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Therapeutic effects of enrofloxacin in an experimental infection with a luminescent Vibrio harveyi in Artemia franciscana Kellog 1906
Aquaculture 220 (2003) 37 – 42 www.elsevier.com/locate/aqua-online
Therapeutic effects of enrofloxacin in an experimental infection with a luminescent Vibrio harveyi in Artemia franciscana Kellog 1906 A. Roque *, B. Gomez-Gil Centro de Investigacio´n en Alimentacio´n y Desarrollo, A.C., Unidad Mazatla´n en Acuicultura y Manejo Ambiental, AP. 711, CP. 82000 Mazatla´n, Sinaloa, Mexico Received 29 November 2001; received in revised form 14 January 2002; accepted 18 June 2002
Abstract Although a wide range and number of chemotherapeutants have been developed and applied in aquaculture, there have been no studies on the absorption of antibiotics by crustacean larvae and, consequently, none has been reported to treat an established infection. The aim of this study was to evaluate the therapeutic effects of enrofloxacin during an experimental infection with the strain PN9801 identified as Vibrio harveyi in Artemia franciscana. Four treatments were used: (1) Nauplii experimentally infected with V. harveyi; (2) Nauplii enriched with enrofloxacin; (3) Nauplii enriched with enrofloxacin and, 4 h later, experimentally infected with V. harveyi; and (4) Nauplii experimentally infected with V. harveyi and 24 h later enriched with enrofloxacin. Survival rates were estimated after 48 h and they were as it follows: 29%, 99%, 88% and 85% for treatments 1, 2, 3 and 4, respectively. This study is the first one to show some evidence that antibiotics can, in fact, withhold the course of a bacterial infection already established in Artemia and possibly in crustaceans. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Artemia franciscana; Enrofloxacin; Vibrio harveyi
1. Introduction Although a wide range and number of chemotherapeutants have been developed and applied in aquaculture; chemotherapeutants for shrimp farming use are limited. Antibiotics *
Corresponding author. Tel.: +52-69-880157; fax: +52-69-880159. E-mail address: [email protected] (A. Roque).
0044-8486/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0044-8486(02)00272-7
38
A. Roque, B. Gomez-Gil / Aquaculture 220 (2003) 37–42
are often used in Asian and Latin American shrimp farms, but information on the use of these compounds is limited. Typically, the treatment of choice, if one exists, has been established by a trial-and-error process with the compounds, which were most readily available at that particular time. Up to now, there have been no studies on the absorption of antibiotics by crustacean larvae and, consequently, none has been reported to treat an established infection although they may slow the infection rate in the tank by reducing the bacterial load present in the water. Existing challenge models for shrimp larvae have not produced an established model to study vibriosis (Gomez-Gil, 1998; Prayitno and Latchford, 1995). This is mainly due to the variability of results. The same bacterium in one replicate kills many larvae; in the other replicate, no mortalities are recorded. The general rule is that very low or no mortalities are produced. However, a technique has been developed to encapsulate bacteria in Artemia nauplii under sterile conditions (Gomez-Gil et al., 1998). The results presented suggest that this technique can be used as quick screening test for finding potentially pathogenic bacteria. The aim of this study was to evaluate the therapeutic effects of enrofloxacin during an experimental infection with Vibrio harveyi in Artemia franciscana.
