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Dietary nicarbazin reduces prevalence and severity of Kudoa thyrsites (Myxosporea: Multivalvulida) in Atlantic salmon Salmo salar post-smolts
Aquaculture 342–343 (2012) 1–6
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Dietary nicarbazin reduces prevalence and severity of Kudoa thyrsites (Myxosporea: Multivalvulida) in Atlantic salmon Salmo salar post-smolts Simon R.M. Jones a,⁎, Ian Forster b, Xiangjun Liao c, Michael G. Ikonomou c a b c
Pacific Biological Station, Fisheries and Oceans Canada, Nanaimo, British Columbia, Canada Centre for Aquaculture and Environmental Research, Fisheries and Oceans Canada, West Vancouver, British Columbia, Canada Institute of Ocean Sciences, Fisheries and Oceans Canada, Sidney, British Columbia, Canada
a r t i c l e
i n f o
Article history: Received 7 December 2011 Received in revised form 26 January 2012 Accepted 31 January 2012 Available online 10 February 2012 Keywords: Atlantic salmon Kudoa thyrsites Soft-flesh Nicarbazin Chemotherapy
a b s t r a c t This experiment investigated a range of dietary nicarbazin concentrations for efficacy against the myxosporean parasite Kudoa thyrsites and for toxicity in seawater-reared Atlantic salmon post-smolts. Nicarbazin was incorporated into diets at 0 (control), 2.5, 5, 10 or 25 g kg− 1 such that each dose was replicated among three tanks and delivered intermittently over 2155 degree-days. Compared with controls, the prevalence and severity of K. thyrsites, determined histologically, were significantly reduced in fish fed diets containing nicarbazin. Nicarbazin residues in skeletal muscle, liver and skin were proportional to dietary concentration between 0 and 10 g kg− 1 and were associated with a red discolouration of the skin. Cumulative mortality ranged from 0% to 12.4% among groups and was significantly elevated in the 10 and 25 g kg− 1 groups, relative to control. Weight and condition factor were reduced because of reduced feeding responses to the medicated diets. Feeding responses and appearance of the skin returned to normal and mortality ceased upon resumption of non-medicated diets. In conclusion, dietary nicarbazin was efficacious against K. thyrsites in Atlantic salmon, when included in diets at concentration of at least 2.5 g kg− 1. Further research will optimise nicarbazin treatment strategies and establish tissue residues following cessation of treatment. Crown Copyright © 2012 Published by Elsevier B.V. All rights reserved.
1. Introduction Infections with myxosporean parasites in commercially valuable species of finfish can have economic consequences resulting from proteolytic damage to skeletal muscle (Kent et al., 2001). Kudoa thyrsites develops as spore-filled plasmodia within skeletal muscle cells of marine finfish (King et al., 2012) and the infections can be severe among Atlantic salmon Salmo salar reared in open seawater netpens in British Columbia, Canada. Following salmon harvest and processing, proteases produced by the parasite cause proteolytic degradation of the fillet in proportion to the severity of the infection (Dawson-Coates et al., 2003; Funk et al., 2008; St-Hilaire et al., 1997). Efforts to manage K. thyrsites infections within populations of cultured salmon have focused on early detection, which may be achieved directly by microscopic examination of fresh or histological muscle preparations (Whitaker and Kent, 1991) or indirectly by serological or molecular methods (Funk et al., 2007; Taylor and Jones, 2005). The risk of infection and its consequences may also be mitigated through site selection and optimising smolt quality and stocking density. Vaccines or chemotherapeutants are not available as management options for the prevention or treatment of K. thyrsites. ⁎ Corresponding author at: 3190 Hammond Bay Road, Nanaimo, British Columbia, Canada V9T 6N7. Tel.: + 1 205 729 8351; fax: + 1 250 756 7053. E-mail address: [email protected] (S.R.M. Jones).
Several orally administered compounds have been shown to be efficacious against myxozoan infections in finfish. Among these, fumagillin (or its analogue TNP-470) was used to treat several species of myxozoan parasites in salmonid and non-salmonid hosts (Athanassopoulou et al., 2004; El-Matbouli and Hoffmann, 1991; Hedrick et al., 1988; Higgins and Kent, 1998; Molnár et al., 1987; Sitjà-Bobadilla and Alvarez-Pellitero, 1992; Székely et al., 1988; Wishkovsky et al., 1990; Yokoyama et al., 1990, 1999). However, elevated doses of dietary fumagillin were toxic in 5–9 g Chinook salmon, 2 g and 100–300 g rainbow trout, mature sea bass and 20 g sharpsnout sea bream (Athanassopoulou et al., 2004; Hedrick et al., 1988; Laurén et al., 1989; Sitjà-Bobadilla and Alvarez-Pellitero, 1992; Wishkovsky et al., 1990). Other compounds (clamoxyquin, proguanil) or mixtures of compounds (amprolium and salinomycin) were efficacious against Myxobolus spp. when delivered orally to finfish (Alderman, 1986; Athanassopoulou et al., 2004). Nicarbazin, an equimolar complex of 4,4′-dinitrocarbanilide (DNC) and 2-hydroxy-4,6-dimethylpyrimidine (DHP), is used in poultry feeds for the prevention of coccidiosis (Chapman, 1993). In an earlier trial, dietary nicarbazin failed to control Myxobolus cerebralis in rainbow trout (Taylor et al., 1973). However in another trial, there was no histological evidence of K. thyrsites among Atlantic salmon smolts fed a diet containing 25 g kg − 1 nicarbazin whereas the prevalence of K. thyrsites among non-medicated controls was 50% (S. Jones, unpublished data). In addition, cumulative mortality among the
0044-8486/$ – see front matter. Crown Copyright © 2012 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2012.01.033
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nicarbazin-treated salmon was 49% compared with 4% mortality among controls (S. Jones, unpublished data). The purpose of the present study was to confirm the efficacy against K. thyrsites and toxicity of 25 g kg − 1 dietary nicarbazin and to determine efficacy and toxicity when the compound is incorporated into a salmon diet at lower concentrations. 2. Materials and methods 2.1. Salmon Atlantic salmon smolts were obtained from a commercial hatchery on Vancouver Island and transported in freshwater to the Pacific Biological Station, Nanaimo, British Columbia. Thirty-five fish were allocated to each of 15 × 400 L flow-through tanks and acclimated to seawater over 42 days by gradually increasing the proportion of seawater. The fish were fed a pelleted commercial diet during the acclimation period. The seawater, pumped from Departure Bay, was sand-filtered but otherwise untreated. During the course of the trial, which began on December 21, 2009, fish were exposed to seawater (12 L min − 1 tank − 1) for 231 days at a mean temperature of 9.4 °C (range 7.7 °C–15.0 °C). The mean dissolved oxygen was 8.6 mg L − 1 and the mean salinity, 32.5‰. 2.2. Diets Four experimental diets were prepared in 50 kg batches in which 40 kg of an Atlantic salmon diet mash (Ewos Canada, Vancouver, BC) was blended with 5 kg anchovy oil. The remaining 5 kg comprised a combination of Alphacel non-nutritive bulk fibre (MP Biomedicals, Solon, OH) and 25% (w/w) nicarbazin premix (Havepharma, Peachtree City, CA) to achieve nicarbazin concentrations in the diets of 2.5 g kg − 1, 5.0 g kg − 1, 10 g kg − 1 or 25 g kg − 1. A 150 kg batch of non-medicated control diet was prepared from 120 kg mash, 15 kg oil and 15 kg Alphacel. Each diet was blended and extruded as 3.0 mm pellets and stored at − 40 °C. Samples of premix and feed were stored at − 20 °C for nicarbazin assay.
