Aquaculture International 10: 399–409, 2002. © 2003 Kluwer Academic Publishers. Printed in the Netherlands.
Treatment of the early life stages of scallop (Pecten maximus) with antimicrobial agents; searching for an alternative to chloramphenicol LISE TORKILDSEN 1,*, ROSIE COYNE 1, OLE BENT SAMUELSEN 1, THOROLF MAGNESEN 2 and ØIVIND BERGH 1 1 Institute of Marine Research, Division of Aquaculture, P.O. Box 1870, Nordnes, N-5817, Bergen, Norway; 2Centre for Studies of Environment and Resources, University of Bergen, P.O. Box 7800, N-5020, Bergen, Norway; *Author for correspondence (e-mail:
[email protected]; phone: +47 55 23 63 61; fax: +47 55 23 63 79)
Received 27 December 2001; accepted in revised form 17 December 2002
Key words: Antibacterial agents, Bacteria, Larvae, Minimum Inhibitory Concentration, Scallop Abstract. High mortality rates are often observed in rearing the early stages of the great scallop, Pecten maximus. The addition of antibacterial agents has been necessary to improve larval survival. However, as one antibacterial agent, chloramphenicol, is banned in Norway and Europe the aim of this study is to investigate alternative antibacterial agents. The therapeutic agents investigated in this study were florfenicol, oxytetracycline, oxolinic acid, neomycin and Pyceze. The mean minimum inhibitory concentration (MIC) values were determined for oxytetracycline, oxolinic acid and Pyceze against bacteria isolated from scallop larvae. Two types of treatment regime were investigated on an intermediate scale (20 L). One regime involved continuous exposure of scallop larvae to the therapeutic agent while the other involved a short exposure lasting two hours. All intermediate scale treatments were performed in parallel to large-scale production (800 L) treatment with chloramphenicol. Of the therapeutants investigated, oxolinic acid was the most effective, although only at high concentrations. The short exposure of two hours was ineffective for all therapeutics.
Introduction The early life stages of scallop are susceptible to high mortality (Nicolas et al. 1996; Robert et al. 1996; Robert and Gérard 1999). Scallop hatcheries in France have used the antibacterial agent chloramphenicol for 15 years, without which outbreaks of disease normally occur (Nicolas et al. 1996). Similarly, the Norwegian experience is that a reduction in larval mortality of up to 30% is achieved by employing chloramphenicol at a concentration of 10 µg ml −1 in the production tanks, indicating bacterial aetiology (Torkildsen et al. 2000). However, the possibility of other causal factors of larval mortality should not be excluded. Recent regulations in Norway and the EU have banned the use of chloramphenicol in the production of organisms intended for human consumption, including scallop production (Anonymous 1990, 1994). A long-term goal for the scallop industry is to develop production strategies that do not use antibacterial agents. The
400 immediate goal is to find a suitable alternative therapeutic to replace chloramphenicol. Oxytetracycline and oxolinic acid, which are available for use in aquaculture in Norway (Markestad and Grave 1997) along with florfenicol, which is similar to chloramphenicol in molecular structure, were therefore investigated as possible alternatives to chloramphenicol. Results from Canada with the scallop Placopecten magellianicus indicate that neomycin might be an appropriate candidate (pers. comm. Dabinet, 1998) and it was therefore included in the investigation. Complicating the search for an alternative is the fact that the exact aetiology of the infection in scallop larvae is unclear. Transmission electron microscopy of scallop larvae revealed inclusions within the larvae, which were tentatively interpreted as fungal-like (Torkildsen, unpublished results). For this reason, Pyceze, an antifungal agent was also included in the investigation. In designing a suitable treatment regime, the minimum inhibitory concentration (MIC) values of bacterial isolates from scallop larvae were established against the therapeutics under investigation and the efficacies of oxytetracycline, florfenicol, oxolinic acid and Pyceze relative to chloramphenicol were examined. Two different treatment regimes on an intermediate scale were investigated; continuous exposure, and two-hour exposure at higher concentrations of the therapeutic agent.