2. Material and methods 2.1. Artemia hatching A. franciscana cysts from the Great Salt Lake Utah were employed for this study. The chorion of the cysts was chemically removed employing the methodology proposed by Sorgeloos et al. (1977), a process known as decapsulation. Hatching of the eggs (decapsulated cysts) was performed overnight in a flask with 500 ml of sterile fullstrength seawater (35x), aerated through an air hose connected to an aquarium air pump and maintained at 28 jC in a reciprocal shaking water bath (Precison Scientific, Chicago, USA). Twenty-four hours after hatching, the nauplii were harvested and transferred to the enrichment system. This one consisted of 16 flasks with 45 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. Artemia nauplii enrichment with antibiotics The enrichment of the Artemia nauplii was performed using the commercial lipid emulsion RichR (Sanders Brine Shrimp Golden, Utah, USA) to which 40% w/w of enrofloxacin (Cheminova de Me´xico, Me´xico) was added. The desired (0.032 g/flask, a total of 0.384 g of enrofloxacin) quantity of antibiotic was added to 100 ml of filtered seawater and dissolved; then, these were added to the RichR (0.08 g/flask according to the manufacturer instructions) and mixed in a domestic mixer for 3 min. This mixture was the enrichment offered to the Artemia nauplii. Five milliliters of this enrichment were added to
A. Roque, B. Gomez-Gil / Aquaculture 220 (2003) 37–42
39
each flask at the time required. Plain RichR-emulsified was added to the Artemia in the cases where no antibiotic was added. 2.3. Preparation of the V. harveyi strain The strain of V. harveyi used was isolated from the lymphoid organ of a diseased juvenile marine shrimp Penaeus monodon in the Philippines and coded PN9801. It was identified and donated to the Collection of Aquacultural Important Microorganisms at CIAD/Mazatlan Unit by Dr. Lavilla-Pitogo in 1999. The V. harveyi strain was preserved at 70 jC in an ultralow mechanical freezer (Revco Scientific, Asheville, NC) in cryovials filled with glass beads (Gherna, 1994). In order to recover the strain from cryopreservation, a bead was obtained from the cryovial and placed in a test tube with tripticasein soy broth (TSB, Bioxon, Me´xico) with 2% sodium chloride (NaCl) and incubated overnight at 30 jC with constant agitation. The following day, 8 ml of the bacterial solution were centrifuged at 3600 g for 10 min at 4 jC. The supernatant was discharged and the bacterial pellet resuspended in sterile saline solution (2.5% NaCl). This bacterial solution was centrifuged again under the same conditions. The supernatant was discharged and the resuspention and centrifugation were repeated. Once the supernatant was discharged, the bacterial pellet was suspended in approximately 5 ml of sterile saline solution and this volume was adjusted to get an optical density of 1.00 at 610 nm in a spectrophotometer (model DR-2000; Hach, Loveland, CO). To estimate the bacterial concentration achieved and to verify its purity, the suspension was serially diluted in sterile saline solution and spread plated in agar thiosulphate citrate bile salt (TCBS, Difco, Detroit, MI). 2.4. Challenge of the Artemia Six hundred sterile nauplii (individually counted) were added to each 100-ml flask with the 5 ml of bacterial suspension at a density 10 7 cells ml 1 and 45 ml of sterile seawater for 48 h and the number of surviving nauplii estimated. In the treatments with Rich, 0.08 g of it were added per flask prepared as described previously. 2.5. Experimental protocol The protocol followed to infect the Artemia nauplii was the one described above. Preliminary work was carried to rule out the possibility of Rich acting as an antimicrobial. 2.6. Final experiment Four treatments were used: 1. Artemia nauplii experimentally infected with strain PN9801; 2. Artemia nauplii enriched with enrofloxacin;
40
A. Roque, B. Gomez-Gil / Aquaculture 220 (2003) 37–42
3. Artemia nauplii enriched with enrofloxacin and experimentally infected with strain PN9801 4 h later; 4. Artemia nauplii experimentally infected with strain PN9801 and enriched with enrofloxacin 24 h later; All treatments had four replicates. The experiment was performed twice. Both experiments lasted 48 h from the moment the bacteria were applied. After this period of time, the survival rates per treatment and replicate were estimated. Survival estimation was performed harvesting all the live Artemia in each flask individually, resuspending them in 10 ml and counting the whole volume by placing drops with a Pasteur pipette on a microscope glass slide and under a stereoscopic microscope counting all the organisms in the sample. Significant differences among treatments were statistically analyzed using a nonparametric One-way ANOVA (Kruskal – Wallis) followed by a Student – Newman– Keuls (SNK) test.
