Biochemical, histopathological and ultra structural profile after pulsed water medication of enrofloxacin in broiler chickens

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1 Veterinary World, EISSN: Available at RESEARCH ARTICLE Open Access Biochemical, histopaological and ultra structural profile after pulsed water medication of enrofloxacin in broiler chickens How to cite is article: Sureshkumar V, Sarachandra G and Ramesh J (2013) Biochemical, histopaological and ultra structural profile after pulsed water medication of enrofloxacin in broiler chickens, Vet World 6(9): , doi: /vetworld Introduction Veerapandian Sureshkumar, Ghadevaru Sarachandra and Jayaramachandran Ramesh Pharmacovigilance Laboratory for Animal Feed and Food Safety, Directorate of Centre for Animal Heal Studies, Tamil Nadu Veterinary and Animal Sciences University, Madhavaram Milk Colony, Chennai , Tamil Nadu, India Corresponding auor: Ghadevaru Sarachandra, gsarachandra@rediffmail.com Received: , Revised: , Accepted: , Published online: Abstract Aim: A pharmacological study was undertaken to evaluate e safety and adverse effects of enrofloxacin administration in broiler chickens by assessing e serum biochemical parameters, associated histopaological and ultra structural changes in liver and kidney. Materials and Meods: Bis in e treatment group were administered wi enrofloxacin at e recommended erapeutic dose 10mg/kg body weight via drinking water for five successive days, while e control group (untreated group) received non medicated water. Serum biochemical parameters viz., total protein, albumin, lactate dehydrogenase, alkaline phosphatase, creatine kinase, lipase, triglyceride, gamma glutamyl transferase, urea, uric acid and creatinine were estimated at 24hour and 48hour intervals during e dosing and widrawal periods, respectively. Liver and kidney tissue samples collected from 1, 3, 5, 7, and 9 days post treatment groups were subjected to histopaological and ultrastructural examinations. Results: There was no significant change (p>0.05) in total protein, albumin, lactate dehydrogenase, alkaline phosphatase, creatine kinase, lipase, triglyceride and urea levels in e enrofloxacin administered broiler chickens at all e time points evaluated. However, a significant increase (p<0.05) in gamma glutamyl transferase, uric acid and creatinine levels were observed after e 4 dose of e enrofloxacin and on day 1 post treatment. During e widrawal period, e elevated levels declined gradually and showed e trend towas control values as evidenced by a statistically insignificant difference on 3, 5, 7 and 9 days post treatment when compared to at of control group. These biochemical changes were substantiated by histopaological and ultrastructural changes elicited in liver and kidney. Conclusion: The reversible trend observed in serum biochemical parameters, histopaological and ultra structural alterations in liver and kidney during e widrawal period suggests at enrofloxacin is safe if administered to broiler chickens at e recommended erapeutic dose and if e stipulated widrawal period is strictly adhered to. Keywos: biochemical parameters, broiler chickens, enrofloxacin, histopaology, ultrastructural, veterinary pharmacovigilance This article is an open access article licensed under e terms of e Creative Commons Attribution License ( org/licenses/by/2.0) which permits unrestricted use, distribution and reproduction in any medium, provided e work is properly cited. In e modern intensive poultry production, antimicrobial agents are being increasingly used to enhance feed efficiency, promote heal, improve productivity, and are also used for disease prophylaxis and treatment [1, 2]. In is context, it is important to note at e fluoroquinolones are synetic anti- microbial agents used extensively in e poultry industry for e control of premature mortality and for e prophylaxis and treatment of respiratory, renal, and digestive infections [3,4]. Enrofloxacin is a fluoroquinolone at was developed exclusively for veterinary use and is currently used in large-scale in poultry, especially for e treatment of chronic respiratory disease, colibacillosis, salmonellosis and fowl cholera [5,6]. In vivo, enrofloxacin is metabolised into pharma- cologically active metabolite ciprofloxacin by liver microsomal enzymes at belong to e cytochrome P450 family [4,7]. The fluoroquinolones are considered relatively safe and well tolerated as indicated by