2.4. Histology After 24 to 48 h, NBF-fixed tissues were transferred to 70% ethanol for storage and eventually dehydrated in an alcohol gradient, cleared in xylene, embedded with paraffin and sectioned at 3 μm. One section from each muscle sample (three per fish) was mounted together onto a glass slide, stained with haematoxylin and eosin stains, coverslipped and examined using a compound microscope (Zeiss Axio Imager) equipped with a digital imaging system (Axiocam MRc5). An infection severity index for each fish was calculated as the arithmetic mean number of K. thyrsites plasmodia mm − 2 from three skeletal muscle sections. Liver samples (n = 15 per treatment) were similarly mounted, stained and cover-slipped and examined by light microscopy. 2.5. Polymerase chain reaction (PCR) DNA was extracted (DNeasy kits, Qiagen Inc.) from approximately 35 mg of ethanol-fixed skeletal muscle into AE buffer and quantified using a Nanodrop-1000 spectrophotometer (v. 3.2.1). PCR was performed using 0.025 U Platinum Taq polymerase (Invitrogen), 1.5 mM MgCl2 (Invitrogen), 1× PCR buffer (Invitrogen), 0.5 μM of each primer (Kt18S3F: 5′-TACCGGAGTAGACCGTAT-3′, KUD3R: 5′GAACTAGGACGGTATCTG-3′) (Jones et al., 2003), 0.2 mM of each dNTP, and 1.0 μL DNA template (≤20 μg mL − 1) to a reaction volume of 25 μL with filter-sterilised (0.22 μm), deionized, and doubledistilled water. Positive (K. thyrsites DNA) and negative (water) controls were included in all reactions. Reactions were conducted in a PTC-200 thermocycler (MJ Research). The amplification protocol was: 94 °C for 2 min, 40 cycles of 94 °C for 1 min, 53 °C for 1 min, 72 °C for 2 min, followed by 72 °C for 10 min. The reaction amplified a 495 base-pair (bp) fragment of the K. thyrsites 18S ribosomal RNA gene which was electrophoretically resolved and visualised in a 1.5% agarose gel with Sybr Safe (Invitrogen) by UVtransillumination (UVP-BioDoc-It System equipped with a 302– 530 nm filter). 2.6. Chemical analysis of nicarbazin
2.3. Experimental procedures Diet treatments were randomly assigned (completely random design) to the 400 L tanks with three-fold replication. Fish were fed at 1% biomass to achieve expected daily nicarbazin doses of 0, 25, 50, 100 or 250 mg kg − 1. The medicated diets were provided during four medicated intervals (41 days for the first, 30 days for the remainder) which alternated with 30 day intervals on the control diet. Revised biomass estimates obtained at the end of each treatment interval were used to adjust the amount of feed provided daily. Fish were visually assessed daily throughout the trial for feeding response and change in appearance. To enhance appetite, all diets were supplemented with approximately 10% krill meal by weight. Dead and moribund fish were counted and removed from the tank twice daily and moribund fish were killed by immersion in 500 mg L − 1 MS-222. Interim samples of five fish from a different tank per treatment group were taken at the end of each of the first three medicated intervals, resulting in samples from all tanks after three intervals. The trial was terminated at the end of the fourth medicated interval and surviving fish were killed. For interim and terminal samples, fork length (L) and weight (W) were measured, condition factor was calculated (W L − 3) and skeletal muscle was assayed for K. thyrsites by histology and polymerase chain reaction (PCR). Freshly collected muscle (skin attached) and liver were wrapped separately in acetone- and methanol-rinsed aluminium foil and stored at −20 °C for nicarbazin assay. Three 1 cm 3 samples of skeletal muscle and one sample of liver were collected from fresh-killed specimens and preserved in cassettes in neutral-buffered 10% formalin (NBF).
DNC concentration was measured in medicated premix, feed and in liver (n = 10 per group), muscle (skin removed, n = 10) and skin (n = 3) samples. Approximately 0.5 g of sample homogenate was mixed with 10 g of anhydrous Na2SO4 dried at room temperature, and subsequently, 8.2 mL of dimethylformamide was added to the mix. The suspension was vortexed for 30 s, ultrasonicated for 20 min and mixed on a horizontal shaker for 1 h before centrifugation at 6000 rpm for 30 min. Approximately 800 μL of the supernatant was spiked with 100 μL of 100 ppb deuterated DNC (DNC-d8, method internal standard), and 100 μL of acetonitrile (ACN). The new mixture was centrifuged at 14,000 rpm for 10 min. Subsequently a 100 μL aliquot was transferred to an autosampler vial and diluted 10-fold with DNC-d8 and ACN prior to instrumental analysis by liquid chromatography tandem mass spectrometry (LC/MS/MS). The instrument used was an Applied Biosystems® API5000 LC/MS/MS system equipped with a Dionex LC system and a Dionex autosampler. The LC column used was an Xterra MS C18 5 μm, 4.6 × 30 mm and 3 μm guard column (10 × 2.1 mm id). The flow rate was 0.30 mL min − 1, and the injection volume was 5 μL. The MS/MS was operated in the multiple reaction monitoring (MRM) mode. The MRM transitions monitored were: for DNC m/z 301.1 to 137.1 and m/z 301.1 to 107.0; and for DNC-d8: m/z 309.1 to 141.1 and m/z 309.1 to 111.0. 2.7. Statistics Fish weight, condition factor and infection severity data were logtransformed and the statistical significance of differences among
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treatment groups determined by 2-way analyses of variance (ANOVA) followed by pairwise Tukey's multiple comparison tests. The statistical significance of differences in nicarbazin concentrations in tissues was calculated by using Kruskal–Wallis tests and the significance of differences in cumulative percent mortality was calculated using Chi-square tests. In all cases, statistical significance was assumed when p ≤ 0.05. 3. Results DNC occurred in the nicarbazin premix at 91% of the expected value and in the medicated diets at levels ranging from 78% to 94% of expected values (Table 1). Compared with salmon on control diets, those provided diets containing 10 or 25 g kg − 1 nicarbazin refused to consume feed pellets, consumed pellets slowly or ingested and rejected pellets. This behaviour was observed sporadically among fish provided diets containing 2.5 or 5 g kg − 1 nicarbazin; the inclusion of krill in all diets lessened the incidence of the altered feeding responses. Cumulative mortality to the end of the trial ranged among treatment groups from 0% (2.5 g kg − 1) to 12.4% (25 g kg − 1) (Fig. 1). Mean mortality within the 25 g kg − 1 groups was greater than that of the 0 g kg − 1 (P = 0.009), 2.5 g kg − 1 (P = 0.000) and 5 g kg − 1 (P = 0.003) groups and was not different from that of the 10 g kg − 1 groups (P = 0.096). However, mean mortality in the latter treatment group was significantly higher than in the 0 g kg − 1 groups. Conversely, cumulative mortality within the control group (0 g kg − 1) did not differ from that in the 2.5 g kg − 1 (P = 0.083), 5 g kg − 1 (P = 0.659) and 10 g kg − 1 (P = 0.293) groups. The altered feeding behaviour and elevated mortality triggered premature cessation of medication, such that on days 13 and 16 of the second and fourth medicated intervals, respectively, fish on 25 g kg − 1 nicarbazin were returned to the control diet. Similarly, during each week of the third and fourth intervals, medicated diets were provided on three alternating days. Mean smolt weight among treatment groups (n = 150 per group) at the beginning of the trial ranged from 67.1 g to 69.5 g. Mean weights at the end of the trial (Table 2), differed significantly among treatment groups (ANOVA, Fratio = 71.97, P = 0.00). Differences in mean weights between fish in the 2.5 g kg − 1 and 5.0 g kg − 1 and between those in the 10 g kg − 1 and 25 g kg − 1 groups were not statistically significant (Tukey, P ≥ 0.99). Mean condition factors at the end of the trial (Table 2) ranged from 1.04 ± 0.01 (10.0 g kg − 1) to 1.14 ± 0.01 (0 g kg − 1) (ANOVA, Fratio = 18.6, P = 0.00). Differences in mean condition factor between the 2.5 or 5.0 and 25 g kg − 1 groups or between the 2.5 and 5.0 g kg − 1 groups were not statistically significant (Tukey, P ≥ 0.99). DNC was detected in liver and skeletal muscle in proportion to dietary concentrations of nicarbazin between 0 and 10 g kg − 1 (Table 3). Residues in both tissues were significantly reduced in the 25 g kg − 1 group compared with 2.5 g kg − 1, 5.0 g kg − 1 and 10 g kg − 1 groups (P b 0.01). At each dietary concentration, DNC residues were significantly higher in liver compared with muscle Table 1 Nicarbazin and 4,4′-dinitrocarbanilide concentrations in experimental diets. Diet group
1 2 3 4 5 Premix a
Total nicarbazin g kg− 1
0 2.5 5.0 10.0 25.0 250.0
Measured by LC/MS/MS.