Materials and methods Isolation and characterization of isolates The scallop eggs and larvae were obtained from a scallop hatchery, Scalpro AS, in Rong on the west coast of Norway. Isolates were obtained from scallop eggs and larvae according to the methods of Torkildsen et al. (2000). The isolates were characterised to genus level (Table 1). Minimum inhibitory concentration determinations The determination of MIC values was performed using an agar dilution method (Samuelsen and Lunestad 1996; Washington 1985). The method included strains with known MIC values. Strains were tested on Mueller Hinton agar/broth supplemented with 2% NaCl. The strains were transferred to 10 ml Mueller Hinton broth and incubated for 48 h at 20 °C, resulting in a final cell density of approximately 5 × 10 8 ml −1. Bacteria were transferred to Mueller Hinton agar using an inoculation loop of 10 µl. The Mueller Hinton agar plates contained increasing concentrations of the antibacterial agents oxolinic acid (0.016–1 µg ml −1), oxytetracycline (0.033–4 µg ml −1) and Pyzece (0.125–32 µg ml −1) in two-fold dilution. The MIC testing was performed in triplicate. The antibacterial agents were obtained from Norsk Medisinaldepot (Bergen, Norway). The agar plates were incubated for 72 h at 20 °C. The lowest concentrations of oxolinic acid, oxytetracycline and Pyzece that produced complete inhibition were recorded as the MIC values. No MIC val-
al. al. al. al. al. al. al.
(2000) (2000) (2000) (2000) (2000) unpubl. res. unpubl. res.
Torkildsen Torkildsen Torkildsen Torkildsen Torkildsen Torkildsen Torkildsen
Vibrio Pseudomonas Aeromonas Aeromonas Pseudomonas Vibrio Vibrio
et et et et et et et
Torkildsen et al. unpubl. res. Torkildsen et al. unpubl. res. Torkildsen et al. unpubl. res.
Eukaryote Vibrio Pseudoalteromonas/Alteromonas Vibrio
LT 02 LT 06 LT 13 LT 21 LT 25 LT 51 LT 54 LT 58 LT 59 LT 62 LT 73 PMV 19
Characterised by
Genus
Strains
Table 1. List of isolates characterised to genus level.
401
402 ues were determined for neomycin since this drug was included only in the last experiment (Experiment 3). Source of scallops All experiments were carried out at Scalpro AS using larvae supplied by the hatchery. Spawning was induced by thermal shock and eggs were fertilised by the method described by Gruffyd and Beaumont (1970). After fertilisation the embryos were allocated to tanks of 800 L and kept in stagnant seawater at 18 °C ± 1. The seawater was obtained from the nearby fjord at 150 m and filtered using a 1 µm bag filter (Gaf Filters, Belgium). Experimental set-up Intermediate scale The experiments were performed in small tanks (20 L) containing 18 L of seawater and were protected from light once the therapeutic agent had been added. The therapeutics are degraded by light (Lunestad et al. 1995). Approximately 250,000 larvae (14 larvae ml −1) were added to each tank and five replicate tanks for each therapeutic were used. The temperature of the water was 18 °C ± 1 and the water was aerated continuously during the experiments. The seawater in each tank was changed three times a week. The larvae were fed monocultures of the algae Isochrysis galbana (Parke) Tahitian strain, Pavlova lutheri (Droop) and Chaetoceros calcitrans p. pumilus (Takano) 1:1:2 at a total concentration of 50 cells µl −1. In the continuous exposure treatment, Experiments 1 and 2, the therapeutants were administrated directly into each 20 L tank. In Experiment 1, chloramphenicol (10 µg ml −1), florfenicol (10 µg ml −1) and oxolinic acid (40 and 80 µg ml −1) were used in addition to unmedicated controls. In Experiment 2, chloramphenicol (10 µg ml −1), oxolinic acid (100 µg ml −1), oxytetracycline (200 µg ml −1) and pyzece (20 µg ml −1) were used in addition to unmedicated controls. In Experiment 3, the short exposure treatment, the larvae were removed from the 20 L tanks and treated in a 1 L container for two hours with high concentrations of either chloramphenicol (300 µg ml −1), oxolinic acid (100 µg ml −1), oxytetracycline (300 µg ml −1), neomycin (300 µg ml −1), or as unmedicated controls. They were then washed and returned to the unmedicated tanks of seawater until the next water change. Large scale Larvae were maintained in the 800 L tanks, and seawater was changed three times a week. Between five and ten million larvae (6–13 larvae ml −1) were in each tank and three to five tanks were used for each therapeutic. The larvae were fed monocultures of the algae as in the intermediate scale experiments. The temperature of the water was 18 °C and the tanks were aerated continuously during the experiments. In the large-scale experiments, the larvae were exposed continuously to chloramphenicol or florfenicol at a concentration of 10 µg ml −1.
