- Email: [email protected]
Immunochemotherapy with doxycycline and CpG-ODN 1668 for treatment of scuticociliatosis in olive flounder (Paralichthys olivaceus)
Aquaculture 435 (2015) 143–145
Contents lists available at ScienceDirect
Aquaculture journal homepage: www.elsevier.com/locate/aqua-online
Short communication
Immunochemotherapy with doxycycline and CpG-ODN 1668 for treatment of scuticociliatosis in olive flounder (Paralichthys olivaceus) Yue Jai Kang a, Ki Hong Kim b,⁎ a b
Department of Aquatic Life Medical Sciences, Sun Moon University, Asan-si, Chungnam 336-708, South Korea Department of Aquatic Life Medicine, Pukyong National University, Busan 608-737, South Korea
a r t i c l e
i n f o
Article history: Received 26 August 2014 Received in revised form 16 September 2014 Accepted 18 September 2014 Available online 26 September 2014 Keywords: Scuticociliatosis Immunochemotherapy Doxycycline CpG-ODN 1668 Olive flounder
a b s t r a c t Immunotherapy along with chemotherapy can be a way to enhance therapeutic efficacy and to reduce amount of chemotherapeutics. So, in the present study, we adopted an immunochemotherapy (CpG-ODN 1668 plus doxycycline) to reduce doxycycline dose to be used while at the same time to get the same treatment efficacy against scuticociliatosis in olive flounder (Paralichthys olivaceus). The survival rates of fish administered with doxycycline 100 mg/kg plus 10 μg of CpG-ODN 1668 were higher than those of fish administered with doxycycline alone at a dose of 200 mg/kg of fish weight, suggesting that coadministration of CpG-ODN 1668 can reduce doxycycline to half the dose. Furthermore, reduction of CpG-ODN 1668 to 5 μg/fish plus doxycycline 100 mg/kg led to high survival rates that were comparable to doxycycline 200 mg/kg, suggesting that the used amount of both immunotherapeutics and chemotherapeutics can be reduced through coadministration. There have been no reports on the treatment of scuticociliatosis by combining chemotherapy and immunotherapy. In the present study, we demonstrated that a combination of chemotherapy (doxycycline) and immunotherapy (CpG-ODN 1668) was effective against scuticociliatosis in olive flounder fingerlings. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Miamiensis avidus (Syn. Philasterides dicentrarchi) is a facultative histophagous parasitic ciliate which causes a devastating disease called scuticociliatosis in cultured marine fish, especially in flat fish such as olive flounder (Paralichthys olivaceus) and turbot (Scophthalmus maximus) (Iglesias et al., 2001; Jung et al., 2007; Kim et al., 2004). Development of effective prophylactic vaccines is required to control scuticociliatosis, however, difficulties in culture of scuticociliates to the commercial scale and presence of several serotypes that are not crossprotective (Budiño et al., 2011; Piazzon et al., 2008) make the vaccine development hard. In the absence of effective vaccines against scuticociliatosis, therapeutic approach is the alternative option to control the disease. Although considerable efforts have been made to develop therapeutic measures against scuticociliatosis, commercialized effective counter-measures are not reported. One of the difficulties in development of therapeutic measures is the ability of the ciliates to invade into all the internal organs of fish. Moreover, since the ciliates can invade into the brain without any distinct outer lesions, which is considered as the potent cause of acute mass mortality of olive flounder, therapeutics against scuticociliatosis should be able to pass through the blood–brain barrier.
⁎ Corresponding author. Tel.: +82 51 629 5943; fax: +82 51 629 5938. E-mail address: [email protected] (K.H. Kim).
http://dx.doi.org/10.1016/j.aquaculture.2014.09.024 0044-8486/© 2014 Elsevier B.V. All rights reserved.