Fig. 1. Mortality among Atlantic salmon (Salmo salar) smolts on nicarbazin medicated (2.5 to 25 g kg− 1) or control (0 g kg− 1) diets during exposure to Kudoa thyrsites in seawater.
(Table 3). In control salmon, DNC was detected at low levels in four liver (≤20 ng g − 1) and one muscle (1.6 ng g − 1) samples. DNC was undetectable in the skin of control salmon and was measured in skin from medicated fish as follows: 1264 to 1988 ng g − 1 (2.5 g kg − 1 diet); 3365 to 5451 ng g − 1 (5 g kg − 1 diet); 4942 to 15,580 ng g − 1 (10 g kg − 1 diet) and 214 to 2375 ng g − 1 (25 g kg − 1 diet) (n = 3 in all groups). K. thyrsites was not detected by PCR or histology (n = 5 per group at 41 and 103 days) until day 165 (1451 degree-days) of exposure, when the parasite was detected by PCR in all 10 samples from fish on 0 and 2.5 g kg − 1 nicarbazin, two of five on 5.0 g kg − 1, one of five on 10 g kg − 1 and in two of five on 25 g kg − 1 nicarbazin. At this time, plasmodia were also detected by histology in all five nonmedicated controls (0.9 ± 0.5 mm − 2) and in three of five samples from the 2.5 g kg − 1 group (0.3 ± 0.1 mm − 2). At the end of the 231 day trial (2155 degree-days) the parasite was detected by PCR in all 20 control samples and the number of positive samples among the medicated groups ranged from 17 to 18 (n = 20 per group). The percentage of histological muscle samples in which plasmodia were observed ranged from 67.7% to 81.3% among fish on 10 g kg − 1 nicarbazin to 97.1% to 100% among those on the non-medicated control diet (Fig. 2A). The mean severity (plasmodia mm − 2) varied significantly among diet groups (ANOVA, Fratio = 26.71, P = 0.00) but not among tanks within treatment groups (Fratio = 0.01, P = 0.99); dose × tank interactions were not statistically significant (ANOVA, Fratio = 0.70, P = 0.69). The mean severity among control salmon was significantly higher (Tukey, P b 0.01) than in any treated group (Fig. 2B). The mean severity among fish on 2.5 g kg − 1 nicarbazin was not different (Tukey, P = 0.209) from those on 5.0 g kg − 1 but was significantly higher than those on 10 or 25 g kg − 1 (Tukey, P b 0.01 both cases). Differences in mean severity among fish on 5,
Table 2 Weight and condition factor of Atlantic salmon fed diets containing nicarbazin. Dietary nicarbazin (g kg− 1)
4,4′-dinitrocarbanilide g kg− 1 Expected
Observeda (% expected)
0 1.8 3.5 7.1 17.7 177.3
0 1.4 (78) 3.3 (94) 6.2 (87) 15.3 (86) 160.6 (91)
Weight (g) Mean1 ± s.e. Range
0
2.5
5
10
25
204.4 ± 3.0a 238–286
175.4± 2.8b 118–270
172.2 ± 3.1b 88–269
143.8 ± 2.0c 98–214
145.5± 3.1c 99–318
1.09± 0.01a 0.79–1.33
1.08± 0.01a 0.89–1.44
1.04 ± 0.01 0.86–1.21
1.08 ± 0.01a 0.94–1.37
Weight length− 3 Mean± s.e. 1.14± 0.01 Range 0.99–1.32
1. The same superscripts indicate a probability less than 5% that mean values are different.
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Table 3 LC/MS/MS measured concentrations of 4,4′-dinitrocarbanilide (ng g− 1) in kidney and liver of Atlantic salmon on experimental diets containing various concentrations of nicarbazin (mean ± std. error and range). Dietary nicarbazin (g kg− 1)
Liver (n = 10)
Muscle (n = 10)
P-valuea
0.0 2.5 5.0 10.0 25.0
4.8 ± 2.2 (0–20.0) 3721.4 ± 365.9 (1336.4–5062.2) 11,168.3 ± 1631.3 (168.5–16,620.0) 19,998.9 ± 2464.7 (5927.9–28,521.7) 87.4 ± 31.0 (14.0–261.4)
0.2 ± 0.2 (0–1.6) 344.7 ± 38.6 (129.9–506.3) 1479.5 ± 266.9 (31.6–2631.2) 2920.8 ± 473.5 (423.9–4635.9) 3.8 ± 0.7 (0–7.3)
0.068 b 0.001 b 0.001 b 0.001 0.023
a
Probability that differences between mean tissue (liver and muscle) concentrations are the same (Kruskal–Wallis).
10 or 25 g kg − 1 nicarbazin (Fig. 2B) were not statistically significant (Tukey, P b 0.01 all cases). Foci of hepatocellular degeneration and necrosis were only observed in histological liver preparation from two of 15 salmon on 10 g kg − 1 nicarbazin diets (Fig. 3). The development of a fibrous capsule was associated with some lesions. A rust-red discolouration of the skin was observed in salmon fed diets containing 5 to 25 g kg − 1 nicarbazin. The discolouration was most evident in naturally unpigmented skin but was also visible on the dorsal surface of the head of swimming fish. The discolouration disappeared between 10 and 29 days after the resumption of feeding on the non-medicated diet. 4. Discussion K. thyrsites was first detected by histology and PCR in Atlantic salmon smolts after a 165-day (1451 degree-days) exposure to
Fig. 2. The effects of dietary nicarbazin concentration on Kudoa thyrsites in histological sections of skeletal muscle from Atlantic salmon (Salmo salar) smolts collected after 231 days exposure to seawater. Bars at each concentration are values from individual tanks (n = 95 to 103 per group). A, Estimated prevalence of K. thyrsites. B, Severity of K. thyrsites (mean ± std. error).
Departure Bay seawater. Previously, the parasite was first detected by light microscopy after 63 and 91 days (Moran et al., 1999) or after 2 months (1000 degree-days) (Moran and Kent, 1999) of exposure to Departure Bay seawater. The delay in the time of first detection in the present study may be related to the absence or low concentration of infective stages in the water at this site between December and May (Moran and Kent, 1999). Despite this, the parasite was eventually detected in almost all non-medicated Atlantic salmon smolts, providing a reference against which the efficacy of nicarbazin was assessed. Two patterns of infection were evident among fish treated with nicarbazin: a delay in the first detection of infection and lower prevalence and severity of infection at the end of the trial. Previously, nicarbazin fed continuously at 6–14 mg kg − 1 or 30–60 mg kg − 1 for 12 months was found to be inefficacious against M. cerebralis in rainbow trout (Taylor et al., 1973). Although the latter
Fig. 3. Light micrographs of histological sections of liver from Atlantic salmon (Salmo salar) following 10 g kg− 1 nicarbazin treatment. Foci of hepatocellular degeneration and necrosis before (A) and during (B) formation of fibrous capsule. Haematoxylin and eosin stain.
