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In vitro and in vivo efficacy of anthelmintic compounds against blood fluke (Cardicola forsteri)
Aquaculture 334–337 (2012) 39–44
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In vitro and in vivo efficacy of anthelmintic compounds against blood fluke (Cardicola forsteri) Paul Hardy-Smith a, 1, David Ellis b, c, 1, John Humphrey a, Mathew Evans c, Daryl Evans c, Kirsten Rough b, Victoria Valdenegro c, Barbara Nowak c,⁎ a b c
Panaquatic® Health Solutions Pty Ltd, 26A Liddiard Street, Hawthorn, Victoria, 3122, Australia ASBTIA Australian Southern Bluefin Tuna Industry Association, PO Box 1146, Port Lincoln, South Australia 5606, Australia NCMCRS, University of Tasmania, Locked Bag 1370, Launceston 7250 Tasmania, Australia
a r t i c l e
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Article history: Received 22 September 2011 Received in revised form 19 December 2011 Accepted 20 December 2011 Available online 30 December 2011 Keywords: Southern Bluefin Tuna Ranching Fish parasite Treatment
a b s t r a c t Blood fluke, Cardicola forsteri, infects Southern Bluefin Tuna, particularly during ranching. Efficacy of four anthelmintics was tested against this parasite. There was an agreement between in vitro and in vivo results. Praziquantel was the only effective anthelmintic. It was the most potent anthelmintic in decreasing fluke responsiveness in vitro, with concentrations ranging between 1.5 μg/mL and 200 μg/mL stopping adult fluke response within less than 5 min. In vivo, both the higher (150 mg/kg) and the lower (75 mg/kg) dose praziquantel treatment resulted in a significant reduction of the number of flukes present in the hearts. A significant effect of treatment on the mean number of blood fluke eggs per cm 2 of tuna myocardium was observed, with fish treated with either of the two doses of praziquantel having at least 6 times lower numbers of eggs in their hearts. Control fish and fish treated with praziquantel (both doses) and lower dose Closal had very low average number of eggs per cm 2 of gill and were significantly lower than in fish treated with fenbendazole. While this research shows that praziquantel is the treatment of choice against blood fluke, C. forsteri, further research is needed to determine optimum dose, best treatment application method, palatability and any potential side effects. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Ranching of Southern Bluefin Tuna (SBT) Thunnus maccoyii commenced in Australia in 1991 as a result of value adding to the wild catch of SBT. Since then SBT ranching has developed into the second most productive (both by volume and value) aquaculture finfish industry in Australia. The industry is based on the capture of wild fish which are conditioned over a few months and sold principally to the Japanese sashimi market (Farwell, 2001). The fish are towed in pontoons from the capture site in the Great Australian Bight to ranching sites near Port Lincoln, South Australia. After approximately 2– 9 months fish are harvested and marketed both domestically and internationally (Aiken et al., 2006; Colquitt et al., 2001). There are few health problems during ranching, possibly due to the age of the fish at capture and the short ranching time (Deveney et al., 2005; Nowak, 2004). Blood fluke, Cardicola forsteri (Trematoda Aporocotylidae) was described from ranched SBT (Cribb et al., 2000), which is the final host, while a marine polychaete Longicarpus modestus is the intermediate host (Cribb et al., 2011). This parasite mostly infects SBT during ⁎ Corresponding author. Tel.: + 61 3 63243814; fax: + 61 3 63243804. E-mail address: [email protected] (B. Nowak). 1 These authors contributed equally to this paper. 0044-8486/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2011.12.037
ranching as the intensity and prevalence of infections are very low in the wild (Aiken et al., 2007), and at transfer (Aiken et al., 2006), and an antibody response against C. forsteri increases only after transfer to the ranching zone from the wild (Aiken et al., 2008). Epizootics of this blood fluke have been reported in ranched SBT (Aiken et al., 2006, 2008, 2009; Colquitt et al., 2001; Cribb et al., 2000; Dennis et al., 2011; Kirchhoff et al., 2011) with the prevalence of infection reaching 100% within 2 months post-transfer to ranching pontoon (Aiken et al., 2006; Kirchhoff et al., 2011). Although in the past infections with C. forsteri did not appear to result in clinical disease in the host (Aiken et al., 2006, 2008; Colquitt et al., 2001), more recently an increase in the intensity of infection was reported, which coincided with peaks of tuna mortalities (Dennis et al., 2011; Hayward et al., 2010). Based on the association between the increased intensity of C. forsteri and the mortalities in SBT, clinical trials to investigate the efficacy of anthelmintics in the treatment of C. forsteri were undertaken. Anthelmintics have been used to treat farmed fish species against monogeneans, digeneans and cestodes. A number of different classes of these compounds were considered as potential candidates for these trials including an isoquinolone, three benzimidazoles and a salicylanilide. Praziquantel, an isoquinolone, causes spastic paralysis in trematodes (flukes) as well as damaging the parasite tegument (Taylor et al., 2007). Praziquantel has been used in fish against
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monogeneans (Schmahl and Mehlhorn, 1985; Sharp et al., 2004; Sitjà-Bobadilla et al., 2006; Williams et al., 2007), digeneans (Björklund and Bylund, 1987) and intestinal cestodes (Sanmartin Duran et al., 1989). Importantly, it is the drug of choice to treat schistosomiasis in man (Doenhoff et al., 2008). Albendazole, fenbendazole and triclabendazole are all benzimidazoles, a class of anthelmintic considered generally to be safe compounds which are used widely in livestock (Taylor et al., 2007). Closantel, a salicylanilide, was also considered for these trials. Albendazole, fenbendazole, triclabendazole and tetramisole have been tested as a treatment against a wide range of fish parasites, triclabendazole and fenbendazole were effective against monogeneans, albendazole was effective against flagellate Hexamita salmonis infection in rainbow trout (Tojo and Santamarina, 1998a, 1998b, 1998c; Tojo et al., 1992). The aim of this study was to determine the in vitro and in vivo efficacy of anthelminthic drugs against C. forsteri infection of SBT and to assess the reliability of in vitro screening. 2. Materials and methods 2.1. In vitro trial 2.1.1. Heart processing and live fluke isolation Up to 150 hearts were collected and stored in a large icebag during SBT commercial harvests. Hearts were dissected lengthwise from apex to base and 3–5 cuts were made in the internal surface to increase the flushing area. Ten to fifteen hearts were placed in individual 2 L bags, along with any remaining blood and approximately 300 mL of saline solution was added to the bags, which were then shaken. Heart flushes and blood were then poured into 500 mL plastic containers and allowed to settle for 20 min. Supernatant was removed and the remaining liquid (approximately 60 mL) decanted into 4–12 Petri dishes, diluted with saline and left to settle for 2 min. Live C. forsteri were observed using a dissecting microscope and transferred to a storage solution (saline solution + 25 μg/mL gentamicin sulphate) for the duration of fluke isolation. 2.1.2. Live fluke preparation and testing To assess toxicity of anthelmintics to flukes, the effect of different concentrations of the tested compounds on responsiveness of the adult flukes was determined over time. C. forsteri were deemed responsive if any movement was observed following 3–5 s of manual plate shaking. Live C. forsteri were isolated from hearts and transferred with a pipette in 100 μL aliquots from the storage solution into a 12-well plate, separated into groups of 10–20 flukes and washed three times in 2 mL Dulbecco's Modified Eagle's Medium (DMEM). The plates were then incubated at 20 °C for 1 h. After 1 h any nonresponsive individuals were removed from the test. Remaining live flukes were re-incubated under the same conditions and any flukes that seemed to have lost responsiveness were replaced with responsive flukes. Toxicity testing was undertaken in 3 mL DMEM containing 25 μg/mL gentamicin sulphate per well. This medium was chosen on the basis of in vitro models for helminths, particularly schistosomes (Keiser, 2010) and our own experience with maintaining C. forsteri adults in vitro (F. Van Ede, P. Crosbie and B. Nowak unpublished). While the use of blood or blood serum has been suggested, it is not recommended for in vitro studies (Keiser, 2010), most likely as it would introduce another source of variability into multiple in vitro experiments. For each anthelmintic, multiple concentrations were assessed to determine their effects on fluke responsiveness, with each concentration having 3 replicate wells, and each replicate well containing 5 flukes, which were responsive at the start of the experiment. Wells were assigned randomly to the anthelmintic concentrations, and 10 μL of each drug concentration was added to wells. Four different anthelmintics were tested: praziquantel, fenbendazole,
