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Standardization of the bioencapsulation of enrofloxacin and oxytetracycline in Artemia franciscana Kellogg, 1906
Aquaculture 196 Ž2001. 1–12 www.elsevier.nlrlocateraqua-online
Standardization of the bioencapsulation of enrofloxacin and oxytetracycline in Artemia franciscana Kellogg, 1906 B. Gomez-Gil ) , J. Cabanillas-Ramos, S. Paez-Brambila, A. Roque Centro de InÕestigacion ´ en Alimentacion ´ y Desarrollo, A.C., Unidad Mazatlan ´ en Acuicultura y Manejo Ambiental. AP.711 CP. 82000 Mazatlan ´ Sinaloa, Mexico Received 1 August 2000; received in revised form 22 October 2000; accepted 4 November 2000
Abstract Bioencapsulation of enrofloxacin and oxytetracycline into Artemia franciscana nauplii was standardized. Both antibacterials were delivered to the nauplii individually and the amounts used in the study were percentages of the lipid emulsion Rich w added as nutrition enrichment for the nauplii. The determination of the amounts of drug incorporated in A. franciscana nauplii was obtained using a bioassay radial diffusion method, standardized in the laboratory and using Escherichia coli as an indicator. The minimum time for full enrichment for both enrofloxacin and oxytetracycline bioencapsulation in A. franciscana nauplii was 4 h after the initial exposure of the nauplii to the antibiotics and this was established sampling nauplii at 1, 2, 3, 4, 8 and 24 h after adding the nauplii to the mix. These experiments were carried out twice and at 4 h, 1.10 and 1.13 ng of enrofloxacin per nauplius and 9.32 and 9.37 ng of oxytetracycline per nauplius were obtained. The optimum percentages of enrichment were 40% of enrofloxacin in relation to Rich w and 80% of oxytetracycline. The percentages tested were 10%, 20%, 30%, 40% for the enrofloxacin and 0%, 40%, 80%, and 160% for the oxytetracycline. The maximum time at which antibiotic was still detected in the Artemia after it had been introduced in seawater was 8 h for both antibacterial agents. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Artemia franciscana; Enrofloxacin; Oxytetracycline; Bioencapsulation
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Corresponding author. Tel.: q52-69-880157; fax: q52-69-880159. E-mail address: [email protected] ŽB. Gomez-Gil..
0044-8486r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 4 - 8 4 8 6 Ž 0 0 . 0 0 5 6 8 - 8
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1. Introduction The delivery of drugs to aquatic animals is problematic. Delivery by medicated food leads to leaching of the drug from the pellets ŽJacobsen and Berglind, 1988.. In bath treatments, a substantial portion of the drug is not used and it can be difficult to dispose of the excess of drug safely. The value of Artemia sp. as a carrier for the delivery of antibacterials to fish and shrimp larvae has been investigated ŽMohney et al., 1990; Chair et al., 1991a; Nelis et al., 1991; Touraki et al., 1991; Verpraet et al., 1992; Roque et al., 1998.. Most of the authors worked with either trimethoprim-sulfamethoxazole or chemical analogous, ormethroprim-sulphadimethoxine ŽMohney et al., 1990; Chair et al., 1991a; Touraki et al., 1991; Verpraet et al., 1992; Gapasin et al., 1996.. The technique though is not standardized and enrichment times varied from 2 h ŽGapasin et al., 1996. to 32 h ŽTouraki et al., 1991, 1995.. The quantity of antibiotic offered is, in most cases, defined as a percentage of the commercial enrichment in which it goes mixed ŽTouraki et al., 1991; Verpraet et al., 1992; Dixon et al., 1995b; Gapasin et al., 1996; Roque et al., 1998. varying from 1% wrv ŽVerpraet et al., 1992. to 40% wrv ŽDixon et al., 1995b; Gapasin et al., 1996; Roque et al., 1998.. An exception to this is the work performed by Mohney et al. Ž1990. who added 3 mg of antibiotic per liter of water. The techniques used to measured the levels of antibiotic incorporated in the Artemia nauplii also varied including radial diffusion bioassay which used either Escherichia coli as the indicator bacterium ŽDixon et al., 1995b.; Vibrio alginolyticus ŽMohney et al., 1990. and Bacillus subtilis ŽRoque et al., 1998.. Other studies have used HPLC as a mean to more accurately measure the amount of antibiotic incorporated ŽTouraki et al., 1991, 1995; Chair et al., 1991b.. In Mexico, enrofloxacin and oxytetracycline ŽOTC. are the antibacterials most commonly used by shrimp hatcheries. Although the possibility of delivering OTC to shrimp larvae by this method has already been investigated the technique was not standardized and the minimum time and quantity of antibiotic to enrich the Artemia, in order to incorporate therapeutic levels of the drug in the Artemia has not been determined. As for the enrofloxacin, the possibility of bioencapsulation into Artemia has not been reported in the literature. Therefore, the aim of this study was to investigate whether enrofloxacin could be incorporated into the Artemia nauplii and to standardize the bioencapsulation technique for both antibacterial agents enrofloxacin and oxytetracycline. 2. Materials and methods 2.1. Artemia hatching Artemia franciscana cysts from the Great Salt Lake Utah were employed for this study. The corion of the cysts was chemically removed by decapsulation ŽSorgeloos et al., 1977.. Hatching of the eggs Ždecapsulated cysts. was performed overnight in a flask with 500 ml of sterile full-strength seawater, aerated through an air-hose connected to an aquarium air-pump and maintained at 288C in a reciprocal-shaking water-bath ŽPrecision Scientific, Chicago, IL, USA.. Twenty-four hours after hatching the nauplii were
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harvested and transferred to the enrichment system. This system consisted of six flasks with 100 ml of clean seawater, placed in the same water bath and individually aerated. For all the experiments the percentage of hatching and the number of nauplii was estimated by taking 10 samples of 0.5 ml of the seawater with nauplii strongly agitated to make it as homogenous as possible. The nauplii were then fixed and counted. 2.2. Enrichment of the Artemia nauplii with antibacterial agents The enrichment of the Artemia nauplii was performed using the commercial lipid emulsion Rich w ŽSanders Brine Shrimp Golden, UT, USA. to which either enrofloxacin ŽCheminova de Mexico, Mexico ´ ´ . or oxytetracycline ŽSigma, Mexico ´ . was added. In all experiments, 0.08 g of Rich w was added per flask, according to the manufacturer instructions. The desired quantity of antibiotic was added to 100 ml of filtered seawater and dissolved, then these were added to the Rich w and mixed in a domestic blender for 3 min. This mixture was the enrichment offered to the experimental Artemia nauplii, to the control nauplii, the enrichment solution was prepared in the same manner but without antibacterial agents. E. coli strain ATCC25922 was employed as an indicator. The strain used was kept frozen at y708C according to the methodology described elsewhere ŽGherna, 1994.. This bacterium was prepared by reviving it into triptic soy broth ŽTSB, Bioxon, Mexico ´ . and after 24 h it was plated onto triptic soy agar ŽTSA, Bioxon, Mexico ´ .. Several colonies were collected with a sterile swab to prepare a bacterial suspension in a sterile solution Ž0.85% NaCl. to achieve an optical density ŽOD. of 1.00 at 610 nm. This suspension was used to measure the quantity of antibiotic in the samples. 