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One hundred and twenty 10-week-old white Leghorn Bovans chickens of average 1.250 Kg body weight were used in the present experiment. The chickens for the experiment were obtained from a commercial broiler flock with no history of health problems. The chicken provider assured that the chicken had been vaccinated against the common known diseases except vaccination against Pasteurella multocida. In addition, chosen chickens were of no history of fowl cholera. One week before the onset of the study, chickens were screened for the presence of P.
multocida by bacteriological examination of swabs from the nasopharyngeal cavity. This examination proved that all birds were free from the bacterial infection.
Chicken were housed in the experimental lab animal house at the Animal Health Research institute, Zagazig, Aharkia, Egypt, in a separate room. The disinfected room was cleaned prior to the commencement of experiment. The room floor was bedded by fresh clean sawdust forming a deep litter of four centimeters depth. Chickens were allowed to acclimatize for two weeks prior to the beginning of the experimentation.
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Birds were maintained at 20 ± 3oC. All chickens were fed with a commercial ration and water was freely available before and during the experiment.
Propolis sample (100 g) was obtained from the Agriculture Research Center (ARC) in Giza. This powder was subjected to a continuous stirring in 70% ethanol for three days. Then, the solution was filtered and the filtrate was evaporated using a Rotary evaporator (Rota Vapor). The remaining residual (extract) was then lyophilized and kept in deep freezer (-10 °C).
At time of propolis injection the lyophilized specimen was allowed to reach the room temperature. The lyophilized extract was dissolved in Dimethyl sulfoxide (DMSO). Chicken were injected with a dose of 50 mg/Kg body weight.
Pasteurella multocida strain was obtained from the Animal Health Research Institute (from Veterinary Serum and Vaccine Research Institute, Abbasia, Cairo) in the form of lyophilized ampoules. It was activated by culturing in nutrient broth, inoculation in Swiss mice, and re-isolation of the organism from heart blood of mice on Tryptic Soya agar plates (Difco) then, plated onto 10% sheep blood agar plate and incubated for 24 hr at 37°C. The inoculated plate was flooded with 5 ml physiological saline and the colonies were removed from the solid medium by gentle rubbing with a glass rod. The density was adjusted to reach the resultant organism suspension an average of 5 ? 109 bacterial cell/mL (Cruickshank et al., 1975) and injected intravenously (wing vein) in birds.
The used vaccine was a mineral oil emulsion containing local inactivated avian strains of Pasteurella multocida [Avian isolates A (5, 8 &9) and D: 2]. This vaccine was obtained from The Veterinary Serum and Vaccine Research Institute in Cairo, Egypt, in a bottle containing 500 ml (to vaccinate 1000 bird with a dose of 0.5 ml/bird). The vaccine is to be injected subcutaneously in the back of the neck of birds at 10 weeks old.
Chicks were randomly assigned into 5 equal groups (15 chicks per group). Each treatment group involved three replicates (5 birds in each replicate) in a completely randomized design. The 5 groups were subjected to the following treatments:
Group 1 (Negative Control G.): Chickens in this group were left without any special treatment and served as negative controls. They were provided with food and water ad libitum. All chickens of this group didn't subject to bacterial challenge test and were sacrificed at the end of the experiment.
Group 2 (Positive Control G.): Chickens were subcutaneously injected (back of the bird's neck) with 0.5 ml sterile phosphate buffer solution (PBS) and served as positive controls and subjected to bacterial (Pasteurella multocida) challenge test.
Group 3 (Vaccinated G.): Chickens were subcutaneously injected (back of the bird's neck) with 0.5 ml of Pasteurella multocida vaccine. These birds were also subjected to bacterial challenge test.
Group 4 (Propolis G.): Chickens were subcutaneously injected with 50 mg/Kg of Propolis. Birds of this group were also subjected to Pasteurella multocida challenge test.
Group 5 (Vaccine & Propolis G.): Chickens were subcutaneously injected with 0.5 ml of Pasteurella multocida vaccine (back of the bird's neck) and 50 mg/Kg of propolis. Following such treatment, chickens of this group were also subjected to bacterial challenge test.
Treatments with the vaccine, propolis and vaccine plus propolis were repeated as a booster dose after 3 weeks in groups 3, 4 and 5, respectively. As for the second group, the phosphate buffer was also repeated after 3 weeks.