earlier reports which demonstrated at enrofloxacin is well tolerated at e recommended dosage in cats [8, 9, 10]. However, it has been reported at fluoroquinolones are also associated wi a low incidence of adverse effects related to gastrointestinal, skin, hepatic, central nervous system functions, and phototoxicity [11]. Furer, ere is evidence to show at enrofloxacin administration in broiler chickens resulted in a significant decline in e lymphocyte count [12] and reduction in haemagglutination inhibition (HI) titre and associated histopao- logical changes in e lymphoid organs [13]. Data on e safety regaing repeated oral administration of fluoroquinolones in poultry are very scarce [14]. Information on e residual effects of enrofloxacin and post market surveillance on serum biochemical parameters, histopaological and ultra structural changes in broiler chickens during e post treatment periods is lacking. Hence, we conducted a pharmacological study in broiler chickens to assess e safety and adverse effects of enrofloxacin administration on serum biochemical Veterinary World, EISSN:

2 Table-1. Effect of enrofloxacin administration (10mg/kg body weight, in drinking water for 5 successive days) on total protein, albumin, lactate dehydrogenase, alkaline phosphatase, creatine kinase, lipase and triglyceride levels (data is shown as Mean±SE, n=6) parameters, and to oroughly evaluate e associated histopaological and ultrastructural alterations in liver and kidney. Materials and Meods TP (g/dl) Albumin (g/dl) LD (U/L) AP (U/L) CK (U/L) Lipase (U/L) TG (mg/dl) Control 2.88± ± ± ± ± ± ±5.90 After 1st dose 3.01± ± ± ± ± ± ±5.53 2nd dose 3.14± ± ± ± ± ± ± dose 3.03± ± ± ± ± ± ± dose 2.94± ± ± ± ± ± ±6.36 Post treatment Day ± ± ± ± ± ± ±8.38 Day ± ± ± ± ± ± ±4.75 Day ± ± ± ± ± ± ±3.13 Day ± ± ± ± ± ± ±8.39 Day ± ± ± ± ± ± ±1.42 TP : Total protein, LD: Lacto dehydrogenase, AP: Alkaline phosphatase, CK: Creatinine kinase, TG: Triglyceride Table-2. Effect of enrofloxacin administration (10mg/kg body weight, in drinking water for 5 successive days) on gamma glutamyl transferase, urea, uric acid and creatinine levels (data is shown as Mean±SE, n=6) Gamma glutamyl transferase(u/l) Urea (mg/dl) Uric acid (mg/dl) Creatinine (mg/dl) Eical approval: Institutional Animal Eics Committee (IAEC), Madras Veterinary College, TANUVAS has accoed permission for conducting is biological trial. Medication wi enrofloxacin containing water: 36 day old broiler chicks (Broiler strain B 1) acquired from Institute of Poultry Production and Management (IPPM), TANUVAS, Chennai-51 were randomly divided into control and treatment groups and were c Control ± ± ± ±0.01 c c c After 1st dose ± ± ± ±0.01 abc bc bc 2nd dose ± ± ± ±0.02 abc abc c 3 dose ± ± ± ±0.02 a ab ab 4 dose ± ± ± ±0.03 Post treatment ab a a Day ± ± ± ±0.03 abc abc bc Day ± ± ± ±0.01 abc c bc Day ± ± ± ±0.02 c bc bc Day ± ± ± ±0.03 c bc bc Day ± ± ± ±0.03 Means bearing different superscripts (a,b,c) wiin e column differ significantly (p<0.05) collected from e wing vein at 24 hour interval during e dosing period and at 48 hour interval during e widrawal period up to 9 days post treatment. Serum samples us obtained were subjected to total protein, albumin, lactate dehydrogenase (LDH), alkaline phosphatase (ALP), creatine kinase (CK), lipase, triglyceride, gamma glutamyl transferase (GGT), urea, uric acid and creatinine estimation by auto analyzer (A-15, Bio-Systems) using standa kits. Statistical analysis was carried out by following One way analysis of variance as described by Snedecor and Cochran [17]. Histopaology: Treatment groups were sacrificed eically on 1, 3, 5, 7 and 9 days post treatment and control bis were sacrificed on day 9 post treatment. maintained under standa management conditions. Liver and kidney tissue samples were collected in 10% Control group (I) received non medicated water while formalin and were subjected to histopaological e treatment groups (II, III, IV, V and VI) were examination [18]. administered wi enrofloxacin at recommended Ultrastructural study: erapeutic dose of 10mg/kg body weight, via drinking Liver and kidney tissue samples water for five successive days from 43 to 47 day of from control and treatment groups (day 1 and day 9 age [13, 15]. The pulsed water medication was given as post treatments) were collected immediately after per e meods described by Charleston et al. [16]. sacrificing e bis and processed. Tissue sections Bis