml water (in a 500 ml glass beaker), respectively. Control groups were treated likewise except for exposure to cercariae. The mortality of the fish in each group was then recorded every six hours for 24 hours. After this period the surviving fish from each group were transferred to 10 litre glass aquaria containing 3 litres of water, with daily water exchange. After 30 days the fish were killed with an overdose of MS 222 (3-aminobenzoic acid ethyl ester) (Sigma A-5040). The fish were dissected and the numbers of metacercariae in their eyes were recorded. All experiments were conducted in a thermostat controlled room at 11-12°C, and a light/darkness interval of 12 hours. Filtration procedure The Diplostomum cercariae were shed naturally from the snail into a 200 ml glass beaker with water. Then, 100 cercariae were randomly taken out using a pipette. They were released into 250 ml de-chlorinated tap water and poured through the filters. However, before the cercariae were poured through the filter, it was checked (using Olympus stereomicroscope, 7–40x magnifications) that they were intact. Subsequently, the filter was rinsed with 300 ml dechlorinated water and examined counting the number of cercariae using a stereomicroscope. Formalin and sodium percarbonate exposure Solutions of formalin and sodium percarbonate (Na2CO3)2;3H2O2) in seven different concentrations each (10, 20, 40, 60, 80, and 100 mg/l) were tested. The cercariae in the control group were placed in dechlorinated tap water. A total of 130 cercariae were used. Each concentration (including the control group) was tested on 10 cercariae. m u m o t s o l p i D m o r f e a i r a c r e c h t g n e L ) . x a m . n i m ( n a e M h t d i W ) . x a m . n i m ( n a e M e a i r a c r e c f o y d o B a i r a l u c i r u a . R 5 5 1 3 3 1 0 . 6 4 1 3 6 0 6 5 . 1 6 s i l a n g a t s . L 8 7 1 3 6 1 5 . 0 7 1 3 6 0 5 5 . 7 5