한수지 52(4), 382-388, 2019 Original Article Korean J Fish Aquat Sci 52(4),382-388,2019 Streptococcus parauberis 의디스크확산법결과에대한 Epidemiological Cut-off Value 의설정 천원경 이윤항 김윤재 노형진 김아란 김남은 서정수 1 권문경 1 이지훈 1 김도형 * 부경대학교수산생명의학과, 1 국립수산과학원방역과 Epidemiological Cut-off Values Generated for Disc Diffusion Data from Streptococcus parauberis Won-kyong Chun, Yoonhang Lee, Yoon-Jae Kim, Heyong Jin Roh, Ahran Kim, Nameun Kim, Jung- Soo Seo 1, Mun-Gyeong Kwon 1, Ji Hoon Lee 1 and Do-Hyung Kim* Department of Aquatic Life Medicine, Pukyong National University, Busan 48513, Korea 1 Aquatic Life Disease Control Division, National Institute of Fisheries Science, Busan 46083, Korea Streptococcosis caused by Streptococcus parauberis is a very important disease in farmed olive flounder Paralichthys olivaceus. For most fish pathogens, including S. parauberis, there are no analytical criteria to distinguish antibioticsusceptible strains from antibiotic-resistant strains. In this study, epidemiological cut-off (CO WT ) values were generated to classify 75 strains of S. parauberis isolated from 1999 to 2018 as wild type (WT) and non-wild type (NWT) using disc diffusion data and normalized resistance interpretation (NRI) analysis. The susceptibility of the isolates to 16 antibiotics was evaluated using CLSI guideline M42-A. The wild-type cut-off values for amoxicillin, erythromycin, oxytetracycline, and florfenicol for S. parauberis were 35, 31, 28, and 27 mm, respectively. The NWT ratios of S. parauberis strains to treatment with GEN, FFC, ENR, SXT, EFT, VAN, and CHL were 17% or less, indicating that these antibiotics may be used to treat streptococcosis caused by S. parauberis. For recent S. parauberis isolates, the NWT ratios for AMX, ERY, OTC and FFC are much higher than for strains isolated from 1999-2007. The CO WT data from this study will assist aquatic animal disease professionals in prescribing appropriate antibiotics for the treatment of streptococcosis caused by S. parauberis, which will help reduce the misuse and abuse of antibiotics in the aquaculture sector. Key words: Streptococcus parauberis, Antimicrobial susceptibility testing, CLSI protocols, Disc diffusion, Epidemiological cut-off values 서론 2017 48% (KOSTAT, 2017). Lactococcus garviae, Streptococcus iniae Streptococcus parauberis, 2000 S. parauberis (Baeck et al., 2006; Park et al., 2016).. (Kanai et al., 2009; Kanai et al., 2015) S. parauberis 4. amoxicillin (AMX), florfenicol (FFC), ampicillin (AMP), erythromycin (ERY) AMX, FFC ERY (Kwon et al., 2014; *Corresponding author: Tel: +82. 51. 629. 5945 Fax: +82. 51. 629. 5938 E-mail address: dhkim@pknu.ac.kr This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial Licens (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. https://doi.org/10.5657/kfas.2019.0382 Korean J Fish Aquat Sci 52(4), 382-388, August 2019 Received 14 May 2019; Revised 10 June 2019; Accepted 6 August 2019 저자직위 : 천원경 ( 대학원생 ), 이윤항 ( 대학원생 ), 김윤재 ( 대학원생 ), 노형진 ( 대학원생 ), 김아란 ( 대학원생 ), 김남은 ( 대학원생 ), 서정수 ( 연구사 ), 권문경 ( 연구관 ), 이지훈 ( 연구사 ), 김도형 ( 교수 ) Copyright 2019 The Korean Society of Fisheries and Aquatic Science 382 pissn:0374-8111, eissn:2287-8815