2.3. Standard curÕe (inhibition zone diameter Õs. antibiotic concentration) A standard curve was determined to know the relation between the diameter of the inhibition halo and the antibiotic concentration. This relationship was later used to calculate the quantity of antibiotic incorporated in the enriched nauplii. The bacterial suspension described earlier was plated on isosensitest agar ŽISA, Oxoid, Basinstoke, UK., after no more than 15 min, sensidiscs ŽOxoid, Basinstoke, UK. with known quantities of the antibacterial agents were placed on it. Concentrations used to calculate the curve were 300.0, 225.0, 150.0, 112.5, 75.0, 56.25, 37.5, 28.13, 18.75, 14.07, and 9.38 mg mly1 for OTC, and for enrofloxacin, 7.03, 4.69, 3.51, and 1.17 mg mly1 were also employed. The plates were incubated at 358C for 24 h and then the diameters of the inhibition zones Žinhibition halos. were measured. A linear regression was calculated. This standard curve was performed three times to confirm no significant differences amongst them. 2.4. Determination of the minimum time of bioencapsulation 2.4.1. Enrofloxacin Twenty-four hours after decapsulation, the hatching percentage of the Artemia nauplii was estimated. The nauplii were equally distributed into six flasks of 250 ml
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with 100 ml of seawater. To each flask 100 ml of the enrichment solution were added: three flasks with enrichment and enrofloxacin Ž40% of the wrv; 0.032 g of enrofloxacin per flask. and three flask with only enrichment emulsified in seawater. The flasks were placed at random in water bath at 288C with constant light and aeration. The experimental units were sampled at 0, 1, 2, 3, 4, 8, and 24 h after the enrichment was added. Ten milliliters were sampled from each flask and the nauplii contained in the sample were filtered and thoroughly washed to remove, as much as possible the enrofloxacin attached to the body of the nauplii. The samples were macerated with a tissue homogenizer with sterile saline solution Ž0.85% NaCl.. Afterwards, 20 ml of the supernatant were added to a sterile sensidisc and left to dry inside a sterile Petri dish, four sensidiscs were prepared per sample. After drying, they were applied on ISA plates inoculated by lawn with E. coli. The Petri dishes were incubated at 358C for 24 h and then the inhibition halos were measured. By means of the formula generated with the standard curve, the quantity of antibiotic present in the sample was calculated. 2.4.2. Oxytetracycline The methodology for the oxytetracycline was similar to the enrofloxacin procedure, but 80% of OTC wrv was used. Both experiments were carried out twice. 2.5. Determination of the optimal percentage of antibiotic in the enrichment solution 2.5.1. Enrofloxacin For this experiment, four treatments were used 0, 10, 20 and 40% enrofloxacin wrv, which corresponds to 0, 0.008, 0.016, and 0.032 g of enrofloxacin per gram of Rich w . The enrichment solutions were prepared as described before and 0.08 g of Rich w was used per flask. Three flasks were used in each treatment and the Artemia nauplii were prepared as before. Sampling times were at 0 h and at the time determined by the previous set of experiments as minimum to achieve a maximum incorporation of antibiotic by the nauplii. The samples were processed as for the preceding bioassay and the experiment was carried out twice. 2.5.2. Oxytetracycline For this antibiotic the quantities used were 0, 40, 80, and 160% wrv of enrichment. Sampling times and samples processing was the same as for the enrofloxacin and the experiments were carried out twice. 2.6. Determination of the loss of antibiotic from the Artemia once it is introduced into clean seawater 2.6.1. Enrofloxacin For this set of experiments 24-h-old Artemia nauplii were enriched for 4 h with 40% wrv of enrofloxacin in the Rich w and the antibioticq Rich w emulsion was prepared in the same manner as described before. After this period of time, samples were taken at
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T0 , as the 4 h were finished. Afterwards the nauplii were introduced in clean seawater trying to imitate a situation where medicated Artemia would be offered to aquatic larvae in a culture system. The sampling times of nauplii were 30 min, 1, 2, 4, 8, and 24 h. This experiment had three flasks per treatment Žwith and without antibiotic. and three sensidiscs were inoculated with the nauplii supernatant taken from macerated samples of each flask at the corresponding sampling times. All the methodology was the same as for the previous experiments. The experiment was carried out twice. 2.6.2. Oxytetracycline This experiment was the same as the previous one but 80% wrv of OTC in Rich w was used. 2.7. Statistical analysis The data from all the experiments was statistically analyzed and parametric or non-parametric tests were applied as needed. For the calibration curves and the determination of the optimal percentage of antibiotic to use an one-way ANOVA was used and for the minimum time of enrichment a Kruskal–Wallis test was used followed by a Student–Newman–Keuls test ŽSNK..
3. Results The hatching percentage was 86.85% ŽStd. Dev. 3.14, Min. 81.50, Max. 92.00. and the total number of nauplii counted were 2130 nauplii mly1 ŽStd. Dev. 121, Min. 1900, Max. 2340.. Therefore, each gram of cyst contained 245,250 cysts and 213,000 nauplii were obtained from that gram. 3.1. Standard curÕes The standard curves, zone diameter ŽZD. vs. concentration ŽConc.. determined by bioassay for the antibiotics employed produced the following equations Žmean of 3 bioassays, Fig. 1.; for enrofloxacin: ZD Žmm. s 9.451Log 10 Conc. Žmg mly1 . q 6.844; with a r 2 s 0.9899; and for OTC: ZD Žmm. s 9.1432Log 10 Conc. Žmg mly1 . y 2.6119; r 2 s 0.9931. These equations were obtained from ZD minus the diameter of the sensidiscs Ž5.5 mm.. The minimum concentration tested that produced an inhibition zone was 3.51 mg mly1 Ž12.278 mm. for enrofloxacin and 14.070 mg mly1 Ž8.333 mm. for OTC. The next lower concentrations tested produced no inhibition halo, 1.170 mg mly1 and 9.380 mg mly1 , respectively. 3.2. Determination of the optimal percentage of antibiotic in the enrichment solution The optimal concentration of antibiotic to be offered to the nauplius was found to be 40% for enrofloxacin; at this time, 1.01 and 1.07 ng per nauplius of the antibiotic was
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Fig. 1. Calibration line determined for enrofloxacin ŽEnro. and oxytetracycline ŽOTC.. Mean and 95% confidence interval. Equations describe the line best fit and with the correlation factor Ž R 2 ..