The experiment lasted for 5 weeks and finally chickens of all groups (except group 1 involving negative controls) were challenged by injecting each chick intraperitoeally with a virulent strain of Pasteurella multocida. One week post challenge, all birds were sacrificed and examined for histopathological lesions.
Preparation of hepatic and lung tissues for light microscopy:
Immediately after sacrificing the chicken by decapitation and dissection, small slices of liver and lung tissues (1 ? 1 ? 0.5?cm) were washed in 0.9% sodium chloride solution and fixed in phosphate buffered formalin (4%, pH 7, 24 h, room temperature). Tissue samples were then dehydrated in a graded series of ethanol and cleared in xylene. After embedding in paraffin, blocks of wax were sectioned at 5 microns. Sections were mounted on glass slides and stained with haematoxylin and eosin (H & E). The stained sections were examined under low and high powered field of Nikon binocular light microscope.
Pieces of 1-mm cubes were cut from the liver and lung samples using sharp blade and quickly fixed in 6% solution of phosphate buffered gluteraldehyde pH 7.4 for 6 h at 4 °C (McDowell and Trump, 1976). After initial fixation, the tissues were washed several times in cold (4°C) 0.1 M phosphate buffer every 15 min for 2 h. The tissues were post fixed in 1% solution of cold osmium tetroxide (OsO 4) buffered with 0.1 M buffer pH 7.2 for 2 h. Then, specimens were rapidly dehydrated through ascending grades of ethyl alcohol and transferred to a 1:1 mixture of propylene oxide and epoxy araldite. Semi- thin sections (0.5?m-1µm) were cut firstly and stained with toluidine blue and viewed with light microscope to select the suitable areas for the ultramicrotomy. The ultrathin sections (60-100 nm) were cut by a glass knife with LKB microtome, and stained with uranyl acetate for 8 minutes followed by lead citrate for 4 minutes (Hayat, 1986). These sections were examined under the transmission electron microscope (JEOL 100cx, Japan) operating at 80 kV.
Phagocytosis was estimated by determining the proportion of macrophages which contain intracellular yeast cells in a random of 300 macrophages and expressed as percentage of phagocytic activity (PA). The number of phagocytized candida cells was counted in the phagocytic cells to calculate the phagocytic index according to the following equations:
Phagocytic activity (PA) = Macrophages containing yeast/Total number of Macrophages X100.
Phagocytic index (PI)= Number of cells phagocytized/Number of phagocytic cells.
Chemicals and reagents for hematological studies:
The sterile heparin solution was obtained as ampoules of 50 IU/ml blood (Nile or Amoun Co. product). The solution was used as anticoagulant for phagocytosis test. The working solution was prepared by dissolving one ampoule of 5000 I.U. in 50 ml sterile phosphate buffered solution (PBS) to obtain 100 I.U. /ml. After fertilization and sterilization, the working solution was stored at 4° C till the time of use.
Chemicals and reagents for phagocytosis and phagocytic index studies:
The disodium salt of Ethylene Diamine Tetra-acetic acid (EDTA) from Al Nasr Pharmaceutical Company was used as anticoagulant for hematological examination. EDTA was used at a dose of one drop of 10% solution/ 5 ml blood for collection of blood samples.
This medium was used to growing and obtaining Candidans albicas in yeast phase only according to the method of Wilkinson (1977).
This solution was used to re-suspension of yeast to obtain a concentration of 5 X 106 cell/ ml (Wilkinson, 1977).
The patent sterile solution was obtained from GIBCO Limited U.K. This solution was used for isolation of mononuclear leukocytic cells from chicken peripheral blood (Goddeeris et al., 1986).
The maintenance of leukocyte activity, the RPMI sterile patent medium with L-glutamine and without sodium bicarbonate (Lucy and Larry, 1982) was used (GIBCO Limited U.K.)
Phosphate buffered saline (PBS):
The buffer were prepared by mixing 8 gm NaCl + 0.2 gm KCl + 1.15 gm Na2HPO4 + 0.2 gm Kh2PO4 and added double distilled water to 1000 and dissolving the above ingredient well and sterilized by autoclaving and keep at 4?C till used.
Neutral buffered formalin 10%:
It was used for preservation of specimens collected from experimentally infected, vaccinated and contact chickens for histopathological studies.
Hematoxylin and Eosin (H&E) stain were used for staining of tissue sections for histopathological examination.
Experimental Chickens. (2019, Nov 25). Retrieved from https://studymoose.com/experimental-chickens-essay
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