Doxycycline, a tetracycline derivative, is known to cross the blood brain barrier and is mostly used to treat bacterial infections (Bahrami et al., 2012; Cunha et al., 1982). Recently, antiprotozoal activity of doxycycline against Plasmodium falciparum has made it as a potent drug for chemoprophylaxis of malaria in human (Draper et al., 2013). In fish parasites, in vitro scuticocidal activity of doxycycline was reported by Iglesias et al. (2002). Previously, we have demonstrated that doxycycline can be used as a therapeutic to treat scuticociliatosis in olive flounder via both oral and bath treatments (Kang et al., 2013). Treatment with chemotherapeutics can induce resistance against the drug in the pathogen. One of the ways to reduce possibility of resistance induction may be increase of treatment efficacy, which can decrease the chance for survived pathogens to acquire resistance. However, increase of chemotherapeutic dose to enhance treatment efficacy can lead to increase of adverse effects. Treatment through immunological methods would be helpful to control infectious diseases without induction of resistance. It has been reported that scuticociliates were susceptible to complement activity of fish (Leiro et al., 2008; Piazzon et al., 2011). Thus, elevation of complement activity can provide one of the treatment measures against scuticociliatosis. Previously, we have demonstrated that administration of olive flounder with an artificially synthesized oligodeoxynucleotides containing unmethylated deoxycytidyl-deoxyguanosine motif (CpG-ODN) 1668 was effective not only in prevention but also in treatment of scuticociliatosis, and the treatment efficiency was positively related to the enhanced complement activity (Kang and Kim, 2012, 2014; Lee and Kim, 2009).
144
Y.J. Kang, K.H. Kim / Aquaculture 435 (2015) 143–145
Immunotherapy along with chemotherapy can be a way to enhance therapeutic efficacy and to reduce amount of chemotherapeutics. So we adopted an immunochemotherapy (CpG-ODN 1668 plus doxycycline) in the present study to reduce doxycycline dose to be used while at the same time to get the same treatment efficacy against scuticociliatosis in olive flounder. 2. Material and methods 2.1. Ciliates M. avidus isolated from diseased olive flounder were grown using Epithelioma papulosum cyprini (EPC) cells in Leibovitz medium (L-15, Sigma) supplemented with penicillin (100 U/ml, Sigma), streptomycin (100 μg/ml, Sigma) and 10% fetal bovine serum (FBS, Gibco) at 20 °C. 2.2. Fish Olive flounder fingerlings (3.5–4.5 g in weight) were obtained from a local hatchery in Korea, and were acclimated at 20–21 °C for 1 week prior to initiating the experiments. During the acclimation period, 10 fish were randomly sampled, and the skin, gill and internal organs were examined for the presence of pathogenic viruses (viral hemorrhagic septicemia virus, Lymphocystis virus), bacteria (Edwardsiella tarda, Streptococcus spp., Vibrio spp.) and parasites by our routine method. From the all examined fish, viruses and bacteria were not detected, however, scuticociliates were found from the skin and/or the gills, and were identified as M. avidus using a species-specific oligonucleotide primers (Kim et al., 2004). We diagnosed the infection state as an early stage, and performed the experiment according to our plan. 2.3. Treatment experiment After 1 week acclimation, a total of 168 fish showing no external signs of scuticociliatosis were selected, and randomly divided into 7 groups with 2 replicates. Fish in each group were immersed in seawater (80 l) containing 1 × 106 ciliates/l for 3 days, then transferred to each corresponding 14 tanks containing 50 l seawater. The fish in the seven experimental groups were administered with doxycycline 100 or 200 mg/kg of fish; 5 or 10 μg/fish of CpG-ODN 1668 (5′-TCCATGACGT TCCTGATGCT-3′; phosphorothioated) synthesized from Bioneer Corporation (Korea); 5 or 10 μg/fish of CpG-ODN 1668 plus doxycycline 100 mg/kg of fish; or phosphate buffered saline (PBS). Doxycycline or PBS was administered through the oral route using a gastric tube at 0, 3, and 6 d post-infection, and CpG-ODN 1668 was intraperitoneally (i.p.) administered at 0 d post-infection. The mortality was monitored for 22 d postinfection, and dead fish were necropsied to confirm the presence of ciliates.
100 mg/kg of fish weight were comparable to those of the fish administered with doxycycline 200 mg/kg of fish.
