doi:10.5455/vetworld.2013.337-342 Effect of enrofloxacin on zootechnical performance, behaviour and immunohistopathological response in broiler chicken

Aim: A safety pharmacology trial was conducted to evaluate the impact of enrofloxacin on zootechnical performance, 
behaviour and immunohistopathological response in Newcastle disease virus vaccinated broiler chicken after pulsed water medication. 
 
Materials and Methods: Experimental group birds were administered with enrofloxacin at recommended therapeutic dose 
10mg/Kg body weight, through drinking water for five consecutive days from 43rd to 47th day of age. Zootechnical 
performance parameters, behavioural and humoral immune response in terms of haemagglutination inhibition (HI) titre were assessed at different time interval during pre-treatment, treatment, post-treatment period. Bursa of Fabricius and spleen tissues collected at each sampling point viz. 1, 3, 5, 7 and 9 days post treatment were subjected to histopathological examination. 
 
Results: A significant reduction in HI titre was noticed in enrofloxacin administered birds. The decreased HI titre was further substantiated by the histopathological changes observed in bursa of Fabricius and spleen which showed a lymphocytic dispersion and depletion with several areas of lymphoblastic degeneration. Conversely, a down regulatory effect on humoral immunity was observed as evidenced by increased HI titre value noticed from 5th day post treatment onwards and a congruent reversible trend in histopathological changes as indicated by repopulation with lymphocytes on 9th day post treatment. However, there was no significant change in body weight, cumulative feed intake, feed efficiency and behaviour in enrofloxacin administered groups. 
 
Conclusion: The present study suggests that the immuno suppressive activity of enrofloxacin may alter the immune response to vaccines, if it is coadministered during vaccination of broilers. On the other hand, enrofloxacin, though it decreased the humoral immune response, it did not have any appreciable effect on broiler\'s performance.


Introduction
immunized with living cells of Salmonella enterica subsp.enterica serovar enteritidis and lipopolysaccharide.In poultry farms, it is a common practice to add Studies conducted in other species revealed that antibiotics in drinking water at the time of vaccination.
the presence of circulating antimicrobial residues, Most farmers administer antibiotics at the time of especially fluoroquinolones, in nestlings of the three vaccination without scientific knowledge of the effects vulture species breeding in central Spain resulted in on the immune response.Database on the effects of impaired cellular and humoral immune systems such practices on the immune system is scarce, and compared with nestlings from the control areas, which more authenticated studies are needed to investigate did not ingest antibiotics [4].the impact of antibiotics on the immune system at the After administration, enrofloxacin is metabolised time of vaccination [1].
in the liver via de-ethylation into pharmacologically Enrofloxacin, a fluoroquinolone developed active metabolite ciprofloxacin [5][6][7].Porchezhian [8] exclusively for veterinary use is advocated in poultry in reported that ciprofloxacin significantly reduced the large-scale for treatment of chronic respiratory disease, antibody titre against Lasota and Sheep Red Blood colibacillosis, salmonellosis and fowl cholera [2].The Cells (SRBC) antigens in broiler chicks.Consequently effect of enrofloxacin on the immune response is not there is every possibility of alteration of immune well documented [1].However, Tokarzewski [3] response after enrofloxacin administration in broiler revealed that enrofloxacin and chloramphenicol chicken.Further, our earlier study [9] speculated that decreased the level of specific IgY in laying hens the reduction in the lymphocyte count at therapeutic dose of enrofloxacin may have influence on the immune response of the broiler chicken.
Hence the present study has been undertaken to investigate the effect of enrofloxacin on humoral and coliform count.Pulsed water medication was used immune response, the key factor that has been because oral administration of drugs is the most underestimated during vaccination programs in the practical way for treating birds being followed in field.Zootechnical performance, behavioural and Indian field condition.immunohistopathological response of broiler chicken Zootechnical performance parameters: Zootechnical were also evaluated after recommended therapeutic performance parameters such as body weight and dosage of enrofloxacin under experimental conditions.cumulative feed intake were recorded at different time and 9 post treatment (48 to 56 day of age).During the starter mash (Moisture-7.76%,Crude protein-22.55%,treatment period blood samples were collected from 6 Crude fibre-2.15%,Ether extract-3.63%,Total ashbirds randomly selected from treatment groups and 5.56% and Gross energy-4037 kcal/Kg) and finisher during the post treatment period blood samples were mash (Moisture-9.07%,Crude protein-17.07%,Crude collected from respective treatment groups.Serum was fibre-4.17%,Ether extract-2.59%,Total ash-4.40%subjected to haemagglutination inhibition (HI) titre and Gross energy-3884 kcal/Kg) ad libitum from 0-2 against NDV vaccine following standard procedures weeks and 3-8 weeks respectively.Non medicated [14].Data were subjected to one way analysis of broiler ration (free from feed additives, antibiotics and variance for their significance as per Snedecor and anticoccodials) was used throughout the study.Broiler Cochran [15].feed was screened for mycotoxins, pesticides, heavy metals and coliform count.
Histopathology: After cessation of the last dose of enrofloxacin, six birds from corresponding treatment Enrofloxacin dosing protocol: Enrofloxacin 10% was group were sacrificed ethically at each sampling point obtained as gratis from M/s. Neospark Drugs and viz. 1, 3, 5, 7 and 9 days post treatment.Control birds Chemicals Private Limited, Hyderabad, India.Chicks were sacrificed on day 9 post treatment.Bursa of were randomly divided on the first day of age into six Fabricius and spleen tissues were collected in 10 % groups.Group I (6 birds) was kept as untreated control, formalin.Paraffin embedded tissues were sectioned to received non medicated water and Groups II, III, IV, V 5 µm thickness and stained by haematoxylin and eosin and VI each 6 birds were administered with (H&E) for histopathological examination [16].enrofloxacin at recommended therapeutic dose 10mg/Kg body weight, through drinking water for five