tetramisole chloride and closantel (Table 1). Depending on the treatment, 10 μL of dimethyl sulfoxide (DMSO) or distilled H2O was added to control wells. All reagents were purchased from Sigma Aldrich Pty Ltd. The commercial praziquantel preparation is a racemate composed of equal parts of “levo” R(−) and “dextro” S(+) isomers (Cioli and Pica-Mattoccia, 2003). Pilot experiment showed that 0.3% DMSO had no effect on responsiveness of the blood flukes for at least 50 h (results not shown). Plates were incubated at 20 °C, and regularly checked for the number of responsive flukes. The final time point for assessment of the effect of treatment was 48 h. 2.2. In vivo treatment 2.2.1. Anthelmintics and experimental doses Four compounds, Prazifish (All Farm Animal Health), Closal® (Coopers Animal Health), Panacur 100® (Intervet Schering Plough Animal Health) and Fasinex 240® (Novartis Animal Health), were used individually and in combination in this trial. Two compounds (Prazifishl and Closal®) were used at two different dose rates. The dose given to each SBT under each treatment regime is shown in Table 2. Treatment was timed to coincide with increasing fluke burdens in the SBT and prior to the mortality peak observed approximately 6–8 weeks post transfer to the grow out cage. SBT were treated 27 days after transfer from the towing pontoon to the ranching pontoon and a total of 63 days post capture. All treatments were administered as a single dose to a total of twenty SBT for each treatment group. None of these compounds are registered in Australia for treatment of fish destined for human consumption. Each treatment was administered by a registered veterinarian. In addition, a license condition of aquaculture farms in South Australia is that any use of an unregistered compound, even under veterinary direction, must be given Ministerial approval. 2.2.2. Experimental fish and administration of anthelmintic Individual fish were caught using a baited barbless hook and handline and placed in a padded capture cradle. A moistened towel was placed over the eyes of the SBT. The length of the SBT was measured and two identification tags were inserted. A Kruuse No. 26 Small Animal plastic stomach tube was used for stomach tubing. Prior to insertion a fiberglass stylet was inserted into the tube to provide increased rigidity. A small amount of vegetable oil was then applied to the tip of the tube and the tube inserted through the mouth using a piece of hard PVC piping (the “bite pipe”) to protect the softer stomach tube from the teeth of the SBT. Once the tip of the stomach tube had entered the oesophagus the fibreglass stylet was withdrawn as the tube was advanced into the stomach. Location of the tube within the stomach was confirmed by stomach ingesta tracking back out of the tube. The syringe containing the appropriate concentration of anthelmintic was then attached to the end of the stomach tube and the treatment administered. A further 10–15 ml of diluted saline was then flushed through the stomach tube to ensure no treatment was left within the dead space of the stomach tube. The stomach tube near where the syringe attached was then crimped and the tube withdrawn back out through the “bite pipe”. The fish was then released into the treatment pontoon. Tuna were medicated based on their individual size at the dose rates shown in Table 2. Mortalities were monitored post treatment. Twenty five percent of fish were not recovered at the end of the trial, with the loss attributed to a combination of mortality, poaching and/or predation by seals. 2.2.3. Sample collection Twenty four days after treatment, all SBT remaining in the treatment pontoon were euthanised. The treatment to which the fish was assigned was identified using the tags inserted at the time of treatment. Immediately after euthanasia, a sample of gill filaments
P. Hardy-Smith et al. / Aquaculture 334–337 (2012) 39–44
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Table 1 Treatments used in the experiments and their effect on blood fluke, Cardicola forsteri, in vitro and in vivo. * Active ingredient tested in vitro and dissolved in DMSO before testing, NT – not tested, ND – not determined – none of the tested concentrations or doses significantly reduced survival of the flukes, LD – low dose, HD – high dose. Treatment
Active ingredient (content)
Distributor
Dose tested in vivo (mg/kg) Concentrations Lowest effective tested in vitro concentration (μg/mL)
Lowest effective dose (mg/kg)
Closal
Albendazole (19 g/L) Closantel (37.5 g/L) Triclabendazole (240 g/L) Fenbendazole (100 g/L) Praziquantel (98%)
Coopers Animal Health
0.5–10*
ND
ND
Novartis Animal Health Australasia Pty Ltd Virbac Australia Allfarm Animal Health
NT
NT
0.625–100* 0.0625–200*
10 0.125
2, 4, 6- Trichlorobenzoyl Sigma Aldrich chloride (TCB)
0.782–100
ND
LD 4.1 mg/kg albendazole, 8.25 mg/kg closantel HD 12.5 mg/kg albendazole 24.75 mg/kg closantel 20 mg/kg in combination with Panacur100 (100 mg/kg fenbendazole) 100 LD 75 HD 150 NT
Tetramiole chloride (TM)
6.25–100
50
NT
Fasinex240 Panacur100 Prazifish 2, 4, 6- Trichlorobenzoyl chloride (TCB) Tetramiole chloride (TM)
Sigma Aldrich
was removed from the left second gill arch of each fish and immediately placed into 10% neutral buffered formalin. The heart was exposed in situ and the ductus arteriosus and the ventral aorta were then clamped using haemostats as close to the heart as possible before the heart was removed intact. This ensured that no fluke escaped from the heart during its removal from the fish. When the heart was placed in its container, the clamps were removed. Heart samples were taken from ventricle, atrium and bulbous arteriosus and placed into neutral buffered formalin and processed for histological examination. 2.2.4. Adult C. forsteri counts in heart flushes The hearts were opened longitudinally and flushed until the rinse was clear with diluted seawater (1:2 seawater to regular water) in order to dislodge any adult C. forsteri. Flushes were poured into petri dishes and allowed to settle for 20 min. Then, the flushes were observed under a dissecting microscope (Olympus SZX12 stereomicroscope, Olympus, Japan) for the presence of adult C. forsteri. Parasite numbers were recorded for each fish. The counts were done blind, with no sample identified back to the treatment. 2.2.5. Histology Samples for histological examination were collected from gills and heart. Three 2 cm wide gill sections were obtained from the second gill arch of the left-side holobranch (cranial, middle and ventral sections). Samples of atrium, bulbus arteriosus and the caudal tip of the ventricle were fixed in 10% neutral buffered formalin for 24–48 h. Subsequently, 3 mm thick sections were cut and placed into a histology cassette. The samples were processed using standard histological techniques. Gills inside the cassettes were decalcified for 1 h using Rapid Decalcifying Fluid (Australian Biostain, Australia) and then washed with water for 1 h. After this initial step, all samples were processed, embedded into paraffin blocks and 5 μm sections were obtained. Slides were then stained with Haematoxylin-Eosin (H&E) and examined under light microscope (Leitz Diaplan microscope, Leica Microsystems, Germany) at 100× magnification and details at 400× or 1000× under oil immersion. 2.2.6. Egg and granulomas counts C. forsteri eggs and granulomatous reactions were assessed in all 3 sections of heart and gill from each fish. Structures counted as eggs had a spherical shape from 19 to 25 μm in diameter as described by Colquitt et al. (2001). Granulomas were identified as encapsulated eggs with a reaction consisting of epithelioid cells and lymphocytes surrounded by fibroblasts and fibrocytes in older lesions following the description provided by the same authors (Colquitt et al., 2001). Counts from the entire area of a section were obtained. The area of