Streptococcus parauberis 에대한 epidemiological cut-off values 결정 383 Jee et al., 2014). AMX (Seo et al., 2015; Kim et al., 2015), Lim et al. (2017) AMX 40 mg/kg body weight fish S. parauberis. Lim et al. (2017) 10 S. parauberis (minimum inhibitory concentration, MIC; μg/ml) 0.5 AMX. AMX. S. parauberis AMX. (antimicrobial susceptibility testing) clinical and laboratory standards institute (CLSI) (Interpretive criteria) Aeromonas salmonicida (CLSI, 2014). Kwon et al. (2016) clinical breakpoint normalized resistance interpretation (NRI) epidemiological cut-off values (wild-type cut-off values; CO WT ) (wild type; WT) (non-wild type; NWT). CO WT WT NWT (Smith, 2012; Kwon et al., 2016). Kronvall (2003) NRI CO WT. A. salmonicida (Smith et al., 2007), Vibrio anguillarum (Smith and Christofilogiannis, 2007), Streptococcus phocae (Avendaño-Herreraa et al., 2011), Flavobacterium psychrophilum (Henríquez-Núñez et al., 2012), Vibrio ichthyoenteri (Lee et al., 2012), Weissella sp. (Figueiredo et al., 2012), Vibrio ordalii (Poblete-Morales et al., 2013), Edwardsiella tarda, Vibrio harveyi (Lim et al., 2016), Piscirickettsia salmonis (Henríquez et al., 2016), Photobacterium damselae (Kwon et al., 2016) Edwardsiella piscicida (Kim et al., 2018) CO WT. NRI (Smith and Kronvall, 2015; Kronvall and Smith, 2016) CO WT. S. parauberis 75 NRI (Kronvall and Smith, 2016) CO WT wild type (WT) non wild-type (NWT) NWT. 재료및방법 사용균주 Streptococcus parauberis 1999 2018,,,,,, 75 (Table 1). 1% NaCl brain heart infusion agar (BHIA) 28 C 18-24. 항생제감수성시험 CLSI M42-A (CLSI, 2006) (Disk diffusion test)., 28 C 18-24 2 10 8 CFU/mL 5% sheep blood mueller-hinton agar (MHA) 28 C. 24-28 6 mm. Quality control (QC) CLSI M42-A (CLSI, 2006) Escherichia coli ATCC 25922 Aeromonas salmonicida ATCC 33658. Oxoid 16. ampicillin (AMP), amoxicillin (AMX), amoxicillin-clavulanic (AMC), doxycycline (DOX), oxytetracycline (OTC), cephalexin (CFL), cefuroxime (CXM), ceftiofur (EFT), enrofloxacin (ENR), erythromycin (ERY), clindamycin (CLI), trimethoprim/sulfamethoxazole (SXT), gentamicin (GEN), vancomycin (VAN), florfenicol; (FFC), chloramphenicol (CHL) (Table 2). Epidemiological cut-off value 의설정 Epidemiological cut-off value (ECOFF) [ Wild-type cut-off values (CO WT )] Kronvall and Smith (2016) NRI. Wild type (normalized) zone probit. CO WT Kronvall and Smith (2016) 2.5. 결과및고찰