403 Table 2. MIC values (µg ml −1) of therapies for isolates from scallop larvae. Strain
Oxytetracycline (µg ml −1)
Oxolinic acid (µg ml −1)
Pyceze (µg ml −1)
LT 02 LT 06 LT 07 LT 13 LT 21 LT 25 LT 51 LT 54 LT 58 LT 59 LT 62 LT 73 PMV 19 Average MIC-values (µg ml −1) Range (µg ml −1)
0.5 0.5 4 1 1 0.83 0.42 0.5 0.5 0.33 0.5 0.38 0.42 0.84 0.33–4
0.13 0.33 0.11 0.03 0.33 0.19 0.25 0.11 0.1 0.02 0.11 0.25 0.25 0.17 0.02–0.33
26.7 16 16 16 24 10.7 10.7 26.7 NT 16 18.7 16 10.7 17.34 10.7–26.7
Three larval groups were used in this study, a different group being used in each of the three experiments. Large-scale tanks were run in parallel with each intermediate scale experiment in order to assess the effect of the quality of each larval group. Statistical analysis The mean survival rates at the end of the experiments were not normally distributed and a non-parametric Kruskal-Wallis test was used to test survival rates with different treatments.
Results The MIC values from bacteria isolated from scallop larvae are presented in Table 2. In Experiment 1, in both the intermediate-scale group (Table 3) and the parallel large-scale group (Table 4) florfenicol-treated larvae had a mortality of 100% on days 15 and 13 respectively and in both experiments the untreated control had a higher survival rate. In all florfenicol-treated tanks a layer of viscous slime appeared. However, no attempts were made to characterise this layer. The results of Experiment 2 (Figure 1) show that a survival rate similar to chloramphenicol at intermediate scale was obtained only in the tanks medicated with oxolinic acid at a concentration of 100 µg ml −1. The survival rate of the groups treated with chloramphenicol was 10% (SD 3.1) of the initial number of larvae and
404 Table 3. Survival of scallop larvae during the larval stage with different antibacterial treatments, in intermediate and large scale. Control Days Larval after survival spawning (%)
SD
Larval survival (%)
SD
Larval survival (%)
SD
Chloramphenicol large scale Larval SD * survival (%)
3 10 13 15 17 20 24 27
0 34 22 – – 16 13 0
100 84 56 30 – 3 0 –
0 55 28 38 – 6 0 –
100 75 54 0 – – – –
0 14 27 0 – – – –
100 88 84 – 72 57 – 41
*
100 92 33 – – 24 26 0
Chloramphenicol
Florfenicol
– – – – – – – –
Standard deviation not calculated, only one parallel
Table 4. Survival of scallop larvae during the larval stage treated with florfenicol and chloramphenicol in large-scale production system. Control large scale Days after spawning
Larval survival (%)
SD
3 8 10 13 15 17 24
100 58.49 52.20 – 22.64 4.40 2.83
0 13.2 4.75 – 5.66 1.09 4.90
Chloramphenicol Large scale Larval surSD vival (%)
Florfenicol large scale Larval survival (%)
SD
100 64.78 66.67 – 51.57 30.19 22.64
100 66.04 23.9 0 – – –
0 4.99 4.75 0 – – –
0 2.88 5.76 – 3.77 3.27 2.67