3. Results
4. Discussion
To investigate the treatment effect of doxycycline-based chemotherapy plus CpG-ODN 1668-based immunotherapy against scuticociliatosis, olive flounder infected with M. avidus were treated with either doxycycline alone, CpG-ODN 1668 alone or doxycycline plus CpG-ODN 1668 (Fig. 1). Fish in the PBS administered control group showed 91.7 and 100% cumulative mortalities. Fish treated with doxycycline alone at a dose of 200 mg/kg of fish weight showed 41.7 and 50% mortalities. Both replicates in the group of fish administered with 10 μg/fish of CpG-ODN 1668 alone showed 50% cumulative mortalities. While coadministration of doxycycline 100 mg/kg of fish weight plus 10 μg/fish of CpG-ODN-1668 led to reduction of cumulative mortality (33.3% in both replicates) in comparison to doxycycline alone or CpG-ODN 1668 alone. Survival rates of fish administered with 5 μg/fish of CpG-ODN-1668 plus doxycycline
One of the important aspects of chemotherapy is the range of dose that can effectively treat disease without inducing (or minimizing) toxic effects. Our previous results showed that feeding a 2% doxycycline diet once a day (approximately corresponding to 200 mg of doxycycline/kg of fish/day) for 3 times was effective in treatment of olive flounder fingerlings infected with M. avidus (Kang et al., 2013). Although the fish did not show any behavioral changes during the experimental period, the dose of doxycycline used in the previous experiment was definitely high, compared to a recommendable dose of doxycycline to treat streptococcosis in olive flounder in Korea (less than 50 mg/kg/day). Thus, it was inevitable to develop other auxiliary therapeutic approaches that can reduce the dose of doxycycline. The prophylactic and therapeutic abilities of CpG-ODN 1668 against scuticociliatosis in olive flounder
Fig. 1. Cumulative mortality rates of olive flounder (Paralichthys olivaceus) fingerlings. A total of 168 fish were randomly divided into 7 groups with 2 replicates (A and B), then, immersed in seawater containing 8 × 107 ciliates/80 l for 3 days. Fish in each group were administered with doxycycline 100 or 200 mg/kg of fish, 5 or 10 μg/fish of CpGODN 1668, 5 or 10 μg/fish of CpG-ODN 1668 plus doxycycline 100 mg/kg (CpG-5 plus Doxy 100 or CpG-10 plus Doxy 100), or phosphate buffered saline (PBS, Control). Doxycycline or PBS was administered through the oral route using a gastric tube at 0, 3, and 6 d post-infection, and CpG-ODN 1668 was intraperitoneally (i.p.) administered at 0 d postinfection.
Y.J. Kang, K.H. Kim / Aquaculture 435 (2015) 143–145
were reported from our previous papers (Kang and Kim, 2012, 2014; Lee and Kim, 2009). Hence, in this study, we investigated whether coadministration of CpG-ODN 1668 can reduce the dose of doxycycline without decrease of treatment efficacy. There have been no reports on the treatment of scuticociliatosis by combining chemotherapy and immunotherapy. In the present study, we demonstrated that a combination of chemotherapy (doxycycline) and immunotherapy (CpG-ODN 1668) was effective against scuticociliatosis in olive flounder fingerlings. In this study, the survival rates of fish administered with doxycycline 100 mg/kg plus 10 μg of CpG-ODN 1668 were higher than those of fish administered with doxycycline alone at a dose of 200 mg/kg. This result suggests that coadministration of CpG-ODN 1668 can reduce doxycycline to half the dose. As the cost for preparation of CpG-ODNs is expensive, reduction of CpG-ODN 1668 amount would be favorable for an economic aspect. In the present results, although the treatment efficacy was lower than the fish administered with doxycycline 100 mg/kg plus 10 μg of CpGODN 1668, reduction of CpG-ODN 1668 to 5 μg/fish plus administration of 100 mg/kg of doxycycline led to high survival rates that were comparable to doxycycline 200 mg/kg, suggesting that the used amount of both immunotherapeutics and chemotherapeutics can be reduced through coadministration. Recently, we have shown that oral administration of auxotrophic E. tarda mutant harboring CpG 1668 motif-enriched plasmids was effective in treatment of scuticociliatosis (Kang et al., 2014). The treatment effects of oral administration of doxycycline and the mutant bacteria containing CpG 1668 motif-enriched plasmids should be further studied to reduce cost and labor related to the preparation and injection of CpGODN 1668. More research is needed to understand the interactive mechanism of the immunochemotherapy, and search for other immunotherapeutics that can enhance scuticocidal activity is also needed. Acknowledgments This research was supported by the Basic Science Research Program through the National Research Foundation (NRF) funded by the Ministry of Science, ICT and Future Planning of South Korea (2013R1A2A2A01067204).