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study did not describe methods for the preparation of medicated diets and nicarbazin residues were not titrated in feed or fish tissues (Taylor et al., 1973), all diets were palatable, indicating that refusal to eat did not explain the absence of efficacy. The tropism of M. cerebralis for cartilage, in contrast with that of K. thyrsites for skeletal muscle, may have reduced exposure to the compound during parasite development. Similarly in another study, dietary nicarbazin (0.1 g kg − 1) had no effect on infection with the ciliate Ichthyophthirius multifiliis in the skin of rainbow trout (Shinn et al., 2003). In contrast, the present data demonstrated the efficacy of nicarbazin, incorporated into the diet at concentrations ranging from 2.5 to 25 g kg − 1, against K. thyrsites in seawater-reared Atlantic salmon. Dinitrocarbanilide (DNC) was detected in skeletal muscle and liver of salmon following the 231-day trial. Mean DNC concentrations in liver were higher than in muscle in salmon on diets ranging from 2.5 to 10 g kg − 1 nicarbazin. Furthermore, the concentration of DNC in both tissues was proportional to that in feed over this range, but was reduced in the 25 g kg − 1 group. Nicarbazin is used as a feed additive for the treatment of coccidiosis in broiler chickens up to 28 days old and as with salmon, the compound is retained in avian liver at concentrations higher than in muscle (Penz et al., 1999). DNC possesses anti-coccidial properties (Chapman, 1993) and is the residue marker for nicarbazin in poultry, with a maximum limit of 200 ng g − 1 in broiler tissues (McCarney et al., 2003). The low mean DNC residues in the 25 g kg − 1 salmon group (87 ng g − 1 liver, 4 ng g − 1 muscle) was probably related to reduced ingestion of nicarbazin because of the reduced palatability at this concentration and to the premature cessation of this diet in two of four medication intervals. However, additional data are required to more precisely determine the rate of reduction of DNC in salmon tissues. The adverse effects of dietary nicarbazin in salmon were dosedependent and included reduced weight, lower condition factor and elevated mortality. Reduced weight gain and the associated loss of condition, particularly among salmon fed diets containing 10 and 25 g kg − 1 nicarbazin, resulted from reduced feed intake, associated with low palatability of nicarbazin at these concentrations, despite supplementation with krill. Normal feeding behaviour was reestablished following the resumption of the non-medicated diet. While there are few published data for nicarbazin in fishes, dietary fumagillin was also associated anorexia and reduced weight in fish (Athanassopoulou et al., 2004; Sitjà-Bobadilla and Alvarez-Pellitero, 1992). In addition, mortality was elevated among salmon within the 10 and 25 g kg − 1 nicarbazin groups which at the latter dose confirmed our earlier observations (S. Jones, unpublished data). The mechanism of mortality may be related to the liver pathology however more research is required to fully document the development of hepatic and possibly other lesions associated with therapeutic and supratherapeutic nicarbazin doses in Atlantic salmon. The rustcoloured discolouration of the skin among salmon on diets containing 5 to 25 g kg − 1 nicarbazin was associated with the occurrence of DNC in skin at levels similar to those in measured in liver. Associated histological changes were not evident in skin and the discolouration disappeared following resumption of control diets. The return to normal colouration was particularly rapid (10 days) in fish on 25 g kg − 1 following the resumption of the control diet and was associated with DNC residues in skin that were significantly reduced. In summary, although its mode of action is not known (Lindsay and Blagburn, 1995), dietary nicarbazin was efficacious against K. thyrsites in seawater-reared Atlantic salmon. In salmon, DNC residues in skin, liver and muscle were proportional to dietary concentrations. The higher concentrations of nicarbazin used here reduced palatability of diets leading to reduced weight gain and condition factor. Mortality was elevated in proportion to nicarbazin concentration in the diet. However, these adverse effects ceased upon resumption of non-medicated diets. Further work is required to develop a treatment regime that retains the efficacy of the compound,
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minimises the adverse effects and provides data on DNC residuals in Atlantic salmon upon resumption of non-medicated diets.
Acknowledgements We thank the support of Marine Harvest Canada, Mainstream Canada, Grieg Seafoods, Ewos Canada and the Aquaculture Collaborative Research and Development Program. Thanks also to E. Kim, A. Yu Xin and H. Jones for maintaining the fish, collecting samples and conducting microscopic examinations and to M. Rowshandeli for mixing and preparing the diets.
References Alderman, D.J., 1986. Whirling disease chemotherapy. Bulletin of the European Association of Fish Pathologists 6, 38–40. Athanassopoulou, F., Karagouni, E., Dotsika, E., Ragias, V., Tavla, J., Christofilloyanis, P., Vatsos, I., 2004. Efficacy and toxicity of orally administered anti-coccidial drugs for innovative treatments of Myxobolus sp. infection in Puntazzo puntazzo. Diseases of Aquatic Organisms 62, 217–226. Chapman, H.D., 1993. A review of the biological activity of the anticoccidial drug nicarbazin and its application for the control of coccidiosis in poultry. Poultry Science Review 5, 231–243. Dawson-Coates, J.A., Chase, J.C., Funk, V., Booy, M.H., Haines, L.R., Falkenberg, C.L., Whitaker, D.J., Olafson, R.W., Pearson, T.W., 2003. The relationship between flesh quality and numbers of Kudoa thyrsites plasmodia and spores in farmed Atlantic salmon, Salmo salar L. Journal of Fish Diseases 26, 451–459. El-Matbouli, M., Hoffmann, R.W., 1991. Prevention of experimentally induced whirling disease in rainbow trout Oncorhynchus mykiss by fumagillin. Diseases of Aquatic Organisms 10, 109–113. Funk, V.A., Raap, M., Sojonky, K., Jones, S., Robinson, J., Falkenberg, C., Miller, K.M., 2007. Development and validation of an RNA- and DNA-based quantitative PCR assay for determination of Kudoa thyrsites infection levels in Atlantic salmon Salmo salar. Diseases of Aquatic Organisms 75, 239–249. Funk, V.A., Olafson, R.W., Raap, M., Smith, D., Aitken, L., Haddow, J.D., Wang, D., Dawson-Coates, J.A., Burke, R.D., Miller, K.M., 2008. Identification, characterization and deduced amino acid sequence of the dominant protease from Kudoa paniformis and K. thyrsites: a unique cytoplasmic cysteine protease. Comparative Biochemistry and Physiology. B 149, 477–489. Hedrick, R.P., Groff, J.M., Foley, P., McDowell, T., 1988. Oral administration of fumagillin DCH protects Chinook salmon Oncorhynchus tshawytscha from experimentallyinduced proliferative kidney disease. Diseases of Aquatic Organisms 4, 165–168. Higgins, M.J., Kent, M.L., 1998. TNP-470, the analogue of fumagillin-DCH, controls PKX in naturally infected sockeye salmon, Oncorhynchus nerka (Walbaum), underyearlings. Journal of Fish Diseases 21, 455–457. Jones, S.R.M., Goh, B., Prosperi-Porta, G., 2003. Duration and method of fixation affects the sensitivity of a digoxygenin-labelled DNA probe in detecting Kudoa thyrsites in Atlantic salmon skeletal muscle. Aquaculture 220, 157–164. Kent, M.L., Andree, K.B., Bartholomew, J.L., El-Matbouli, M., Desser, S.S., Devlin, R.H., Feist, S.W., Hedrick, R.P., Hoffmann, R.W., Khattra, J., Hallett, S.L., Lester, R.J.G., Longshaw, M., Palenzuela, O., Siddall, M.E., Xiao, C., 2001. Recent advances in our knowledge of the Myxozoa. Journal of Eukaryotic Microbiology 48, 395–413. King, J.R., McFarlane, G.A., Jones, S.R.M., Gilmore, S.R., Abbott, C.L., 2012. Stock delineation of migratory and resident Pacific hake in Canadian waters. Fisheries Research 14, 19–30. Laurén, D.J., Wishkovsky, A., Groff, J.M., Hedrick, R.P., Hinton, D.E., 1989. Toxicity and pharmacokinetics of the antibiotic fumagillin in yearling rainbow trout (Salmo gairdneri). Toxicology and Applied Pharmacology 98, 444–453. Lindsay, D.S., Blagburn, B.L., 1995. Antiprotozoan drugs, In: Adams, H.R. (Ed.), Veterinary Pharmacology and Therapeutics, 7th Edition. Iowa University Press, Ames, pp. 955–983. McCarney, B., Traynor, I.M., Fodey, T.L., Crooks, S.R.H., Elliott, C.T., 2003. Surface plasmon resonance biosensor screening of poultry liver and eggs for nicarbazin residues. Analytica Chimica Acta 483, 165–169. Molnár, K., Baska, F., Székely, C., 1987. Fumagillin, an efficacious drug against renal sphaerosporosis of the common carp Cyprinus carpio. Diseases of Aquatic Organisms 2, 187–190. Moran, J.D.W., Kent, M.L., 1999. Kudoa thyrsites (Myxozoa: Myxosporea) infections in pen-reared Atlantic salmon in the northeast Pacific Ocean with a survey of potential nonsalmonid reservoir hosts. Journal of Aquatic Animal Health 11, 101–109. Moran, J.D.W., Margolis, L., Webster, J.M., Kent, M.L., 1999. Development of Kudoa thyrsites (Myxozoa: Myxosporea) in netpen-reared Atlantic salmon determined by light microscopy and a polymerase chain reaction test. Diseases of Aquatic Organisms 37, 185–193. Penz Jr., A.M., Vieira, S.L., Ludke, J.V., 1999. Nicarbazin residues in broiler tissue and litter. Journal of Applied Poultry Research 8, 292–297. Shinn, A.P., Wootten, R., Côté, I., Sommerville, C., 2003. Efficacy of selected oral chemotherapeutants against Ichthyophthirius multifiliis (ciliophora: ophyroglenidae) infecting rainbow trout Oncorhynchus mykiss. Diseases of Aquatic Organisms 55, 17–22.