ND ND 75 NT
NT
the sections was measured using Image J Image processing and analysis in java software (Maryland, USA). An average number of eggs per cm 2 of gill or heart was then obtained from the total area of the 3 independent (each from a different block) sections of each organ. All samples were coded to avoid observer bias. 2.3. Statistical analyses All data for both in vitro and in vivo trials were analysed by comparing treatments using a one-way ANOVA followed by a Tuckey's HSD post-hoc test. Levene's test was used to test if the variances were homogenous and normality plots to determine if the data were normally distributed. Data from the in vivo trial violated assumptions of the ANOVA test, therefore data sets for number of eggs per cm 2 of heart and C. forsteri counts in heart flushes were analysed after being transformed by square root. Number of eggs per cm 2 of gill had to be transformed by log10. As a result statistical analyses were carried out with transformed data, but results are presented with raw data. All statistical analyses were performed using the free software R: a language and environment for statistical computing (R Development Core Team, Vienna, Austria). P-value b0.05 was considered a significant difference. 3. Results 3.1. In vitro tests Praziquantel was the most potent anthelmintic in decreasing fluke responsiveness in vitro. Upon addition of praziquantel concentrations ranging between 1.5 μg/mL and 200 μg/mL to wells containing vigorous flukes, the flukes stopped responding to the shaking of the plate within less than 5 min. The threshold at which praziquantel affected Table 2 In vivo treatments and their doses, and the final number of fish examined at the end of the experiment for presence of adult blood flukes and eggs in heart and gills. Treatment
Active component
Dose (mg/kg of Number of fish at SBT biomass) sampling
Prazifish lower dose Praziquantel 75 Prazifish higher dose Praziquantel 150 Closal lower dose Closantel albendazole 8.25 4.1 Closal higher dose Closantel 24.75 Albendazole 12.5 Panacur 100 Fenbendazole 100 Panacur 100 + Fenbendazole 100 Fasinex 240 Triclabendazole 20 Control Saline 30 mL per SBT
13 16 13 15 14 12 25
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C. forsteri was established between 0.0625 μg/mL and 0.125 μg/mL. Concentrations of 0.125 μg/mL and above resulted in a significant reduction in the mean number of responsive flukes after 48 h of exposure (F = 58.440; df 5, 12; P b 0.001), while 0.0625 μg/mL showed no significant difference on the average number of responsive flukes when compared to the control (0 μg/mL). Fenbendazole did not show an immediate effect on responsiveness of flukes as did praziquantel. After two days of exposure, however, concentrations of 10 μg/mL and above significantly affected fluke responsiveness (F = 23.880; df 5, 12; P b 0.001). Tetramisole chloride at concentrations of 50 μg/mL and above significantly decreased the responsiveness of the flukes at the 48 h end time point, compared to concentrations equal to or less than 12.5 μg/mL (F = 31.125; df 5, 12; P b 0.001). The testing with Closantel was inconclusive due to the small number of flukes available for these assays, resulting in large variation between replicates. No significant differences in the responsiveness of the flukes to Closantel could be found (F = 2.846, df 2, 6, P = 0.135).
Average number of Cardicola forsteri eggs per cm2 in hearts of Thunnus maccoyii + SE
42
7 a
6 5 4
a a
a a
3 2 1
b b
0
3.2. In vivo experiment
Average number of Cardicola forsteri in heart flushes from Thunnus maccoyii SE
The mean number of blood flukes C. forsteri observed in heart flushes of SBT was significantly different among treatments (F = 17.46, df 6,101, P b 0.001). Both the higher and the lower dose praziquantel treatment resulted in a significant reduction of the number of flukes present in the hearts, with heart flushes obtained from these fish showing on average 2 blood flukes or fewer. Comparing to the controls the reduction in adult blood flukes in the praziquantel treated fish ranged from 91 to 95%. There was no significant difference between the high and low concentrations. Heart flushes from fish subjected to the other anthelmintic treatments and the controls had average fluke counts 9 times or higher than those observed in heart flushes from fish treated with the higher and lower praziquantel (Fig. 1). A significant difference was observed in the mean number of blood fluke eggs per cm 2 of SBT myocardium (F = 15.26, df 6,101, P b 0.001). The fish which were subjected to either of the two doses 35
a a
30
a
a
25 a
20 15 10 5
b b
Treatments Fig. 2. Effect of treatment on the mean number of blood fluke (Cardicola forsteri) eggs per cm2 of myocardium obtained from SBT (Thunnus maccoyii). Averages with different small letters (a, b) are significantly different from one another by one-way ANOVA. Closal LD – 4.1 g/kg albendazole and 8.25 g/kg closantel, Closal HD – 12.5 g/kg albendazole and 24.75 g/kg closantel, Prazifish LD – 75 mg/kg praziquantel, Prazifish HD – 150 mg/kg praziquantel, Panacur 100–100 mg/kg fenbendazole, Panacur 100 + Fasinex 240–100 mg/kg fenbendazole and 20 mg/kg triclabendazole.
of praziquantel had a significantly reduced number of eggs per cm 2, and these numbers were at least 6 times lower than those observed in fish treated with other anthelmintics or control group (Fig. 2). The number of eggs per cm 2 of gill was significantly affected by treatment (F = 4.32, df 6,101, P b 0.001). However, the pattern showed by the number of fluke eggs in the gills was different to what was observed for the eggs in myocardium and for the flukes found in the heart flushes. Control fish and fish treated with praziquantel (both doses) and lower dose Closal presented very low average number of eggs per cm 2 of gill – the highest being an average of 8 eggs per cm 2 – and these means were different from the mean observed in fish treated with fenbendazole (Fig. 3). Fish treated with fenbendazole showed the largest mean number of eggs per cm 2 of gill, and although this average was 1.5 and 3 times larger than the average number of eggs in gills for fish treated with fenbendazole/triclabendazole combined and the higher dose Closal respectively, these three treatments were not significantly different from each other. In contrast to the even distribution of blood flukes eggs in the myocardium, eggs in the gills are found in “patches”. The angle of sectioning and the depth of sectioning could affect results from the gills more than from heart due to the complex three dimensional structure of the gills.
0
4. Discussion
Treatments Fig. 1. Effect of treatment on the mean number of blood flukes (Cardicola forsteri) in heart flushes obtained from SBT (Thunnus maccoyii). Averages with different small letters (a,b) are significantly different from one another by one-way ANOVA. Closal LD – 4.1 g/kg albendazole and 8.25 g/kg closantel, Closal HD – 12.5 g/kg albendazole and 24.75 g/kg closantel, Prazifish LD – 75 mg/kg praziquantel, Prazifish HD – 150 mg/kg praziquantel, Panacur 100–100 mg/kg fenbendazole, Panacur 100 + Fasinex 240– 100 mg/kg fenbendazole and 20 mg/kg triclabendazole.