145
References Bahrami, F., Morris, D.L., Pourgholami, M.H., 2012. Tetracyclines: drugs with huge therapeutic potential. Mini-Rev. Med. Chem. 12, 44–52. Budiño, B., Lamas, J., Pata, M.P., Arranz, J.A., Sanmartín, M.L., Leiro, J., 2011. Intraspecific variability in several isolates of Philasterides dicentrarchi (syn Miamiensis avidus), a scuticociliate parasite of farmed turbot. Vet. Parasitol. 175, 260–272. Cunha, B.A., Comer, J.B., Jonas, M., 1982. The tetracyclines. Med. Clin. North Am. 66, 293–302. Draper, M.P., Bhatia, B., Assefa, H., Honeyman, L., Garrity-Ryan, L.K., Verma, A.K., Gut, J., Larson, K., Donatelli, J., Macone, A., Klausner, K., Leahy, R.G., Odinecs, A., Ohemeng, K., Rosenthal, P.J., Nelson, M.L., 2013. In vitro and in vivo antimalarial efficacies of optimized tetracyclines. Antimicrob. Agents Chemother. 57, 3131–3136. Iglesias, R., Paramá, A., Alvarez, M.F., Leiro, J., Fernández, J., Sanmartín, M.L., 2001. Philasterides dicentrarchi (Ciliophora, Scuticociliatida) as the causative agent of scuticociliatosis in farmed turbot Scophthalmus maximus in Galicia (NW Spain). Dis. Aquat. Org. 46, 47–55. Iglesias, R., Paramá, A., Alvarez, M.F., Leiro, J., Sanmartín, M.L., 2002. Antiprotozoals effective in vitro against the scuticociliate fish pathogen Philasterides dicentrarchi. Dis. Aquat. Org. 49, 191–197. Jung, S.J., Kitamura, S.I., Song, J.Y., Oh, M.J., 2007. Miamiensis avidus (Ciliophora: Scuticociliatida) causes systemic infection of olive flounder Paralichthys olivaceus and is a senior synonym of Philasterides dicentrarchi. Dis. Aquat. Org. 73, 227–234. Kang, Y.J., Kim, K.H., 2012. Effect of CpG-ODNs belonging to different classes on resistance of olive flounder (Paralichthys olivaceus) against viral hemorrhagic septicemia virus (VHSV) and Miamiensis avidus (Ciliata; Scuticociliatia) infections. Aquaculture 324–325, 39–43. Kang, Y.J., Kim, K.H., 2014. Therapeutic potential of CpG-ODN 1668 against scuticociliatosis in olive flounder (Paralichthys olivaceus). Aquaculture 430, 17–20. Kang, Y.J., Kim, D.S., Kim, K.H., 2013. Evaluation of treatment efficacy of doxycycline and albendazole against scuticociliatosis in olive flounder (Paralichthys olivaceus). Aquaculture 416–417, 192–195. Kang, Y.J., Choi, S.H., Kim, K.H., 2014. Preventive and therapeutic effects of auxotrophic Edwardsiella tarda mutant harboring CpG 1668 motif-enriched plasmids against scuticociliatosis in olive flounder (Paralichthys olivaceus). Exp. Parasitol. 144, 34–38. Kim, S.M., Cho, J.B., Kim, S.K., Nam, Y.K., Kim, K.H., 2004. Occurrence of scuticociliatosis in olive flounder Paralichthys olivaceus by Philasterides dicentrarchi (Ciliophora: Scuticociitida). Dis. Aquat. Org. 62, 233–238. Lee, E.H., Kim, K.H., 2009. CpG-ODN increases resistance of olive flounder (Paralichthys olivaceus) against Philasterides dicentrarchi (Ciliophora: Scuticociliatia) infection. Fish Shellfish Immunol. 26, 29–32. Leiro, J., Piazzón, M.C., Budiño, B., Sanmartín, M.L., Lamas, J., 2008. Complement-mediated killing of Philasterides dicentrarchi (Ciliophora) by turbot serum: relative importance of alternative and classical pathways. Parasite Immunol. 30, 535–543. Piazzon, C., Lamas, J., Castro, R., Budiño, B., Cabaleiro, S., Sanmartín, M., Leiro, J., 2008. Antigenic and cross-protection studies on two turbot scuticociliate isolates. Fish Shellfish Immunol. 25, 417–424. Piazzon, M.C., Wiegertjes, G.F., Leiro, J., Lamas, J., 2011. Turbot resistance to Philasterides dicentrarchi is more dependent on humoral than on cellular immune responses. Fish Shellfish Immunol. 30, 1339–1347.