Results
rd th consecutive days from 43 to 47 day of age [10].Birds

Effect on Zootechnical performance parameters:
received their freshly prepared daily medication during There was no significant change in body weight (Table-1), a 4 hours period in the morning, and their water was cumulative feed intake (Table -2) and feed efficiency antibiotic free for the remaining 20 hours of each day.
(Table -3) in enrofloxacin administered groups when The concentration of enrofloxacin in the water to give compared to that of control.the required dose per Kilogram of body weight was calculated by determining the water consumption and Effect on behaviour: All the birds were active and body weight of each bird on the day of medication [11].
interested in feed, water and the surroundings Drinking water was free from pesticides, heavy metals throughout the experiment period.Birds did not show pain on palpation of joints, no clinical signs and group showed normal medullary areas populated with mortality were observed during the study period.
lymphocytes (Fig- 1).Histopathological lesions in the bursa of fabricius of 1 day post treatment group Effect on humoral immune response: There was revealed the presence of lymphocyte depletion significant reduction in HI titre of enrofloxacin especially in the central portion of the lymphoid administered birds when compared to that of control follicles in the medullary areas and increased (Table-4).The reduction in HI titre noticed during the rd lymphophagocytosis were also noticed (Fig- 2). 3 day th treatment period i.e. after 4 dose of enrofloxacin was post treatment group showed dispersion and depletion not statistically significant, whereas, it was significant of lymphocytes in medullary areas and few areas of th (p<0.05) after 5 dose and during the post treatment lymphoblastic degeneration (Fig- 3).Whereas, period.However, a tendency in increased HI titre was lymphocyte depletion was mild with lymphocytosis th noticed from 5 day post treatment onwards.treatment group showed mild depletion and areas of improvement on growth performance under cleaner repopulation with lymphocytes (Fig- 5).
environment despite continuous administration in low Spleen of control group revealed normal doses.Further Porchezhian [8] also documented that architecture (Fig- 6).Whereas, severe lymphocyto-ciprofloxacin did not have any influence on body lysis, depletion of lymphocytes with several areas of weight in healthy broilers and suggested that haemorrhages were noticed in spleen of 1 day post fluoroquinolones has influence in increasing body rd weight only in infection, not in healthy condition.treatment group (Fig- 7). 3 day post treatment group Indeed, Ahmad et al.
[18] also described that showed depletion of lymphocytes around arterioles enrofloxacin (25%) administration through drinking with mild haemorrhagic areas.In few birds blast cell water did not have any effect on mean body weights proliferation were also noticed (Fig- 8).Mild depletion and feed conversion ratio. of lymphocytes with lot of blast cell proliferation is the th th In the present study, enrofloxacin administration only appreciable lesion observed in 5 day and 7 day did not have any influence on the behaviour of the post treatment groups (Fig- 9).Repopulation of th broilers and all the birds showed normal activity.lymphocytes was observed in 9 day post treatment Droppings of all the birds were semisolid, dark green group (Fig- 10).