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Sitjà-Bobadilla, A., Alvarez-Pellitero, P., 1992. Effect of fumagillin treatment on sea bass Dicentrarchus labrax parasitized by Sphaerospora testicularis (Myxosporea: Bivalvulida). Diseases of Aquatic Organisms 14, 171–178. St-Hilaire, S., Hill, M., Kent, M.L., Whitaker, D.J., Ribble, C., 1997. A comparative study of muscle texture and intensity of Kudoa thyrsites infection in farm-reared Atlantic salmon Salmo salar on the Pacific coast of Canada. Diseases of Aquatic Organisms 31, 221–225. Székely, C., Molnár, K., Baska, F., 1988. Efficacy of fumagillin against Myxidium giardi Cépede, 1906 infection of the European eel (Anguilla Anguilla): new observations of myxidiosis of imported glass eels. Acta Veterinaria Hungarica 36, 239–246. Taylor, K., Jones, S., 2005. An enzyme linked immunosorbent assay for the detection of Kudoa thyrsites in Atlantic salmon Salmo salar. Aquaculture 250, 8–15. Taylor, R.E.L., Coli, S.J., Junell, D.R., 1973. Attempts to control whirling disease by continuous drug feeding. Journal of Wildlife Diseases 9, 302–305.
Whitaker, D.J., Kent, M.L., 1991. Myxosporean Kudoa thyrsites: a cause of soft flesh in farm-reared Atlantic salmon. Journal of Aquatic Animal Health 3, 291–294. Wishkovsky, A., Groff, J.M., Lauren, D.J., Toth, R.J., Hedrick, R.P., 1990. Efficacy of fumagillin against proliferative kidney disease and its toxic side effects in rainbow trout (Oncorhynchus mykiss) fingerlings. Fish Pathology 25, 141–146. Yokoyama, H., Ogawa, K., Wakabayashi, H., 1990. Chemotherapy with fumagillin and toltrazuril against kidney enlargement disease of goldfish caused by Hoferellus carassii. Fish Pathology 25, 157–163. Yokoyama, H., Liyanagi, Y.S., Sugai, A., Wakabayashi, H., 1999. Efficacy of fumagillin against haemorrhagic thelohanellosis caused by Thelohanellus hovorkai (Myxosporea: Myxozoa) in coloured carp, Cyprinus carpio L. Journal of Fish Diseases 22, 243–245.
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Dietary nicarbazin reduces prevalence and severity of Kudoa thyrsites (Myxosporea: Multivalvulida) in Atlantic salmon Salmo salar post-smolts Simon R.M. Jones a,⁎, Ian Forster b, Xiangjun Liao c, Michael G. Ikonomou c a b c
Pacific Biological Station, Fisheries and Oceans Canada, Nanaimo, British Columbia, Canada Centre for Aquaculture and Environmental Research, Fisheries and Oceans Canada, West Vancouver, British Columbia, Canada Institute of Ocean Sciences, Fisheries and Oceans Canada, Sidney, British Columbia, Canada
a r t i c l e
i n f o
Article history: Received 7 December 2011 Received in revised form 26 January 2012 Accepted 31 January 2012 Available online 10 February 2012 Keywords: Atlantic salmon Kudoa thyrsites Soft-flesh Nicarbazin Chemotherapy
a b s t r a c t This experiment investigated a range of dietary nicarbazin concentrations for efficacy against the myxosporean parasite Kudoa thyrsites and for toxicity in seawater-reared Atlantic salmon post-smolts. Nicarbazin was incorporated into diets at 0 (control), 2.5, 5, 10 or 25 g kg− 1 such that each dose was replicated among three tanks and delivered intermittently over 2155 degree-days. Compared with controls, the prevalence and severity of K. thyrsites, determined histologically, were significantly reduced in fish fed diets containing nicarbazin. Nicarbazin residues in skeletal muscle, liver and skin were proportional to dietary concentration between 0 and 10 g kg− 1 and were associated with a red discolouration of the skin. Cumulative mortality ranged from 0% to 12.4% among groups and was significantly elevated in the 10 and 25 g kg− 1 groups, relative to control. Weight and condition factor were reduced because of reduced feeding responses to the medicated diets. Feeding responses and appearance of the skin returned to normal and mortality ceased upon resumption of non-medicated diets. In conclusion, dietary nicarbazin was efficacious against K. thyrsites in Atlantic salmon, when included in diets at concentration of at least 2.5 g kg− 1. Further research will optimise nicarbazin treatment strategies and establish tissue residues following cessation of treatment. Crown Copyright © 2012 Published by Elsevier B.V. All rights reserved.
1. Introduction Infections with myxosporean parasites in commercially valuable species of finfish can have economic consequences resulting from proteolytic damage to skeletal muscle (Kent et al., 2001). Kudoa thyrsites develops as spore-filled plasmodia within skeletal muscle cells of marine finfish (King et al., 2012) and the infections can be severe among Atlantic salmon Salmo salar reared in open seawater netpens in British Columbia, Canada. Following salmon harvest and processing, proteases produced by the parasite cause proteolytic degradation of the fillet in proportion to the severity of the infection (Dawson-Coates et al., 2003; Funk et al., 2008; St-Hilaire et al., 1997). Efforts to manage K. thyrsites infections within populations of cultured salmon have focused on early detection, which may be achieved directly by microscopic examination of fresh or histological muscle preparations (Whitaker and Kent, 1991) or indirectly by serological or molecular methods (Funk et al., 2007; Taylor and Jones, 2005). The risk of infection and its consequences may also be mitigated through site selection and optimising smolt quality and stocking density. Vaccines or chemotherapeutants are not available as management options for the prevention or treatment of K. thyrsites. ⁎ Corresponding author at: 3190 Hammond Bay Road, Nanaimo, British Columbia, Canada V9T 6N7. Tel.: + 1 205 729 8351; fax: + 1 250 756 7053. E-mail address: [email protected] (S.R.M. Jones).