This study provides evidence that the drug praziquantel may be used as an efficacious anthelmintic for the treatment or control of blood fluke C. forsteri in SBT. This is in agreement with the success of the experimental use of oral praziquantel administration to treat blood fluke infections in Pacific Bluefin Tuna (Shirakashi et al., 2012). While the effective doses from this study and reported by Shirakashi et al. (2012) are different, the products used, parasites, hosts and application methods differed. This study provides a mechanism for clinical field trials to evaluate praziquantel against C. forsteri infections. Praziquantel offers an effective control against C. forsteri with 91–95% reduction in the infection
Average number of Cardicola forsteri eggs per cm2 in gills of Thunnus maccoyii SE
P. Hardy-Smith et al. / Aquaculture 334–337 (2012) 39–44
180 160
a
140 120
ab
100 80 ab
60 40 20
b b
b
b
0
Treatments Fig. 3. Effect of treatment on the mean number of blood fluke (Cardicola forsteri) eggs per cm2 of gills obtained from SBT (Thunnus maccoyii). Averages with different letters are significantly different from one another by one-way ANOVA. Closal LD – 4.1 g/kg albendazole and 8.25 g/kg closantel, Closal HD – 12.5 g/kg albendazole and 24.75 g/kg closantel, Prazifish LD – 75 mg/kg praziquantel, Prazifish HD – 150 mg/kg praziquantel, Panacur 100–100 mg/kg fenbendazole, Panacur 100 + Fasinex 240–100 mg/kg fenbendazole and 20 mg/kg triclabendazole.
at the tested doses. The next step will be to determine the lowest effective dose and establish pharmacodynamics of praziquantel. Praziquantel was the only anthelmintic efficacious against adult C. forsteri at the concentrations tested in vitro and doses applied in vivo, while other compounds were efficacious in vitro after 48 h they were slower acting and the effective concentrations were greater. No other anthelmintic tested showed promising results in vivo. This suggests that praziquantel could be used against C. forsteri infections of ranched SBT. However, development of resistance should be monitored as resistance against praziquantel has been reported in some schistosomes (Doenhoff et al., 2008, Melman et al., 2009). The demonstration that praziquantel was efficacious in both in vitro and in vivo trials suggests that in vitro trials can be used for future investigations of the effects of treatment on C. forsteri, including screening of novel treatments or investigation of development of resistance to different treatments. Similar approach has been used with other parasites, including those affecting humans (Keiser, 2010). However, the use of in vitro treatment has to be applied with caution as some medications may require to be metabolised before they can be effective. Concentrations of the active compound should be determined and compared between serum of treated animals and media for in vitro testing. This was however outside the scope of this study. In the current study, a single treatment with praziquantel was used. Praziquantel is, however, rapidly cleared from treated fish (Kim et al., 2001; Tubbs and Tingle, 2006) and treatments may need to be repeated if the tuna are exposed more than once to the infective stages of blood flukes during their ranching period. Two major C. forsteri infection events in the first year of SBT ranching have been suggested by stochastic modelling (Aiken et al., 2009). Reinfection with adult C. opisthorchis was suggested in the experimental praziquantel treatment of Pacific Bluefin Tuna (Shirakashi et al., 2012). Praziquantel was effective against adult C. forsteri at a concentration of 0.125 μg/mL in vitro. This is a higher toxicity than reported against other parasites, however equivalent low concentrations were not tested in the previous studies. Praziquantel was reported to be
43
efficacious in vitro against monogeneans, including Heterobothrium okamatoi larvae at 20 μg/mL (Hirazawa et al., 2000) and Pseudodactylogyrus bini at 10 μg/mL (Buchmann, 1987). Similarly, Xiao et al. (2009) demonstrated the effective praziquantel concentration against adult schistosomes as 1–30 μg/mL in vitro. However, it has been reported that if praziquantel was replaced with drug-free medium after 1 or 4 h, adult schistosomes could recover (Xiao et al., 2009). This potential for recovery should be further investigated for C. forsteri, especially given the rapid clearance of praziquantel from SBT. While the reduction in the number of eggs in the heart and gills in SBT treated with praziquantel was related to the reduction in the number of adults in heart, the additional significant reduction of number of eggs in the gills in the control tuna and tuna treated with Closal LD was unexpected and could have been due to the patchiness of egg distribution in the gills observed in our study. Overall, the numbers of adults recovered from the heart corresponded much better to the density of eggs in the heart than to the density of eggs in the gills. Further work is required to investigate the distribution and identification of the eggs in the gills. Immature stages of Schistosoma mansoni were shown to be insensitive to praziquantel (Xiao et al., 1985). Praziquantel is highly effective in killing adult schistosome worms, but it is unable to kill developing schistosomes and so does not prevent reinfection (Gray et al., 2010). While there was a reduction in the number of eggs in gills and hearts of SBT treated with praziquantel, it is possible that this could have been due to the reduction of the number of adults or a lower production of eggs and not due to a direct toxic effect of praziquantel on the eggs themselves. Eggs in gills remained viable after treatment with praziquantel suggesting that the treatment did not kill eggs or miracidia of blood flukes affecting T. orientalis (Shirakashi et al., 2012). Reinfection rates post-treatment should be investigated for ranched SBT. In vitro trials used only one life stage of the parasite (adults), sensitivity of other life stages is currently unknown and while eggs or miracidia could be also tested. The most relevant life stages, for example cercariae are currently not available for experiments. Adult parasites can be obtained from infected SBT, however as most of the fish are harvested after the peak of infection the availability of the adults for in vitro testing can be limited. Viability of the adult flukes limited the scope of in vitro testing. Oral intubation allows delivery of an exact dose to each animal and provides an accurate means of ensuring similar dosages for comparative experimental studies. However, this administration method is not viable on a commercial scale (Williams et al., 2007). Commercial oral treatment will require in-feed medication. Further research is needed to determine the optimum dose, best treatment application method, palatability and any potential side effects as well as potential development of resistance of the parasite against the treatment. Role of funding body Fisheries Research and Development Corporation has funded this project. SBT Research Council approved milestone reports and the final version of this manuscript. Acknowledgements We are grateful to Southern Bluefin Tuna industry for their support, including fish husbandry. We would like to thank Daniel Pountney, Karine Cadoret and Claire Webber for their assistance with sampling and initial processing of samples. We are grateful to Francoise van Ede and Phil Crosbie for their contributions to the development of the in vitro method. We would like to thank Rob Jones for providing valuable advice on the stomach tubing of large fish. This project was funded by Fisheries Research and Development Corporation.
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Kirchhoff, N., Leef, M.J., Ellis, D., Purser, J., Nowak, B.F., 2011. Effects of the first two months of ranching on the health of southern bluefin tuna Thunnus maccoyii. Aquaculture 315, 207–212. Melman, S.D., Steinauer, M.L., Cunningham, C., Kubatko, L.S., Mwangi, I.N., Wynn, N.B., Mutuku, M.W., Karanja, D.M.S., Colley, D.G., Black, C.L., Secor, W.E., Mkoji, G.M., Loker, E.S., 2009. Reduced susceptibility to praziquantel among naturally occurring Kenyan isolates of Schistosoma mansoni. PLoS Neglected Tropical Diseases 3 (8) Art No. e504. Nowak, B.F., 2004. Assessment of health risks to southern bluefin tuna under current culture conditions. Bulletin of European Association of Fish Pathologists 24, 45–51. Sanmartin Duran, M.L., Caamano-Garcia, F., Fernandez, Casal J., Leiro, J., Ubeira, F.M., 1989. Anthelmintic activity of praziquantel, niclosamide, netobimin and mebendazole against Bothriocephalus scorpii naturally infecting turbot (Scophthalmus maximus). Aquaculture 76, 199–201. Schmahl, G., Mehlhorn, H., 1985. Treatment of fish parasites. 1. Praziquantel effective against monogenea (Dactylogyrus vastator, Dactylogyrus extensus, Diplozoon paradoxum). Parasitology Research 71, 727–737. Sharp, N.J., Diggles, B.K., Poortenaar, C.W., Willis, T.J., 2004. Efficacy of Aqui-S, formalin and praziquantel against the monogeneans, Benedenia seriolae and Zeuxapta seriolae, infecting yellowtail kingfish Seriola lalandi lalandi in New Zealand. Aquaculture 236, 67–83. doi:10.1016/j.aquaculture.2004.02.005. Shirakashi, S., Andrews, M., Kishimoto, Y., Ishimaru, K., Okada, T., Sawada, Y., Ogawa, K., 2012. Oral treatment of Praziquantel as an effective control measure against blood fluke 3 infection in Pacific bluefin tuna (Thunnus orientalis). Aquaculture 326–329, 15–19. Sitjà-Bobadilla, A., Conde de Felipe, M., Alvarez-Pellitero, P., 2006. In vivo and in vitro treatments against Sparicotyle chrysophrii (Monogenea: Microtylidae) parasitizing the gills of gilthead sea bream (Sparus auratus L.). Aquaculture 26, 856–864. Taylor, M.A., Coop, R.L., Wall, R.L., 2007. Veterinary Parasitology, 3rd Edition. Blackwell Oxford. Tojo, J.L., Santamarina, M.T., 1998a. Oral pharmacological treatments for parasitic diseases of rainbow trout Oncorhynchus mykiss. I: Hexamita salmonis. Diseases of Aquatic Organisms 33, 51–56. Tojo, J.L., Santamarina, M.T., 1998b. Oral pharmacological treatments for parasitic diseases of rainbow trout Oncorhynchus mykiss. II: Gyrodactylus sp. Diseases of Aquatic Organisms 33, 187–193. Tojo, J.L., Santamarina, M.T., 1998c. Oral pharmacological treatments for parasitic diseases of rainbow trout Oncorhynchus mykiss. III: Ichthyobodo necator. Diseases of Aquatic Organisms 33, 195–199. Tojo, J.L., Santamarina, M.T., Ubeira, F.M., Estevez, J., Sanmartin, M.L., 1992. Anthelmintic activity of benzimidazoles against Gyrodactylus sp. infecting rainbow trout Onchorhynchus mykiss. Diseases of Aquatic Organisms 12, 185–189. Tubbs, L.A., Tingle, M.D., 2006. Bioavailability and pharmacokinetics of a praziquantel bolus in kingfish, Seriola lalandi. Diseases of Aquatic Organisms 69, 233–238. Williams, R.E., Ernst, I., Chambers, C.B., Whittington, I.D., 2007. Efficacy of orally administered praziquantel against Zeuxapta seriolae and Benedenia seriolae (Monogenea) in yellowtail kingfish Seriola lalandi. Diseases of Aquatic Organisms 77, 199–205. Xiao, S.H., Catto, B.A., Webster Jr., L.T., 1985. Effects of praziquantel on different developmental stages of Schistosoma mansoni in vitro and in vivo. Journal of Infectious Diseases 151, 1130–1137. Xiao, S.H., Mei, J.Y., Jiao, P.Y., 2009. The in vitro effect of mefloquine and praziquantel against juvenile and adult Schistosoma japonicum. Parasitology Research 106, 237–246.