received eir freshly-prepared daily medication were examined under a transmission electron during a 4 hour period in e morning, and eir water microscope (Philips 2010, Neerlands) operated at was antibiotic free for e remaining 20 hours in each 60KVA and photographed [18]. day. The concentration of enrofloxacin in e water, to Results administer e required dose per kg of body weight, Serum biochemical parameters: It is discernible from was calculated by determining e water consumption our results at ere was no significant change and body weight of each bi on e day of medication. (p>0.05) in total protein, albumin, LDH, ALP, CK, Serum biochemical parameters: Blood samples were lipase, triglyceride and urea levels in enrofloxacin Veterinary World, EISSN: c c

3 Figure-1. Liver of control group showing normal architecture. H & E, x10. Figure-2. Liver of 1 day post treatment group showing degeneration of hepatocytes and areas of congestion. H&E, x10. Figure-3. Liver of 3 day post treatment group showing no sinusoidal spaces indicating swollen hepatocytes and few areas of vacuolar degeneration. H&E, x20. Figure-4. Liver of 5 day post treatment chicken showing focal areas of infiltrated inflammatory cells. H&E, x20. Figure-5. Liver of 9 day post treatment group showing mild vacuolation of hepatocytes. H&E, x10. Figure-6. Liver of control group, normal hepatocytes wi nucleus and mitochondria. Uranyl acetate & Reynold's solution, x Figure-7. Liver of day 1 post treatment group showing hepatocytes wi atrophied nucleus, vacuolated cytoplasm and swollen mitochondria. Uranyl acetate & Reynold's solution, x Figure-8. Liver of 9 day post treatment group wi hepatocytes showing mitochondrial swelling. Uranyl acetate & Reynold's solution, x administered broiler chickens at all e time points on day 1 post treatment, hepatocytes showed atrophied evaluated (Table-1). However, a significant increase nucleus, cytoplasm was vacuolated wi scattered (p<0.05) in GGT, uric acid and creatinine levels were cytoplasmic organelles and swollen mitochondria observed after e 4 dose of e enrofloxacin and on (Fig-7). While, in 9 day post treatment group day 1 post treatment. The elevated levels declined hepatocytes showed normal nucleus but few cells had gradually during e widrawal period as evidenced shrunken nucleus. Mitochondrial swelling was e by a statistically insignificant change starting from day only appreciable lesion observed in few cases (Fig-8). 3 post treatment (Table-2). Histopaology of e kidney: Control group showed Histopaology and ultra structural study normal features of kidney (Fig-9). Kidney from day 1 Histopaology of e liver: Liver of control group post treatment group revealed degeneration and revealed no histological aberrations (Fig-1). On e necrosis of tubular epielial cells wi granular oer hand, on day 1 post treatment, hepatocytes were eosinophilic cytoplasm (Fig-10). On day 3 post swollen and showed vacuolated cytoplasm. treatment, degeneration of tubular epielial cells wi Degeneration of hepatocytes and areas of congestion haemorrhagic areas were noticed. However, in few were also observed (Fig-2). Whereas, on day 3 post cases, cytoplasm of swollen tubular epielial cells was treatment no sinusoidal space could be observed filled wi eosinophilic material (Fig-11). Degeneration indicating at hepatocytes were highly swollen and of tubular epielium persisted in day 5 post treatment also exhibited few areas of vacuolar degeneration wi group; however it was only mild and was also observed vacuolated cytoplasm (Fig-3). However, on day 5 post in only few cases (Fig-12). Indeed, mild swollen treatment sinusoidal spaces reemerged and samples tubular epielial cells were e only appreciable from only a few bis showed focal areas of infiltrated lesions observed on day 7 and 9 post treatment (Figinflammatory cells (Fig-4). Mild vacuolation of 13). hepatocytes was e only appreciable lesion noticed on Ultrastructural changes in e kidney: day 9 post treatment (Fig-5). Control group revealed normal glomeruli and tubular epielial cells Ultrastructural changes in e liver: Control group wi unaltered brush boer (Fig-14). In 1 day post showed normal hepatocyte morphology pertaining to treatment group, normal architecture of glomeruli and organelles such as nucleus, mitochondria and tubular epielial cells vanished. Prominent features of endoplasmic reticulum (Fig-6). In e treatment group, e tubular epielium include shrunken nucleus, Veterinary World, EISSN:

