الفهرس 
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Abstract
The present study was conducted to investigate the immuno-pharmacological effects of tylvalosin, florfenicol and Vit E alone or in combination on the cellular and humoral immune in New Castle and Avian influenza vaccinated broiler chickens in addition to, the pharmacokinetics of the mentioned drugs and their combination were also evaluated and trying to connect between the pharmacokinetics (serum concentration) and their effects on the immune system in relation to their blood concentration besides Vit E effects on these drugs. Moreover, the genotoxic effect on the chicken’s blood leucocytes and the histopathological changes in lymphoid organs were also studies as well as image analysis of the microscopic histopathology were also determined as a representative to the cellular percentage affects by each treatment(Area percentage).
a- Immunological study:
A total of 180 one-day old broiler chicks have been used for the immunological part of this study. Chicks have been reared until 10th day of age in a clean disinfected room, fed with commercial chick basal diets and watered ad libitum. At the 11th day of age, birds were divided into 6 equal groups and each was further subdivided into 2 equal repeats. from 11th through 13th day of age, calculation of water consumption for each repeat was done on both 8 and 24 h level to accurately adjust the amount of water in which the drugs to be dissolved and the rate of daily increase in water consumption per group. Birds vaccinated with New Castle and Avian influenza vaccinated broiler chickens at 7th and 10th day of age respectively.
- Birds in each group and repeats were treated with the target drug according to the following schedule:
group 1 (G1): vaccinated and treated orally for 24 h for 3 successive days with tylvalosin at dose of 25 mg/kg b.wt.
G2: vaccinated and treated orally for 24 h for 5 successive days with florfenicol at a dose of 30 mg/kg b.wt. To ensure complete solubility of florfenicol the calculated daily amount was diluted with water at the rate of 1:5 followed by addition of 300 µl DMF.
G3: vaccinated and treated orally for 24 h for 3 successive days with Vit E at a dose of 2 mg/kg b.wt.
G4: vaccinated and treated orally for 3 successive days with Vit E (16 h) then tylvalosin (8 h) at a dose of 2 and 25 mg/kg b.wt. respectively.
G5: vaccinated and treated orally for 5 successive days with Vit E (16 h) then florfenicol (8 h) at a dose of 2 and 30 mg/kg b.wt. respectively.
G6: birds were left as vaccinated non treated control.
- Drug dosing, started from the 14th day through the 16th day of age in G1, G3 and G4 receiving tylvalosin, Vit E and tylvalosin-Vit E, respectively, meanwhile the dosing was continued until the 19th day of age in G2 and G5 receiving florfenicol, florfenicol-Vit E, respectively.
Two blood samples were collected from the wing vein and from the heart of each chickens or by slaughtering at the zero day of the experiment and subsequently weekly after vaccination at 14th, 18th, 21th , 28th , 35th and 42th day of age for humoral immunity evaluation through HI technique.
a) Sample 1:
Whole blood (2-3 ml) was collected from the wing vein in a sterile Wasserman tube containing EDTA (0.5 mg/ml of blood) to be used for determination of total and differential leucocytic counts, and tubes with Heparin anticoagulant for assessment of genotoxicity by the comet assay.
b) Sample 2:
In a sterile Wasserman tube, 3-5 ml of blood was collected from the wing vein without an anticoagulant.The samples were allowed to coagulate, and then the serum was separated by centrifugation at 3000 r.p.m. for 10 minutes and stored at -20 Co in sterile Eppendorf tubes until used for estimation of the humoral immune response, and for determination of serum nitric oxide, Lysozyme activity and Liver enzymes.
At the end of the experiment at about 45th day of age 4 chickens of each group were slaughtered and the lymphoid organs (spleen and bursa) were collected to be used for studying the histopathological changes.
b- Pharmacological study:
The pharmacokinetic aspects of tylvalosin, florfenicol alone or pre-treated with Vit E in healthy brolier chickens. the pharmacokinetics parameters were determined after single and muliple oral adminstration of the different alone or pre-treated with Vit E.The protien binding percent of the drugs to serum was also estimated.
The total number of chickens (24) were divided into four groups each group six (6) chickens, The all groups received different drugs to each group and they were given the different drugs after single oral dose (intra-crop route) and multiple adminstration orally to the same groups.
The chickens were calssified according to the treatment into four different groups as the follow:
Group(1): Birds adminstered orally tylvalosin (Aivlosin) 625mg/gm at s dose of 25mg/ kg b.wt.
group (2):chickens administered orally with florfenicol (pure powder) 98% at a dose of 30 mg/kg b.wt.
group (3): chickens administered orally withtylvalosin pre-treated by 2h with Vit E orally Vit E (2 mg/ kg b.wt) and (tylvalosin) at a dose of 25 mg/ kg b.wt. for three successive days.