384 천원경ㆍ이윤항ㆍ김윤재ㆍ노형진ㆍ김아란ㆍ김남은ㆍ서정수ㆍ권문경ㆍ이지훈ㆍ김도형 (Aoki et al., 1990),. Lim et al. (2016) WT NWT.. OIE aquatic code ( ) wild type (CO WT ). S. parauberis 75 (mm) Table 2. QC E. coli ATCC 25922 A. salmonicida ATCC 33658 CLSI M42-A (2006). 16 S. parauberis 75 (mm) NRI. NRI CO WT WT Table 3. NRI CO WT AMP, AMX, AMC, DOX, OTC, CFL, CXM, EFT, EFT, ENR, ERY, CLI, SXT, GEN, VAN, FFC CHL 35, 35, 36, 25, 28, 29, 36, 30, 24, 31, 30, 22, 15, 21, 27 24 (Table 3). NRI (e.g., Kwon et al., 2016). NRI (Kronvall and Smith, 2016). CO WT CO WT, 30 WT (Smith et al., 2009; CLSI, 2011). WT 30 CO WT. AMP, AMX, AMC, DOX, OTC ERY NWT 47, 49, 48, 49, 51 51%. AMX, ERY FFC (Jee et al., 2014), FFC S. parauberis NWT 11% AMX ERY NWT 50% FFC. GEN, FFC, ENR, SXT, EFT, VAN, CHL S. parauberis NWT 17%. S. parauberis Table 1. Streptoccocus parauberis strains used in this study Isolate code Isolation source Isolation location Isolation year KSP44-46 (n=3) Olive flounder Jejudo 1999 KSP47 Olive flounder Gyeongsangbuk-do 1999 KSP1-4 (n=4) Olive flounder Jejudo 2003 KSP5-10 (n=6) Olive flounder Jejudo 2004 KSP11-21 (n=11) Olive flounder Jejudo 2005 KSP22-23 (n=2) Olive flounder Haenam 2005 KSP24-43 (n=20) Olive flounder Wando 2005 PH0710-0711 (n=2) Starry flounder Pohang 2007 SpOF3k, 04151K, 04161K (n=3) Olive flounder Geojedo 2013 HFTC0023, HFTC0059-0060, HFTC0063-0064 (n=5) Olive flounder Pohang 2014 HFTC0076 Starry flounder Yeongdeok 2014 HFTC0083 Starry flounder Yeongdeok 2015 HFTC0086, HFTC0091 (n=2) Starry flounder Uljin 2015 HFTC0157 Starry flounder Pohang 2015 HFTC0237 Starry flounder Yeongdeok 2015 SPOF18J1-J11 (n=11) Olive flounder Jejudo 2018 SPOF18G1 Starry flounder Uljin 2018
Streptococcus parauberis 에대한 epidemiological cut-off values 결정 385 Table 2. Distribution zone diameters (mm) for 75 strains of Streptococcus parauberis Zone diameter (mm) Number of isolates AMP AMX AMC DOX OTC CFL CXM EFT ENR ERY CLI SXT GEN VAN FFC CHL 6 28 1 18 4 2 7 1 1 2 8 1 1 9 1 2 10 4 2 2 11 1 1 1 12 3 1 2 2 13 10 1 2 14 8 2 3 15 6 1 5 16 5 2 5 17 1 1 2 1 16 18 1 1 1 2 8 19 1 5 1 14 3 20 2 1 1 2 14 3 21 2 1 3 1 5 3 10 1 22 1 1 3 1 4 5 1 9 23 1 1 2 1 3 1 1 9 6 1 24 1 6 5 1 4 1 4 10 1 25 7 10 1 7 1 6 2 12 13 1 1 26 2 4 2 1 7 3 1 4 1 7 1 12 4 5 27 3 1 2 1 5 2 1 2 1 11 7 1 5 28 2 2 3 2 4 2 6 1 8 7 5 29 1 2 4 1 4 2 2 2 1 2 3 2 8 30 9 3 5 7 3 5 7 12 5 4 3 1 7 12 31 4 4 8 4 5 5 1 9 1 6 1 8 9 32 3 3 9 8 10 3 3 10 13 1 3 16 10 33 1 5 7 6 3 3 1 4 3 9 7 34 1 4 5 6 5 5 13 4 7 35 1 2 2 6 6 7 7 11 3 36 3 1 1 2 10 2 7 1 7 7 2 37 1 3 1 1 5 4 7 3 1 1 38 2 8 4 3 1 9 1 5 1 39 6 8 5 2 2 8 1 3 40 12 6 10 5 5 2 1 41 1 8 8 2 1 42 6 6 8 3 43 5 1 2 5 44 5 1 7 45 1 2 46 1 4 47 3 48 1 Abbreviations for antibiotic agent are as in the materials and methods. Areas highlight in grey indicate the range of zones for isolates categorized as WT. Penicillin (AMP, ampicillin; AMX, amoxicillin; AMC, amoxicillin-clavulanic); Tetracyclines (DOX, doxycycline; OTC, oxytetracycline); Cephalosporins (CFL, cephalexin; CXM, cefuroxime; EFT, ceftiofur); Fluoroquinolones (ENR, enrofloxacin); Macrolides(ERY, erythromycin); Lincosamides (CLI, clindamycin); Sulfonamides (SXT, trimethoprim/sulfamethoxazole); Aminoglycosides (GEN, gentamicin); Glycopeptide antibiotic (VAN, vancomycin); Amphenicols (FFC, Florfenicol; CHL, chloramphenicol).