4 Figure-9. Kidney of control group showing normal glomeruli and tubular epielial cells. H&E, x10. Figure-10. Kidney of 1 day post t r e a t m e n t g r o u p s h o w i n g degeneration and necrosis of tubular epielial cells wi granular eosinophilic cytoplasm. H&E, x20. Figure-11. Kidney of 3 day post t r e a t m e n t g r o u p s h o w i n g degeneration of tubular epielial cells wi haemorrhagic areas. H&E, x20. Figure-12. Kidney of 5 day post treatment chicken showing mild tubular degeneration. H&E, x20. Figure-13. Kidney of 9 day post treatment group showing mild tubular degenerative changes. H&E, x10. Figure-14. Kidney of control group showing normal tubular epielial cells wi unaltered brush boer. Uranyl acetate & Reynold's solution, x Figure-15. Kidney of day 1 post treatment group showing tubular epielium wi shrunken nucleus, balonised mitochondria, vacuolated cytoplasm and attrition of brush boer. Uranyl acetate & Reynold's solution, x Figure-.16. Kidney of 9 day post treatment group showing tubular epielial cells wi normal nucleus, brush boer, but wi mild mitochondrial swelling. Uranyl acetate & Reynold's solution, x swollen mitochondria, vacuolated cytoplasm and recommended dose of 5 mg/kg IM, once daily to attrition of brush boer (Fig-15). However, mild healy dogs for 14 days, caused transient increases in vacuolation in cytoplasm and swollen mitochondria AST and mean corpuscular volume levels. Similar were e only considerable lesions found in e 9 day effects are reported in humans receiving ciprofloxacin post treatment group (Fig-16). and norfloxacin [22, 23]. Discussion Serum GGT is a highly specific and indicative enzyme for liver function and more sensitive an In e present study, e serum biochemical transaminases (AST and ALT) and ALP. A significant parameters revealed insignificant changes in total elevation in serum GGT level was observed after e protein, albumin, LDH, ALP, CK, lipase, triglyceride 4 dose of enrofloxacin administration and on day 1 and urea levels in enrofloxacin administered broiler post treatment GGT levels showed a tendency to chickens at all e time points evaluated. This finding is decline gradually during e widrawal period. These in accoance wi Elkholy et al. [19], who showed at findings were supported by Ellakany et al. [24] who administration of Enrotryl and Baytril at a dose also reported a similar trend at enrofloxacin at equivalent to 10 mg enrofloxacin base/kg body weight 100mg/kg body weight in broiler chicken for 5 daily for five successive days, to laying hens induced consecutive days caused elevation of serum ALT, but 6 insignificant changes in serum glucose, cholesterol, days after e widrawal e values returned to e creatinine levels, serum alanine amino transferase control levels. Similarly, e auors furer reported (ALT), aspartate amino transferase (AST) and ALP at serum AST level was found to be higher an e activities. control group, albeit not significant, in ose receiving However, Moustafa et al. [20] reported at 100mg/kg body weight or 10mg/kg body weight administration of enrofloxacin in high doses (100, 200 enrofloxacin, en decreased after widrawal of e and 400 ppm rough drinking water) for long periods drug. (6 weeks) was accompanied wi adverse effects on In e present study, e elevated levels of GGT different organs along wi elevated levels of AST, can be correlated wi e histopaological changes in ALT, ALP, urea, uric acid and creatinine, hypopro- e liver observed during e widrawal period. For teinaemia and hypoalbuminaemia. In addition, Tras et example, on day 1 post treatment hepatocytes were al. [21] showed at enrofloxacin injection at e swollen, degenerated wi vacuolated cytoplasm and Veterinary World, EISSN:

5 showed areas of congestion. Vacuolar degeneration Conclusion was persistent till 3 day post treatment, however, Enrofloxacin administration at recommended during e subsequent widrawal period e lesions erapeutic dose caused insignificant changes in total were mild as indicated by clear sinusoidal spaces and protein, albumin, LDH, ALP, CK, lipase, triglycerides focal areas of infiltrated inflammatory cells wi only a and urea levels. A significant but transient elevation in mild vacuolation of hepatocytes. These observations GGT, uric acid and creatinine was noticed and ese are in conformity wi Ellakany et al. [24]. Furer, biochemical alterations were suggestive of histopao- Shawky et