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Egyptian Journal of Aquatic Research (2013) 39, 241–248

National Institute of Oceanography and Fisheries

Egyptian Journal of Aquatic Research http://ees.elsevier.com/ejar www.sciencedirect.com

Eﬀect of oxytetracycline and ﬂorfenicol as growth promoters on the health status of cultured Oreochromis niloticus Rasha M. Reda *, R.E. Ibrahim, El-Nobi G. Ahmed, Z.M. El-Bouhy Department of Fish Disease and Management, Faculty of Veterinary Medicine, Zagazig University, 44511 Zagazig, Egypt Received 23 October 2013; revised 27 November 2013; accepted 2 December 2013 Available online 22 December 2013

KEYWORDS ALT; Antibiotic; IgM; Urea; Oreochromis niloticus

Abstract Several classes of antibiotics are commonly used in aquaculture; among them the most used are oxytetracycline (OTC) and ﬂorfenicol (FLO) due to their high potency against bacterial diseases. Sixty Oreochromis niloticus (20 ± 0.09 g) were divided into 3 groups (20 ﬁsh/group) in duplicate. Group 1 was fed a basal diet as the control group, while group 2 and 3 were fed basal diet supplemented with Oxytetracycline at 100 mg kg 1 diet and Florfenicol at 5 mg kg 1 body weight, respectively. After 12 weeks of feeding, group 3 showed a signiﬁcant increase in the ﬁnal body weight, and weight gain when compared with the control group. Group 3 did not show signiﬁcant differences in immunoglobulin M (IgM) total levels with an insigniﬁcant decrease in phagocytic activity when compared to the control group. All groups showed signiﬁcant increase in Lysozyme activity. Group 3 did not show signiﬁcant difference in Alanine aminotransferase (ALT) activity compared to the control group, while group 2 showed the highest values of Aspartate Transaminase (AST) activity than other groups. The highest value of urea was shown in group 2, while there was a decrease in the value of creatinine in the treated groups than the control. Histopathological examination revealed pathological alterations in liver and kidney of the treated groups. After a withdrawal period of 15 days the OTC and FLO residues in O. niloticus muscle were within the permissible limit of Commission Regulation, EU (2010). In conclusion, Florfenicol at 5 mg kg 1 body weight is more safe in its effect on Oreochromis niloticus. ª 2013 Production and hosting by Elsevier B.V. on behalf of National Institute of Oceanography and Fisheries.

Introduction * Corresponding author. Tel.: +20 1006223528; fax: +20 55/2283683. E-mail address: [email protected] (R.M. Reda). Peer review under responsibility of National Institute of Oceanography and Fisheries.

Production and hosting by Elsevier

Fish aquaculture constitutes a rapidly growing industry worldwide. This has led to a concomitant increase in aquaculture intensity methods, which increase the susceptibility to disease outbreaks and the necessity use of the medicated diet (Jerbi et al., 2011). But the most dangerous is that, a large proportion of the world’s antimicrobial industrial production is used as

1687-4285 ª 2013 Production and hosting by Elsevier B.V. on behalf of National Institute of Oceanography and Fisheries. http://dx.doi.org/10.1016/j.ejar.2013.12.001