incorporated in both experiments. With 20%, 0.66 and 0.65 ng per nauplius of enrofloxacin was bioencapsulated, and with 10% no inhibition halo was observed. 40% was also the maximum concentration offered, since a higher concentration would render an antibacterial concentration that far exceeds the determined MIC for strains of Vibrio, as discussed later. Highly significant differences were observed between all the treatments Ž p - 0.01.. The enrofloxacin concentrations available in the 20% and 40% enrichment solutions were equal to 80.0 and 160.0 mg mly1 and, because the nauplii density was 2,130 nauplius mly1 , the maximum antibacterial agent that the nauplii could ingest was 37.56 Ž"2.14. ng per nauplius for the 20% and 75.117 ng per nauplius for the 40%. A significant difference Ž p - 0.001. was observed between the percentages of antibacterial agent bioencapsulated in the solutions; 1.77% of the available antibacterial agent was bioencapsulated in the 20% solution and 1.38% in the 40% solution.
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Fig. 2. Mean amount of enrofloxacin ŽEnro. and oxytetracycline ŽOTC. bioencapsulated per nauplius of Artemia during a 24-h period. Data from experiment 1. Mean and 95% confidence interval.
For OTC, 80% was the optimum with 9.32 ng per nauplius in both experiments. 160% produced 9.23 and 9.32 ng per nauplius, and with the 40%, no inhibition halos were observed. Significant differences were encountered among the different treatments Ž p - 0.01.. The 40% treatment was significantly different than the 80 and 160% treatments Ž p - 0.05.; between treatments 80% and 160%, no significant differences were obtained Ž p ) 0.05..
Table 1 Results of the multiple comparison test SNK amongst the inhibition zones for each sampling time and experiment for the enrofloxacin. Values with the same letter are not significantly different Ž p- 0.05. within the same row. SDsStandard deviation Time Žh.
1
2
3
4
8
24
Ž1. Mean ŽSD. Ž2. Mean ŽSD.
7.5 Ž0.00.a 7.5 Ž0.00.a
8.13 Ž0.00.b 8.21 Ž0.07.b
8.75 Ž0.25.c 9.08 Ž0.14.c
10.21 Ž0.19.d 10.10 Ž0.18.d
10.13 Ž0.38.d 10.02 Ž0.20.d
9.49 Ž0.39.d 10.00 Ž0.43.d
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Table 2 Results of the multiple comparison test SNK amongst the inhibition zones for each sampling time and experiment for the oxytetracycline. Values with the same letter are not significantly different Ž p- 0.05. within the same row. SDsStandard deviation Time Žh.
3
4
8
24
Exp. 1. Mean ŽSD. Exp. 2. Mean ŽSD.
8.29 Ž0.19.a 7.96 Ž0.04.a
9.34 Ž0.03.b 9.33 Ž0.04.b
9.37 Ž0.13.bc 9.29 Ž0.04.b
9.25 Ž0.00.c 9.31 Ž0.17.b
The OTC concentrations available in the 80% and 160% enrichment solutions were equal to 320.0 and 640.0 mg mly1 , respectively. Since we had 2,130 nauplii mly1 , the maximum antibiotic that the nauplii could ingest was 150.2 Ž"8.56. ng per nauplius for the 80% and 300.47 Ž"17.12. ng per nauplius for the 160% solution. A significant
Fig. 3. Mean amount of enrofloxacin ŽEnro. and oxytetracycline ŽOTC. per nauplius of bioencapsulated Artemia after placing the nauplii in sterile seawater. Data from experiment 1. Mean and 95% confidence interval.
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difference Ž p - 0.001. was observed between the percentages of antibiotic bioencapsulated in the solutions; 6.20% of the available antibiotic was bioencapsulated in the 80% solution and 3.09% in the 160% solution. 3.3. Determination of the minimum time of bioencapsulation The best time of bioencapsulation of enrofloxacin was 4 h with an average concentration of 1.1 ng of enrofloxacin per nauplius ŽFig. 2.. Significant differences were obtained between the inhibition zone diameters of the samples taken at various time intervals Ž p - 0.01.. Significant differences ŽSNK test. were observed between the values at 1, 2, 3, and 4 h, but no differences obtained between the 4, 8, and 24 h values ŽTable 1.. As for the OTC, the optimal time of bioencapsulation was also at 4 h where an average concentration of 9.3 ng of OTC was incorporated per nauplius. The Kruskal– Wallis test followed by SNK analysis showed differences from 3 to 4 h but no differences were found from 4 to 8 h, nor from 8 to 24 h, analysis are presented in Table 2 and the OTC levels of incorporation are presented in Fig. 2. 3.4. Determination of the loss of antibiotic from the bioencapsulated Artemia nauplii once they were introduced into clean seawater In just 15 min, a considerable loss of both antibiotics was observed. After 1 h, the nauplii bioencapsulated with OTC, reached a concentration of antibiotics not detectable with confidence with this method, around 0.2 ng per nauplius ŽFig. 3.; for the nauplii bioencapsulated with enrofloxacin, after 2 h this level was achieved.
4. Discussion The amount of antibiotic bioencapsulated in Artemia nauplii in this work was very similar to the results obtained by Touraki et al. Ž1999.. They incorporated 0.46 ng of trimethoprim ŽTMP. and 1.1 ng of sulfamethoxazole ŽSMX. per nauplius after 24 h, whereas in this work, an average of 1.04 ng per nauplius of enrofloxacin was obtained in just 4 h. Mohney et al. Ž1990. under similar conditions Ž4 h with rice bran as carrier. reported an uptake of 100 ng of Romet-30 Ž80% sulfadimethoxine-20% ormetoprim. per nauplius. Dixon et al. Ž1995b. bioencapsulated approximately 66 ng of sarafloxacin per nauplius with only 15% of the antibiotic in Super Selco for 6 h. Significant differences were encountered with previous reports ŽRoque et al., 1998. on the bioencapsulation of OTC, they achieved a maximum of 450 ng per nauplius whereas we found that the nauplii could not ingest and accumulate more than 9.6 ng per nauplius. This huge difference could be attributed to several factors, lack of thorough rinsing of the nauplii after the bioencapsulation period. When this procedure is not done, a higher level of antibiotic is detected, as occurred in this study when the rinsing was forgotten Žunpublished data.. Another explanations could be varying microbiological assessment techniques and bioencapsulation times.