Contents lists available at SciVerse ScienceDirect
Aquaculture journal homepage: www.elsevier.com/locate/aqua-online
In vitro and in vivo efficacy of anthelmintic compounds against blood fluke (Cardicola forsteri) Paul Hardy-Smith a, 1, David Ellis b, c, 1, John Humphrey a, Mathew Evans c, Daryl Evans c, Kirsten Rough b, Victoria Valdenegro c, Barbara Nowak c,⁎ a b c
Panaquatic® Health Solutions Pty Ltd, 26A Liddiard Street, Hawthorn, Victoria, 3122, Australia ASBTIA Australian Southern Bluefin Tuna Industry Association, PO Box 1146, Port Lincoln, South Australia 5606, Australia NCMCRS, University of Tasmania, Locked Bag 1370, Launceston 7250 Tasmania, Australia
a r t i c l e
i n f o
Article history: Received 22 September 2011 Received in revised form 19 December 2011 Accepted 20 December 2011 Available online 30 December 2011 Keywords: Southern Bluefin Tuna Ranching Fish parasite Treatment
a b s t r a c t Blood fluke, Cardicola forsteri, infects Southern Bluefin Tuna, particularly during ranching. Efficacy of four anthelmintics was tested against this parasite. There was an agreement between in vitro and in vivo results. Praziquantel was the only effective anthelmintic. It was the most potent anthelmintic in decreasing fluke responsiveness in vitro, with concentrations ranging between 1.5 μg/mL and 200 μg/mL stopping adult fluke response within less than 5 min. In vivo, both the higher (150 mg/kg) and the lower (75 mg/kg) dose praziquantel treatment resulted in a significant reduction of the number of flukes present in the hearts. A significant effect of treatment on the mean number of blood fluke eggs per cm 2 of tuna myocardium was observed, with fish treated with either of the two doses of praziquantel having at least 6 times lower numbers of eggs in their hearts. Control fish and fish treated with praziquantel (both doses) and lower dose Closal had very low average number of eggs per cm 2 of gill and were significantly lower than in fish treated with fenbendazole. While this research shows that praziquantel is the treatment of choice against blood fluke, C. forsteri, further research is needed to determine optimum dose, best treatment application method, palatability and any potential side effects. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Ranching of Southern Bluefin Tuna (SBT) Thunnus maccoyii commenced in Australia in 1991 as a result of value adding to the wild catch of SBT. Since then SBT ranching has developed into the second most productive (both by volume and value) aquaculture finfish industry in Australia. The industry is based on the capture of wild fish which are conditioned over a few months and sold principally to the Japanese sashimi market (Farwell, 2001). The fish are towed in pontoons from the capture site in the Great Australian Bight to ranching sites near Port Lincoln, South Australia. After approximately 2– 9 months fish are harvested and marketed both domestically and internationally (Aiken et al., 2006; Colquitt et al., 2001). There are few health problems during ranching, possibly due to the age of the fish at capture and the short ranching time (Deveney et al., 2005; Nowak, 2004). Blood fluke, Cardicola forsteri (Trematoda Aporocotylidae) was described from ranched SBT (Cribb et al., 2000), which is the final host, while a marine polychaete Longicarpus modestus is the intermediate host (Cribb et al., 2011). This parasite mostly infects SBT during ⁎ Corresponding author. Tel.: + 61 3 63243814; fax: + 61 3 63243804. E-mail address: [email protected] (B. Nowak). 1 These authors contributed equally to this paper. 0044-8486/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2011.12.037
ranching as the intensity and prevalence of infections are very low in the wild (Aiken et al., 2007), and at transfer (Aiken et al., 2006), and an antibody response against C. forsteri increases only after transfer to the ranching zone from the wild (Aiken et al., 2008). Epizootics of this blood fluke have been reported in ranched SBT (Aiken et al., 2006, 2008, 2009; Colquitt et al., 2001; Cribb et al., 2000; Dennis et al., 2011; Kirchhoff et al., 2011) with the prevalence of infection reaching 100% within 2 months post-transfer to ranching pontoon (Aiken et al., 2006; Kirchhoff et al., 2011). Although in the past infections with C. forsteri did not appear to result in clinical disease in the host (Aiken et al., 2006, 2008; Colquitt et al., 2001), more recently an increase in the intensity of infection was reported, which coincided with peaks of tuna mortalities (Dennis et al., 2011; Hayward et al., 2010). Based on the association between the increased intensity of C. forsteri and the mortalities in SBT, clinical trials to investigate the efficacy of anthelmintics in the treatment of C. forsteri were undertaken. Anthelmintics have been used to treat farmed fish species against monogeneans, digeneans and cestodes. A number of different classes of these compounds were considered as potential candidates for these trials including an isoquinolone, three benzimidazoles and a salicylanilide. Praziquantel, an isoquinolone, causes spastic paralysis in trematodes (flukes) as well as damaging the parasite tegument (Taylor et al., 2007). Praziquantel has been used in fish against
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P. Hardy-Smith et al. / Aquaculture 334–337 (2012) 39–44
monogeneans (Schmahl and Mehlhorn, 1985; Sharp et al., 2004; Sitjà-Bobadilla et al., 2006; Williams et al., 2007), digeneans (Björklund and Bylund, 1987) and intestinal cestodes (Sanmartin Duran et al., 1989). Importantly, it is the drug of choice to treat schistosomiasis in man (Doenhoff et al., 2008). Albendazole, fenbendazole and triclabendazole are all benzimidazoles, a class of anthelmintic considered generally to be safe compounds which are used widely in livestock (Taylor et al., 2007). Closantel, a salicylanilide, was also considered for these trials. Albendazole, fenbendazole, triclabendazole and tetramisole have been tested as a treatment against a wide range of fish parasites, triclabendazole and fenbendazole were effective against monogeneans, albendazole was effective against flagellate Hexamita salmonis infection in rainbow trout (Tojo and Santamarina, 1998a, 1998b, 1998c; Tojo et al., 1992). The aim of this study was to determine the in vitro and in vivo efficacy of anthelminthic drugs against C. forsteri infection of SBT and to assess the reliability of in vitro screening. 2. Materials and methods 2.1. In vitro trial 2.1.1. Heart processing and live fluke isolation Up to 150 hearts were collected and stored in a large icebag during SBT commercial harvests. Hearts were dissected lengthwise from apex to base and 3–5 cuts were made in the internal surface to increase the flushing area. Ten to fifteen hearts were placed in individual 2 L bags, along with any remaining blood and approximately 300 mL of saline solution was added to the bags, which were then shaken. Heart flushes and blood were then poured into 500 mL plastic containers and allowed to settle for 20 min. Supernatant was removed and the remaining liquid (approximately 60 mL) decanted into 4–12 Petri dishes, diluted with saline and left to settle for 2 min. Live C. forsteri were observed using a dissecting microscope and transferred to a storage solution (saline solution + 25 μg/mL gentamicin sulphate) for the duration of fluke isolation. 