3. Results Previous work was carried out with bacterium to evaluate its in vitro susceptibility to enrofloxacin. The diameter of the inhibition halum caused by 5 Ag of enrofloxacin to PN9801 was 21 mm and the minimum inhibitory concentration in Mueller Hinton Broth was 18.5 Ag ml 1. For the preliminary experiments, RichR did not seem to affect the results; the average mortality from the three replicates where RichR was used was 8 F 10.6% and for where RichR was not used was 22.6 F 37.5%, which when analyzed by Student’s t-test showed no significant statistical difference ( p < 0.005). Bacterial counts showed the nauplii were offered a bacterial density of 3.8 106 and 6.15 106 CFU ml 1 for Experiments 1 and 2, respectively. Significant differences were observed between the treatments in both experiments; significant mortalities were obtained when the Artemia nauplii were challenged with the Table 1 Percentage of survival of Artemia nauplii challenged with strain PN9801, V. harveyi and treated with enrofloxacin after 48 h Experiment 1
Mean S.D. n
Experiment 2
Enro
Strain PN9801
Enro 4 h
Enro 24 h
Enro
Strain PN9801
Enro 4 h
Enro 24 h
98.75a 1.60 4
9.79b 10.68 4
83.33a 12.83 3
83.33a 11.03 4
100a 0 4
48.33b 10.45 4
92.17a 11.65 4
86.50a 8.35 3
Treatments: Enro = Artemia nauplii enriched with enrofloxacin, Enro 4 h = Artemia nauplii enriched with Enrofloxacin and experimentally infected with V. harveyi 4 h later, Enro 24 h = Artemia nauplii experimentally infected with V. harveyi and enriched with enrofloxacin 24 h later. SD = Standard deviation, n = number of replicates. Different superscript letters in the mean of each treatment represent statistically different results (Kruskal – Wallis H-test, p < 0.05), within the experiment.
A. Roque, B. Gomez-Gil / Aquaculture 220 (2003) 37–42
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strain PN9801. When enrofloxacin was administered with RichR at 4 and 24 h after the bacterial challenge, the nauplii mortalities were prevented. The final survival for each treatment and experiment are shown in Table 1. The maximum survival for the nauplii challenged and not treated with enrofloxacin was 60.0%, whereas the minimum survival in any treatment where the enrofloxacin was added after the bacterial challenge was 71.7%. In both experiments, the treatments where bacteria and antibiotic were added to the water according to different sequences were not significantly different from each other.
4. Discussion The aim of this study was to evaluate the capacity of enrofloxacin to withhold an experimental infection with a V. harveyi pathogenic strain. The results presented here indicate that this is possible. The mortalities caused by the Vibrio strain in Artemia nauplii during 48 h can go up to 100% of the nauplii challenged. With the addition of enrofloxacin before or after the challenge, an average survival of more than 83.3% was achieved. A decision was taken to apply the antibiotic before and after the challenge in order to verify whether there was a real possibility that the antibiotic reverses the course of infection even if infection was suspected only after the first mortalities were observed. The mortalities found in the treatments where only bacteria were added reproduce other works done with the same bacterium (Soto-Rodrı´guez et al., submitted to Aquaculture), where several bioluminescent vibrios were used to experimentally challenge Artemia nauplii for a virulence screening. Other similar results were obtained with a pathogenic strain of V. proteolyticus (Verschuere et al., 2000) and with several V. parahaemolyticus strains (Rico-Mora and Voltolina, 1995; Gomez-Gil et al., 1998). In a study by Verschuere et al. (2000), it was found that Artemia nauplii fed with dry feed died before the ones not fed and cultured in axenic conditions. The authors suggest that this is because the nauplii also were fed with the Vibrio sp. This was not the case in this study possibly because the enrichment with RichR may affect the bacterial strain employed. In the preliminary trials, even though there was no statistical significant difference between the cumulative mortalities of challenged Artemia nauplii fed and not fed with Rich, there was a higher survival rate for the ones fed with Rich. The results obtained from using the antibiotic to stop the course of infection seem very promising and can probably be upscaled to Artemia adults. The industrial production of Artemia has an important role in the aquaculture industry to be used as live feed for fish and crustaceans (Lavens and Sorgeloos, 2000). As for other aquatic species, Artemia suffers from bacterial infections (Verschuere et al., 1999), and up to now, no treatment has been established even though some solutions have been tested at experimental levels such as the use of probiotic bacteria (Verschuere et al., 2000; Gomez-Gil, 1998). This study shows evidence that antibiotics can, in fact, withold the course of a bacterial infection already established in Artemia.
42
A. Roque, B. Gomez-Gil / Aquaculture 220 (2003) 37–42
Acknowledgements We would like to thank Carmen Bola´n Mejı´a, Franciss Marrujo and Dr. Celia LavillaPitogo. This work was funded by SIMAC grant no. SIMAC980106073.