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Evaluation of the antibiotic present in the nauplii has been performed with bioassay techniques Žmodified plate diffusions. by several authors ŽMohney et al., 1990; Dixon et al., 1995b; Roque et al., 1998. with reproducible and confident results. Although the lowest limit of detection with this method is around 3.5 mg mly1 for enrofloxacin and 14.0 mg mly1 for OTC. For sarafloxacin at 15% in a lipid emulsion, 6 h was found to be enough to bioencapsulate a maximum of this antibiotic ŽDixon et al., 1995a,b.; in the present work, 4 h were optimal to bioencapsulate enrofloxacin at 40% and OTC at 80%. Touraki et al. Ž1991, 1996. found an increase in trimethoprim and sulfamethoxazole concentration with time Žfrom 8 to 32 h., suggesting that the nauplii can incorporate and accumulate high quantities of the antibiotic-lipid emulsion without reaching equilibrium soon. These findings are in contrast to the data presented here and elsewhere ŽDixon et al., 1995a,b. where an equilibrium is rapidly achieved, not only regarding the incorporation of a antibiotic-lipid emulsion, but also in the uptake of live bacteria ŽGomez-Gil et al., 1998. and formalin-killed bacteria ŽCampbell et al., 1993.. It is apparent, that Artemia nauplii can bioencapsulate a maximum concentration of particles in a period ranging from less than an 1 h to around 8 h, depending on the substance ingested. After that time, it is possible that no more substance will be bioencapsulated as the organism has reached equilibrium between the ingestion of the substances and the excretion of the waste products. Therefore, exceeding this limit could be a waste of time. The Artemia nauplii could only incorporate as much as 1.77% of the potential enrofloxacin available to be bioencapsulated, and 6.2% of the OTC. A probable explanation could be that they have a limited capacity of incorporation andror storage of the antibiotics in the tissues; and the majority detected remains in the gut. It was also demonstrated that employing a more concentrated enrichment solution does not necessarily mean that the nauplii will encapsulate a higher percentage of the available antibiotic. An optimum has to be found between the concentration and the efficiency of bioencapsulation. Rinsing the Artemia nauplii after bioencapsulation with either antibiotic employed significantly lowers the concentration rapidly. Therefore, it is suggested that the nauplii should only be collected in an appropriate mesh and delivered to the larvae without any further treatment. Storing of the nauplii for periods longer than 2 h at 258C significantly reduces the concentration of trimethoprim and sulfamethoxazole ŽTouraki et al., 1999.; even at 58C, after just 4 h, the concentration of sulfamethoxazole is significantly reduced. It is important to calculate if the antibiotic bioencapsulated is sufficient to achieve more than the recommended MIC in the target organism, this has to take into account the number of nauplii that the target organism can consume in a period of time. Touraki et al. Ž1999. administered Artemia nauplii with TMPrSMX in 10 doses to seabass larvae during 5 days at 12-h intervals, which exceeded several times the MIC value of 0.8 mg mly1 reported for a Vibrio anguillarum strain ŽSamuelsen et al., 1997.. In shrimps, a 0.05 g postlarvae can consume an average of 2500 Artemia nauplii per day Žunpublished data., with the data presented here, a total amount of 55.6 mg of enrofloxacin per gram of postlarvae could be achieved. The MIC of enrofloxacin for 100
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Vibrio strains isolated from diseased shrimps is 3.0 mg mly1 ŽRoque et al., submitted for publication., therefore, a crude estimation results in a concentration almost 18 times de MIC if administered daily. Following the same reasoning but for the case of OTC, 466.0 mg could be achieved per gram of postlarvae. This value is just over 1.5 times the MIC for OTC, therefore, the use of OTC is not recommended bioencapsulated in Artemia nauplii against vibrios isolated from diseased shrimps from northwestern Mexico.
Acknowledgements QFB. Carmen Bolan-Mejıa, ´ ´ Ing. Cesareo Cabrera, Dr. Perez-Lopez ´ ´ of Cheminova de Mexico. This research was funded by FOSIMAC 98016073. ´
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Roque, A., Molina-Aja, A., Bolan-Mejıa, ´ ´ C., Gomez-Gil, B., submitted for publication. In vitro susceptibilities of Vibrio strains isolated from penaeid shrimps in Northwestern Mexico to 15 antibiotics. Intl. J. Antimicrob. Agents. Samuelsen, O.B., Lunestad, B.T., Jelmert, A., 1997. Pharmacokinetic and efficacy studies on bath-administering potentiated sulphonamides in Atlantic halibut, Hippoglossus hippoglossus L. J. Fish. Dis. 20, 287–296. Sorgeloos, P., Bossuyt, E., Lavina, ˜ E., Baeza-Mesa, M., Persoone, G., 1977. Descapsulation of Artemia cysts: a simple technique for improvement of the use of brine shrmip in aquaculture. Aquaculture 12, 311–312. Touraki, M., Rigas, P., Pergantas, P., Abatzopoulos, T., Kastritsis, C., 1991. Optimizing bioencapsulation of the antibiotics trimethoprim and sulfamethoxazole in Artemia nauplii. In: Lavens, P., Sorgeloos, P., Jaspers, E., Ollevier, F. ŽEds.., Larvi’91, vol. 15, pp. 415–418. Touraki, M., Rigas, P., Kastritsis, C., 1995. Liposome mediated delivery of water soluble antibiotics to the larvae of aquatic animals. Aquaculture 136, 1–10. Touraki, M., Mourelatus, S., Karamanlidou, G., Kalaitzopoulou, S., Kastritsis, C., 1996. Bioencapsulation of chemotherapeutics in Artemia as a means of prevention and treatment of infectious diseases of marine fish fry. Aquacult. Eng. 15, 133–147. Touraki, M., Niopas, I., Kastritsis, C., 1999. Bioaccumulation of trimethoprim, sulfamethoxazole and N-acetyl-sulfamethoxazole in Artemia nauplii and residual kinetics in seabass larvae after repeated oral dosing of medicated nauplii. Aquaculture 175, 15–30. Verpraet, R., Chair, M., Leger, P., Nelis, H., Sorgeloos, P., Leenheer, A.D., 1992. Live-food mediated drug delivery as a tool for disease treatment in larviculture. The enrichment of therapeutics in rotifers and Artemia nauplii. Aquacult. Eng. 11, 133–139.
