NAAS Score 2018

                   5.36

Declaration Format

Please download Declaration Form and submit along with manuscript.

Submit News Items

You can submit news items via contact us

Symposium

conference

Seminar

Summer/Winter School

Awards

UserOnline

Flag Counter

Previous Next

Effect of Amoxicillin on Haematobiochemical Parameters in Poultry

Madhuchhanda Khan Uttam Sarkar Tapan Kumar Mandal
Vol 8(4), 170-179
DOI- http://dx.doi.org/10.5455/ijlr.20170704045203

Amoxicillin, a broad-spectrum, β-lactam antibiotic is extensively used for empirical treatment of bacterial infections in poultry industry. Amoxicillin was administered orally to layer hen (Rhode Island Red) at therapeutic @20 mg kg -1 and at subtherapeutic (@10 mg kg -1 dose level for consecutive 14 days and the activities of some antioxidant enzymes and malondialdehyde (MDA) blood levels were studied. Amoxicillin produced a nonsignificant increase of haemoglobin , total leucocyte count and heterophil count in layer hens of different groups at different days compared to control group following daily oral administration for consecutive 14 days. On the other hand, Amoxicillin caused a nonsignificant decrease of lymphocyte and eosinophil count in layer hens of different groups at different days compared to control group. Values of blood glucose level and serum protein level of amoxicillin treated group of birds decreased nonsignificantly as compared to control birds. Plasma ALT, Plasma AST and serum lipid peroxidation activity in treated groups of layer poultry were increased significantly (P< 0.05) as compared to control birds. Erythrocyte catalase activity in treated groups of layer hens were decreased significantly (P< 0.05) on day 15 as compared to control birds. Erythrocyte superoxide dismutase activity in treated groups of layer hens were decreased significantly as compared to control birds (P<0.05) on day 15.


Keywords : Amoxicillin haematological parameter Antioxidant enzyme Malondialdehyde Reactive Oxygen Species

Introduction

Amoxicillin, a broad -spectrum, β-lactam antibiotic  is an analog of ampicillin, derived from the basic penicillin nucleus, 6-aminopenicillanic acid. Chemically, amoxicillin is (2S, 5R, 6R)-6-[(R) ¬ (-)-2-Amino-2-(p-hydroxyphenyl) acetamido]-3, 3-dimethyl-7-oxo-4-thia-1-azabicyclo (3.2.0) heptane -2-carboxylic acid trihydrate . Good oral absorption and potent broad-spectrum antimicrobial activity of amoxicillin (Rolinson, 1979; Comber et al., 1980)   leads to its extensive use for empirical treatment of bacterial infections in poultry industry. Amoxicillin’s mechanism of action involves the inhibition of stage III of the bacterial cell wall biosynthesis, preventing cross-linking of peptidoglycan. It is an alternative substrate for transpeptidases because of its structural similarity to the transition state of the Ala-Ala terminal during cross-linking. The inability to synthesizes the cell wall leads to cell lysis and thus amoxicillin is bactericidal (William et al., 2002). Hematological profile in animals is an important indicator of physiological or pathophysiological status of the body (Khan et al., 2005) and  toxicity of many xenobiotics is associated with the production of free radicals, which are not only toxic themselves, but are also implicated in the pathophysiology of many diseases. They may produce oxidative stress by generating free radicals and inducing or altering enzymatic and non-enzymatic anti-oxidant systems (Hybertson et al., 2011). Keeping in view, the present study was carried out to explore the effects of the Amoxicillin using status of haemogram, antioxidants defense mechanism, oxidative stress and some biochemical indices.

Materials and Method

Drugs and Chemicals

Amoxicillin trihydrate (Zovex®) was purchased from market (oral powder 50% W/W, Dey’s Medical Stores Ltd). All other chemicals were used in this study were obtained from E.Merck (India), Sigma Chemicals Co. (USA).