References Gherna, L.R., 1994. Culture preservation. In: Gerhardt, P. (Ed.), Methods for General and Molecular Bacteriology. American Society for Microbiology, Washington, pp. 278 – 292. Gomez-Gil, B., 1998. Evaluation of potential probionts for use in penaeid shrimp larval culture. PhD thesis, University of Stirling, Scotland, p. 259. 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 (Artemia franciscana). Appl. Environ. Microbiol. 64, 2318 – 2322. Lavens, P., Sorgeloos, P., 2000. The history, present status and prospects of the availability of Artemia cysts for aquaculture. Aquaculture 181, 397 – 403. Prayitno, S.B., Latchford, J.W., 1995. Experimental infections of Crustaceans with luminous bacteria related to Photobacterium and Vibrio—effect of salinity and pH on infectiosity. Aquaculture 132, 105 – 112. Rico-Mora, R., Voltolina, D., 1995. Effects of bacterial isolates from Skeletonema costatum cultures on the survival of Artemia franciscana nauplii. J. Invertebr. Pathol. 66, 203 – 204. Sorgeloos, P., Bossuyt, E., Lavin˜a, E., Baeza-Mesa, M., Persoone, G., 1977. Decapsulation of Artemia cysts: a simple technique for the improvement of the use of brine shrimp in aquaculture. Aquaculture 12, 311 – 315. Soto-Rodrı´guez, S., Roque, A., Liza´rraga-Partida, M.L., Gomez-Gil, B., submitted for publication. Virulence of luminous vibrios isolated from shrimp culture systems to Artemia franciscana Kellog 1906 nauplii. Aquaculture. Verschuere, L., Rombaut, G., Huys, G., Dhont, J., Sorgeloos, P., Verstraete, W., 1999. Microbial control of the culture of Artemia juveniles through preemptive colonization by selected bacterial strains. App. Environ. Microbiol. 65, 2527 – 2533. Verschuere, L., Heang, H., Criel, G., Sorgeloos, P., Verstraete, W., 2000. Selected bacterial strains protect Artemia spp. from the pathogenic effects of Vibrio proteolyticus CW8T2. App. Environ. Microbiol. 66, 1139 – 1146.
Therapeutic effects of enrofloxacin in an experimental infection with a luminescent Vibrio harveyi in Artemia franciscana Kellog 1906 A. Roque *, B. Gomez-Gil Centro de Investigacio´n en Alimentacio´n y Desarrollo, A.C., Unidad Mazatla´n en Acuicultura y Manejo Ambiental, AP. 711, CP. 82000 Mazatla´n, Sinaloa, Mexico Received 29 November 2001; received in revised form 14 January 2002; accepted 18 June 2002
Abstract Although a wide range and number of chemotherapeutants have been developed and applied in aquaculture, there have been no studies on the absorption of antibiotics by crustacean larvae and, consequently, none has been reported to treat an established infection. The aim of this study was to evaluate the therapeutic effects of enrofloxacin during an experimental infection with the strain PN9801 identified as Vibrio harveyi in Artemia franciscana. Four treatments were used: (1) Nauplii experimentally infected with V. harveyi; (2) Nauplii enriched with enrofloxacin; (3) Nauplii enriched with enrofloxacin and, 4 h later, experimentally infected with V. harveyi; and (4) Nauplii experimentally infected with V. harveyi and 24 h later enriched with enrofloxacin. Survival rates were estimated after 48 h and they were as it follows: 29%, 99%, 88% and 85% for treatments 1, 2, 3 and 4, respectively. This study is the first one to show some evidence that antibiotics can, in fact, withhold the course of a bacterial infection already established in Artemia and possibly in crustaceans. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Artemia franciscana; Enrofloxacin; Vibrio harveyi
1. Introduction Although a wide range and number of chemotherapeutants have been developed and applied in aquaculture; chemotherapeutants for shrimp farming use are limited. Antibiotics *
Corresponding author. Tel.: +52-69-880157; fax: +52-69-880159. E-mail address: [email protected] (A. Roque).