386 천원경ㆍ이윤항ㆍ김윤재ㆍ노형진ㆍ김아란ㆍ김남은ㆍ서정수ㆍ권문경ㆍ이지훈ㆍ김도형. Lee et al. (2018) quinolone, sulfonamide, tetracycline, penicillin, erythromycin CLSI M100 (CLSI, 2017) 13 mm. Lee et al. (2018) Staphylococcus. Table 3. Summary of the results of NRI analysis Antibiotics Table 4. Ratio of non-wild strains of Streptococcus parauberis Isolation year Number of strains Streptococcus parauberis (n=75) CO WT (mm) Sd 1 WT 2 AMP 35 2.42 40 AMX 35 1.90 38 AMC 36 1.85 39 DOX 25 2.46 38 OTC 28 1.96 37 CFL 29 3.13 41 CXM 36 2.78 41 EFT 30 2.67 63 ENR 24 2.63 65 ERY 31 2.00 37 CLI 30 1.64 47 SXT 22 1.90 62 GEN 15 1.48 68 VAN 21 1.49 68 FFC 27 2.55 67 CHL 24 2.84 75 Abbreviations for antibiotic agent are as in the materials and methods. 1 Standard deviation of the normalized distribution of WT observations. 2 Number of isolates categorized by the application of the relevant CO WT value. AMP, ampicillin; AMX, amoxicillin; AMC, amoxicillin-clavulanic; DOX, doxycycline; OTC, oxytetracycline; CFL, cephalexin; CXM, cefuroxime; EFT, ceftiofur; ENR, enrofloxacin; ERY, erythromycin; CLI, clindamycin; SXT, trimethoprim/sulfamethoxazole; GEN, gentamicin; VAN, vancomycin; FFC, Florfenicol; CHL, chloramphenicol. Ratio (%) of NWT AMX ERY OTC FFC 1999-2007 49 22% 49% 31% 0% 2013-2018 26 100% 100% 88% 31% NWT, non-wild type; AMX, amoxicillin; ERY, erythromycin; OTC, oxytetracycline; FFC, Florfenicol.. 5 S. parauberis NWT 1999-2007 NWT (Table 4). (Meng et al., 2009; Park et al., 2009) tet (s) erm (B) gene S. parauberis erythromycin tetracycline. S. parauberis, NWT. S. parauberis WT NWT. S. parauberis NRI. NWT. 사사 (R2019058). References Aoki T, Takami K and Kitao T. 1990. Drug resistance in a nonhemolytic Streptococcus sp. isolated from cultured yellowtail Seriola quinqueradiata. Dis aquat Org 8, 171-177. http://doi.org/10.3354/dao008171. Avendaño-Herrera R, Molina A, Magariños B, Toranzo AE and Smith P. 2011. Estimation of epidemiological cut-off values for disk diffusion susceptibility test data for Streptococcus phocae. Aquaculture 314, 44-48. https://doi.org/10.1016/j. aquaculture.2011.01.049. Baeck GW, Kim JH, Gomez DK and Park SC. 2006. Isolation and characterization of Streptococcus sp. from diseased flounder (Paralichthys olivaceus) in Jeju Island. J Vet Sci 7, 53-58. https://doi.org/10.4142/jvs.2006.7.1.53. CLSI (Clinical and Laboratory Standards Institute). 2006. Methods for Antimicrobial Disk Susceptibility Testing of Bacteria Isolated From Aquatic Animals; Approved Guideline. CLSI document M42-A. Clinical and Laboratory Standards
Streptococcus parauberis 에대한 epidemiological cut-off values 결정 387 Institute, Wayne, Pennsylvania, U.S.A. CLSI (Clinical and Laboratory Standards Institute). 2011. Generation, presentation and application of antimicrobial susceptibility test data for bacteria of animal origin: a report. Clinical and laboratory standards institute document X08-R. Clinical and Laboratory Standards Institute, Wayne, Pennsylvania, U.S.A. CLSI (Clinical and Laboratory Standards Institute). 2014. Performance standards for antimicrobial susceptibility testing of bacteria isolated from aquatic animals; Second informational supplement. CLSI document VET03/VET04-S2. Clinical and Laboratory Standards Institute, Wayne, Pennsylvania, U.S.A. CLSI (Clinical and Laboratory Standards Institute). 2017. Performance Standards for Antimicrobial Susceptibility Testing; Twentieth Informational Supplement. CLSI document M100-S28. Clinical and Laboratory Standards Institute, Wayne, Pennsylvania, U.S.A. Figueiredo HCP, Costa FAA, Leal CAG, Carvalho-Castro GA and Leite RC. 2012. Weissella sp. outbreaks in commercial rainbow trout (Oncorhynchus mykiss) farms in Brazil. Vet Microbiol 156, 359-366. https://doi.org/10.1016/j.vetmic.2011.11.008. Henríquez P, Kaiser M, Bohle H, Bustos P and Mancilla M. 2016. Comprehensive antibiotic susceptibility profiling of Chilean Piscirickettsia salmonis field isolates. J Fish Dis 39, 441-448. https://doi.org/10.1111/jfd.12427. Henríquez-Núñez