Contents lists available at ScienceDirect
Aquaculture journal homepage: www.elsevier.com/locate/aqua-online
Short communication
Immunochemotherapy with doxycycline and CpG-ODN 1668 for treatment of scuticociliatosis in olive flounder (Paralichthys olivaceus) Yue Jai Kang a, Ki Hong Kim b,⁎ a b
Department of Aquatic Life Medical Sciences, Sun Moon University, Asan-si, Chungnam 336-708, South Korea Department of Aquatic Life Medicine, Pukyong National University, Busan 608-737, South Korea
a r t i c l e
i n f o
Article history: Received 26 August 2014 Received in revised form 16 September 2014 Accepted 18 September 2014 Available online 26 September 2014 Keywords: Scuticociliatosis Immunochemotherapy Doxycycline CpG-ODN 1668 Olive flounder
a b s t r a c t Immunotherapy along with chemotherapy can be a way to enhance therapeutic efficacy and to reduce amount of chemotherapeutics. So, in the present study, we adopted an immunochemotherapy (CpG-ODN 1668 plus doxycycline) to reduce doxycycline dose to be used while at the same time to get the same treatment efficacy against scuticociliatosis in olive flounder (Paralichthys olivaceus). The survival rates of fish administered with doxycycline 100 mg/kg plus 10 μg of CpG-ODN 1668 were higher than those of fish administered with doxycycline alone at a dose of 200 mg/kg of fish weight, suggesting that coadministration of CpG-ODN 1668 can reduce doxycycline to half the dose. Furthermore, reduction of CpG-ODN 1668 to 5 μg/fish plus doxycycline 100 mg/kg led to high survival rates that were comparable to doxycycline 200 mg/kg, suggesting that the used amount of both immunotherapeutics and chemotherapeutics can be reduced through coadministration. There have been no reports on the treatment of scuticociliatosis by combining chemotherapy and immunotherapy. In the present study, we demonstrated that a combination of chemotherapy (doxycycline) and immunotherapy (CpG-ODN 1668) was effective against scuticociliatosis in olive flounder fingerlings. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Miamiensis avidus (Syn. Philasterides dicentrarchi) is a facultative histophagous parasitic ciliate which causes a devastating disease called scuticociliatosis in cultured marine fish, especially in flat fish such as olive flounder (Paralichthys olivaceus) and turbot (Scophthalmus maximus) (Iglesias et al., 2001; Jung et al., 2007; Kim et al., 2004). Development of effective prophylactic vaccines is required to control scuticociliatosis, however, difficulties in culture of scuticociliates to the commercial scale and presence of several serotypes that are not crossprotective (Budiño et al., 2011; Piazzon et al., 2008) make the vaccine development hard. In the absence of effective vaccines against scuticociliatosis, therapeutic approach is the alternative option to control the disease. Although considerable efforts have been made to develop therapeutic measures against scuticociliatosis, commercialized effective counter-measures are not reported. One of the difficulties in development of therapeutic measures is the ability of the ciliates to invade into all the internal organs of fish. Moreover, since the ciliates can invade into the brain without any distinct outer lesions, which is considered as the potent cause of acute mass mortality of olive flounder, therapeutics against scuticociliatosis should be able to pass through the blood–brain barrier.
⁎ Corresponding author. Tel.: +82 51 629 5943; fax: +82 51 629 5938. E-mail address: [email protected] (K.H. Kim).
http://dx.doi.org/10.1016/j.aquaculture.2014.09.024 0044-8486/© 2014 Elsevier B.V. All rights reserved.