Group(4):chickens administered orally withflorfenicol pre-treated by 2h with Vit E orally Vit E (2 mg/kg b.wt) and (florfenicol) at a dose of 30 mg/kg b.wt. for five 5 successive days as a single dose and for another five days in the same chickens for multiple dosing.
- Vit E administered first (before the second drug) to the chickens in the drug combination groups by 2 hours.
Sampling:
Blood samples (1ml each) were collected from each chicken in a sterile Wasserman tube without anticoagulant from the wing vein just after 5,10,15,30 minutes,1,2,4,6,8,12,24,48 hours post single administration and for another72 hour for multipledosing in tylvalosin and tylvalosin-Vit Ewhile till 96 h in florfenicol and florfenicol pre-treated with Vit E groups after single dosing and for another 96 hour in the same groups after multiple adminstration.
I-Effect on the cellular immunity:
1- Effect on total leucocytic count:
Both tylvalosin and florfenicol had been evoked a significant decrease in the total leucocytic count at 21th and 35th day against New Castle disease virus while,chickens administered Vit E drug showed a significant increase in the total leucocytic count along the 2 weeks. In tylvalosin and florfenicol pre-treated with Vit E groups showed a significant increase in the total leucocytic count at 21th and 35th day of age.
2- Effect on differential leucocytic count:
There were significant decrease in both lymphocytes and neutrophils percentage at 21th and 35th day post drug administration in tylvalosin and florfenicol administered groups, On the other hand Vit E, TVN/Vit E and FFC/Vit E treated chickens showed a significant increase but no significant effects were determined in the other differential cell types.
3- Effect on serum nitric oxide level:
Nitric oxide level was significantly decreased in the groups treated with tylvalosin and florfenicol at 21th day and 35th day while, Vit E treated chickens showed the most significant increase in nitric oxide level compared with the other groups .Moreover, groups of chickens treated with TVN/Vit E and FFC/Vit E groups showed no significant increase in nitric oxide level at 21th and 35th day of the experiment.
4- Effect on serum Lysozyme activity:
Administration of TVN and Vit E to vaccinated broiler chickens showed a significant increase in the serum lysozyme activity at 21th and 35th day of the experiment; On the other hand administration of florfenicol elicited a significant decrease in the serum lysozyme level. Meanwhile, TVN/Vit E (revealed the highest serum lysozyme in between the all groups) and FFC/Vit Etreated groups showed a significant increase in the serum lysozyme activity.
II – Effect of tylvalosin (TVN), florfenicol (FFC) alone or pre-treated with Vit E and Vit E on humoral immunity:
1- Effects on haemagglutination inhibition (HI) antibody titers:
Both tylvalosin and florfenicol administered groups showed decrease in the antibody titer in vaccinated chickens at 18th day, 21th day, 28th day 35th day and 42th day of age, On the other hand there was a significant increase in the antibody titers in Vit E administered group. Meanwhile, in TVN/Vit E group,Vit E make significant modulation to the effect of tylvalosin on antibody titer as it causes increase in the antibody titer well observed at 21th day and 28th day .group of chickens administered FFC/Vit E also caused decrease in the antibody titer as Vit E, not significantly modulate the decrease of florfenicol in the antibody titer.
2- Effect on serum total protein, albumin and globulin levels:
Administration of tylvalosin, florfenicol, or TVN/Vit E, FFC/Vit E combination evoked non-significant changes in total serum protein level but serum albumin level was significantly increased in groups of chickens administered tylvalosin and florfenicol , On the other hand group of chickens administered Vit E showed a significant decrease in the serum albumin level but tylvalosin and florfenicol treated chickens showed decrease in the serum globulin , On the other hand the other treated groups as Vit E, TVN/Vit E and FFC/Vit E revealed a higher significant increase in serum globulin.
III- Effect on tail moment (Genotoxicity using the comet assay):
A remarkable significant increase in tail moment was recorded in TVN and TVN/Vit E administered group, On the other hand no significant difference were recorded for the other treatments on the DNA of the leucocytes.
IV-Histopathological findings in lymphoid organs:
a-Microscopic (morphological analysis):
In tylvalosin and florfenicol treated chickens showed severe lymphoid depletion in the lymphoid follicles of bursa and spleen with severe shrinkage of both covering epithelium and follie as well as highly increasing in the inter-follicular spaces On the other hand, Vit E treated chickens showed hyperplasia in the lymphoid follicles, highly aggregation of lymphocytes in the germinal center and the covering epithelium appear tall and ciliated. Meanwhile, TVN/Vit E and FFC/Vit E treated chickens showed modulatory effects on the depleted lymphoid follicles.
b- Image Analysis (Area of cellular percentage (%) by numbers:
Image analysis had been done to the histopathological (morphological analysis) of the immune organs through calculation the cellular percentages of each treatment in this study and expressed by the area percentages of the cellular mass in each treatment, The analysis of the microscopic morphology had been revealed significance effect between the different groups specially between tylvalosin and the control groups.