al. [25] also showed at enrofloxacin logical and ultrastructural changes observed in liver administered at 400 ppm for 6 weeks produced and kidney. The restoration towas e control values histopaological changes in liver. Vaccaro et al. [26] of biochemical parameters accompanied by reversal of opined at e major cause of enrofloxacin-induced histopaological and ultra structural lesions towas hepato toxicity can be attributed to e inhibition of normal contour during e widrawal period clearly hepatic cytochrome P450 enzymes which are suggests at enrofloxacin is extremely safe if responsible for drug metabolism. administered at recommended erapeutic dose and if Hepatocellular injury represented by significant e stipulated widrawal period is strictly followed. elevations in e serum GGT activities was furer Adhering to ese precautions can prevents any ascertained by e electron microscopic examination of adverse drug reactions and untowa residual effects. liver samples of broiler chickens administered wi enrofloxacin. Shrunken nucleus, swollen mitochondria Auors contributions and vacuolated cytoplasm wi scattered cytoplasmic This study is a significant component of e work organelles are clearly indicative of hepatocyte towas e Ph.D. esis of e first auor VS, degeneration and ese changes were observed on day submitted to Tamil Nadu Veterinary and Animal Sciences 1 post treatment group. However, in day 9 post University, Chennai-51. GS was e Chairman and treatment group mitochondrial swelling is e only designed e trial. VS conducted e experimental trial. appreciable lesion found in few cases. Similar JR performed e sampling. GS and VS analysed and observations using electron microscopy were made by interpreted e data, organised and oroughly revised El Daly [27] in e livers of rats at were administered e manuscript. All auors read and approved e final wi 10% enrofloxacin wi a daily dose of 75mg/kg version of e manuscript. body weight for 10 days intraperitoneally. The auors Acknowledgements demonstrated at enrofloxacin induces e production of free radicals at cause oxidative stress to hepatocytes The Auors are highly grateful to Drugs and and its organelles, particularly to mitochondria and cell Pharmaceutical Research Programme, Department of membranes. Science and Technology (DST), Government of India, A significant but transient elevation in serum uric New Delhi, for e financial support as part of e DST acid and creatinine was noticed after e 4 dose and on scheme entitled A National Facility for Pharmaco- day 1 post treatment of enrofloxacin and evinced vigilance on Drug Residue and oer Toxic Xenobiotics similar trend as at of GST during e widrawal including Genetically Manipulated Organisms in period. These biochemical changes were in harmony Veterinary Products at Pharmacovigilance Laboratory wi histopaological alterations in kidneys which for Animal Feed and Food Safety, DCAHS, TANUVAS, showed degeneration and necrosis of tubular epielial Chennai The auors express profound anks cells wi granular eosinophilic cytoplasm in day 1 to Dr. S. Vairamuu, Ph.D., Associate Professor and post treatment group. However, a reversible tendency Head, Centralised Clinical Laboratory, Madras Veterinary College, Chennai for providing Auto analyser facility was noticed as evidenced by mild swollen tubular for e biochemical estimation of various parameters. epielial cells which were e only appreciable lesions The auors also ank Mrs. Rita Rajan, Technician, observed on day 7 and 9 post treatment. Ellakany et al. Wellcome Research Laboratory, Department of [24] also found congestion, tubular degeneration, and Gastrointestinal Sciences, Christian Medical College, areas of hemorrhage in kidney of broiler chicken Vellore, for help wi Transmission Electron administered wi enrofloxacin at 100mg/kg body Microscopy. weight rough drinking water for 5 consecutive days. Kidney injury as evidenced by significant Competing interests elevation in e serum uric acid and creatinine levels was furer bolstered by results from e electron The auors declare at ey have no competing interests. microscopic examination of kidneys isolated from References enrofloxacin administered broiler chickens. Indeed, 1. Johnston, A.M. (1998) Use of antimicrobial drugs in mild vacuolation and mitochondrial swelling were e veterinarypractice. Br. Med. J. 317: Prescott, J.F., Baggot, J.D. and Walker, R.D. 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