H, Evrard O, Kronvall G and Avendaño- Herrera R. 2012. Antimicrobial susceptibility and plasmid profiles of Flavobacterium psychrophilum strains isolated in Chile. Aquaculture 354, 38-44. https://doi.org/10.1016/j. aquaculture.2012.04.034. Jee BY, Shin KW, Lee DW, Kim YJ and Lee MK. 2014. Monitoring of the mortalities and medications in the inland farms of olive flounder, Paralichthys olivaceus, in South Korea. J Fish Pathol 27, 77-83. https://doi.org/10.7847/ jfp.2014.27.1.077. Kanai K, Tu C, Katayama N and Suga K. 2015. Existence of subserotypes in Streptococcus parauberis serotype I. Japanese Soc Fish Pathol 50, 75-80. https://doi.org/10.3147/ jsfp.50.75. Kanai K, Yamada M, Meng F, Takahashi I, Nagano T, Kawakami H, Yamashita A, Matsuoka S, Fukuda Y, Miyoshi Y, Takami I, Nakano H, Hirae T, Shutou K and Honma T. 2009. Serological differentiation of Streptococcus parauberis strains isolated from cultured Japanese flounder in Japan. Japanese Soc Fish Pathol 44, 33-39. https://doi.org/10.3147/ jsfp.44.33. Kim A, Lim Y, Kim N, Luan Nguyen T, Roh HJ, Park CI, Han HJ, Jung SH, Cho MY, Kim DH and Smith P. 2018. A comparison of genotypic and phenotypic methods for analyzing the susceptibility to sulfamethoxazole and trimethoprim in Edwardsiella piscicida. Microb Drug Resist 24, 1226-1235. https://doi.org/10.1089/mdr.2017.0137. Kim JS, Lee JH, Lee SJ and Park KH. 2015. Pharmacokinetics of amoxicillin after intramuscular injection at different temperatures to cultured olive flounder, Paralichthys olivaceus. J Fish Pathol 28, 43-51. https://doi.org/10.7847/ jfp.2015.28.1.043. KOSTAT (Statistics Korea). 2017. Republic of Korea: Survey on the status of fish culture. 2017, Media release, Statistics Korea webpage, KOSIS. Retrieved from http://kostat.go.kr/ portal/eng/index.action on May 08, 2019. Kronvall G and Smith P. 2016. Normalized resistance interpretation, the Nri method: Review of Nri disc test applications and guide to calculations. Apmis 124, 1023-1030. https:// doi.org/10.1111/apm.12624. Kronvall G. 2003. Determination of the real standard distribution of susceptible strains in zone histograms. Internat J Antimic Agent 22, 7-13. http://doi.org/10.1016/s0924-8579(03)00107-9. Kwon MG, Hwang JY and Jung SH. 2014. The Efficacy and safety on combination vaccines: Edwardsiella tarda, Streptococcus iniae and S. parauberis, in Olive flounder, Paralichthys olivaceus. J Fish Mari Sci Edu 26, 1193-1200. https:// doi.org/10.13000/jfmse.2014.26.6.1193. Kwon MG, Lim YJ, Kim MS, Seo JS, Kim DH. 2016. Epidemiological cut-off values gnerated for disc diffusion data from Photobacterium damselae. Korean J Fish Aquat Sci 49, 838-844. https://doi.org/10.5657/kfas.2016.0838 Lee DC, Han HJ, Choi SY, Kronvall G, Park CI and Kim DH. 2012. Antibiograms and the estimation of epidemiological cut off values for Vibrio ichthyoenteri isolated from larval olive flounder, Paralichthys olivaceus. Aquaculture 342, 31-35. https://doi.org/10.1016/j.aquaculture.2012.02.011. Lee DW, Jun LJ, Kim SM and Jeong JB. 2018. Analysis of erythromycin resistance gene in pathogenic bacteria isolates from cultured Olive flounder Paralichthys olivaceus in Jeju. Korean J Fish Aquat Sci 51, 397-403. https://doi. org/10.5657/kfas.2018.0397. Lim JW, Jung MH, Jung SJ, Kim DH, Park KH and Kang SY. 2017. The efficacy of amoxicillin sodium against streptococcosis in cultured olive flounder Paralichthys olivaceus and its pharmacokinetics. J Vet Pharmacol Ther 40, 77-87. https://doi.org/10.1111/jvp.12321. Lim YJ, Kim DH, Roh HJ, Park MA, Park CI and Smith P. 2016. Epidemiological cut-off values for disc diffusion data generated by standard test protocols from Edwardsiella tarda and Vibrio harveyi. Aquaculture Int 24, 1153-1161. https://doi. org/10.1007/s10499-016-9977-0. Meng F, Kanai K and Yoshikoshi K. 2009. Characterization of drug resistance in Streptococcus parauberis isolated from Japanese flounder. Japanese Soc Fish Pathol 44, 40-46. https://doi.org/10.3147/jsfp.44.40.