-Pharmacokinetic aspects of a single and multiple oral administration of tylvalosin , florfenicolalone or pre-treated with Vit E:
1- Single oral administration of tylvalosin in control healthy broiler chickens :
The drug was firstly detected (0.05) after 15 minutes and the maximum serum level (2.25 µg/ml) was reached at 2 hour after the drug administration. The peak concentration (Cmax) was 2.11µg/ml and the calculated value of tmax was 2.06hour. The drug was absorbed from healthy broilers gut with absorption half-life (t0.5ab 0.94h) and eliminated with a mean half-life (t0.5 el 1.61h).
2- Repeated oral administration of tylvalosin in control healthybroiler chickens:
The drug was firstly detected (0.07 µg/ml) after 5 minutes and the maximum serum level (5.01µg/ml) was reached at 2hpostTVN administration. The peak concentration (Cmax) was 4.1µg/ml and the calculated value of (tmax2.19 hour). The drug was absorbed from healthy broilers gut with absorption half-life (t0.5ab 1.39h) and eliminated with a mean half-life (t0.5 el 2.78h).
3-Single oral administration of TVN/Vit E in healthy broiler chickens:
The drug was firstly detected (0.23ug/ml) after 15 minutes and the maximum serum level (3.77µg/ml) was reached at 2h after the drug administration. The peak concentration (Cmax) was 3.27µg/ml and the calculated value of (tmax 2.18h). The drug was absorbed from healthy broilers gut with absorption half-life (t0.5ab 0.88h) and eliminated with a mean half-life (t0.5el 2.42h).
4-Repeated oral administration of TVN/Vit E in healthy broiler chickens:
The drug was firstly detected (0.15µg/ml) after 5 minutes and the maximum serum level (5.9 µg/ml) was reached at 2h after the drug administration. The peak concentration (Cmax) was 5.67µg/ml and the calculated value of (tmax2.25h). The drug was absorbed from healthy broilers gut with absorption half-life (t0.5ab0.86h) and eliminated with a mean half-life (t0.5 el3.05h).
5-Single oral administration of florfenicol in control healthy broiler chickens :
The drug was firstly detected (0.73µg/ml) after 10 minutes and the maximum serum level (6.46 µg/ml) was reached at 1h after the drug administration. The peak concentration (Cmax) was 5.9µg/ml and the calculated value of (tmax1.40 h). The drug was absorbed from healthy broilers gut with absorption half-life (t0.5ab 0.51h) and eliminated with a mean half- life (t0.5 el2.72 h).
6- Multiple oral administration of florfenicol in healthy broiler chickens :
The drug was firstly detected (0.62 µg/ml) after 5 minutes and the maximum serum level (7.47 µg/ml) was reached at 2h after the drug administration. The peak concentration (Cmax) was 7.4µg/ml and the calculated value of (tmax2.3h). The drug was absorbed from healthy broilers gut with absorption half-life (t0.5ab 0.82h) and eliminated with a mean half -life (t0.5el 3.77h).
7- Single oral administration of FFC/Vit E in control healthy broiler chickens:
The drug was firstly detected (0.61µg/ml) after 5 minutes and the maximum serum level (7.55µg/ml) was reached at 2h after the drug administration. The peak concentration (Cmax) was 7.48 µg/ml and the calculated value of (tmax2.42h). The drug was absorbed from healthy broilers gut with absorption half-life (t0.5ab 0.71h) and eliminated with a mean half-life (t0.5 el4.34h).
8-Multiple oral administration of FFC/VitE in control healthy broiler chickens:
The drug was firstly detected (0.74 µg/ml) after 5 minutes and the maximum serum level (8.38 µg/ml) was reached at 2h after the drug administration. The peak concentration (Cmax) was 8.07µg/ml and the calculated value of (tmax2.5h). The drug was absorbed from healthy broilers gut with absorption half-life (t0.5ab 0.81h) and eliminated with a mean half-life (t0.5 el4.3 h).
In conclusion; both tylvalosin and florfenicol drugs evoke immunoinhibitory effects whereas, Vit E showed immunostimulatory response. In addition; Vit E not completely modulate the immunosuppressive effects of the mentioned drugs when combined with them and this may attributed to the lower dose of Vit E used in this study or due to Vit E increased the serum concentrations of the drugs when used in combination in the pharmacokinetic studies so increasing their immunosuppressive effects.
In the pharmacokinetic studies both tylvalosin and florfenicol had serum cumulative effects leading to an increase in the time of their elimination besides their combination with Vit E also increasing their serum concentration and prolongation the duration of their action for more 12 and 24 h after single and multiple administration which reflects their serum efficacy.