Doxycycline, a tetracycline derivative, is known to cross the blood brain barrier and is mostly used to treat bacterial infections (Bahrami et al., 2012; Cunha et al., 1982). Recently, antiprotozoal activity of doxycycline against Plasmodium falciparum has made it as a potent drug for chemoprophylaxis of malaria in human (Draper et al., 2013). In fish parasites, in vitro scuticocidal activity of doxycycline was reported by Iglesias et al. (2002). Previously, we have demonstrated that doxycycline can be used as a therapeutic to treat scuticociliatosis in olive flounder via both oral and bath treatments (Kang et al., 2013). Treatment with chemotherapeutics can induce resistance against the drug in the pathogen. One of the ways to reduce possibility of resistance induction may be increase of treatment efficacy, which can decrease the chance for survived pathogens to acquire resistance. However, increase of chemotherapeutic dose to enhance treatment efficacy can lead to increase of adverse effects. Treatment through immunological methods would be helpful to control infectious diseases without induction of resistance. It has been reported that scuticociliates were susceptible to complement activity of fish (Leiro et al., 2008; Piazzon et al., 2011). Thus, elevation of complement activity can provide one of the treatment measures against scuticociliatosis. Previously, we have demonstrated that administration of olive flounder with an artificially synthesized oligodeoxynucleotides containing unmethylated deoxycytidyl-deoxyguanosine motif (CpG-ODN) 1668 was effective not only in prevention but also in treatment of scuticociliatosis, and the treatment efficiency was positively related to the enhanced complement activity (Kang and Kim, 2012, 2014; Lee and Kim, 2009).
144
Y.J. Kang, K.H. Kim / Aquaculture 435 (2015) 143–145
Immunotherapy along with chemotherapy can be a way to enhance therapeutic efficacy and to reduce amount of chemotherapeutics. So we adopted an immunochemotherapy (CpG-ODN 1668 plus doxycycline) in the present study to reduce doxycycline dose to be used while at the same time to get the same treatment efficacy against scuticociliatosis in olive flounder. 2. Material and methods 2.1. Ciliates M. avidus isolated from diseased olive flounder were grown using Epithelioma papulosum cyprini (EPC) cells in Leibovitz medium (L-15, Sigma) supplemented with penicillin (100 U/ml, Sigma), streptomycin (100 μg/ml, Sigma) and 10% fetal bovine serum (FBS, Gibco) at 20 °C. 2.2. Fish Olive flounder fingerlings (3.5–4.5 g in weight) were obtained from a local hatchery in Korea, and were acclimated at 20–21 °C for 1 week prior to initiating the experiments. During the acclimation period, 10 fish were randomly sampled, and the skin, gill and internal organs were examined for the presence of pathogenic viruses (viral hemorrhagic septicemia virus, Lymphocystis virus), bacteria (Edwardsiella tarda, Streptococcus spp., Vibrio spp.) and parasites by our routine method. From the all examined fish, viruses and bacteria were not detected, however, scuticociliates were found from the skin and/or the gills, and were identified as M. avidus using a species-specific oligonucleotide primers (Kim et al., 2004). We diagnosed the infection state as an early stage, and performed the experiment according to our plan. 2.3. Treatment experiment After 1 week acclimation, a total of 168 fish showing no external signs of scuticociliatosis were selected, and randomly divided into 7 groups with 2 replicates. Fish in each group were immersed in seawater (80 l) containing 1 × 106 ciliates/l for 3 days, then transferred to each corresponding 14 tanks containing 50 l seawater. The fish in the seven experimental groups were administered with doxycycline 100 or 200 mg/kg of fish; 5 or 10 μg/fish of CpG-ODN 1668 (5′-TCCATGACGT TCCTGATGCT-3′; phosphorothioated) synthesized from Bioneer Corporation (Korea); 5 or 10 μg/fish of CpG-ODN 1668 plus doxycycline 100 mg/kg of fish; or phosphate buffered saline (PBS). Doxycycline or PBS was administered through the oral route using a gastric tube at 0, 3, and 6 d post-infection, and CpG-ODN 1668 was intraperitoneally (i.p.) administered at 0 d post-infection. The mortality was monitored for 22 d postinfection, and dead fish were necropsied to confirm the presence of ciliates.
100 mg/kg of fish weight were comparable to those of the fish administered with doxycycline 200 mg/kg of fish.