Experimental Birds

Vaccinated and dewormed layer (Rhode Island Red) hens of six months old were purchased from a commercial farm .The birds were housed in deep litter system with optimum lighting facility. The birds were fed with layer feed as per Bureau of Indian Standard (BIS), 1992. Fresh water and feed were supplied ad libitum. Prior to start of this experiment, the animal room was cleaned and fumigated with potassium permanganate and commercial formaldehyde solution (1:10). All the feeders and watering troughs were properly cleaned and disinfected with potassium permanganate solution (5%) 24 hr before start of the experiment.

Experimental Design and Mode of Administration

Eighteen layer hens were divided into three groups viz. I, II, III consisted of six birds in each group. Group I was considered as control group while Amoxicillin was administered orally to group II birds at therapeutic dose of 20 mg kg-1 and to group III birds at subtherapeutic dose of 10 mg kg-1 dose level for consecutive 14 days. Group I (Control group) birds were not treated with any antibacterial.  The experimental protocol was approved by the Institutional Animal Ethical Committee (IAEC).

Collection of Samples

Blood                        

Blood samples (5 ml) were collected from layer hen before administration of antibiotic (0 day) from wing vein and thereafter on day 15 and day 22 post dosing. One ml of blood was kept for haematological study, 2 ml of blood for collection of plasma and preparation of haemolysate for analysis of enzyme and 2 ml of blood without any anticoagulant was used for serum collection for analysis of glucose and protein. The birds of each group were sacrificed on day 22 and a piece of liver was collected for histopathological study.

Analytical Method

Haemoglobin level was determined by indirect acid-haematin method described by Coffin (1953) and expressed as gm/dl. Total leucocyte count was estimated by the method of Natt and Herrick (1952) and expressed as SI unit and differential count (DLC %) were estimated following right modified Giemsa stain method described by Campbell (1988). Blood glucose was determined by Glucose Assay kit by GOD-POD method using in vitro Diagnostic kit (Trinders methodology manufactured by Span Diagnostic Ltd.). Plasma aspartate and alanine transaminase was determined by Reitman and Frankel (1957) method using in vitro Diagnostic kit (Manufactured by Span Diagnostic Ltd). Serum Protein was estimated by Biuret method (Wooton, 1974) and was expressed as gm /dl. The total amount of lipid peroxidation products in serum was assayed using the thiobarbturic acid (TBA) method measuring malondialdehyde (MDA) and expressed as n mole of malondialdehyde per ml of serum according to the method described by Buege and Ausf (1976). The red blood cells were washed twice with equal volume of normal saline and after further centrifugation at 2500 rpm for 15 min 5% cell lysate was prepared. Superoxide dismutase was estimated according to the method of Misera et al., 1972. Further 1:500 dilution of haemolysate with phosphate buffer was prepared and Catalase activity was estimated according to the method of Aebi et al. (1974).

 

Histopathological Study

Small pieces of liver was collected and preserved in 10% (V/V) formal saline for histopathological study by the method as described by Lillie and Fullmer (1976).

Statistical Analysis

The results were analysed by One Way ANOVA using general linear model with invariable data in SPSS 20.0 version of software. The results were expressed as mean ± standard error (S.E).

Results and Discussion

The variation of haemoglobin level, total leucocyte count and differential leucocyte count in the blood of Rhode Island Red hen after administration of Amoxicillin trihydrate orally  at therapeutic (20 mg kg-1) and  subtherapeutic (10 mg kg-1) dose level for consecutive 14 days are illustrated in Table 1 ,2 and 3 respectively.

Table 1: Effect of Amoxicillin on Haemoglobin level (gm dl-1) in layer poultry following consecutive daily oral administration at two dose level for 14 days (Mean value of six replicates with S.E.)