significantly higher than in the untreated control groups (p < 0.05). The survival rate of the oxolinic acid-treated groups was 12% (SD 13.7). However, this was not significantly higher than the control groups, due to high variance between the tanks. Oxytetracycline treatment resulted in slightly, but not significantly, better survival than the unmedicated control groups. Treatment with Pyceze resulted in 100% mortality by day 13. Comparisons of various concentrations of oxolinic acid showed that only 100 µg ml −1 increased the survival rate to a level comparable to groups treated with chloramphenicol (Table 5). Data from Experiment 3, in which the larvae were medicated with high concentrations of various therapeutics for two hours between every water change, three times a week, show low survival rates for all drugs in all groups (0–10%) while
405 Table 5. Survival rates of scallop larvae treated with different concentrations of oxolinic acid. Oxolinic acid 40 ppm Oxolinic acid 80 ppm Days after spawning Larval survival (%) SD Larval survival (%) SD
Oxolinic acid 100 ppm Larval survival (%) SD
3 10 13 15 17 21 24
100 49.1 35.2 – 27.2 30.4 10.1
100 15 – – – – –
0 17 – – – – –
100 73 61 10 0 – –
0 43 27.5 7.4 0 – –
0 5.5 19 – 10.1 13.8 13.1
good survival was noted in the large-scale group treated with 10 µg ml −1 chloramphenicol (55% survival) (Figure 2). The low survival rate for all drugs was not related to the quality of the larvae used in these investigations. The survival rate of the parallel large-scale chloramphenicol group result was similar to that of previous large-scale groups (Tables 3 and 4, Figure 1).
Discussion Experiment 1 with its parallel large-scale group showed that florfenicol had no positive effect on the larval survival, though the reason for this is unknown. Since florfenicol is similar to chloramphenicol in molecular structure and antibacterial action, this finding was somewhat surprising. In the tanks with florfenicol, a viscosus slime layer emerged around the edge of the tank, perhaps consisting of dead algae. This effect was observed in both the large- and intermediate-scale groups, and starvation of the larvae could be a plausible explanation for their high and rapid mortality. However, this finding was not pursued. In Experiment 2, chloramphenicol was shown to be the most efficient of all the drugs tested and was the only treatment, which resulted in significantly lower mortality than the untreated control groups. The proportion of larvae that survived in the oxolinic acid-treated groups (12%, SD 13.7) was comparable to that in the tanks medicated with chloramphenicol (10%, SD 3.1). However, the number of larvae surviving in the chloramphenicol-treated groups was quite similar between groups and therefore more reproducible in comparison with the treatment with oxolinic acid, where survivors were found in only three of five tanks. In the tanks treated with Pyceze, larval survival was zero, which was even lower than the unmedicated control groups. It may be that Pyceze had a negative effect on the larvae. Treatment with oxytetracycline resulted in only slightly higher survival than the unmedicated controls. Oxytetracycline may prove more effective at higher concentrations than used in this study but the use of high concentrations in a commercial hatchery would involve very large quantities of antibiotics. In the Scalpro hatchery, a single
406
Figure 1. a) Average survival (%) of scallop larvae during the larval stage and b) average survival rates (%) of scallop larvae on day 24, with different treatment on intermediate scale and chloramphenicol on large scale.