2.1.2. Live fluke preparation and testing To assess toxicity of anthelmintics to flukes, the effect of different concentrations of the tested compounds on responsiveness of the adult flukes was determined over time. C. forsteri were deemed responsive if any movement was observed following 3–5 s of manual plate shaking. Live C. forsteri were isolated from hearts and transferred with a pipette in 100 μL aliquots from the storage solution into a 12-well plate, separated into groups of 10–20 flukes and washed three times in 2 mL Dulbecco's Modified Eagle's Medium (DMEM). The plates were then incubated at 20 °C for 1 h. After 1 h any nonresponsive individuals were removed from the test. Remaining live flukes were re-incubated under the same conditions and any flukes that seemed to have lost responsiveness were replaced with responsive flukes. Toxicity testing was undertaken in 3 mL DMEM containing 25 μg/mL gentamicin sulphate per well. This medium was chosen on the basis of in vitro models for helminths, particularly schistosomes (Keiser, 2010) and our own experience with maintaining C. forsteri adults in vitro (F. Van Ede, P. Crosbie and B. Nowak unpublished). While the use of blood or blood serum has been suggested, it is not recommended for in vitro studies (Keiser, 2010), most likely as it would introduce another source of variability into multiple in vitro experiments. For each anthelmintic, multiple concentrations were assessed to determine their effects on fluke responsiveness, with each concentration having 3 replicate wells, and each replicate well containing 5 flukes, which were responsive at the start of the experiment. Wells were assigned randomly to the anthelmintic concentrations, and 10 μL of each drug concentration was added to wells. Four different anthelmintics were tested: praziquantel, fenbendazole,
tetramisole chloride and closantel (Table 1). Depending on the treatment, 10 μL of dimethyl sulfoxide (DMSO) or distilled H2O was added to control wells. All reagents were purchased from Sigma Aldrich Pty Ltd. The commercial praziquantel preparation is a racemate composed of equal parts of “levo” R(−) and “dextro” S(+) isomers (Cioli and Pica-Mattoccia, 2003). Pilot experiment showed that 0.3% DMSO had no effect on responsiveness of the blood flukes for at least 50 h (results not shown). Plates were incubated at 20 °C, and regularly checked for the number of responsive flukes. The final time point for assessment of the effect of treatment was 48 h. 2.2. In vivo treatment 2.2.1. Anthelmintics and experimental doses Four compounds, Prazifish (All Farm Animal Health), Closal® (Coopers Animal Health), Panacur 100® (Intervet Schering Plough Animal Health) and Fasinex 240® (Novartis Animal Health), were used individually and in combination in this trial. Two compounds (Prazifishl and Closal®) were used at two different dose rates. The dose given to each SBT under each treatment regime is shown in Table 2. Treatment was timed to coincide with increasing fluke burdens in the SBT and prior to the mortality peak observed approximately 6–8 weeks post transfer to the grow out cage. SBT were treated 27 days after transfer from the towing pontoon to the ranching pontoon and a total of 63 days post capture. All treatments were administered as a single dose to a total of twenty SBT for each treatment group. None of these compounds are registered in Australia for treatment of fish destined for human consumption. Each treatment was administered by a registered veterinarian. In addition, a license condition of aquaculture farms in South Australia is that any use of an unregistered compound, even under veterinary direction, must be given Ministerial approval. 2.2.2. Experimental fish and administration of anthelmintic Individual fish were caught using a baited barbless hook and handline and placed in a padded capture cradle. A moistened towel was placed over the eyes of the SBT. The length of the SBT was measured and two identification tags were inserted. A Kruuse No. 26 Small Animal plastic stomach tube was used for stomach tubing. Prior to insertion a fiberglass stylet was inserted into the tube to provide increased rigidity. A small amount of vegetable oil was then applied to the tip of the tube and the tube inserted through the mouth using a piece of hard PVC piping (the “bite pipe”) to protect the softer stomach tube from the teeth of the SBT. Once the tip of the stomach tube had entered the oesophagus the fibreglass stylet was withdrawn as the tube was advanced into the stomach. Location of the tube within the stomach was confirmed by stomach ingesta tracking back out of the tube. The syringe containing the appropriate concentration of anthelmintic was then attached to the end of the stomach tube and the treatment administered. A further 10–15 ml of diluted saline was then flushed through the stomach tube to ensure no treatment was left within the dead space of the stomach tube. The stomach tube near where the syringe attached was then crimped and the tube withdrawn back out through the “bite pipe”. The fish was then released into the treatment pontoon. Tuna were medicated based on their individual size at the dose rates shown in Table 2. Mortalities were monitored post treatment. Twenty five percent of fish were not recovered at the end of the trial, with the loss attributed to a combination of mortality, poaching and/or predation by seals. 2.2.3. Sample collection Twenty four days after treatment, all SBT remaining in the treatment pontoon were euthanised. The treatment to which the fish was assigned was identified using the tags inserted at the time of treatment. Immediately after euthanasia, a sample of gill filaments
P. Hardy-Smith et al. / Aquaculture 334–337 (2012) 39–44
41
Table 1 Treatments used in the experiments and their effect on blood fluke, Cardicola forsteri, in vitro and in vivo. * Active ingredient tested in vitro and dissolved in DMSO before testing, NT – not tested, ND – not determined – none of the tested concentrations or doses significantly reduced survival of the flukes, LD – low dose, HD – high dose. Treatment
Active ingredient (content)
Distributor
Dose tested in vivo (mg/kg) Concentrations Lowest effective tested in vitro concentration (μg/mL)
Lowest effective dose (mg/kg)
Closal
Albendazole (19 g/L) Closantel (37.5 g/L) Triclabendazole (240 g/L) Fenbendazole (100 g/L) Praziquantel (98%)
Coopers Animal Health
0.5–10*
ND
ND
Novartis Animal Health Australasia Pty Ltd Virbac Australia Allfarm Animal Health
NT
NT
0.625–100* 0.0625–200*
10 0.125
2, 4, 6- Trichlorobenzoyl Sigma Aldrich chloride (TCB)
0.782–100
ND
LD 4.1 mg/kg albendazole, 8.25 mg/kg closantel HD 12.5 mg/kg albendazole 24.75 mg/kg closantel 20 mg/kg in combination with Panacur100 (100 mg/kg fenbendazole) 100 LD 75 HD 150 NT
Tetramiole chloride (TM)
6.25–100
50
NT
Fasinex240 Panacur100 Prazifish 2, 4, 6- Trichlorobenzoyl chloride (TCB) Tetramiole chloride (TM)
Sigma Aldrich
was removed from the left second gill arch of each fish and immediately placed into 10% neutral buffered formalin. The heart was exposed in situ and the ductus arteriosus and the ventral aorta were then clamped using haemostats as close to the heart as possible before the heart was removed intact. This ensured that no fluke escaped from the heart during its removal from the fish. When the heart was placed in its container, the clamps were removed. Heart samples were taken from ventricle, atrium and bulbous arteriosus and placed into neutral buffered formalin and processed for histological examination. 2.2.4. Adult C. forsteri counts in heart flushes The hearts were opened longitudinally and flushed until the rinse was clear with diluted seawater (1:2 seawater to regular water) in order to dislodge any adult C. forsteri. Flushes were poured into petri dishes and allowed to settle for 20 min. Then, the flushes were observed under a dissecting microscope (Olympus SZX12 stereomicroscope, Olympus, Japan) for the presence of adult C. forsteri. Parasite numbers were recorded for each fish. The counts were done blind, with no sample identified back to the treatment. 