Group
Day Gr. I Gr. II Gr. III
0 10.28±0.53 10±0.49 9.72±0.30
15 10.08±0.34 11.01±0.69 10.64±0.66
22 10.00±0.37 10.59±0.62 10.35±0.42

I Control group received no drug

III Administered Amoxicillin at therapeutic dose level (20 mg/Kg) orally for consecutive 14  days

III Administered Amoxicillin at subtherapeutic dose level (10 mg/Kg) orally for consecutive 14 days       

Table 2: Effect of Amoxicillin on Total leucocyte count (x103µl-1) in layer poultry following consecutive daily oral administration at two dose level for 14 days (Mean value of six replicates with S.E)

Day Gr I Gr II Gr III
0 13.68±0.69 13.95±0.43 13.37±0.71
15 13.82±0.63 14.74±0.53 13.97±0.67
22 13.65±0.48 14.23±0.36 13.66±0.50

I Control group received no drug

II Administered Amoxicillin at therapeutic dose level (20 mg/Kg) orally for consecutive 14 days

III Administered Amoxicillin at subtherapeutic dose level (10 mg/Kg) orally for consecutive 14 days       

Haemoglobin level of group II & group III birds increased nonsignificantly compared to control birds which corroborates with the findings of Turcu et al. (2012) who reported that haemoglobin level was increased non significantly in layer hen following Amoxicillin soluble powder (50%) treatment. There was nonsignificant increase of total leucocyte count of group II and group III birds compared to control batch within the normal physiological limit. Similar finding was also observed by Turcu et al. (2012) in broiler chicken following Amoxicillin soluble powder (50%) treatment.

Table 3: Effect of Amoxicillin on Differential Leucocyte Count (%) in layer poultry following consecutive daily oral administration at two dose level for 14 days (Mean value of six replicates with S.E)

Day Leucocyte Differential count(DC%) of leucocyte
Gr. I Gr. II Gr. III
0 Heterophil 24.1±1.14 24.8±0.87 24±1.40
Lymphocyte 66.4±1.48 67.5±1.74 67.7±1.56
Eosinophil 3.6±0.38 3.7±0.31 3.9±0.26
Monocyte 2.6±0.21 2.2±0.30 2.8±0.40
Basophil 1.5±0.22 1.8±0.40 1.6±0.34
Heterophil 23.8±1.20 26.5±1.26 26.1±1.05
15 Lymphocyte 67.4±1.60 65.4±1.48 65.9±1.63
Eosinophil 4±0.39 3.5±0.34 3.2±0.28
Monocyte 2.8±0.22 2.6±0.33 2.0±0.25
Basophil 1.3±0.30 2±0.44 1.8±0.20
22 Heterophil 23.6±1.10 26.2±1.33 26.5±1.19
Lymphocyte 68.3±1.63 67±1.50 66.3±1.58
Eosinophil 4.2±0.30 3.0±0.26 3.5±0.39
Monocyte 2.2±0.30 2.5±0.22 2.2±0.20
Basophil 1.7±0.21 1.3±0.33 1.5±0.34

I Control group received no drug

II Administered Amoxicillin at therapeutic dose level (20 mg/Kg) orally for consecutive 14 days

III Administered Amoxicillin at subtherapeutic dose level (10 mg/Kg) orally for consecutive 14 days       

Lymphocyte and Eosinophil count were less in group II and group III birds compared to control birds whereas Heterophil count was increased nonsignificantly in amoxicillin treated groups. Turcu et al. (2012) reported similar finding in broiler chicken following amoxicillin soluble powder (50%) treatment. Values of blood glucose level of group II and III birds decreased nonsignificantly as compared to control birds (Table 4). Hypoglycemia is a result of any disorder that causes abnormal restrictions in the production of glucose by the liver or kidneys, or that causes an abnormal increase in glucose uptake by the cells.

Table 4: Effect of Amoxicillin on Serum glucose (mg/dl) level in layer poultry following consecutive daily oral administration at two dose level for 14 days (Mean value of six replicates with S.E)

Day Group
Gr. I Gr. II Gr. III
0 204.59±9.03 205.71±10.16 203.02±11.12
15 205.83±10.22 198.43±9.39 200.81±8.95
22 206.34±9.78 197.36±9.65 199.31±9.25

I Control group received no drug

II Administered Amoxicillin at therapeutic dose level (20 mg/Kg) orally for consecutive 14 days

III Administered Amoxicillin at subtherapeutic dose level (10 mg/Kg) orally for consecutive 14 days       

It is revealed from Table 5 that no significant alterations in values of serum protein level were observed in treated birds of different groups as compared to control.