spawning occupies around 10 larval tanks (800 L) and the larval stage lasts for about 24 days, with oxytetracycline (200 ppm) being added ten times during the larval development. This would mean a consumption of about 16 kg of oxytetracycine per spawning. The results of the oxolinic acid investigations (Table 5) demonstrate a dose-response effect with higher larval survival at 100 ppm than at 60 or 80 ppm. The results support the hypothesis of a bacterial aetiology. The concentration required to obtain an effect was large compared with the MIC values. However, seawater is known to considerably lower the antibacterial effect of quinolones like
407
Figure 2. a) Average survival rates (%) of scallop larvae during the larval stage and b) average survival rates (%) on day 24, following treatment for a short period with various antibacterial agents.
flumequine and oxolinic acid (Barnes et al. 1995; Torkildsen et al. 2000; Lunestad and Samuelsen 2001) In Experiment 4, the scallop larvae were treated with high concentrations of various antibacterial agents for a short time between each water change. This was initiated in order to test a treatment strategy that would potentially reduce the amount of antibacterials used by a substantial amount, and make it possible to treat and destroy the bath treatment effluent in a controlled manner. However, the results show that this treatment strategy did not reduce mortality. Similar results were reported by Robert et al. (1996) after dipping the larvae for one hour in 8 mg L −1 chloramphenicol.
408 As transmission electron microscopy revealed fungal-like inclusions in the larvae, the treatment with Pyceze tested the possibility of a fungal aetiology of the infection in scallop larvae. However, in both the large- and intermediate-scale experiments, Pyceze was found to be ineffective, indicating that this was not a treatable fungal infection. Since the survival of larvae in large tanks was similar in all the experiments, the quality of the larval groups could not be regarded as a causal factor for the differences in the results of the intermediate-scale experiments. In parallel investigations the survival was higher in large-scale tanks than intermediate tanks, indicating that there was a negative effect on survival rates when the scale of the experiments was reduced from from large to intermediate. The results of this and a previous investigation by Torkildsen et al. (2000) show that mean MIC values are lower than 3 µg ml −1 for all the antibacterial agents tested. Hence, the concentrations used in the tanks were within a range within which an effect on the bacteria would be expected. An exception to this was the fungicide Pyzece, whose MIC value is 27 µg ml −1, demonstrating low antibacterial activity. The improved survival rate of scallop larvae in tanks containing antibiotics suggests that the aetiology is partly microbial in nature. However, the aetiology has yet to be fully determined since mortality was approximately 70% at the end of the treatment period. If the mortality were predominantly microbiological in nature, it would be reasonable to expect survival rates to be higher. Other investigators have considered the problems associated with scallop larval mortalities, and Robert et al. (1996) argue that previous studies have shown that neither a decrease in larval density (to 1 larva ml −1) nor an increase in the frequency of seawater changes (to one per day) had any positive effects on survival. Furthermore, these authors reported that elective substances such as sugars (erythriol, xylose, melibiose, D galacturonate and rhamnose) were not suitable as treatments and that the use of another antibiotic, erythromycin, led to inconsistent results.
Conclusions Florfenicol should not be regarded as an alternative to chloramphenicol. Short-term treatment (two hours) with high concentrations of antibiotics between water exchanges did not increase survival rate. The concentration of oxytetracycline needed to obtain an effect on survival is high, possibly too high for practical purposes. While it is recognised that on an intermediate scale oxolinic acid was useful at a high concentration, 100 µg mg −1, scaling up to production volume might have a positive effect on survival rates, thus permitting a reduction in the concentration required. There was no indication of a treatable fungal infection.
409 Acknowledgements We thank the personnel of the hatchery Scalpro AS for technical assistance. We also thank Audun Høylandsskjær of the Department of Pharmacology, University of Bergen, and Heidi Kongshaug of the Department of Aquaculture of the Institute of Marine Research for technical assistance. This study was financially supported by Research Council of Norway grants 1294061/120 and European Commission grant no QLK2-CT-2000-51162.
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