3. Results
4. Discussion
To investigate the treatment effect of doxycycline-based chemotherapy plus CpG-ODN 1668-based immunotherapy against scuticociliatosis, olive flounder infected with M. avidus were treated with either doxycycline alone, CpG-ODN 1668 alone or doxycycline plus CpG-ODN 1668 (Fig. 1). Fish in the PBS administered control group showed 91.7 and 100% cumulative mortalities. Fish treated with doxycycline alone at a dose of 200 mg/kg of fish weight showed 41.7 and 50% mortalities. Both replicates in the group of fish administered with 10 μg/fish of CpG-ODN 1668 alone showed 50% cumulative mortalities. While coadministration of doxycycline 100 mg/kg of fish weight plus 10 μg/fish of CpG-ODN-1668 led to reduction of cumulative mortality (33.3% in both replicates) in comparison to doxycycline alone or CpG-ODN 1668 alone. Survival rates of fish administered with 5 μg/fish of CpG-ODN-1668 plus doxycycline
One of the important aspects of chemotherapy is the range of dose that can effectively treat disease without inducing (or minimizing) toxic effects. Our previous results showed that feeding a 2% doxycycline diet once a day (approximately corresponding to 200 mg of doxycycline/kg of fish/day) for 3 times was effective in treatment of olive flounder fingerlings infected with M. avidus (Kang et al., 2013). Although the fish did not show any behavioral changes during the experimental period, the dose of doxycycline used in the previous experiment was definitely high, compared to a recommendable dose of doxycycline to treat streptococcosis in olive flounder in Korea (less than 50 mg/kg/day). Thus, it was inevitable to develop other auxiliary therapeutic approaches that can reduce the dose of doxycycline. The prophylactic and therapeutic abilities of CpG-ODN 1668 against scuticociliatosis in olive flounder
Fig. 1. Cumulative mortality rates of olive flounder (Paralichthys olivaceus) fingerlings. A total of 168 fish were randomly divided into 7 groups with 2 replicates (A and B), then, immersed in seawater containing 8 × 107 ciliates/80 l for 3 days. Fish in each group were administered with doxycycline 100 or 200 mg/kg of fish, 5 or 10 μg/fish of CpGODN 1668, 5 or 10 μg/fish of CpG-ODN 1668 plus doxycycline 100 mg/kg (CpG-5 plus Doxy 100 or CpG-10 plus Doxy 100), or phosphate buffered saline (PBS, Control). Doxycycline or PBS was administered through the oral route using a gastric tube at 0, 3, and 6 d post-infection, and CpG-ODN 1668 was intraperitoneally (i.p.) administered at 0 d postinfection.
Y.J. Kang, K.H. Kim / Aquaculture 435 (2015) 143–145
were reported from our previous papers (Kang and Kim, 2012, 2014; Lee and Kim, 2009). Hence, in this study, we investigated whether coadministration of CpG-ODN 1668 can reduce the dose of doxycycline without decrease of treatment efficacy. There have been no reports on the treatment of scuticociliatosis by combining chemotherapy and immunotherapy. In the present study, we demonstrated that a combination of chemotherapy (doxycycline) and immunotherapy (CpG-ODN 1668) was effective against scuticociliatosis in olive flounder fingerlings. In this study, the survival rates of fish administered with doxycycline 100 mg/kg plus 10 μg of CpG-ODN 1668 were higher than those of fish administered with doxycycline alone at a dose of 200 mg/kg. This result suggests that coadministration of CpG-ODN 1668 can reduce doxycycline to half the dose. As the cost for preparation of CpG-ODNs is expensive, reduction of CpG-ODN 1668 amount would be favorable for an economic aspect. In the present results, although the treatment efficacy was lower than the fish administered with doxycycline 100 mg/kg plus 10 μg of CpGODN 1668, reduction of CpG-ODN 1668 to 5 μg/fish plus administration of 100 mg/kg of doxycycline led to high survival rates that were comparable to doxycycline 200 mg/kg, suggesting that the used amount of both immunotherapeutics and chemotherapeutics can be reduced through coadministration. Recently, we have shown that oral administration of auxotrophic E. tarda mutant harboring CpG 1668 motif-enriched plasmids was effective in treatment of scuticociliatosis (Kang et al., 2014). The treatment effects of oral administration of doxycycline and the mutant bacteria containing CpG 1668 motif-enriched plasmids should be further studied to reduce cost and labor related to the preparation and injection of CpGODN 1668. More research is needed to understand the interactive mechanism of the immunochemotherapy, and search for other immunotherapeutics that can enhance scuticocidal activity is also needed. Acknowledgments This research was supported by the Basic Science Research Program through the National Research Foundation (NRF) funded by the Ministry of Science, ICT and Future Planning of South Korea (2013R1A2A2A01067204).