Table 5: Effect of Amoxicillin on Serum protein (gm/dl) level in layer poultry following consecutive daily oral administration at two dose level for 14 days (Mean value of six replicates with S.E)

Day Group
Gr. I Gr. II Gr. III
0 3.47±0.32 3.38±0.34 3.60±0.26
15 3.40±0.30 3.42±0.38 3.56±0.29
22 3.30±0.35 3.33±0.40 3.65±0.43

I Control group received no drug

II Administered Amoxicillin at therapeutic dose level (20 mg/Kg) orally for consecutive 14 days

III Administered Amoxicillin at subtherapeutic dose level (10 mg/Kg) orally for consecutive 14 days       

Table 6 shows that mean value of plasma ALT in layer poultry of groups II and III were increased significantly (P<0.05) as compared to control birds. Table 7 shows that mean values of plasma AST activity in layer hens of (group II) were increased significantly (P<0.05) on day 22 as compared to control birds. Mean values of plasma AST activity in layer poultry of (group III) were increased nonsignificantly on 15 day and 22 day as compared to control birds.

Table 6: Effect of Amoxicillin on plasma ALT activity (IU/L) in layer poultry following consecutive daily oral administration at two dose level for 14 days (Mean value of six replicates with S.E)

Day Group
Gr. I Gr. II Gr. III
0 14.32±1.28 13.89±1.20 14.10±1.10
15 15.43±1.44 19.12±1.30 18.68±1.45
22 14.49a±1.26 19.02b±1.39 18.78 bc±1.35

I Control group received no drug

II Administered Amoxicillin at therapeutic dose level (20 mg/Kg) orally for consecutive 14 days

III Administered Amoxicillin at subtherapeutic dose level (10 mg/Kg) orally for consecutive 14 days       

*Mean values bearing different superscript in a row vary significantly (P<0.05)

Table 7: Effect of Amoxicillin on plasma AST (IU/L) activity in layer poultry following consecutive daily oral administration at two dose level for 14 days (Mean value of six replicates with S.E)

Day Group
Gr. I Gr. II Gr. III
0 169.03±5.10 168.92±4.39 168.18±4.73
15 167.37±4.43 179.26±3.89 176.26±4.48
22 166.95a±3.05 181.52b±4.82 177.20ab±3.65

I Control group received no drug

II Administered Amoxicillin at therapeutic dose level (20 mg/Kg) orally for consecutive 14 days

III Administered Amoxicillin at subtherapeutic dose level (10 mg/Kg) orally for consecutive 14 days       

*Mean values bearing different superscript in a row vary significantly (P<0.05)

Similarly Olayinka and Olukowade (2010) observed an increase in the activities of plasma AST and ALT following (amoxycillin/clavulanic acid) treatment in rats. These enzymes elevation in the plasma by the drug might be as a result of release of the enzymes from some tissues indicating tissue damage. Increase in plasma ALT and AST has been reported in conditions involving necrosis of hepatocytes (Macfarlane et al., 2000). Acute cellular swelling and vacuolar degeneration with mild coagulative necrosis of hepatocytes were observed in the present study (Plate: 1 & 2) which might be responsible for the increase of plasma AST and ALT activity.

Plate 1: Section of Liver of Layer poultry showing acute cellular swelling and vacuolar degeneration following daily oral administration of Amoxicillin at 10 mg/kgbw for 14 days (H&E,10x)

Plate 2: Section of Liver of Layer poultry showing coagulative necrosis following daily oral administration of Amoxicillin at 20 mg/kgbw for 14 days (H&E, 10x)

It is adduced from Table 8 that mean values of serum lipid peroxidation activity in group II and III layer hens were increased significantly(P<0.05) as compared to control birds on day 15. Sherbiny et al. (2009) reported that administration of either Amoxicillin or Clavulinic acid and their combination resulted in marked elevation of oxidative stress marker as evidenced by increased lipid peroxidation product MDA, reduction in antioxidant molecules content such as reduced glutathione (GSH). Lipid peroxidation is one of the best parameters indicative for the level of reactive oxygen species (ROS)-induced systemic biological damage (Georgieva et al., 2006).