Standardization of the bioencapsulation of enrofloxacin and oxytetracycline in Artemia franciscana Kellogg, 1906 B. Gomez-Gil ) , J. Cabanillas-Ramos, S. Paez-Brambila, A. Roque Centro de InÕestigacion ´ en Alimentacion ´ y Desarrollo, A.C., Unidad Mazatlan ´ en Acuicultura y Manejo Ambiental. AP.711 CP. 82000 Mazatlan ´ Sinaloa, Mexico Received 1 August 2000; received in revised form 22 October 2000; accepted 4 November 2000
Abstract Bioencapsulation of enrofloxacin and oxytetracycline into Artemia franciscana nauplii was standardized. Both antibacterials were delivered to the nauplii individually and the amounts used in the study were percentages of the lipid emulsion Rich w added as nutrition enrichment for the nauplii. The determination of the amounts of drug incorporated in A. franciscana nauplii was obtained using a bioassay radial diffusion method, standardized in the laboratory and using Escherichia coli as an indicator. The minimum time for full enrichment for both enrofloxacin and oxytetracycline bioencapsulation in A. franciscana nauplii was 4 h after the initial exposure of the nauplii to the antibiotics and this was established sampling nauplii at 1, 2, 3, 4, 8 and 24 h after adding the nauplii to the mix. These experiments were carried out twice and at 4 h, 1.10 and 1.13 ng of enrofloxacin per nauplius and 9.32 and 9.37 ng of oxytetracycline per nauplius were obtained. The optimum percentages of enrichment were 40% of enrofloxacin in relation to Rich w and 80% of oxytetracycline. The percentages tested were 10%, 20%, 30%, 40% for the enrofloxacin and 0%, 40%, 80%, and 160% for the oxytetracycline. The maximum time at which antibiotic was still detected in the Artemia after it had been introduced in seawater was 8 h for both antibacterial agents. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Artemia franciscana; Enrofloxacin; Oxytetracycline; Bioencapsulation
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Corresponding author. Tel.: q52-69-880157; fax: q52-69-880159. E-mail address: [email protected] ŽB. Gomez-Gil..
0044-8486r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 4 - 8 4 8 6 Ž 0 0 . 0 0 5 6 8 - 8
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1. Introduction The delivery of drugs to aquatic animals is problematic. Delivery by medicated food leads to leaching of the drug from the pellets ŽJacobsen and Berglind, 1988.. In bath treatments, a substantial portion of the drug is not used and it can be difficult to dispose of the excess of drug safely. The value of Artemia sp. as a carrier for the delivery of antibacterials to fish and shrimp larvae has been investigated ŽMohney et al., 1990; Chair et al., 1991a; Nelis et al., 1991; Touraki et al., 1991; Verpraet et al., 1992; Roque et al., 1998.. Most of the authors worked with either trimethoprim-sulfamethoxazole or chemical analogous, ormethroprim-sulphadimethoxine ŽMohney et al., 1990; Chair et al., 1991a; Touraki et al., 1991; Verpraet et al., 1992; Gapasin et al., 1996.. The technique though is not standardized and enrichment times varied from 2 h ŽGapasin et al., 1996. to 32 h ŽTouraki et al., 1991, 1995.. The quantity of antibiotic offered is, in most cases, defined as a percentage of the commercial enrichment in which it goes mixed ŽTouraki et al., 1991; Verpraet et al., 1992; Dixon et al., 1995b; Gapasin et al., 1996; Roque et al., 1998. varying from 1% wrv ŽVerpraet et al., 1992. to 40% wrv ŽDixon et al., 1995b; Gapasin et al., 1996; Roque et al., 1998.. An exception to this is the work performed by Mohney et al. Ž1990. who added 3 mg of antibiotic per liter of water. The techniques used to measured the levels of antibiotic incorporated in the Artemia nauplii also varied including radial diffusion bioassay which used either Escherichia coli as the indicator bacterium ŽDixon et al., 1995b.; Vibrio alginolyticus ŽMohney et al., 1990. and Bacillus subtilis ŽRoque et al., 1998.. Other studies have used HPLC as a mean to more accurately measure the amount of antibiotic incorporated ŽTouraki et al., 1991, 1995; Chair et al., 1991b.. In Mexico, enrofloxacin and oxytetracycline ŽOTC. are the antibacterials most commonly used by shrimp hatcheries. Although the possibility of delivering OTC to shrimp larvae by this method has already been investigated the technique was not standardized and the minimum time and quantity of antibiotic to enrich the Artemia, in order to incorporate therapeutic levels of the drug in the Artemia has not been determined. As for the enrofloxacin, the possibility of bioencapsulation into Artemia has not been reported in the literature. Therefore, the aim of this study was to investigate whether enrofloxacin could be incorporated into the Artemia nauplii and to standardize the bioencapsulation technique for both antibacterial agents enrofloxacin and oxytetracycline. 2. Materials and methods 2.1. Artemia hatching Artemia franciscana cysts from the Great Salt Lake Utah were employed for this study. The corion of the cysts was chemically removed by decapsulation ŽSorgeloos et al., 1977.. Hatching of the eggs Ždecapsulated cysts. was performed overnight in a flask with 500 ml of sterile full-strength seawater, aerated through an air-hose connected to an aquarium air-pump and maintained at 288C in a reciprocal-shaking water-bath ŽPrecision Scientific, Chicago, IL, USA.. Twenty-four hours after hatching the nauplii were
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harvested and transferred to the enrichment system. This system consisted of six flasks with 100 ml of clean seawater, placed in the same water bath and individually aerated. For all the experiments the percentage of hatching and the number of nauplii was estimated by taking 10 samples of 0.5 ml of the seawater with nauplii strongly agitated to make it as homogenous as possible. The nauplii were then fixed and counted. 2.2. Enrichment of the Artemia nauplii with antibacterial agents The enrichment of the Artemia nauplii was performed using the commercial lipid emulsion Rich w ŽSanders Brine Shrimp Golden, UT, USA. to which either enrofloxacin ŽCheminova de Mexico, Mexico ´ ´ . or oxytetracycline ŽSigma, Mexico ´ . was added. In all experiments, 0.08 g of Rich w was added per flask, according to the manufacturer instructions. The desired quantity of antibiotic was added to 100 ml of filtered seawater and dissolved, then these were added to the Rich w and mixed in a domestic blender for 3 min. This mixture was the enrichment offered to the experimental Artemia nauplii, to the control nauplii, the enrichment solution was prepared in the same manner but without antibacterial agents. E. coli strain ATCC25922 was employed as an indicator. The strain used was kept frozen at y708C according to the methodology described elsewhere ŽGherna, 1994.. This bacterium was prepared by reviving it into triptic soy broth ŽTSB, Bioxon, Mexico ´ . and after 24 h it was plated onto triptic soy agar ŽTSA, Bioxon, Mexico ´ .. Several colonies were collected with a sterile swab to prepare a bacterial suspension in a sterile solution Ž0.85% NaCl. to achieve an optical density ŽOD. of 1.00 at 610 nm. This suspension was used to measure the quantity of antibiotic in the samples. 2.3. Standard curÕe (inhibition zone diameter Õs. antibiotic concentration) A standard curve was determined to know the relation between the diameter of the inhibition halo and the antibiotic concentration. This relationship was later used to calculate the quantity of antibiotic incorporated in the enriched nauplii. The bacterial suspension described earlier was plated on isosensitest agar ŽISA, Oxoid, Basinstoke, UK., after no more than 15 min, sensidiscs ŽOxoid, Basinstoke, UK. with known quantities of the antibacterial agents were placed on it. Concentrations used to calculate the curve were 300.0, 225.0, 150.0, 112.5, 75.0, 56.25, 37.5, 28.13, 18.75, 14.07, and 9.38 mg mly1 for OTC, and for enrofloxacin, 7.03, 4.69, 3.51, and 1.17 mg mly1 were also employed. The plates were incubated at 358C for 24 h and then the diameters of the inhibition zones Žinhibition halos. were measured. A linear regression was calculated. This standard curve was performed three times to confirm no significant differences amongst them. 2.4. Determination of the minimum time of bioencapsulation 2.4.1. Enrofloxacin Twenty-four hours after decapsulation, the hatching percentage of the Artemia nauplii was estimated. The nauplii were equally distributed into six flasks of 250 ml