0044-8486/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0044-8486(02)00272-7
38
A. Roque, B. Gomez-Gil / Aquaculture 220 (2003) 37–42
are often used in Asian and Latin American shrimp farms, but information on the use of these compounds is limited. Typically, the treatment of choice, if one exists, has been established by a trial-and-error process with the compounds, which were most readily available at that particular time. Up to now, there have been no studies on the absorption of antibiotics by crustacean larvae and, consequently, none has been reported to treat an established infection although they may slow the infection rate in the tank by reducing the bacterial load present in the water. Existing challenge models for shrimp larvae have not produced an established model to study vibriosis (Gomez-Gil, 1998; Prayitno and Latchford, 1995). This is mainly due to the variability of results. The same bacterium in one replicate kills many larvae; in the other replicate, no mortalities are recorded. The general rule is that very low or no mortalities are produced. However, a technique has been developed to encapsulate bacteria in Artemia nauplii under sterile conditions (Gomez-Gil et al., 1998). The results presented suggest that this technique can be used as quick screening test for finding potentially pathogenic bacteria. The aim of this study was to evaluate the therapeutic effects of enrofloxacin during an experimental infection with Vibrio harveyi in Artemia franciscana.
2. Material and methods 2.1. Artemia hatching A. franciscana cysts from the Great Salt Lake Utah were employed for this study. The chorion of the cysts was chemically removed employing the methodology proposed by Sorgeloos et al. (1977), a process known as decapsulation. Hatching of the eggs (decapsulated cysts) was performed overnight in a flask with 500 ml of sterile fullstrength seawater (35x), aerated through an air hose connected to an aquarium air pump and maintained at 28 jC in a reciprocal shaking water bath (Precison Scientific, Chicago, USA). Twenty-four hours after hatching, the nauplii were harvested and transferred to the enrichment system. This one consisted of 16 flasks with 45 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. Artemia nauplii enrichment with antibiotics The enrichment of the Artemia nauplii was performed using the commercial lipid emulsion RichR (Sanders Brine Shrimp Golden, Utah, USA) to which 40% w/w of enrofloxacin (Cheminova de Me´xico, Me´xico) was added. The desired (0.032 g/flask, a total of 0.384 g of enrofloxacin) quantity of antibiotic was added to 100 ml of filtered seawater and dissolved; then, these were added to the RichR (0.08 g/flask according to the manufacturer instructions) and mixed in a domestic mixer for 3 min. This mixture was the enrichment offered to the Artemia nauplii. Five milliliters of this enrichment were added to
A. Roque, B. Gomez-Gil / Aquaculture 220 (2003) 37–42
39
each flask at the time required. Plain RichR-emulsified was added to the Artemia in the cases where no antibiotic was added. 2.3. Preparation of the V. harveyi strain The strain of V. harveyi used was isolated from the lymphoid organ of a diseased juvenile marine shrimp Penaeus monodon in the Philippines and coded PN9801. It was identified and donated to the Collection of Aquacultural Important Microorganisms at CIAD/Mazatlan Unit by Dr. Lavilla-Pitogo in 1999. The V. harveyi strain was preserved at 70 jC in an ultralow mechanical freezer (Revco Scientific, Asheville, NC) in cryovials filled with glass beads (Gherna, 1994). In order to recover the strain from cryopreservation, a bead was obtained from the cryovial and placed in a test tube with tripticasein soy broth (TSB, Bioxon, Me´xico) with 2% sodium chloride (NaCl) and incubated overnight at 30 jC with constant agitation. The following day, 8 ml of the bacterial solution were centrifuged at 3600 g for 10 min at 4 jC. The supernatant was discharged and the bacterial pellet resuspended in sterile saline solution (2.5% NaCl). This bacterial solution was centrifuged again under the same conditions. The supernatant was discharged and the resuspention and centrifugation were repeated. Once the supernatant was discharged, the bacterial pellet was suspended in approximately 5 ml of sterile saline solution and this volume was adjusted to get an optical density of 1.00 at 610 nm in a spectrophotometer (model DR-2000; Hach, Loveland, CO). To estimate the bacterial concentration achieved and to verify its purity, the suspension was serially diluted in sterile saline solution and spread plated in agar thiosulphate citrate bile salt (TCBS, Difco, Detroit, MI). 