Table 8: Effect of Amoxicillin on Serum lipid peroxidation activity (n mole/ml) in layer poultry following consecutive daily oral administration at two dose level for 14 days (Mean value of six replicates with S.E)

Day Group
Gr. I Gr. II Gr. III
0 10.03±1.12 10.5±1.40 10.40±1.37
15 10.9a±1.41 15.18b±1.23 14.76bc±1.20
22 10.16±1.14 12.12±1.17 12.54±1.09

I Control group received no drug

II Administered Amoxicillin at therapeutic dose level (20 mg/Kg) orally for consecutive 14 days

III Administered Amoxicillin at subtherapeutic dose level (10 mg/Kg) orally for consecutive 14 days       

*Mean values bearing different superscript in a row vary significantly (P<0.05)

In this present study the elevated MDA serum concentration is probably due to the oxidative stress that occurred after administration of amoxicillin trihydrate orally , which may be attributed to increased ROS production and reduced antioxidant defence system, resulting in lipid peroxidation (Sarban et al., 2005).

In Table 9 erythrocyte catalase activity in group II and III layer hens were decreased significantly (P<0.05) on day 15 as compared to control birds.

Table 9: Effect of Amoxicillin erythrocyte super oxide dismutase activity (U/mg Hb)  in layer poultry following consecutive daily oral administration at two dose level for 14 days  (Mean value of six replicates with S.E)

Day Group
Gr. I Gr. II Gr. III
0 1.72±0.10 1.69±0.08 1.60±0.11
15 1.67a±0.12 1.16b±0.14 1.24bc±0.07
22 1.63±0.10 1.51±0.09 1.49±0.13

I Control group received no drug

II Administered Amoxicillin at therapeutic dose level (20 mg/Kg) orally for consecutive 14 days

III Administered Amoxicillin at subtherapeutic dose level (10 mg/Kg) orally for consecutive 14 days       

*Mean values bearing different superscript in a row vary significantly (P<0.05)

Table 10: Effect of Amoxicillin on erythrocyte catalase activity (K/g.Hb) in layer poultry following consecutive daily oral administration at two dose level for 14 days (Mean value of six replicates with S.E)

Day Group
Gr. I Gr. II Gr. III
0 0.56±0.06 0.54±0.08 0.53±0.05
15 0.54a±0.07 0.38b±0.04 0.34bc±0.03
22 0.50±0.09 0.49±0.05 0.53±0.06

I Control group received no drug

II Administered Amoxicillin at therapeutic dose level (20 mg/Kg) orally for consecutive 14 days

III Administered Amoxicillin at subtherapeutic dose level (10 mg/Kg) orally for consecutive 14 days       

*Mean values bearing different superscript in a row vary significantly (P<0.05)

Table 10 shows that mean values of erythrocyte superoxide dismutase activity in layer hens of Gr II and III were decreased significantly as compared to control birds(P<0.05) on day 15. The changes in the level of SOD activity in the treated groups in this study is probably due to enhanced ROS production as a result of the prolonged oral application of amoxicillin trihydrate.

Conclusion

From the present study it may be concluded that haemoglobin, total leucocyte count and heterophil count in experimental group of layer hens at different days as compared to control group following daily oral administration of Amoxicillin trihydrate for consecutive 14 days showed nonsignificant increase. Values of blood glucose level and serum protein level of amoxicillin treated group of birds were decreased nonsignificantly as compared to control birds. In the experimental group, Amoxicillin produced a significant increase (P<0.05) in plasma ALT, plasma AST on day 22 and serum lipid peroxidation activity as compared to control birds on day 15. Erythrocyte catalase activity and Erythrocyte superoxide dismutase activity in treated groups of layer hens were decreased significantly as compared to control birds (P<0.05) on day 15.