Several orally administered compounds have been shown to be efficacious against myxozoan infections in finfish. Among these, fumagillin (or its analogue TNP-470) was used to treat several species of myxozoan parasites in salmonid and non-salmonid hosts (Athanassopoulou et al., 2004; El-Matbouli and Hoffmann, 1991; Hedrick et al., 1988; Higgins and Kent, 1998; Molnár et al., 1987; Sitjà-Bobadilla and Alvarez-Pellitero, 1992; Székely et al., 1988; Wishkovsky et al., 1990; Yokoyama et al., 1990, 1999). However, elevated doses of dietary fumagillin were toxic in 5–9 g Chinook salmon, 2 g and 100–300 g rainbow trout, mature sea bass and 20 g sharpsnout sea bream (Athanassopoulou et al., 2004; Hedrick et al., 1988; Laurén et al., 1989; Sitjà-Bobadilla and Alvarez-Pellitero, 1992; Wishkovsky et al., 1990). Other compounds (clamoxyquin, proguanil) or mixtures of compounds (amprolium and salinomycin) were efficacious against Myxobolus spp. when delivered orally to finfish (Alderman, 1986; Athanassopoulou et al., 2004). Nicarbazin, an equimolar complex of 4,4′-dinitrocarbanilide (DNC) and 2-hydroxy-4,6-dimethylpyrimidine (DHP), is used in poultry feeds for the prevention of coccidiosis (Chapman, 1993). In an earlier trial, dietary nicarbazin failed to control Myxobolus cerebralis in rainbow trout (Taylor et al., 1973). However in another trial, there was no histological evidence of K. thyrsites among Atlantic salmon smolts fed a diet containing 25 g kg − 1 nicarbazin whereas the prevalence of K. thyrsites among non-medicated controls was 50% (S. Jones, unpublished data). In addition, cumulative mortality among the
0044-8486/$ – see front matter. Crown Copyright © 2012 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2012.01.033
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nicarbazin-treated salmon was 49% compared with 4% mortality among controls (S. Jones, unpublished data). The purpose of the present study was to confirm the efficacy against K. thyrsites and toxicity of 25 g kg − 1 dietary nicarbazin and to determine efficacy and toxicity when the compound is incorporated into a salmon diet at lower concentrations. 2. Materials and methods 2.1. Salmon Atlantic salmon smolts were obtained from a commercial hatchery on Vancouver Island and transported in freshwater to the Pacific Biological Station, Nanaimo, British Columbia. Thirty-five fish were allocated to each of 15 × 400 L flow-through tanks and acclimated to seawater over 42 days by gradually increasing the proportion of seawater. The fish were fed a pelleted commercial diet during the acclimation period. The seawater, pumped from Departure Bay, was sand-filtered but otherwise untreated. During the course of the trial, which began on December 21, 2009, fish were exposed to seawater (12 L min − 1 tank − 1) for 231 days at a mean temperature of 9.4 °C (range 7.7 °C–15.0 °C). The mean dissolved oxygen was 8.6 mg L − 1 and the mean salinity, 32.5‰. 2.2. Diets Four experimental diets were prepared in 50 kg batches in which 40 kg of an Atlantic salmon diet mash (Ewos Canada, Vancouver, BC) was blended with 5 kg anchovy oil. The remaining 5 kg comprised a combination of Alphacel non-nutritive bulk fibre (MP Biomedicals, Solon, OH) and 25% (w/w) nicarbazin premix (Havepharma, Peachtree City, CA) to achieve nicarbazin concentrations in the diets of 2.5 g kg − 1, 5.0 g kg − 1, 10 g kg − 1 or 25 g kg − 1. A 150 kg batch of non-medicated control diet was prepared from 120 kg mash, 15 kg oil and 15 kg Alphacel. Each diet was blended and extruded as 3.0 mm pellets and stored at − 40 °C. Samples of premix and feed were stored at − 20 °C for nicarbazin assay.
2.4. Histology After 24 to 48 h, NBF-fixed tissues were transferred to 70% ethanol for storage and eventually dehydrated in an alcohol gradient, cleared in xylene, embedded with paraffin and sectioned at 3 μm. One section from each muscle sample (three per fish) was mounted together onto a glass slide, stained with haematoxylin and eosin stains, coverslipped and examined using a compound microscope (Zeiss Axio Imager) equipped with a digital imaging system (Axiocam MRc5). An infection severity index for each fish was calculated as the arithmetic mean number of K. thyrsites plasmodia mm − 2 from three skeletal muscle sections. Liver samples (n = 15 per treatment) were similarly mounted, stained and cover-slipped and examined by light microscopy. 2.5. Polymerase chain reaction (PCR) DNA was extracted (DNeasy kits, Qiagen Inc.) from approximately 35 mg of ethanol-fixed skeletal muscle into AE buffer and quantified using a Nanodrop-1000 spectrophotometer (v. 3.2.1). PCR was performed using 0.025 U Platinum Taq polymerase (Invitrogen), 1.5 mM MgCl2 (Invitrogen), 1× PCR buffer (Invitrogen), 0.5 μM of each primer (Kt18S3F: 5′-TACCGGAGTAGACCGTAT-3′, KUD3R: 5′GAACTAGGACGGTATCTG-3′) (Jones et al., 2003), 0.2 mM of each dNTP, and 1.0 μL DNA template (≤20 μg mL − 1) to a reaction volume of 25 μL with filter-sterilised (0.22 μm), deionized, and doubledistilled water. Positive (K. thyrsites DNA) and negative (water) controls were included in all reactions. Reactions were conducted in a PTC-200 thermocycler (MJ Research). The amplification protocol was: 94 °C for 2 min, 40 cycles of 94 °C for 1 min, 53 °C for 1 min, 72 °C for 2 min, followed by 72 °C for 10 min. The reaction amplified a 495 base-pair (bp) fragment of the K. thyrsites 18S ribosomal RNA gene which was electrophoretically resolved and visualised in a 1.5% agarose gel with Sybr Safe (Invitrogen) by UVtransillumination (UVP-BioDoc-It System equipped with a 302– 530 nm filter). 2.6. Chemical analysis of nicarbazin
2.3. Experimental procedures Diet treatments were randomly assigned (completely random design) to the 400 L tanks with three-fold replication. Fish were fed at 1% biomass to achieve expected daily nicarbazin doses of 0, 25, 50, 100 or 250 mg kg − 1. The medicated diets were provided during four medicated intervals (41 days for the first, 30 days for the remainder) which alternated with 30 day intervals on the control diet. Revised biomass estimates obtained at the end of each treatment interval were used to adjust the amount of feed provided daily. Fish were visually assessed daily throughout the trial for feeding response and change in appearance. To enhance appetite, all diets were supplemented with approximately 10% krill meal by weight. Dead and moribund fish were counted and removed from the tank twice daily and moribund fish were killed by immersion in 500 mg L − 1 MS-222. Interim samples of five fish from a different tank per treatment group were taken at the end of each of the first three medicated intervals, resulting in samples from all tanks after three intervals. The trial was terminated at the end of the fourth medicated interval and surviving fish were killed. For interim and terminal samples, fork length (L) and weight (W) were measured, condition factor was calculated (W L − 3) and skeletal muscle was assayed for K. thyrsites by histology and polymerase chain reaction (PCR). Freshly collected muscle (skin attached) and liver were wrapped separately in acetone- and methanol-rinsed aluminium foil and stored at −20 °C for nicarbazin assay. Three 1 cm 3 samples of skeletal muscle and one sample of liver were collected from fresh-killed specimens and preserved in cassettes in neutral-buffered 10% formalin (NBF).
