ANTI-INFLAMMATORY, IMMUNOMODULATORY AND ANTIOXIDANT EFFECTS OF LYCOPENE AND VITAMIN E IN E. COLI INFECTED BROILERS.

Khalid. M. Fararh 1 , Adel. M. Abd El-Aziz 2 and Nagwan. A. Alhelbawy 2 . 1. Clinical Pathology Department, Faculty of Veterinary Medicine, Banha University, Egypt. 2. Animal Health Research Institute, Tanta, Egypt. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History Received: 22 May 2019 Final Accepted: 24 June 2019 Published: July 2019


Serum inflammatory markers:
In table (1): LYC treated non infected group when compared to control one showed a significant decrease in CRP and haptoglobin 4 days and 2 weeks PI as well as significantly decreased IL-1β only 2 weeks PI and significantly increased IL-10 at both collections. VE treated group showed non significant changes in all above mentioned parameters when compared to negative control group. A significant increase in CRP and haptoglobin was observed in E. coli infected non treated group 4 days and 2 weeks PI in addition to significant increases in both IL-1β and IL-10 only 4 days PI when compared to negative control group. LYC treated E. coli infected group comparing to infected non treated group had significantly decreased CRP and haptoglobin 4 days and 2 weeks PI and a significant decrease in IL-1β only 4 days PI and a significant increase in IL-10 at both collections. VE treated infected group comparing to infected non treated group had significantly decreased CRP only 4 days PI and significantly decreased haptoglobin at both collections. Also non significant changes in IL-1β and IL-10 were noticed. More improved picture was observed in lycopene treated group than VE treated one. Table (2) revealed non significant changes in LYC and VE treated groups (gps 2 and 3) comparing to negative control group 4 days and 2 weeks PI except a significant increase in phagocytic activity in LYC treated group 4 days PI. On the other hand the infected non treated group (gp 4) when compared to control group showed non significant changes in these parameters 4 days PI turned to significant decreases at 2 weeks PI. LYC treated E. coli infected group (gp5) when compared to infected non treated group (gp 4) revealed a statistical increase in phagocytic activity and phagocytic index 2 weeks PI (normalization toward control). VE treated E. coli infected group (gp 6) when compared to infected non treated group (gp 4) had a significantly increased phagocytic activity and non significantly increased phagocytic index. Table (3) showed a significant decrease in the liver MDA and a significant increase in liver CAT, GPX and SOD in treated groups (2 and 3) 4 days and 2 weeks PI while E.coli infected non treated group (gp4) showed a significant increase in MDA and a significant decrease in CAT at both collections in addition to non significant changes GPX and SOD 4 days PI turned to a significant decrease 2 weeks PI when compared to negative control group. Concerning to LYC treated E.coli infected group (gp5) comparing to E coli infected non treated group (gp 4) showed a significant decrease in MDA and a significant increase in CAT, GPX and SOD at both collections while VE treated E.coli infected group (6) had a significant decrease in MDA and a significant increase in CAT 4 days and 2 weeks PI as well as non significant increase in GPX and SOD 4 days PI turned to a significant increase 2 weeks PI. More prominent antioxidant effect was in LYC treated group than VE treated one.

Serum biochemical parameters:
As in table (4) serum AST, GGT, uric acid, creatinine and TC revealed non significant changes in treated groups (2 and 3) at both collections while E.coli infected non treated group (gp4) showed a significant increase serum AST, GGT uric acid and TC at both collections and a significant increase in serum creatinine at the second collection only when compared to control group. LYC treated E.coli infected group (gp5) when compared to E coli infected non treated group (gp4) represented a significant decrease in serum AST and GGT at both collections and non significant changes in TC and creatinine at first collection turned to a significant decrease at second collection while uric acid revealed non significant changes at both collections (but not statistically differ from control at second collection). VE treated E.coli infected group (gp 6) showed non significant changes in all above mentioned parameters except AST at second collection and GGT at both collections revealed significant decreases when compared to E coli infected non treated group (gp 4). LYC treated birds showed more prominent improvements in biochemical parameters than VE treated ones.