145
References Bahrami, F., Morris, D.L., Pourgholami, M.H., 2012. Tetracyclines: drugs with huge therapeutic potential. Mini-Rev. Med. Chem. 12, 44–52. Budiño, B., Lamas, J., Pata, M.P., Arranz, J.A., Sanmartín, M.L., Leiro, J., 2011. Intraspecific variability in several isolates of Philasterides dicentrarchi (syn Miamiensis avidus), a scuticociliate parasite of farmed turbot. Vet. Parasitol. 175, 260–272. Cunha, B.A., Comer, J.B., Jonas, M., 1982. The tetracyclines. Med. Clin. North Am. 66, 293–302. Draper, M.P., Bhatia, B., Assefa, H., Honeyman, L., Garrity-Ryan, L.K., Verma, A.K., Gut, J., Larson, K., Donatelli, J., Macone, A., Klausner, K., Leahy, R.G., Odinecs, A., Ohemeng, K., Rosenthal, P.J., Nelson, M.L., 2013. In vitro and in vivo antimalarial efficacies of optimized tetracyclines. Antimicrob. Agents Chemother. 57, 3131–3136. Iglesias, R., Paramá, A., Alvarez, M.F., Leiro, J., Fernández, J., Sanmartín, M.L., 2001. Philasterides dicentrarchi (Ciliophora, Scuticociliatida) as the causative agent of scuticociliatosis in farmed turbot Scophthalmus maximus in Galicia (NW Spain). Dis. Aquat. Org. 46, 47–55. Iglesias, R., Paramá, A., Alvarez, M.F., Leiro, J., Sanmartín, M.L., 2002. Antiprotozoals effective in vitro against the scuticociliate fish pathogen Philasterides dicentrarchi. Dis. Aquat. Org. 49, 191–197. Jung, S.J., Kitamura, S.I., Song, J.Y., Oh, M.J., 2007. Miamiensis avidus (Ciliophora: Scuticociliatida) causes systemic infection of olive flounder Paralichthys olivaceus and is a senior synonym of Philasterides dicentrarchi. Dis. Aquat. Org. 73, 227–234. Kang, Y.J., Kim, K.H., 2012. Effect of CpG-ODNs belonging to different classes on resistance of olive flounder (Paralichthys olivaceus) against viral hemorrhagic septicemia virus (VHSV) and Miamiensis avidus (Ciliata; Scuticociliatia) infections. Aquaculture 324–325, 39–43. Kang, Y.J., Kim, K.H., 2014. Therapeutic potential of CpG-ODN 1668 against scuticociliatosis in olive flounder (Paralichthys olivaceus). Aquaculture 430, 17–20. Kang, Y.J., Kim, D.S., Kim, K.H., 2013. Evaluation of treatment efficacy of doxycycline and albendazole against scuticociliatosis in olive flounder (Paralichthys olivaceus). Aquaculture 416–417, 192–195. Kang, Y.J., Choi, S.H., Kim, K.H., 2014. Preventive and therapeutic effects of auxotrophic Edwardsiella tarda mutant harboring CpG 1668 motif-enriched plasmids against scuticociliatosis in olive flounder (Paralichthys olivaceus). Exp. Parasitol. 144, 34–38. Kim, S.M., Cho, J.B., Kim, S.K., Nam, Y.K., Kim, K.H., 2004. Occurrence of scuticociliatosis in olive flounder Paralichthys olivaceus by Philasterides dicentrarchi (Ciliophora: Scuticociitida). Dis. Aquat. Org. 62, 233–238. Lee, E.H., Kim, K.H., 2009. CpG-ODN increases resistance of olive flounder (Paralichthys olivaceus) against Philasterides dicentrarchi (Ciliophora: Scuticociliatia) infection. Fish Shellfish Immunol. 26, 29–32. Leiro, J., Piazzón, M.C., Budiño, B., Sanmartín, M.L., Lamas, J., 2008. Complement-mediated killing of Philasterides dicentrarchi (Ciliophora) by turbot serum: relative importance of alternative and classical pathways. Parasite Immunol. 30, 535–543. Piazzon, C., Lamas, J., Castro, R., Budiño, B., Cabaleiro, S., Sanmartín, M., Leiro, J., 2008. Antigenic and cross-protection studies on two turbot scuticociliate isolates. Fish Shellfish Immunol. 25, 417–424. Piazzon, M.C., Wiegertjes, G.F., Leiro, J., Lamas, J., 2011. Turbot resistance to Philasterides dicentrarchi is more dependent on humoral than on cellular immune responses. Fish Shellfish Immunol. 30, 1339–1347.