DNC concentration was measured in medicated premix, feed and in liver (n = 10 per group), muscle (skin removed, n = 10) and skin (n = 3) samples. Approximately 0.5 g of sample homogenate was mixed with 10 g of anhydrous Na2SO4 dried at room temperature, and subsequently, 8.2 mL of dimethylformamide was added to the mix. The suspension was vortexed for 30 s, ultrasonicated for 20 min and mixed on a horizontal shaker for 1 h before centrifugation at 6000 rpm for 30 min. Approximately 800 μL of the supernatant was spiked with 100 μL of 100 ppb deuterated DNC (DNC-d8, method internal standard), and 100 μL of acetonitrile (ACN). The new mixture was centrifuged at 14,000 rpm for 10 min. Subsequently a 100 μL aliquot was transferred to an autosampler vial and diluted 10-fold with DNC-d8 and ACN prior to instrumental analysis by liquid chromatography tandem mass spectrometry (LC/MS/MS). The instrument used was an Applied Biosystems® API5000 LC/MS/MS system equipped with a Dionex LC system and a Dionex autosampler. The LC column used was an Xterra MS C18 5 μm, 4.6 × 30 mm and 3 μm guard column (10 × 2.1 mm id). The flow rate was 0.30 mL min − 1, and the injection volume was 5 μL. The MS/MS was operated in the multiple reaction monitoring (MRM) mode. The MRM transitions monitored were: for DNC m/z 301.1 to 137.1 and m/z 301.1 to 107.0; and for DNC-d8: m/z 309.1 to 141.1 and m/z 309.1 to 111.0. 2.7. Statistics Fish weight, condition factor and infection severity data were logtransformed and the statistical significance of differences among
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3
treatment groups determined by 2-way analyses of variance (ANOVA) followed by pairwise Tukey's multiple comparison tests. The statistical significance of differences in nicarbazin concentrations in tissues was calculated by using Kruskal–Wallis tests and the significance of differences in cumulative percent mortality was calculated using Chi-square tests. In all cases, statistical significance was assumed when p ≤ 0.05. 3. Results DNC occurred in the nicarbazin premix at 91% of the expected value and in the medicated diets at levels ranging from 78% to 94% of expected values (Table 1). Compared with salmon on control diets, those provided diets containing 10 or 25 g kg − 1 nicarbazin refused to consume feed pellets, consumed pellets slowly or ingested and rejected pellets. This behaviour was observed sporadically among fish provided diets containing 2.5 or 5 g kg − 1 nicarbazin; the inclusion of krill in all diets lessened the incidence of the altered feeding responses. Cumulative mortality to the end of the trial ranged among treatment groups from 0% (2.5 g kg − 1) to 12.4% (25 g kg − 1) (Fig. 1). Mean mortality within the 25 g kg − 1 groups was greater than that of the 0 g kg − 1 (P = 0.009), 2.5 g kg − 1 (P = 0.000) and 5 g kg − 1 (P = 0.003) groups and was not different from that of the 10 g kg − 1 groups (P = 0.096). However, mean mortality in the latter treatment group was significantly higher than in the 0 g kg − 1 groups. Conversely, cumulative mortality within the control group (0 g kg − 1) did not differ from that in the 2.5 g kg − 1 (P = 0.083), 5 g kg − 1 (P = 0.659) and 10 g kg − 1 (P = 0.293) groups. The altered feeding behaviour and elevated mortality triggered premature cessation of medication, such that on days 13 and 16 of the second and fourth medicated intervals, respectively, fish on 25 g kg − 1 nicarbazin were returned to the control diet. Similarly, during each week of the third and fourth intervals, medicated diets were provided on three alternating days. Mean smolt weight among treatment groups (n = 150 per group) at the beginning of the trial ranged from 67.1 g to 69.5 g. Mean weights at the end of the trial (Table 2), differed significantly among treatment groups (ANOVA, Fratio = 71.97, P = 0.00). Differences in mean weights between fish in the 2.5 g kg − 1 and 5.0 g kg − 1 and between those in the 10 g kg − 1 and 25 g kg − 1 groups were not statistically significant (Tukey, P ≥ 0.99). Mean condition factors at the end of the trial (Table 2) ranged from 1.04 ± 0.01 (10.0 g kg − 1) to 1.14 ± 0.01 (0 g kg − 1) (ANOVA, Fratio = 18.6, P = 0.00). Differences in mean condition factor between the 2.5 or 5.0 and 25 g kg − 1 groups or between the 2.5 and 5.0 g kg − 1 groups were not statistically significant (Tukey, P ≥ 0.99). DNC was detected in liver and skeletal muscle in proportion to dietary concentrations of nicarbazin between 0 and 10 g kg − 1 (Table 3). Residues in both tissues were significantly reduced in the 25 g kg − 1 group compared with 2.5 g kg − 1, 5.0 g kg − 1 and 10 g kg − 1 groups (P b 0.01). At each dietary concentration, DNC residues were significantly higher in liver compared with muscle Table 1 Nicarbazin and 4,4′-dinitrocarbanilide concentrations in experimental diets. Diet group
1 2 3 4 5 Premix a
Total nicarbazin g kg− 1
0 2.5 5.0 10.0 25.0 250.0
Measured by LC/MS/MS.
Fig. 1. Mortality among Atlantic salmon (Salmo salar) smolts on nicarbazin medicated (2.5 to 25 g kg− 1) or control (0 g kg− 1) diets during exposure to Kudoa thyrsites in seawater.
(Table 3). In control salmon, DNC was detected at low levels in four liver (≤20 ng g − 1) and one muscle (1.6 ng g − 1) samples. DNC was undetectable in the skin of control salmon and was measured in skin from medicated fish as follows: 1264 to 1988 ng g − 1 (2.5 g kg − 1 diet); 3365 to 5451 ng g − 1 (5 g kg − 1 diet); 4942 to 15,580 ng g − 1 (10 g kg − 1 diet) and 214 to 2375 ng g − 1 (25 g kg − 1 diet) (n = 3 in all groups). K. thyrsites was not detected by PCR or histology (n = 5 per group at 41 and 103 days) until day 165 (1451 degree-days) of exposure, when the parasite was detected by PCR in all 10 samples from fish on 0 and 2.5 g kg − 1 nicarbazin, two of five on 5.0 g kg − 1, one of five on 10 g kg − 1 and in two of five on 25 g kg − 1 nicarbazin. At this time, plasmodia were also detected by histology in all five nonmedicated controls (0.9 ± 0.5 mm − 2) and in three of five samples from the 2.5 g kg − 1 group (0.3 ± 0.1 mm − 2). At the end of the 231 day trial (2155 degree-days) the parasite was detected by PCR in all 20 control samples and the number of positive samples among the medicated groups ranged from 17 to 18 (n = 20 per group). The percentage of histological muscle samples in which plasmodia were observed ranged from 67.7% to 81.3% among fish on 10 g kg − 1 nicarbazin to 97.1% to 100% among those on the non-medicated control diet (Fig. 2A). The mean severity (plasmodia mm − 2) varied significantly among diet groups (ANOVA, Fratio = 26.71, P = 0.00) but not among tanks within treatment groups (Fratio = 0.01, P = 0.99); dose × tank interactions were not statistically significant (ANOVA, Fratio = 0.70, P = 0.69). The mean severity among control salmon was significantly higher (Tukey, P b 0.01) than in any treated group (Fig. 2B). The mean severity among fish on 2.5 g kg − 1 nicarbazin was not different (Tukey, P = 0.209) from those on 5.0 g kg − 1 but was significantly higher than those on 10 or 25 g kg − 1 (Tukey, P b 0.01 both cases). Differences in mean severity among fish on 5,
Table 2 Weight and condition factor of Atlantic salmon fed diets containing nicarbazin. Dietary nicarbazin (g kg− 1)
4,4′-dinitrocarbanilide g kg− 1 Expected
Observeda (% expected)
0 1.8 3.5 7.1 17.7 177.3
0 1.4 (78) 3.3 (94) 6.2 (87) 15.3 (86) 160.6 (91)