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with 100 ml of seawater. To each flask 100 ml of the enrichment solution were added: three flasks with enrichment and enrofloxacin Ž40% of the wrv; 0.032 g of enrofloxacin per flask. and three flask with only enrichment emulsified in seawater. The flasks were placed at random in water bath at 288C with constant light and aeration. The experimental units were sampled at 0, 1, 2, 3, 4, 8, and 24 h after the enrichment was added. Ten milliliters were sampled from each flask and the nauplii contained in the sample were filtered and thoroughly washed to remove, as much as possible the enrofloxacin attached to the body of the nauplii. The samples were macerated with a tissue homogenizer with sterile saline solution Ž0.85% NaCl.. Afterwards, 20 ml of the supernatant were added to a sterile sensidisc and left to dry inside a sterile Petri dish, four sensidiscs were prepared per sample. After drying, they were applied on ISA plates inoculated by lawn with E. coli. The Petri dishes were incubated at 358C for 24 h and then the inhibition halos were measured. By means of the formula generated with the standard curve, the quantity of antibiotic present in the sample was calculated. 2.4.2. Oxytetracycline The methodology for the oxytetracycline was similar to the enrofloxacin procedure, but 80% of OTC wrv was used. Both experiments were carried out twice. 2.5. Determination of the optimal percentage of antibiotic in the enrichment solution 2.5.1. Enrofloxacin For this experiment, four treatments were used 0, 10, 20 and 40% enrofloxacin wrv, which corresponds to 0, 0.008, 0.016, and 0.032 g of enrofloxacin per gram of Rich w . The enrichment solutions were prepared as described before and 0.08 g of Rich w was used per flask. Three flasks were used in each treatment and the Artemia nauplii were prepared as before. Sampling times were at 0 h and at the time determined by the previous set of experiments as minimum to achieve a maximum incorporation of antibiotic by the nauplii. The samples were processed as for the preceding bioassay and the experiment was carried out twice. 2.5.2. Oxytetracycline For this antibiotic the quantities used were 0, 40, 80, and 160% wrv of enrichment. Sampling times and samples processing was the same as for the enrofloxacin and the experiments were carried out twice. 2.6. Determination of the loss of antibiotic from the Artemia once it is introduced into clean seawater 2.6.1. Enrofloxacin For this set of experiments 24-h-old Artemia nauplii were enriched for 4 h with 40% wrv of enrofloxacin in the Rich w and the antibioticq Rich w emulsion was prepared in the same manner as described before. After this period of time, samples were taken at
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T0 , as the 4 h were finished. Afterwards the nauplii were introduced in clean seawater trying to imitate a situation where medicated Artemia would be offered to aquatic larvae in a culture system. The sampling times of nauplii were 30 min, 1, 2, 4, 8, and 24 h. This experiment had three flasks per treatment Žwith and without antibiotic. and three sensidiscs were inoculated with the nauplii supernatant taken from macerated samples of each flask at the corresponding sampling times. All the methodology was the same as for the previous experiments. The experiment was carried out twice. 2.6.2. Oxytetracycline This experiment was the same as the previous one but 80% wrv of OTC in Rich w was used. 2.7. Statistical analysis The data from all the experiments was statistically analyzed and parametric or non-parametric tests were applied as needed. For the calibration curves and the determination of the optimal percentage of antibiotic to use an one-way ANOVA was used and for the minimum time of enrichment a Kruskal–Wallis test was used followed by a Student–Newman–Keuls test ŽSNK..
3. Results The hatching percentage was 86.85% ŽStd. Dev. 3.14, Min. 81.50, Max. 92.00. and the total number of nauplii counted were 2130 nauplii mly1 ŽStd. Dev. 121, Min. 1900, Max. 2340.. Therefore, each gram of cyst contained 245,250 cysts and 213,000 nauplii were obtained from that gram. 3.1. Standard curÕes The standard curves, zone diameter ŽZD. vs. concentration ŽConc.. determined by bioassay for the antibiotics employed produced the following equations Žmean of 3 bioassays, Fig. 1.; for enrofloxacin: ZD Žmm. s 9.451Log 10 Conc. Žmg mly1 . q 6.844; with a r 2 s 0.9899; and for OTC: ZD Žmm. s 9.1432Log 10 Conc. Žmg mly1 . y 2.6119; r 2 s 0.9931. These equations were obtained from ZD minus the diameter of the sensidiscs Ž5.5 mm.. The minimum concentration tested that produced an inhibition zone was 3.51 mg mly1 Ž12.278 mm. for enrofloxacin and 14.070 mg mly1 Ž8.333 mm. for OTC. The next lower concentrations tested produced no inhibition halo, 1.170 mg mly1 and 9.380 mg mly1 , respectively. 3.2. Determination of the optimal percentage of antibiotic in the enrichment solution The optimal concentration of antibiotic to be offered to the nauplius was found to be 40% for enrofloxacin; at this time, 1.01 and 1.07 ng per nauplius of the antibiotic was
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Fig. 1. Calibration line determined for enrofloxacin ŽEnro. and oxytetracycline ŽOTC.. Mean and 95% confidence interval. Equations describe the line best fit and with the correlation factor Ž R 2 ..
incorporated in both experiments. With 20%, 0.66 and 0.65 ng per nauplius of enrofloxacin was bioencapsulated, and with 10% no inhibition halo was observed. 40% was also the maximum concentration offered, since a higher concentration would render an antibacterial concentration that far exceeds the determined MIC for strains of Vibrio, as discussed later. Highly significant differences were observed between all the treatments Ž p - 0.01.. The enrofloxacin concentrations available in the 20% and 40% enrichment solutions were equal to 80.0 and 160.0 mg mly1 and, because the nauplii density was 2,130 nauplius mly1 , the maximum antibacterial agent that the nauplii could ingest was 37.56 Ž"2.14. ng per nauplius for the 20% and 75.117 ng per nauplius for the 40%. A significant difference Ž p - 0.001. was observed between the percentages of antibacterial agent bioencapsulated in the solutions; 1.77% of the available antibacterial agent was bioencapsulated in the 20% solution and 1.38% in the 40% solution.