2.4. Challenge of the Artemia Six hundred sterile nauplii (individually counted) were added to each 100-ml flask with the 5 ml of bacterial suspension at a density 10 7 cells ml 1 and 45 ml of sterile seawater for 48 h and the number of surviving nauplii estimated. In the treatments with Rich, 0.08 g of it were added per flask prepared as described previously. 2.5. Experimental protocol The protocol followed to infect the Artemia nauplii was the one described above. Preliminary work was carried to rule out the possibility of Rich acting as an antimicrobial. 2.6. Final experiment Four treatments were used: 1. Artemia nauplii experimentally infected with strain PN9801; 2. Artemia nauplii enriched with enrofloxacin;
40
A. Roque, B. Gomez-Gil / Aquaculture 220 (2003) 37–42
3. Artemia nauplii enriched with enrofloxacin and experimentally infected with strain PN9801 4 h later; 4. Artemia nauplii experimentally infected with strain PN9801 and enriched with enrofloxacin 24 h later; All treatments had four replicates. The experiment was performed twice. Both experiments lasted 48 h from the moment the bacteria were applied. After this period of time, the survival rates per treatment and replicate were estimated. Survival estimation was performed harvesting all the live Artemia in each flask individually, resuspending them in 10 ml and counting the whole volume by placing drops with a Pasteur pipette on a microscope glass slide and under a stereoscopic microscope counting all the organisms in the sample. Significant differences among treatments were statistically analyzed using a nonparametric One-way ANOVA (Kruskal – Wallis) followed by a Student – Newman– Keuls (SNK) test.
3. Results Previous work was carried out with bacterium to evaluate its in vitro susceptibility to enrofloxacin. The diameter of the inhibition halum caused by 5 Ag of enrofloxacin to PN9801 was 21 mm and the minimum inhibitory concentration in Mueller Hinton Broth was 18.5 Ag ml 1. For the preliminary experiments, RichR did not seem to affect the results; the average mortality from the three replicates where RichR was used was 8 F 10.6% and for where RichR was not used was 22.6 F 37.5%, which when analyzed by Student’s t-test showed no significant statistical difference ( p < 0.005). Bacterial counts showed the nauplii were offered a bacterial density of 3.8 106 and 6.15 106 CFU ml 1 for Experiments 1 and 2, respectively. Significant differences were observed between the treatments in both experiments; significant mortalities were obtained when the Artemia nauplii were challenged with the Table 1 Percentage of survival of Artemia nauplii challenged with strain PN9801, V. harveyi and treated with enrofloxacin after 48 h Experiment 1
Mean S.D. n
Experiment 2
Enro
Strain PN9801
Enro 4 h
Enro 24 h
Enro
Strain PN9801
Enro 4 h
Enro 24 h
98.75a 1.60 4
9.79b 10.68 4
83.33a 12.83 3
83.33a 11.03 4
100a 0 4
48.33b 10.45 4
92.17a 11.65 4
86.50a 8.35 3
Treatments: Enro = Artemia nauplii enriched with enrofloxacin, Enro 4 h = Artemia nauplii enriched with Enrofloxacin and experimentally infected with V. harveyi 4 h later, Enro 24 h = Artemia nauplii experimentally infected with V. harveyi and enriched with enrofloxacin 24 h later. SD = Standard deviation, n = number of replicates. Different superscript letters in the mean of each treatment represent statistically different results (Kruskal – Wallis H-test, p < 0.05), within the experiment.
A. Roque, B. Gomez-Gil / Aquaculture 220 (2003) 37–42
41
strain PN9801. When enrofloxacin was administered with RichR at 4 and 24 h after the bacterial challenge, the nauplii mortalities were prevented. The final survival for each treatment and experiment are shown in Table 1. The maximum survival for the nauplii challenged and not treated with enrofloxacin was 60.0%, whereas the minimum survival in any treatment where the enrofloxacin was added after the bacterial challenge was 71.7%. In both experiments, the treatments where bacteria and antibiotic were added to the water according to different sequences were not significantly different from each other.