References

  1. Aebi, H. 1974. Catalase. In: Bergmayer HU, editor. Methods in Enzymatic Analysis. Academic Press; 673–678.
  2. Buege, JA and Ausf, SD. 1976. The thiobarbiturate assay in microsomal lipid peroxidation .Method of enzymology, 52: 306-307.
  3. Campbell, TW. 1988.Avian hematology and cytology.2nd Chapter 9.Iowa State Press.Ames. pp. 177-200.
  4. Comber, KR, Horton, R, Mizen , L. 1980. Activity of amoxicillin/clavulanic acid (2:1) [BRL 25000, Augmentin] in vitro and in vivo. In : Current Chemotherapy and Infectious Disease. Proceedings of the Eleventh International Congress of Chemotherapy and the Nineteenth Interscience Conference on Antimicrobial Agents and Chemo therapy. American Society for Microbiology, Washington, DC, U.S.A. pp. 343-4.
  5. Georgieva,NV, Koinarski, B and Gadjeva, V. 2006. Antioxidant status during the course of Eimeria tenella infection in broiler chickens. Veterinary Journal. 172 ( 3): 488–492
  6. Hybertson, BM., Gao, B, Bose, SK, McCord, JM. 2011. Oxidative stress in health and disease: the therapeutic potential of Nrf2 activation. Mol Aspects Med 32: 234-246.
  7. Khan, AT. and Zafar, F. 2005. Haematological Study in response of varying doses of estrogen in broiler chicken. International Journal of Poultry Science. 4 (10):748:751.
  8. Macfarlane ,WM , Shepherd, RM, Cosgrove, KE, James, RF, Dunne, MJ and Docherty, 2000.Glucose modulation of insulin mRNA levels is dependent on transcription factor PDX-1 and occurs independently of changes in intracellular Ca2+.Diabetes 49: 418-423
  9. Misera, HP and Fridovich, I.1972.The role of superoxide dismutase anion in the auto-oxidation of epinephrine and a simple assay for superoxide dismutase.J. Biol.Chem,247(10):3170-3175
  10. Natt, MP. and Herrick, CA.1952. A new blood diluent for counting erythrocytes and leucocytes of the chicken. Poultry Science, 31: 735-738.
  11. Sarban, A., Sezgin, A, Kocyigit, B, Mithat, Y, Ugur, A and Isikan, E. 2005. Plasma total antioxidant capacity, lipid peroxidation, and erythrocyte antioxidant enzyme activities in patients with rheumatoid arthritis and osteoarthritis. Clinical Biochemistry, 38: 981 – 986.
  12. Trinder, P 1969. Determination of blood glucose using an oxidase-peroxidase system with a non-carginogenic chemogen. J Clin Pathol: 22(2):158–161.
  13. Turcu,D., Oporanu,M., Grigorescu, P. and Roman,M.2011. Studies on hematological parameters in broiler chicken treated with Amoxidem 50% Medicamentul Veterinar  .5(1): 93-98.
  14. Olayinka, ET and Olukowade IL. 2010 . Effect of amoxycillin/clavulanic acid on antioxidant indices and markers of renal and hepatic damage in rats. Journal of Toxic. and Env. Health. Sci, 2(6) : 85-92.
  15. Reitman, S and Frankel, S. 1957. A colorimetric method for determination of serum glutamic oxaloacetic and glutamic pyruvic transaminase. Am J Clin Pathol; 28: 56-63.
  16. Rolinson, GN. 1979. 6-APA and the development of the beta-lactam antibiotics. J  Antimicrob Chemother. 5(1):7-14.
  17. Sherbiny, GAEL, Taye, A and Raheem,ITA, 2009. Role of ursodeoxycholic acid in prevention of hepatotoxicity caused by amoxicillin-clavulanic acid in rats.of. Hepat , 8(2) : 134-140.
  18. Wooton , IDP. 1974. Estimation of protein by biuret method. In Microanalysis in medical biochemistry .5th edn. Churchill Livingstone, Edinburgh and London: 156-158.
Abstract Read : 79 Downloads : 26
Previous Next
Book Promotion

Submit Jobs

You can submit Jobs (JRF/SRF/Others relevant) via contact us This will help to find right talent.

Close