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Fig. 2. Mean amount of enrofloxacin ŽEnro. and oxytetracycline ŽOTC. bioencapsulated per nauplius of Artemia during a 24-h period. Data from experiment 1. Mean and 95% confidence interval.
For OTC, 80% was the optimum with 9.32 ng per nauplius in both experiments. 160% produced 9.23 and 9.32 ng per nauplius, and with the 40%, no inhibition halos were observed. Significant differences were encountered among the different treatments Ž p - 0.01.. The 40% treatment was significantly different than the 80 and 160% treatments Ž p - 0.05.; between treatments 80% and 160%, no significant differences were obtained Ž p ) 0.05..
Table 1 Results of the multiple comparison test SNK amongst the inhibition zones for each sampling time and experiment for the enrofloxacin. Values with the same letter are not significantly different Ž p- 0.05. within the same row. SDsStandard deviation Time Žh.
1
2
3
4
8
24
Ž1. Mean ŽSD. Ž2. Mean ŽSD.
7.5 Ž0.00.a 7.5 Ž0.00.a
8.13 Ž0.00.b 8.21 Ž0.07.b
8.75 Ž0.25.c 9.08 Ž0.14.c
10.21 Ž0.19.d 10.10 Ž0.18.d
10.13 Ž0.38.d 10.02 Ž0.20.d
9.49 Ž0.39.d 10.00 Ž0.43.d
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Table 2 Results of the multiple comparison test SNK amongst the inhibition zones for each sampling time and experiment for the oxytetracycline. Values with the same letter are not significantly different Ž p- 0.05. within the same row. SDsStandard deviation Time Žh.
3
4
8
24
Exp. 1. Mean ŽSD. Exp. 2. Mean ŽSD.
8.29 Ž0.19.a 7.96 Ž0.04.a
9.34 Ž0.03.b 9.33 Ž0.04.b
9.37 Ž0.13.bc 9.29 Ž0.04.b
9.25 Ž0.00.c 9.31 Ž0.17.b
The OTC concentrations available in the 80% and 160% enrichment solutions were equal to 320.0 and 640.0 mg mly1 , respectively. Since we had 2,130 nauplii mly1 , the maximum antibiotic that the nauplii could ingest was 150.2 Ž"8.56. ng per nauplius for the 80% and 300.47 Ž"17.12. ng per nauplius for the 160% solution. A significant
Fig. 3. Mean amount of enrofloxacin ŽEnro. and oxytetracycline ŽOTC. per nauplius of bioencapsulated Artemia after placing the nauplii in sterile seawater. Data from experiment 1. Mean and 95% confidence interval.
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difference Ž p - 0.001. was observed between the percentages of antibiotic bioencapsulated in the solutions; 6.20% of the available antibiotic was bioencapsulated in the 80% solution and 3.09% in the 160% solution. 3.3. Determination of the minimum time of bioencapsulation The best time of bioencapsulation of enrofloxacin was 4 h with an average concentration of 1.1 ng of enrofloxacin per nauplius ŽFig. 2.. Significant differences were obtained between the inhibition zone diameters of the samples taken at various time intervals Ž p - 0.01.. Significant differences ŽSNK test. were observed between the values at 1, 2, 3, and 4 h, but no differences obtained between the 4, 8, and 24 h values ŽTable 1.. As for the OTC, the optimal time of bioencapsulation was also at 4 h where an average concentration of 9.3 ng of OTC was incorporated per nauplius. The Kruskal– Wallis test followed by SNK analysis showed differences from 3 to 4 h but no differences were found from 4 to 8 h, nor from 8 to 24 h, analysis are presented in Table 2 and the OTC levels of incorporation are presented in Fig. 2. 3.4. Determination of the loss of antibiotic from the bioencapsulated Artemia nauplii once they were introduced into clean seawater In just 15 min, a considerable loss of both antibiotics was observed. After 1 h, the nauplii bioencapsulated with OTC, reached a concentration of antibiotics not detectable with confidence with this method, around 0.2 ng per nauplius ŽFig. 3.; for the nauplii bioencapsulated with enrofloxacin, after 2 h this level was achieved.
4. Discussion The amount of antibiotic bioencapsulated in Artemia nauplii in this work was very similar to the results obtained by Touraki et al. Ž1999.. They incorporated 0.46 ng of trimethoprim ŽTMP. and 1.1 ng of sulfamethoxazole ŽSMX. per nauplius after 24 h, whereas in this work, an average of 1.04 ng per nauplius of enrofloxacin was obtained in just 4 h. Mohney et al. Ž1990. under similar conditions Ž4 h with rice bran as carrier. reported an uptake of 100 ng of Romet-30 Ž80% sulfadimethoxine-20% ormetoprim. per nauplius. Dixon et al. Ž1995b. bioencapsulated approximately 66 ng of sarafloxacin per nauplius with only 15% of the antibiotic in Super Selco for 6 h. Significant differences were encountered with previous reports ŽRoque et al., 1998. on the bioencapsulation of OTC, they achieved a maximum of 450 ng per nauplius whereas we found that the nauplii could not ingest and accumulate more than 9.6 ng per nauplius. This huge difference could be attributed to several factors, lack of thorough rinsing of the nauplii after the bioencapsulation period. When this procedure is not done, a higher level of antibiotic is detected, as occurred in this study when the rinsing was forgotten Žunpublished data.. Another explanations could be varying microbiological assessment techniques and bioencapsulation times.