2.2.5. Histology Samples for histological examination were collected from gills and heart. Three 2 cm wide gill sections were obtained from the second gill arch of the left-side holobranch (cranial, middle and ventral sections). Samples of atrium, bulbus arteriosus and the caudal tip of the ventricle were fixed in 10% neutral buffered formalin for 24–48 h. Subsequently, 3 mm thick sections were cut and placed into a histology cassette. The samples were processed using standard histological techniques. Gills inside the cassettes were decalcified for 1 h using Rapid Decalcifying Fluid (Australian Biostain, Australia) and then washed with water for 1 h. After this initial step, all samples were processed, embedded into paraffin blocks and 5 μm sections were obtained. Slides were then stained with Haematoxylin-Eosin (H&E) and examined under light microscope (Leitz Diaplan microscope, Leica Microsystems, Germany) at 100× magnification and details at 400× or 1000× under oil immersion. 2.2.6. Egg and granulomas counts C. forsteri eggs and granulomatous reactions were assessed in all 3 sections of heart and gill from each fish. Structures counted as eggs had a spherical shape from 19 to 25 μm in diameter as described by Colquitt et al. (2001). Granulomas were identified as encapsulated eggs with a reaction consisting of epithelioid cells and lymphocytes surrounded by fibroblasts and fibrocytes in older lesions following the description provided by the same authors (Colquitt et al., 2001). Counts from the entire area of a section were obtained. The area of
ND ND 75 NT
NT
the sections was measured using Image J Image processing and analysis in java software (Maryland, USA). An average number of eggs per cm 2 of gill or heart was then obtained from the total area of the 3 independent (each from a different block) sections of each organ. All samples were coded to avoid observer bias. 2.3. Statistical analyses All data for both in vitro and in vivo trials were analysed by comparing treatments using a one-way ANOVA followed by a Tuckey's HSD post-hoc test. Levene's test was used to test if the variances were homogenous and normality plots to determine if the data were normally distributed. Data from the in vivo trial violated assumptions of the ANOVA test, therefore data sets for number of eggs per cm 2 of heart and C. forsteri counts in heart flushes were analysed after being transformed by square root. Number of eggs per cm 2 of gill had to be transformed by log10. As a result statistical analyses were carried out with transformed data, but results are presented with raw data. All statistical analyses were performed using the free software R: a language and environment for statistical computing (R Development Core Team, Vienna, Austria). P-value b0.05 was considered a significant difference. 3. Results 3.1. In vitro tests Praziquantel was the most potent anthelmintic in decreasing fluke responsiveness in vitro. Upon addition of praziquantel concentrations ranging between 1.5 μg/mL and 200 μg/mL to wells containing vigorous flukes, the flukes stopped responding to the shaking of the plate within less than 5 min. The threshold at which praziquantel affected Table 2 In vivo treatments and their doses, and the final number of fish examined at the end of the experiment for presence of adult blood flukes and eggs in heart and gills. Treatment
Active component
Dose (mg/kg of Number of fish at SBT biomass) sampling
Prazifish lower dose Praziquantel 75 Prazifish higher dose Praziquantel 150 Closal lower dose Closantel albendazole 8.25 4.1 Closal higher dose Closantel 24.75 Albendazole 12.5 Panacur 100 Fenbendazole 100 Panacur 100 + Fenbendazole 100 Fasinex 240 Triclabendazole 20 Control Saline 30 mL per SBT
13 16 13 15 14 12 25
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C. forsteri was established between 0.0625 μg/mL and 0.125 μg/mL. Concentrations of 0.125 μg/mL and above resulted in a significant reduction in the mean number of responsive flukes after 48 h of exposure (F = 58.440; df 5, 12; P b 0.001), while 0.0625 μg/mL showed no significant difference on the average number of responsive flukes when compared to the control (0 μg/mL). Fenbendazole did not show an immediate effect on responsiveness of flukes as did praziquantel. After two days of exposure, however, concentrations of 10 μg/mL and above significantly affected fluke responsiveness (F = 23.880; df 5, 12; P b 0.001). Tetramisole chloride at concentrations of 50 μg/mL and above significantly decreased the responsiveness of the flukes at the 48 h end time point, compared to concentrations equal to or less than 12.5 μg/mL (F = 31.125; df 5, 12; P b 0.001). The testing with Closantel was inconclusive due to the small number of flukes available for these assays, resulting in large variation between replicates. No significant differences in the responsiveness of the flukes to Closantel could be found (F = 2.846, df 2, 6, P = 0.135).
Average number of Cardicola forsteri eggs per cm2 in hearts of Thunnus maccoyii + SE
42
7 a
6 5 4
a a
a a
3 2 1
b b
0
3.2. In vivo experiment
Average number of Cardicola forsteri in heart flushes from Thunnus maccoyii SE
The mean number of blood flukes C. forsteri observed in heart flushes of SBT was significantly different among treatments (F = 17.46, df 6,101, P b 0.001). Both the higher and the lower dose praziquantel treatment resulted in a significant reduction of the number of flukes present in the hearts, with heart flushes obtained from these fish showing on average 2 blood flukes or fewer. Comparing to the controls the reduction in adult blood flukes in the praziquantel treated fish ranged from 91 to 95%. There was no significant difference between the high and low concentrations. Heart flushes from fish subjected to the other anthelmintic treatments and the controls had average fluke counts 9 times or higher than those observed in heart flushes from fish treated with the higher and lower praziquantel (Fig. 1). A significant difference was observed in the mean number of blood fluke eggs per cm 2 of SBT myocardium (F = 15.26, df 6,101, P b 0.001). The fish which were subjected to either of the two doses 35
a a
30
a
a
25 a
20 15 10 5
b b
Treatments Fig. 2. Effect of treatment on the mean number of blood fluke (Cardicola forsteri) eggs per cm2 of myocardium obtained from SBT (Thunnus maccoyii). Averages with different small letters (a, b) are significantly different from one another by one-way ANOVA. Closal LD – 4.1 g/kg albendazole and 8.25 g/kg closantel, Closal HD – 12.5 g/kg albendazole and 24.75 g/kg closantel, Prazifish LD – 75 mg/kg praziquantel, Prazifish HD – 150 mg/kg praziquantel, Panacur 100–100 mg/kg fenbendazole, Panacur 100 + Fasinex 240–100 mg/kg fenbendazole and 20 mg/kg triclabendazole.
of praziquantel had a significantly reduced number of eggs per cm 2, and these numbers were at least 6 times lower than those observed in fish treated with other anthelmintics or control group (Fig. 2). The number of eggs per cm 2 of gill was significantly affected by treatment (F = 4.32, df 6,101, P b 0.001). However, the pattern showed by the number of fluke eggs in the gills was different to what was observed for the eggs in myocardium and for the flukes found in the heart flushes. Control fish and fish treated with praziquantel (both doses) and lower dose Closal presented very low average number of eggs per cm 2 of gill – the highest being an average of 8 eggs per cm 2 – and these means were different from the mean observed in fish treated with fenbendazole (Fig. 3). Fish treated with fenbendazole showed the largest mean number of eggs per cm 2 of gill, and although this average was 1.5 and 3 times larger than the average number of eggs in gills for fish treated with fenbendazole/triclabendazole combined and the higher dose Closal respectively, these three treatments were not significantly different from each other. In contrast to the even distribution of blood flukes eggs in the myocardium, eggs in the gills are found in “patches”. The angle of sectioning and the depth of sectioning could affect results from the gills more than from heart due to the complex three dimensional structure of the gills.