4. Discussion The aim of this study was to evaluate the capacity of enrofloxacin to withhold an experimental infection with a V. harveyi pathogenic strain. The results presented here indicate that this is possible. The mortalities caused by the Vibrio strain in Artemia nauplii during 48 h can go up to 100% of the nauplii challenged. With the addition of enrofloxacin before or after the challenge, an average survival of more than 83.3% was achieved. A decision was taken to apply the antibiotic before and after the challenge in order to verify whether there was a real possibility that the antibiotic reverses the course of infection even if infection was suspected only after the first mortalities were observed. The mortalities found in the treatments where only bacteria were added reproduce other works done with the same bacterium (Soto-Rodrı´guez et al., submitted to Aquaculture), where several bioluminescent vibrios were used to experimentally challenge Artemia nauplii for a virulence screening. Other similar results were obtained with a pathogenic strain of V. proteolyticus (Verschuere et al., 2000) and with several V. parahaemolyticus strains (Rico-Mora and Voltolina, 1995; Gomez-Gil et al., 1998). In a study by Verschuere et al. (2000), it was found that Artemia nauplii fed with dry feed died before the ones not fed and cultured in axenic conditions. The authors suggest that this is because the nauplii also were fed with the Vibrio sp. This was not the case in this study possibly because the enrichment with RichR may affect the bacterial strain employed. In the preliminary trials, even though there was no statistical significant difference between the cumulative mortalities of challenged Artemia nauplii fed and not fed with Rich, there was a higher survival rate for the ones fed with Rich. The results obtained from using the antibiotic to stop the course of infection seem very promising and can probably be upscaled to Artemia adults. The industrial production of Artemia has an important role in the aquaculture industry to be used as live feed for fish and crustaceans (Lavens and Sorgeloos, 2000). As for other aquatic species, Artemia suffers from bacterial infections (Verschuere et al., 1999), and up to now, no treatment has been established even though some solutions have been tested at experimental levels such as the use of probiotic bacteria (Verschuere et al., 2000; Gomez-Gil, 1998). This study shows evidence that antibiotics can, in fact, withold the course of a bacterial infection already established in Artemia.
42
A. Roque, B. Gomez-Gil / Aquaculture 220 (2003) 37–42
Acknowledgements We would like to thank Carmen Bola´n Mejı´a, Franciss Marrujo and Dr. Celia LavillaPitogo. This work was funded by SIMAC grant no. SIMAC980106073.
References Gherna, L.R., 1994. Culture preservation. In: Gerhardt, P. (Ed.), Methods for General and Molecular Bacteriology. American Society for Microbiology, Washington, pp. 278 – 292. Gomez-Gil, B., 1998. Evaluation of potential probionts for use in penaeid shrimp larval culture. PhD thesis, University of Stirling, Scotland, p. 259. 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 (Artemia franciscana). Appl. Environ. Microbiol. 64, 2318 – 2322. Lavens, P., Sorgeloos, P., 2000. The history, present status and prospects of the availability of Artemia cysts for aquaculture. Aquaculture 181, 397 – 403. Prayitno, S.B., Latchford, J.W., 1995. Experimental infections of Crustaceans with luminous bacteria related to Photobacterium and Vibrio—effect of salinity and pH on infectiosity. Aquaculture 132, 105 – 112. Rico-Mora, R., Voltolina, D., 1995. Effects of bacterial isolates from Skeletonema costatum cultures on the survival of Artemia franciscana nauplii. J. Invertebr. Pathol. 66, 203 – 204. Sorgeloos, P., Bossuyt, E., Lavin˜a, E., Baeza-Mesa, M., Persoone, G., 1977. Decapsulation of Artemia cysts: a simple technique for the improvement of the use of brine shrimp in aquaculture. Aquaculture 12, 311 – 315. Soto-Rodrı´guez, S., Roque, A., Liza´rraga-Partida, M.L., Gomez-Gil, B., submitted for publication. Virulence of luminous vibrios isolated from shrimp culture systems to Artemia franciscana Kellog 1906 nauplii. Aquaculture. Verschuere, L., Rombaut, G., Huys, G., Dhont, J., Sorgeloos, P., Verstraete, W., 1999. Microbial control of the culture of Artemia juveniles through preemptive colonization by selected bacterial strains. App. Environ. Microbiol. 65, 2527 – 2533. Verschuere, L., Heang, H., Criel, G., Sorgeloos, P., Verstraete, W., 2000. Selected bacterial strains protect Artemia spp. from the pathogenic effects of Vibrio proteolyticus CW8T2. App. Environ. Microbiol. 66, 1139 – 1146.