Enrofloxacin, a fluoroquinolone developed exclusively for veterinary use, is advocated in poultry in large-scale for treatment of chronic respiratory disease, colibacillosis, salmonellosis and fowl cholera (Papich and Riviere, 2009). It has been reported that fluoroquinolones are associated with a low incidence of adverse effects related to gastrointestinal, skin, hepatic, central nervous system functions, and phototoxicity (Hooper and Wolfson, 1985), significant fall in lymphocyte count (Sureshkumar et al., 2012), reduction in haemagglutination inhibition (HI) titre and associated histopathological changes in lymphoid organs (Sureshkumar et al., 2013a) and residual effects on biochemical status in time dependent manner after nine weeks of enrofloxacin administration (Sinha et al., 2018). Despite several studies have explored the mechanisms of quinolone arthropathy in other species (Kato and Onodera, 1988a; Stahlmann et al., 1990; Cavusoglu et al., 2000), literature appraisal revealed that reports on quinolone arthropathy and the reversibility of lesions in broiler chicken are limited and very little is known about the toxicodynamics of these toxic effects (Gurbay et al., 2001).

On the other hand, there is some evidence that free radical formation might play a role in the pathogenesis of fluoroquinolone induced cartilage defects (Thuong-Guyot et al., 1994). Further, Martinez-Cayuela (1998) found that enrofloxacin residues may occur in meat, milk and eggs, generate free radicals owing to its metabolism and interaction with other medicated drugs (Ershov et al., 2001; Carrreras et al., 2004; Sureshkumar et al., 2004). Our earlier findings revealed that there was significant decrease in glutathione S-transferase, catalase and glutathione (GSH) levels in serum, muscle and liver on 1st and 5th day post treatment with enrofloxacin (Sureshkumar et al., 2013b) and speculated that free radical formation might play a role in quinolone arthropathy. Hence, present study has been undertaken to explore the potential development of quinolone arthropathy after enrofloxacin administration and its reversibility if any during the withdrawal period in broiler chicken.

Materials and Methods

Day old broiler chicks (broiler strain B₁) of thirty numbers obtained from Institute of Poultry Production and Management, Madhavaram Milk Colony, Chennai-600 051 were randomly grouped into control (I) and treatment (II, III, IV and V) and maintained under standard management conditions. Treatment groups were medicated with enrofloxacin at recommended therapeutic dose 10mg/kg body weight, in drinking water for 5 successive days from 43rd to 47th day of age, while control birds received non medicated water (Knoll et al., 1999). Institutional Animal Ethics Committee, Madras Veterinary College, TANUVAS has accorded permission for the biological trial. After cessation of the last dose of enrofloxacin, six birds from corresponding treatment group were sacrificed ethically at each sampling point viz. 1, 3, 5, and 9 days post treatment. Control birds were sacrificed at the end of the experiment i.e. on day 9 post treatment. Juvenile cartilages (articular cartilage from head of the femur) were collected in 10 percent formalin and subjected to histopathological examination (Bancroft and Gamble, 2008).

Results and Discussion

In the present study, articular cartilage collected from head of the femur of control group revealed normal chondrocytes with prominent nucleus and cytoplasm (Plate 1). Degeneration of chondrocytes was noticed as early as in day 1 post treatment group (Plate 2).

The lesions were persisted till 5^th day post treatment and chondrocytes were shrunken, degenerated with pyknotic nuclei (Plate 3) and articular cartilage revealed cavity formation with eosinophilic content (Plate 4). These observations are in line with Kato and Onodera (1988b) who studied the histogenesis of cavity formation in rats. They have shown that condensation of chondrocyte nuclei was the first finding 5 hour after a single dose (1000-3000mg/kg) of ofloxacin. In another study, Kato and Onodera (1988a) showed that the [³H] thymidine binding capacity, an indicator of DNA synthesis was decreased by 80% 5 hour after a single dose of ofloxacin, when compared with control and concluded that ofloxacin is suppressing the DNA synthesis which results in the degeneration of chondrocytes.

Toxicodynamics of quinolone arthropathy remains poorly understood. Quinolones act by inhibiting microbial DNA gyrase (type II topoisomerase) responsible for coiling of circular DNA and thereby interfere with bacterial replication (Shen et al., 1989). Mammalian cells have type II topoisomerases and mitochondria contain circular DNA (Castora et al., 1983), and thus it is postulated that quinolones might be directly toxic to chondrocyte mitochondrial DNA (Stahlmann et al., 1990; Stahlmann, 1991). Interference with DNA synthesis is a plausible explanation for degenerative cellular changes evident in quinolone arthropathy (Kato and Onodera, 1988a). However,  clumping of chondrocytes were observed on day 5 post treatment (Plate 4) and a few cases in 9 day post treatment group showed degenerated chondrocytes around the blood vessels (Plate 5).

Clumping of chondrocytes observed in the present study is indicative of cell replication and regeneration, which is a reparative process relative to healing of other tissues (Gough et al., 1992). Supporting our findings, Cavusoglu et al. (2000) demonstrated the repair response of the cartilage by intensive staining with safranin-O and toluidine blue around the chondrocyte clusters. Further, Kato and Onodera (1988a) showed that 24 hours after a single dose of ofloxacin, [³H] thymidine binding capacity increased by 160% in comparison to the control group, attributed to increased proliferation of chondrocytes. The restoration of histopathological changes observed during 5th and 9th day of withdrawal period could be attributed to the depletion of the enrofloxacin residues from the body during the withdrawal period. This proposition is substantiated by San Martin et al. (2010) who demonstrated that based on the European Union maximum residue limits the withdrawal time was 5 days and Chattha et al. (2008), who observed that enrofloxacin residues were washed out in 9 days whilst its major metabolite ciprofloxacin was washed out in 8 days in chicken meat.

Conclusion

In the present study, quinolone arthropathy was manifested in juvenile cartilages as evidenced by histopathological alteration in articular cartilages. However, the regeneration and reparative process observed in the 5th and 9th day of withdrawal period is indicative of reversal in chondrocyte lesions and suggests that enrofloxacin is safe if administered at recommended therapeutic dose and stipulated withdrawal period is adhered.

Acknowledgements

The authors are highly grateful to Drugs and Pharmaceutical Research Programme, Department of Science and Technology, Government of India, New Delhi, for the financial support as part of the DST scheme entitled A National Facility for Pharmacovigilance on Drug Residue and other Toxic Xenobiotics including Genetically Manipulated Organisms in Veterinary Products at Pharmacovigilance Laboratory for Animal Feed and Food Safety, DCAHS, TANUVAS, Chennai-600 051.

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