242 prophylactics and as growth promoters that far outweigh their use as therapeutics (Bush et al., 2011; Cabello et al., 2013). Especially, antibiotics are still used until now as growth promoters in some of the ﬁsh farms in Egypt. The most used antibiotics in ﬁsh farms are oxytetracyline (OTC) and ﬂorfenicol (FLO) (Rigos and Troisi, 2005). OTC is a tetracycline broad-spectrum antibiotic with bacteriostatic action produced by Streptomyces spp. fungi, used to treat systemic bacterial infections of ﬁsh (Jerbi et al., 2011). FLO is a structural analog of chloramphenicol similar to thiamphenicol, but is more active against some bacteria than chloramphenicol (Cannon et al., 1990). Antibiotics are one of the most common groups used as feed additives in the frame of growth promoter. Several antibiotics have been in use as growth promoters in ﬁsh farms ever since. Sanchez-Martinez et al. (2008) studied the effect of supplementing channel cat ﬁsh (Ictalurus punctatus) feeds with oxytetracycline. Treated ﬁsh exhibit a signiﬁcant increase in weight gain suggesting a growth promotion action of this antibiotic agent. Previous studies stated in the literatures showed the negative effects of antibiotics on the immune system of ﬁsh (Lunden and Bylund, 2000; Guardiola et al., 2012). OTC interferes with humoral innate immune parameters and increases the cellular parameters in gilthead sea bream (Guardiola et al., 2012). FLO induced suppression of immune response in Cyprinus carpio fry and in rainbow trout (Sieroslawska et al., 1998; Lunden and Bylund, 2000). Few studies elucidated the side effects of long term antibiotic use on ﬁsh which can induce nephrotoxicity and liver damage (Horsberg and Berge, 1986; Hentschel et al., 2005). Moreover, the use of large amount of antibiotics may lead to the presence of residual antibiotics in ﬁsh tissue and ﬁsh products (Samanidou and Evaggelopoulou, 2007). The aim of the present study was to determine the effects of dietary supplementation of OTC and FLO for 12 weeks on Oreochromis niloticus growth performance, immunity, liver and kidney functions, histopathological ﬁndings and the residues of these antibiotics after cessation of treatment by 15 days. Materials and methods

R.M. Reda et al. temperature 28 ± 2 C, pH 7.5, nitrite 0.05 mg/L, nitrate 10 mg/L, and ammonium 0.4 mg/L. Growth performance Fish were weighed every two weeks to assess the growth performance. The ﬁnal body weight (g), weight gain (g) and food conversion ratio (FCR) were determined (Siddiqui et al., 1988). Blood collection Blood samples were collected at the end of the experiment from the caudal vessels in clean sterilized tubes containing EDTA as an anticoagulant for determination of phagocytic index. Other blood samples were collected in plain centrifuge tubes and centrifuged at 3000 r.p.m for 15 min for serum separation for determination of liver function tests, kidney function tests, immunoglobulin M (IgM) and lyzosyme activity. Determination of immunological parameters Immunoglobulin M (IgM) was determined using an ELISA Kit (Cuesta et al., 2004); the lysozyme activity was measured using the turbidity assay (Parry et al., 1965). Phagocytic activity was measured using heat-inactivated Candida albicans (Kumari and Sahoo, 2006). Determination of liver and kidney function Estimation of aspartate transaminase (AST) and alanine aminotransferase (ALT) was obtained according to Reitman and Frankel (1957). Determination of serum urea and creatinine levels was performed according to Henry et al. (1974) and Patton and Crouch (1977). Histopathological examination Tissues from liver and kidney of ﬁsh were ﬁxed on 10% neutralbuffered formalin for 24 h, dehydrated in ascending alcohol and cleared in xylene before embedding in parafﬁn. Four to ﬁve micron sections were prepared according to Bancroft et al. (1996).

Fish and experimental design Sixty (60) O. niloticus apparently healthy with an average body weight 20 ± 0.09 g were obtained from the Abbassa Fish hatchery, Sharkia Province, Egypt. Fish were kept in two cement ponds (3 · 1 · 1 m) provided with an aerator for 2 weeks for acclimation before the start of the experiment. Fish were allocated into three groups (20 ﬁsh/group) in duplicate. Group 1 (G1) was fed a basal diet (contained 30% crude protein and 3000 k Cal/kg metabolizable energy) as the control group; while group 2 (G2) and 3 (G3) were fed basal diet supplemented with Oxytetracycline at 100 mg kg 1 diet (Khouraiba et al., 2008) and Florfenicol at 5 mg kg 1 body weight (Lewbart et al., 2005), respectively. The ﬁsh of all groups were fed 5% from biomass regularly three times daily for 12 weeks. Water parameters were measured periodically with an average dissolved oxygen (D.O) 5.2 mg/L, water

Detection of oxytetracycline and ﬂorfenicol residues using HPLC Sample preparation, extraction and determination of Oxytetracycline residue were applied using a high-pressure liquid chromatography machine (HPLC) according to Senyuva et al. (2000). While, Florfenicol residue was carried out according to Hao and Hai (2005). Statistical analysis The data were analyzed by one-way analysis of variance (ANOVA) to determine the signiﬁcant variations among the various parameters in the experimental groups. All of the statistical analyses were performed using SPSS version 14 (SPSS, Chicago, IL, USA). A P-value of <0.05 was considered statistically signiﬁcant.