and with a white uric acid deposition on the top.These Discussion findings were in conformity with Patel et al. [13], who studied the safety evaluation of levofloxacin following In the present study, the enrofloxacin treated repeated oral dose administration in White Leg Horn group showed no significant change in body weight, layer birds and reported similar observations.cumulative feed intake and feed efficiency when There was a significant reduction in humoral compared to that of control.These observations are in immune response as measured by haemagglutination accordance with Roura et al. (NDV) antigen of enrofloxacin dosed birds when carried out the research work.JR involved in conduct compared to that of control.These findings which are of the biological trial and collection of blood and tissue indicative of immunosuppressive action of enrofloxacin samples during the experiment.GS and VS analysed are in accordance with the observations of Khalifeh et the data, prepared and revised the manuscript.All al.
[1], who reported that enrofloxacin reduced the authors read and approved the final manuscript.production of Newcastle Disease (ND) antibody in the th th (histiocyte proliferation) in 5 and 7 day post th Histopathological study: Bursa of Fabricius of control treatment groups (Fig-4).Few birds from 9 day post Means bearing different superscript (a) within the column differ significantly (p<0.05),Mean±SE, n=6

Fig. 1 .
Fig.1.Bursa of Fabricius of control group showing normal medullary areas populated with lymphocytes.H&E, 100X Fig.2.Bursa of st Fabricius of 1 day post treatment group showing lymphocyte depletion from the medullary areas and increased lymphophagord cytosis.H&E, 100X Fig.3.Bursa of Fabricius of 3 day post treatment group showing dispersion and depletion of lymphocytes in medullary areas and areas of lymphoblastic degeneration H&E, 100X Fig.4.th Bursa of Fabricius of 7 day post treatment group showing mild depletion of lymphocytes with lymphocytosis (histiocyte th proliferation), H&E, 100X Fig.5.Bursa of Fabricius of 9 day post treatment group showing mild depletion of lymphocytes and areas of repopulation with lymphocytes, H&E, 100X Fig.6.Spleen of control st group showing normal architecture.H&E, 200X Fig.7.Spleen of 1 day post treatment group showing severe lymphocytolysis and rd depletion with areas of haemorrhages.H&E, 100X Fig.8.Spleen of 3 day post treatment group showing depletion of lymphocytes and th proliferation of few blast cells.H&E, 100X Fig.9.Spleen of 7 day post treatment group showing mild depletion of lymphocytes and more th proliferation of blast cells.H&E, 100X Fig.10.Spleen of 9 day post treatment group repopulated with lymphocytes.H&E, 200X

Table - 1
. Effect of enrofloxacin administration (@ 10mg/Kg body weight, in drinking water for 5 consecutive days) on body weight in broiler chicken (expressed as grams, Mean±SE, n=6) Table-3.Effect of enrofloxacin administration (@ 10mg/Kg body weight, in drinking water for 5 consecutive days) on feed efficiency in broiler chicken (Mean±SE, n=6)