Weight (g) Mean1 ± s.e. Range
0
2.5
5
10
25
204.4 ± 3.0a 238–286
175.4± 2.8b 118–270
172.2 ± 3.1b 88–269
143.8 ± 2.0c 98–214
145.5± 3.1c 99–318
1.09± 0.01a 0.79–1.33
1.08± 0.01a 0.89–1.44
1.04 ± 0.01 0.86–1.21
1.08 ± 0.01a 0.94–1.37
Weight length− 3 Mean± s.e. 1.14± 0.01 Range 0.99–1.32
1. The same superscripts indicate a probability less than 5% that mean values are different.
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Table 3 LC/MS/MS measured concentrations of 4,4′-dinitrocarbanilide (ng g− 1) in kidney and liver of Atlantic salmon on experimental diets containing various concentrations of nicarbazin (mean ± std. error and range). Dietary nicarbazin (g kg− 1)
Liver (n = 10)
Muscle (n = 10)
P-valuea
0.0 2.5 5.0 10.0 25.0
4.8 ± 2.2 (0–20.0) 3721.4 ± 365.9 (1336.4–5062.2) 11,168.3 ± 1631.3 (168.5–16,620.0) 19,998.9 ± 2464.7 (5927.9–28,521.7) 87.4 ± 31.0 (14.0–261.4)
0.2 ± 0.2 (0–1.6) 344.7 ± 38.6 (129.9–506.3) 1479.5 ± 266.9 (31.6–2631.2) 2920.8 ± 473.5 (423.9–4635.9) 3.8 ± 0.7 (0–7.3)
0.068 b 0.001 b 0.001 b 0.001 0.023
a
Probability that differences between mean tissue (liver and muscle) concentrations are the same (Kruskal–Wallis).
10 or 25 g kg − 1 nicarbazin (Fig. 2B) were not statistically significant (Tukey, P b 0.01 all cases). Foci of hepatocellular degeneration and necrosis were only observed in histological liver preparation from two of 15 salmon on 10 g kg − 1 nicarbazin diets (Fig. 3). The development of a fibrous capsule was associated with some lesions. A rust-red discolouration of the skin was observed in salmon fed diets containing 5 to 25 g kg − 1 nicarbazin. The discolouration was most evident in naturally unpigmented skin but was also visible on the dorsal surface of the head of swimming fish. The discolouration disappeared between 10 and 29 days after the resumption of feeding on the non-medicated diet. 4. Discussion K. thyrsites was first detected by histology and PCR in Atlantic salmon smolts after a 165-day (1451 degree-days) exposure to
Fig. 2. The effects of dietary nicarbazin concentration on Kudoa thyrsites in histological sections of skeletal muscle from Atlantic salmon (Salmo salar) smolts collected after 231 days exposure to seawater. Bars at each concentration are values from individual tanks (n = 95 to 103 per group). A, Estimated prevalence of K. thyrsites. B, Severity of K. thyrsites (mean ± std. error).
Departure Bay seawater. Previously, the parasite was first detected by light microscopy after 63 and 91 days (Moran et al., 1999) or after 2 months (1000 degree-days) (Moran and Kent, 1999) of exposure to Departure Bay seawater. The delay in the time of first detection in the present study may be related to the absence or low concentration of infective stages in the water at this site between December and May (Moran and Kent, 1999). Despite this, the parasite was eventually detected in almost all non-medicated Atlantic salmon smolts, providing a reference against which the efficacy of nicarbazin was assessed. Two patterns of infection were evident among fish treated with nicarbazin: a delay in the first detection of infection and lower prevalence and severity of infection at the end of the trial. Previously, nicarbazin fed continuously at 6–14 mg kg − 1 or 30–60 mg kg − 1 for 12 months was found to be inefficacious against M. cerebralis in rainbow trout (Taylor et al., 1973). Although the latter
Fig. 3. Light micrographs of histological sections of liver from Atlantic salmon (Salmo salar) following 10 g kg− 1 nicarbazin treatment. Foci of hepatocellular degeneration and necrosis before (A) and during (B) formation of fibrous capsule. Haematoxylin and eosin stain.
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study did not describe methods for the preparation of medicated diets and nicarbazin residues were not titrated in feed or fish tissues (Taylor et al., 1973), all diets were palatable, indicating that refusal to eat did not explain the absence of efficacy. The tropism of M. cerebralis for cartilage, in contrast with that of K. thyrsites for skeletal muscle, may have reduced exposure to the compound during parasite development. Similarly in another study, dietary nicarbazin (0.1 g kg − 1) had no effect on infection with the ciliate Ichthyophthirius multifiliis in the skin of rainbow trout (Shinn et al., 2003). In contrast, the present data demonstrated the efficacy of nicarbazin, incorporated into the diet at concentrations ranging from 2.5 to 25 g kg − 1, against K. thyrsites in seawater-reared Atlantic salmon. Dinitrocarbanilide (DNC) was detected in skeletal muscle and liver of salmon following the 231-day trial. Mean DNC concentrations in liver were higher than in muscle in salmon on diets ranging from 2.5 to 10 g kg − 1 nicarbazin. Furthermore, the concentration of DNC in both tissues was proportional to that in feed over this range, but was reduced in the 25 g kg − 1 group. Nicarbazin is used as a feed additive for the treatment of coccidiosis in broiler chickens up to 28 days old and as with salmon, the compound is retained in avian liver at concentrations higher than in muscle (Penz et al., 1999). DNC possesses anti-coccidial properties (Chapman, 1993) and is the residue marker for nicarbazin in poultry, with a maximum limit of 200 ng g − 1 in broiler tissues (McCarney et al., 2003). The low mean DNC residues in the 25 g kg − 1 salmon group (87 ng g − 1 liver, 4 ng g − 1 muscle) was probably related to reduced ingestion of nicarbazin because of the reduced palatability at this concentration and to the premature cessation of this diet in two of four medication intervals. However, additional data are required to more precisely determine the rate of reduction of DNC in salmon tissues. The adverse effects of dietary nicarbazin in salmon were dosedependent and included reduced weight, lower condition factor and elevated mortality. Reduced weight gain and the associated loss of condition, particularly among salmon fed diets containing 10 and 25 g kg − 1 nicarbazin, resulted from reduced feed intake, associated with low palatability of nicarbazin at these concentrations, despite supplementation with krill. Normal feeding behaviour was reestablished following the resumption of the non-medicated diet. While there are few published data for nicarbazin in fishes, dietary fumagillin was also associated anorexia and reduced weight in fish (Athanassopoulou et al., 2004; Sitjà-Bobadilla and Alvarez-Pellitero, 1992). In addition, mortality was elevated among salmon within the 10 and 25 g kg − 1 nicarbazin groups which at the latter dose confirmed our earlier observations (S. Jones, unpublished data). The mechanism of mortality may be related to the liver pathology however more research is required to fully document the development of hepatic and possibly other lesions associated with therapeutic and supratherapeutic nicarbazin doses in Atlantic salmon. The rustcoloured discolouration of the skin among salmon on diets containing 5 to 25 g kg − 1 nicarbazin was associated with the occurrence of DNC in skin at levels similar to those in measured in liver. Associated histological changes were not evident in skin and the discolouration disappeared following resumption of control diets. The return to normal colouration was particularly rapid (10 days) in fish on 25 g kg − 1 following the resumption of the control diet and was associated with DNC residues in skin that were significantly reduced. In summary, although its mode of action is not known (Lindsay and Blagburn, 1995), dietary nicarbazin was efficacious against K. thyrsites in seawater-reared Atlantic salmon. In salmon, DNC residues in skin, liver and muscle were proportional to dietary concentrations. The higher concentrations of nicarbazin used here reduced palatability of diets leading to reduced weight gain and condition factor. Mortality was elevated in proportion to nicarbazin concentration in the diet. However, these adverse effects ceased upon resumption of non-medicated diets. Further work is required to develop a treatment regime that retains the efficacy of the compound,
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minimises the adverse effects and provides data on DNC residuals in Atlantic salmon upon resumption of non-medicated diets.
Acknowledgements We thank the support of Marine Harvest Canada, Mainstream Canada, Grieg Seafoods, Ewos Canada and the Aquaculture Collaborative Research and Development Program. Thanks also to E. Kim, A. Yu Xin and H. Jones for maintaining the fish, collecting samples and conducting microscopic examinations and to M. Rowshandeli for mixing and preparing the diets.
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