Effect of oxytetracycline and ﬂorfenicol as growth promoters Results Growth parameters The O. niloticus in group 3 (G3) showed signiﬁcant increase in ﬁnal body weight (g), weight gain (g) and signiﬁcant decrease in food conversion ratio (FCR) followed by O. niloticus in group 2 (G2) with respect to the values obtained in the control groups (Fig. 1-A and B). Immunological parameters The results of immunoglobulin M (IgM) level, phagocytes and lysozyme activity of O. niloticus fed on the OTC and FLO supplemented diets for 12 weeks are presented in Fig. 2. G3 did not show signiﬁcant differences in IgM total levels compared to the control group while there were signiﬁcant decreases in G2 (Fig. 2A). G3 had an insigniﬁcant decrease in the phagocytic activity than the control group (Fig. 2A). Groups 2 and 3 showed signiﬁcant increase in lysozyme activity compared to the control group (Fig. 2B). Liver and kidney function In comparison to the control group, G3 had insigniﬁcant differences in ALT activity but G2 had a signiﬁcant decrease (Fig. 3). The highest values of AST activity were obtained in G2 however, the lowest was obtained in G3 compared to the control group (Fig. 3). G2 showed a signiﬁcant increase in urea with respect to the values of control ﬁsh (Fig. 4A). Creatinine values were signiﬁcantly decreased in all treated groups compared to the control (Fig. 4B). Histopathological ﬁndings Liver tissue specimens of the control group show normal hepatocytes and sinusoidal architectures (Fig. 5A). Liver tissue samples of G2 had congestion (arrowhead), severe fatty change

243 and vacuolations in the hepatocytes (arrow) (Fig. 5B). Liver tissue samples of G3 showed a diffused hydropic degeneration in the hepatocytes (arrow), and activation of melanomacrophage center (arrowhead) (Fig. 5C). Kidney tissue samples of the control group showed normal nephron structure (Fig. 6A). Histopathological alterations in kidney tissue samples of G2 manifested periglomerular lymphocytes aggregation (arrow) (Fig. 6B). Kidney tissue specimens of G3 showed shrunken glomerular tufts and dilation of Bowman’s capsule (arrows) (Fig. 6C). Antibiotic residues Fifteen days after cessation of Oxytetracycline and Florfenicol feeding, ﬁsh samples showed 0.05 and0.04 lg g 1 muscle, respectively. Discussion Intensiﬁcation of aquaculture has led to the promotion of conditions that favor the use of a wide range of chemicals, including antibiotics (Rodgers and Furones, 2009). In the present study, the O. niloticus in G3 and G2 showed signiﬁcant increase in ﬁnal body weight (g), weight gain (g) and signiﬁcant decrease in food conversion ratio (FCR) when compared to the control group. A similar trend was found by Sanchez-Martinez et al. (2008) who studied the effect of supplemented Channel Catﬁsh (I. punctatus) feeds with oxytetracycline, they observed that oxytetracycline-treated catﬁsh exhibited a signiﬁcant increase in weight gain to values higher than those of controls, suggesting a growth promotion action of this antibiotic agent. The attribution of improvement of growth and feed efﬁciency as a result of dietary antibiotic supplementation has been subjected to a wide speculation by many investigators. Jukes et al. (1956) mentioned that, the reduction in the gut wall and villus lamina propria has been used to explain the enhanced nutrient digestibility observed with antimicrobial growth promoters. Waibel et al. (1991), Caston and Leeson (1992) reported that

Figure 1 Effect of oxytetracycline and ﬂorfenicol on O. niloticus growth performance (A) Bars indicate initial body weight, ﬁnal body, weight gain (g) and (B) Bars indicate food conversion ratio (FCR). Data presented as mean ± SD. Different letters stand for statistically signiﬁcant differences at P < 0.05.