0
4. Discussion
Treatments Fig. 1. Effect of treatment on the mean number of blood flukes (Cardicola forsteri) in heart flushes obtained from SBT (Thunnus maccoyii). Averages with different small letters (a,b) are significantly different from one another by one-way ANOVA. Closal LD – 4.1 g/kg albendazole and 8.25 g/kg closantel, Closal HD – 12.5 g/kg albendazole and 24.75 g/kg closantel, Prazifish LD – 75 mg/kg praziquantel, Prazifish HD – 150 mg/kg praziquantel, Panacur 100–100 mg/kg fenbendazole, Panacur 100 + Fasinex 240– 100 mg/kg fenbendazole and 20 mg/kg triclabendazole.
This study provides evidence that the drug praziquantel may be used as an efficacious anthelmintic for the treatment or control of blood fluke C. forsteri in SBT. This is in agreement with the success of the experimental use of oral praziquantel administration to treat blood fluke infections in Pacific Bluefin Tuna (Shirakashi et al., 2012). While the effective doses from this study and reported by Shirakashi et al. (2012) are different, the products used, parasites, hosts and application methods differed. This study provides a mechanism for clinical field trials to evaluate praziquantel against C. forsteri infections. Praziquantel offers an effective control against C. forsteri with 91–95% reduction in the infection
Average number of Cardicola forsteri eggs per cm2 in gills of Thunnus maccoyii SE
P. Hardy-Smith et al. / Aquaculture 334–337 (2012) 39–44
180 160
a
140 120
ab
100 80 ab
60 40 20
b b
b
b
0
Treatments Fig. 3. Effect of treatment on the mean number of blood fluke (Cardicola forsteri) eggs per cm2 of gills obtained from SBT (Thunnus maccoyii). Averages with different letters are significantly different from one another by one-way ANOVA. Closal LD – 4.1 g/kg albendazole and 8.25 g/kg closantel, Closal HD – 12.5 g/kg albendazole and 24.75 g/kg closantel, Prazifish LD – 75 mg/kg praziquantel, Prazifish HD – 150 mg/kg praziquantel, Panacur 100–100 mg/kg fenbendazole, Panacur 100 + Fasinex 240–100 mg/kg fenbendazole and 20 mg/kg triclabendazole.
at the tested doses. The next step will be to determine the lowest effective dose and establish pharmacodynamics of praziquantel. Praziquantel was the only anthelmintic efficacious against adult C. forsteri at the concentrations tested in vitro and doses applied in vivo, while other compounds were efficacious in vitro after 48 h they were slower acting and the effective concentrations were greater. No other anthelmintic tested showed promising results in vivo. This suggests that praziquantel could be used against C. forsteri infections of ranched SBT. However, development of resistance should be monitored as resistance against praziquantel has been reported in some schistosomes (Doenhoff et al., 2008, Melman et al., 2009). The demonstration that praziquantel was efficacious in both in vitro and in vivo trials suggests that in vitro trials can be used for future investigations of the effects of treatment on C. forsteri, including screening of novel treatments or investigation of development of resistance to different treatments. Similar approach has been used with other parasites, including those affecting humans (Keiser, 2010). However, the use of in vitro treatment has to be applied with caution as some medications may require to be metabolised before they can be effective. Concentrations of the active compound should be determined and compared between serum of treated animals and media for in vitro testing. This was however outside the scope of this study. In the current study, a single treatment with praziquantel was used. Praziquantel is, however, rapidly cleared from treated fish (Kim et al., 2001; Tubbs and Tingle, 2006) and treatments may need to be repeated if the tuna are exposed more than once to the infective stages of blood flukes during their ranching period. Two major C. forsteri infection events in the first year of SBT ranching have been suggested by stochastic modelling (Aiken et al., 2009). Reinfection with adult C. opisthorchis was suggested in the experimental praziquantel treatment of Pacific Bluefin Tuna (Shirakashi et al., 2012). Praziquantel was effective against adult C. forsteri at a concentration of 0.125 μg/mL in vitro. This is a higher toxicity than reported against other parasites, however equivalent low concentrations were not tested in the previous studies. Praziquantel was reported to be
43
efficacious in vitro against monogeneans, including Heterobothrium okamatoi larvae at 20 μg/mL (Hirazawa et al., 2000) and Pseudodactylogyrus bini at 10 μg/mL (Buchmann, 1987). Similarly, Xiao et al. (2009) demonstrated the effective praziquantel concentration against adult schistosomes as 1–30 μg/mL in vitro. However, it has been reported that if praziquantel was replaced with drug-free medium after 1 or 4 h, adult schistosomes could recover (Xiao et al., 2009). This potential for recovery should be further investigated for C. forsteri, especially given the rapid clearance of praziquantel from SBT. While the reduction in the number of eggs in the heart and gills in SBT treated with praziquantel was related to the reduction in the number of adults in heart, the additional significant reduction of number of eggs in the gills in the control tuna and tuna treated with Closal LD was unexpected and could have been due to the patchiness of egg distribution in the gills observed in our study. Overall, the numbers of adults recovered from the heart corresponded much better to the density of eggs in the heart than to the density of eggs in the gills. Further work is required to investigate the distribution and identification of the eggs in the gills. Immature stages of Schistosoma mansoni were shown to be insensitive to praziquantel (Xiao et al., 1985). Praziquantel is highly effective in killing adult schistosome worms, but it is unable to kill developing schistosomes and so does not prevent reinfection (Gray et al., 2010). While there was a reduction in the number of eggs in gills and hearts of SBT treated with praziquantel, it is possible that this could have been due to the reduction of the number of adults or a lower production of eggs and not due to a direct toxic effect of praziquantel on the eggs themselves. Eggs in gills remained viable after treatment with praziquantel suggesting that the treatment did not kill eggs or miracidia of blood flukes affecting T. orientalis (Shirakashi et al., 2012). Reinfection rates post-treatment should be investigated for ranched SBT. In vitro trials used only one life stage of the parasite (adults), sensitivity of other life stages is currently unknown and while eggs or miracidia could be also tested. The most relevant life stages, for example cercariae are currently not available for experiments. Adult parasites can be obtained from infected SBT, however as most of the fish are harvested after the peak of infection the availability of the adults for in vitro testing can be limited. Viability of the adult flukes limited the scope of in vitro testing. Oral intubation allows delivery of an exact dose to each animal and provides an accurate means of ensuring similar dosages for comparative experimental studies. However, this administration method is not viable on a commercial scale (Williams et al., 2007). Commercial oral treatment will require in-feed medication. Further research is needed to determine the optimum dose, best treatment application method, palatability and any potential side effects as well as potential development of resistance of the parasite against the treatment. Role of funding body Fisheries Research and Development Corporation has funded this project. SBT Research Council approved milestone reports and the final version of this manuscript. Acknowledgements We are grateful to Southern Bluefin Tuna industry for their support, including fish husbandry. We would like to thank Daniel Pountney, Karine Cadoret and Claire Webber for their assistance with sampling and initial processing of samples. We are grateful to Francoise van Ede and Phil Crosbie for their contributions to the development of the in vitro method. We would like to thank Rob Jones for providing valuable advice on the stomach tubing of large fish. This project was funded by Fisheries Research and Development Corporation.
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