Discussion:-
The E. coli infected chicks showed anorexia, ruffled feathers, dullness, depression and diarrhea with pasted vent at 3 days PI followed by respiratory signs in the form of rhinitis, sneezing, cough, wet eyes and nasal discharge at fourth day. Similar clinical signs were reported by Abd El- Tawab  Our study revealed that serum CRP and haptoglobin were significantly decreased in LYC treated group (gp2) and non significantly changed in VE treated group (gp3) while significant increases in these parameters were observed in E. coli infected non treated group (gp4) when compared to control group 4 days and 2 weeks PI. Regarding to cytokines our study revealed that LYC treatment significantly decreased IL-1β at 2 weeks PI and significantly increased IL-10 4 days and 2 weeks PI while VE treatment had non-significant effect on both IL-1β and IL-10. On the other hand a significant increase in both IL-1β and IL-10 4 days PI turned to non significant increase after 2 weeks was observed in E. coli infected non treated birds comparing to control ones. About our results of phagocytic activity and phagocytic index a significant increase in phagocytic activity was observed in LYC treated group (gp2) comparing to negative control group at first collection. On the other hand, infected non treated group (gp 4) when compared to control group showed non-significant changes in these parameters 4 days PI turned to significant decreases 2 weeks PI. Our results come in agreement with Abd-El-Tawab et al., (2015). These changes may be attributed to E. coli infection that causes impairment of polymorph nuclear leukocytes function decreasing its phagocytic activity and resulted in in effective opsonization (Van Dijk et al.,  1980). LYC treated E. coli infected group (gp 5) when compared to infected non treated group (gp 4) revealed a statistical increase in phagocytic activity and phagocytic index 2 weeks PI. Our results coincide with Yonar, (2012) who recorded that LYC treatment associated with a significant increase in the oxy tetracyclin-suppressed phagocytic activity in rainbow trout. Improvements in immunological parameters may be due to immunostimulant effect of LYC which stimulates  LYC induced changes may be attributed to its potent antioxidant effects related to higher singlet oxygen quenching ability which is twice as high as that of β-carotene and 10 times higher than that of α-tocopherol. It scavenges the free radicals via three different mechanisms: adduct formation, electron transfer, and hydrogen atom transfer (El -Agamey et al., 2004). Consequently LYC prevents lipid peroxidation and DNA damage simultaneously, it induces enzymes of the cellular antioxidant defense systems by activating the antioxidant response element transcription (Kelkel et al., 2011). VE induced changes may be resulted from its antioxidant effect donating hydroxyl group on its ring structure to free radicals, preventing lipid peroxidation thereby prolonging the biological life of 795 polyunsaturated fatty acids in the cell membranes by slowing the formation of free radicals and hyper-peroxides (Traber and Atkinson, 2007).
The results of AST and GGT indicated non-significant change in LYC and VE treated groups (2&3) compared with the control healthy group reflecting their safety usage with no harmful effect on the liver. Our results agreed with Ibrahim and Banaee, (2014) who found non significant changes in serum activities of AST in LYC and VE supplemented fish. The infected non-treated group showed a significant increase in the serum activities of AST and GGT when compared to the control one. These results come Uric acid and creatinine revealed non-significant changes in LYC and VE treated groups (2&3) compared with the control healthy group indicating their safety usage on the kidney. Safety usage of LYC and VE on the kidney also stated by Ibrahim and Banaee, (2014). Infected non-treated chicks displayed a significant increase in the levels of serum uric acid 4 days and 2 weeks PI and creatinine at 2 weeks only. These findings come in agreement with those of Abd El-Ghany and Ismail, (2014) and could be due to the effect of the microorganisms and it's toxin on the kidneys (Abd-Allah et al., 2018)). LYC treated infected group (gp5) when compared to infected non treated group (gp4) showed non significant changes in serum uric acid (but also not statistically differ from normal control values at second collection) beside a significant decrease in serum creatinine at 2 weeks PI. Decreased creatinine level by LYC treatment in hypercholesteremic rats was also documented by Basuny et al., (2009). On contrary Pektaş et al., (2014) observed non significant improvement in serum creatinine in rats with renal ischemia when treated with LYC. Difference may be due to treatment regimen (4 mg/kg per day for 2 days) or species difference (rats). Improvement may be attributed to partial protective effect of LYC from oxidative renal damage by the organism and its endotoxin.VE treated infected group (gp 6) when compared to E. coli infected non treated group showed non significant changes in serum uric and creatinine. This agreed with results of Biwas et al., (2018) who found non significant decrease in serum urea and creatinine in aluminium phosphide toxicated rats treated with VE (1.5 mg/g) Regarding to TC, our results showed that LYC and VE treated groups (2&3) revealed non significant changes while infected non treated group (gp 4) showed a significant increase in serum concentrations of TC 4 days and 2 weeks PI comparing to negative control birds. These results agreed with those of Mekkawy, (2016) and may be due to severe liver damage caused by bacteria that leading to inadequate cholesterol utilization as liver is responsible for biotransformation of cholesterol into bile acids). LYC treated infected group (gp 5) when compared to E. coli infected non treated group (gp 4) showed a significant decrease in serum TC at the second collections (reached to normal control limits). Our results come in the same side with Abdel-Rahman et al., (2018) who found normalization of TC in bisphenol intoxicated rats treated with 10 mg /kg lycopene for 30 days. The normalization of TC in our study may be due to the ability of LYC in protecting LDL from oxidation, to its role in inhibiting HMG-CoA reductase activity and to up-regulate LDL receptor activity in macrophages (Heber and Lu, 2002). VE treated infected group when compared to infected non treated one showed non significant change in serum TC at the two collections. Similar to our results Leonard et al., (2007) found that VE (400 IU for 8 weeks) didn't alter TC in hypercholesterolemic patients.

Conclusion:-
It could be concluded that lycopene and vitamin E might be helpful in reducing the harmful effect of E. coli infection via their anti-inflammatory, immunomodulatory and antioxidant effects with higher improvement in case of lycopene treatment.