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Figure 2 Effect of oxytetracycline and ﬂorofenicol on O. niloticus immunological status (A) Serum IgM and phagocytic activity, (B) serum lysozyme activity. Data presented as mean ± SD. Different letters stand for statistically signiﬁcant differences at P < 0.05.

Figure 3 Effect of oxytetracycline and ﬂorofenicol on ALT and AST activity of O. niloticus. Data presented as mean ± SD. Different letters stand for statistically signiﬁcant differences at P < 0.05.

antibiotics limit microbial population numbers and their production of toxins and by-products which reduce the competition with the host for vital nutrients, and they enhance the absorption and utilization of nutrients due to a thinning of the intestinal wall. Gaskins et al. (2002) demonstrated that the effect of oxytetracycline in growth promotion depends on the reduction of the gastrointestinal tract bacteria. Dibner and Richards (2005) demonstrated that the reduction in microﬂora, and its consequences, may be the underlying mechanism for the beneﬁcial effect of antibiotics. Herein the immunological study showed insigniﬁcant differences between G3 and a control group in the immunoglobulin M (IgM) total level while there was a signiﬁcant decrease in G2. The phagocytic activity of G3 had an insigniﬁcant decrease than the control group. Lysozyme activities of all treated groups signiﬁcantly increased compared to the control group. Other works emphasized the same results and found that OTC suppresses the antibody production, the level of circulating leukocytes as well as phagocytic activity in rainbow trout, sea bream and Koi carp (Lunden et al., 1998; Serezli

Figure 4 Effect of oxytetracycline and ﬂorofenicol on (A) urea and (B) creatinine value of O. niloticus. Data presented as mean ± SD Different letters stand for statistically signiﬁcant differences at P < 0.05.

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Figure 5 Histopathological sections of liver of O. niloticus (A) fed on basal diet (B) fed on OTC at 100 mg kg 1 diet showed congestion (arrow head), severe fatty change and vacuolations in the hepatocytes (arrow) and (C) fed on FLO at 5 mg kg 1 body weight showed diffused hydropic degeneration in the hepatocytes (arrow) and activation of melanomacrophage center (arrow head) (HE, Bar = 100 lm).

et al., 2005; Kasagala and Pathiratne, 2008). Salmonid speciﬁc and nonspeciﬁc immune responses were depressed by administration of tetracycline at 10 mg kg 1 diet (Siwicki et al., 1989). Sea bream were fed diets supplemented with OTC at 4 and 8 mg g 1 feed for 21 days inducing a decrease in the humoral innate immune parameters and an increase in the cellular parameters (Guardiola et al., 2012). In contrary of our ﬁndings, the oral administration of OTC in turbot (Scophthalmus maximus L.) had no effect on the respiratory burst and phagocytosis activities (Taffalla et al., 1999). Sea bream’s total erythrocyte, leukocyte and nitroblue tetrazolium (+) cells were increased by administration of OTC at 75 mg kg 1 body weight for 10 days and lasted around 21 days after ceasing the administration, then returned to normal levels (Serezli et al., 2005). On the other hand, FLO did not signiﬁcantly inﬂuence the immune parameters of rainbow trout at 20 mg kg 1 BW day 1 despite a slight effect on the phagocyte function being indicated (Lunden et al., 1999). Similarly,

hybrid tilapia fed on FLO supplemented diet at 0.02 g kg 1 BW for 16 weeks were unaffected by serum lysozyme activity, head kidney macrophage phagocytic index (He et al., 2011). The conﬂicting results may be due to different conditions used in various studies beside ﬁsh species, temperature, antibiotic dose and route of administration which affect drug absorption (Bjo¨rklund and Bylund, 1990). Lunden et al. (2002) found that OTC and FLO are retained in pronephros which is an important lymphoid organ of ﬁsh; accordingly, the OTC and FLO could interact with the immune cells and have an inﬂuence on immune responses. OTC suppression of immune response may be attributed to the penetration of OTC to immune cells and impair their function (Hand et al., 1990). Reduction in the phagocytic index may be attributed to OTC induced suppression of total neutrophils in the blood stream which is responsible for enhancement of the phagocytic activity of monocytes (Evelyn, 2002). Unlike OTC, FLO therapy did not suppress circulating leukocyte levels or antibody