388 천원경ㆍ이윤항ㆍ김윤재ㆍ노형진ㆍ김아란ㆍ김남은ㆍ서정수ㆍ권문경ㆍ이지훈ㆍ김도형 Park SB, Nho SW, Jang HB, Cha IS, Kim MS, Lee WJ and Jung TS. 2016. Development of three-valent vaccine against streptococcal infections in olive flounder, Paralichthys olivaceus. Aquaculture 461, 25-31. https://doi.org/10.1016/j. aquaculture.2016.04.022. Park YK, Nho SW, Shin GW, Park SB, Jang HB, Cha IS, Ha MA, Kim YR, Dalvi RS, Kang BJ and Jung TS. 2009. Antibiotic susceptibility and resistance of Streptococcus iniae and Streptococcus parauberis isolated from olive flounder (Paralichthys olivaceus). Vet Microbiol 136, 76-81. https:// doi.org/10.1016/j.vetmic.2008.10.002. Poblete-Morales M, Irgang R, Henríquez-Núñez H, Toranzo AE, Kronvall G and Avendaño-Herrera R. 2013. Vibrio ordalii antimicrobial susceptibility testing Modified culture conditions required and laboratory-specific epidemiological cut-off values. Vet Microbiol 165, 434-442. https://doi. org/10.1016/j.vetmic.2013.04.024. Seo JS, Jeon EJ, Jung SH, Park MA and Kim NY. 2015. Pharmacokinetics of amoxicillin trihydrate in cultured olive flounder (Paralichthys olivaceus). J Vet Pharmacol Ther 38, 86-92. https://doi.org/10.1111/jvp.12155. Smith P and Christofilogiannis P. 2007. Application of normalised resistance interpretation to the detection of multiple low-level resistance in strains of Vibrio anguillarum obtained from Greek fish farms. Aquaculture 272, 223-230. https://doi.org/10.1016/j.aquaculture.2007.07.232. Smith P and Kronvall G. 2015. Effect of incubation temperature and time on the precision of data generated by antibiotic disc diffusion assays. J Fish Dis 38, 629-636. https://doi. org/10.1111/jfd.12278. Smith P, Douglas I, McMurray J and Carroll C. 2009. A rapid method of improving the criteria being used to interpret disc diffusion antimicrobial susceptibility test data for bacteria associated with fish diseases. Aquaculture 290, 172-178. https://doi.org/10.1016/j.aquaculture.2009.02.017. Smith P, Ruane NM, Douglas I, Carroll C, Kronvall G and Fleming GT. 2007. Impact of inter-lab variation on the estimation of epidemiological cut-off values for disc diffusion susceptibility test data for Aeromonas salmonicida. Aquaculture 272, 168-179. https://doi.org/10.1016/j.aquaculture. 2007.07.216. Smith P. 2012. Antimicrobial resistance: complexities and difficulties of determination. Improving biosecurity through prudent and responsible use of veterinary medicines in aquatic food production, 99-118. Retrieved from http://www.fao. org/3/ba0056e/ba0056e.pdf#page=117 on May 07, 2019.