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Evaluation of the antibiotic present in the nauplii has been performed with bioassay techniques Žmodified plate diffusions. by several authors ŽMohney et al., 1990; Dixon et al., 1995b; Roque et al., 1998. with reproducible and confident results. Although the lowest limit of detection with this method is around 3.5 mg mly1 for enrofloxacin and 14.0 mg mly1 for OTC. For sarafloxacin at 15% in a lipid emulsion, 6 h was found to be enough to bioencapsulate a maximum of this antibiotic ŽDixon et al., 1995a,b.; in the present work, 4 h were optimal to bioencapsulate enrofloxacin at 40% and OTC at 80%. Touraki et al. Ž1991, 1996. found an increase in trimethoprim and sulfamethoxazole concentration with time Žfrom 8 to 32 h., suggesting that the nauplii can incorporate and accumulate high quantities of the antibiotic-lipid emulsion without reaching equilibrium soon. These findings are in contrast to the data presented here and elsewhere ŽDixon et al., 1995a,b. where an equilibrium is rapidly achieved, not only regarding the incorporation of a antibiotic-lipid emulsion, but also in the uptake of live bacteria ŽGomez-Gil et al., 1998. and formalin-killed bacteria ŽCampbell et al., 1993.. It is apparent, that Artemia nauplii can bioencapsulate a maximum concentration of particles in a period ranging from less than an 1 h to around 8 h, depending on the substance ingested. After that time, it is possible that no more substance will be bioencapsulated as the organism has reached equilibrium between the ingestion of the substances and the excretion of the waste products. Therefore, exceeding this limit could be a waste of time. The Artemia nauplii could only incorporate as much as 1.77% of the potential enrofloxacin available to be bioencapsulated, and 6.2% of the OTC. A probable explanation could be that they have a limited capacity of incorporation andror storage of the antibiotics in the tissues; and the majority detected remains in the gut. It was also demonstrated that employing a more concentrated enrichment solution does not necessarily mean that the nauplii will encapsulate a higher percentage of the available antibiotic. An optimum has to be found between the concentration and the efficiency of bioencapsulation. Rinsing the Artemia nauplii after bioencapsulation with either antibiotic employed significantly lowers the concentration rapidly. Therefore, it is suggested that the nauplii should only be collected in an appropriate mesh and delivered to the larvae without any further treatment. Storing of the nauplii for periods longer than 2 h at 258C significantly reduces the concentration of trimethoprim and sulfamethoxazole ŽTouraki et al., 1999.; even at 58C, after just 4 h, the concentration of sulfamethoxazole is significantly reduced. It is important to calculate if the antibiotic bioencapsulated is sufficient to achieve more than the recommended MIC in the target organism, this has to take into account the number of nauplii that the target organism can consume in a period of time. Touraki et al. Ž1999. administered Artemia nauplii with TMPrSMX in 10 doses to seabass larvae during 5 days at 12-h intervals, which exceeded several times the MIC value of 0.8 mg mly1 reported for a Vibrio anguillarum strain ŽSamuelsen et al., 1997.. In shrimps, a 0.05 g postlarvae can consume an average of 2500 Artemia nauplii per day Žunpublished data., with the data presented here, a total amount of 55.6 mg of enrofloxacin per gram of postlarvae could be achieved. The MIC of enrofloxacin for 100
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Vibrio strains isolated from diseased shrimps is 3.0 mg mly1 ŽRoque et al., submitted for publication., therefore, a crude estimation results in a concentration almost 18 times de MIC if administered daily. Following the same reasoning but for the case of OTC, 466.0 mg could be achieved per gram of postlarvae. This value is just over 1.5 times the MIC for OTC, therefore, the use of OTC is not recommended bioencapsulated in Artemia nauplii against vibrios isolated from diseased shrimps from northwestern Mexico.
Acknowledgements QFB. Carmen Bolan-Mejıa, ´ ´ Ing. Cesareo Cabrera, Dr. Perez-Lopez ´ ´ of Cheminova de Mexico. This research was funded by FOSIMAC 98016073. ´
References Campbell, R., Adams, A., Tatner, M.F., Chair, M., Sorgeloos, P., 1993. Uptake of Vibrio anguillarum vaccine by Artemia salina as a potential oral delivery system to fish fry. Fish. Shellfish Immunol. 3, 451–459. Chair, M., Romdhane, M., Dehasque, M., Nelis, H., De Leenheer, A.P., Sorgeloos, P., 1991a. Live-food mediated drug delivery as a tool for disease treatment in larviculture. 2: a case study with European seabass. LARVI’91—Special Publication of the European Aquaculture Society 15, 412–414. Chair, M., Nelis, H.J., Leger, P., Sorgeloos, P., De Leenheer, A.P., 1991b. Accumulation of trimethoprim, sulfamethoxazole and N-Acetylsulfxazole in fish and shrimp fed medicated Artemia franciscana. Antimicrob. Agents Chemother. 40, 1649–1652. Dixon, B.A., Van Poucke, S.O., Kawahigashi, D., Chair, M., Dehasque, M., Nelis, H.J., Sorgeloss, P., De Leenheer, A.P., 1995a. Bioencapsulation of antibacterial drugs in nauplii and adult brine shrimp, Artemia franciscana. In: Lavens, P., Jaspers, E., Roelants, I. ŽEds.., Larvi’95—Fish and Shellfish Larviculture Symposium. Special Publication vol. 24. European Aquaculture Society, Ghent, Belgium, pp. 508–509. Dixon, B.A., Van Poucke, S.O., Chair, M., Dehasque, M., Nelis, H.J., Sorgeloos, P., De Leenheer, A.P., 1995b. Bioencapsulation of the antibacterial drug sarafloxacin in nauplii of the brine shrimp Artemia franciscana. J. Aquat. Anim. Health 7, 42–45. Gapasin, R.S.J., Nelis, H.J., Chair, M., Sorgeloos, P., 1996. Drug assimilation in the tissue of European sea bass Ž Dicentrarchus labrax . fry delivered orally through bioencapsulation. J. Appl. Ichthyol. Z. Angew. Ichthyol. 12, 39–42. Gherna, L.R., 1994. Culture preservation. In: Gerhardt, P. ŽEd.., Methods for General and Molecular Bacteriology. American Society for Microbiology, Washington, D.C., pp. 278–292. Gomez-Gil, B., Herrera-Vega, M.A., Abreu-Grobois, F.A., Roque, A., 1998. Bioencapsulation of two different Vibrio species in nauplii of the brine shrimp Ž Artemia franciscana.. App. Environ. Microbiol. 64, 2318–2322. Jacobsen, P., Berglind, L., 1988. Persistence of oxytetracycline in sediments from fish farms. Aquaculture 70, 365–370. Mohney, L.L., Lightner, D.V., Williams, R.R., Bauerlein, M., 1990. Bioencapsulation of therapeutic quantities of the antibacterial Romet-30 in nauplii of the brine shrimp Artemia and in the nematode Panagrellus rediÕiÕus. J. World Aquacult. Soc. 21, 186–191. Nelis, H.J., Leger, F., Sorgeloos, P., De Leenheer, A.P., 1991. Liquid chromatographic determination of efficacy of incorporation of trimethoprim and sulfamethoxazole in brine shrimp Ž Artemia spp.. used for prophylactic chemotherapy of fish. Antimicrob. Agents Chemother. 35, 2486–2489. Roque, A., Turnbull, J.F., Gomez-Gil, B., 1998. Delivery of bioencapsulated oxytetracycline to the marine shrimp Penaeus monodon. J. World Aquacult. Soc. 29, 249–251.
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