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Figure 6 Histopathological sections of kidney of O. niloticus (A) fed on basal diet (B) fed on OTC at 100 mg kg 1 diet showed periglomerular lymphocytic aggregation (arrow) and (C) fed on FLO at 5 mg kg 1 body weight showed shrunken glomerular tufts and dilation of Bowman’s capsule (arrows). (HE, Bar = 100 lm).

production in rainbow trout (Lunden et al., 1998). Reports concerning FLO are even more controversial, but there is a consensus that its effects appear to be less pronounced than those demonstrated for OTC (Romero et al., 2012). The biochemical results corroborated the demonstration to the histopathological ﬁnding of both liver and kidney. The signiﬁcant changes in the activities of aspartate transaminase (AST) and alanine aminotransferase (ALT) enzymes in blood indicated tissue impairment caused by stress, toxicity and liver damage produced by drugs and antibiotics (Kori-Siakpere et al., 2010). Alternatively, this disrupted the cell membranes, allowing the enzymes to leak out of the cells. Oxytetraycline greatly impair the liver regeneration and decrease the mitochondrial protein synthesis after causing deﬁciency in cytochrome oxidase C and ATP synthetase enzymes (Den Bogert et al., 1983). Serum creatinine and uric acid can be used as a rough index of the glomerular ﬁltration rate (GFR) (Hernandez and Coulson, 1967). The results of urea and creatinine

may be due to pathological alterations of the treated ﬁsh kidney. The previous studies on the effect of OTC and FLO on the histopathological ﬁndings in ﬁsh species are conﬂicting. In Tinca tinca on the 7th day of OTC intramuscular injection it showed severe necrosis of hematopoietic renal tissue with fatty degeneration in the liver (Soler et al., 1996). Also, the OTC medicated feed which was administered at 15 g kg 1 live weight diffuses cytoplasmic vacuolization of the renal duct epithelium in kidney of C. carpio (Svobodova et al., 2006). FLO administration in the feed of tilapia spp. at 15, 45, or 75 mg/kg body weight for 20 days led to increased glycogen-type and lipid-type with hepatocellular vacuolation in the liver, decreased lymphocytes, increased blast cells, and increased individual cell necrosis in the anterior kidney, and tubular epithelial degeneration and mineralization in the posterior kidney (Gaikowski et al., 2012). Similarly, FLO administration in channel catﬁsh at doses of 10, 30, or 50 mg/kg BW/d for 20 days, showed a

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dose-dependent decrease in hematopoietic/lymphatic tissue in the anterior kidney, posterior kidney, and spleen (Gaikowski et al., 2003). Conversely, FLO administration in feed to Atlantic salmon parr at 100 mg/kg BW/d for 10 days or at 50 mg/ kg BW/d for 10 days on/off cycles did not induce treatment-related histopathologic lesions in the liver, or kidney (Inglis et al., 1991). Also, FLO administration in feed at up to 75 mg/kg BW/d for 20 days to Sunshine bass did not show any hepatic or nephric tissue alteration (Straus et al., 2012). Oxytetracycline and Florfenicol residues in muscles of ﬁsh samples showed 0.05 and 0.04 lg g 1 muscle, respectively after ﬁfteen days of feeding cessation. The obtained results were lower than the maximum residue limits (MRLs) of Oxytetracycline and Florfenicol (0.1 and 1 lg g 1, respectively) according to Commission regulation, EU (2010).

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Conclusion From the previous study, it can be concluded that addition of FLO at 5 mg kg 1 body weight is more safe with its effect on growth performance, immunity, liver and kidney functions of O. niloticus and for human food safety. Besides great care on the excessive use of antibiotics in the aquatic environment to avoid the terrible effects of them it resulted in the emergence of antibiotic-resistant bacteria in aquaculture as well as the transfer of these resistance determinants of human pathogens and antibiotic residues in ﬁsh tissue and ﬁsh products.

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Aquatic Mammals 31(4).indd - Aquatic Mammals Journal