Abstract: This study was designed to determine the effect of garlic powder on humoral immune response of broilers against NDV (Newcastle Disease Virus) vaccine. Two hundred and forty, two-day-old, Ross chicks were randomly assigned into 4 groups of 60 birds each. Chicks in groups 1 and 2 were given control mash diet during the experiment (6 week), but those in groups 3 and 4 were fed on control diet supplemented with 1 and 3% garlic powder, respectively. All groups except number 1 were vaccinated by eye-drop with B1 strain (Pestikal, Croatia) at 9 and 18 days of age. Ten blood samples were taken from each group on days 0, 14, 24 and 34 after first vaccination. The serum antibody level against NDV was measured by both HI and ELISA tests. The EDTA-mixed blood samples were examined for total and differential leukocyte count. The results showed that antibody titers in vaccinated chicks were significantly more than in non-vaccinated chicks (p<0.05), but not influenced by the diet (p>0.05). A significant increase of total leukocyte and percentage of lymphocytes was observed in vaccinated chicks 14 days after vaccination, but there were no difference (p>0.05) among vaccinated groups. It is concluded that inclusion of garlic powder to the diet of broilers don`t have any beneficial effect on humoral immune response to live NDV vaccine.
INTRODUCTION
Garlic was believed in the folklore of many cultures to be effective in curing ailments (Amagase et al., 2001). In the last decades, several clinical reports, including meta-analysis, have described the hypocholesterolemic effect of garlic in humans (Stevinson et al., 2000). In vitro studies have also shown that various garlic preparations have antibacterial (Bakri and Douglas, 2005), antiviral (Weber et al., 1992), antifungal (Lawson, 1996) and antiparasitic (Ankri et al., 1997) properties against human`s pathogens.
Animal studies suggested that allicin, the main biologically active component in garlic, has the potential to exhibit anti-tumor activity in mice (Patya et al., 2004). Also, Ghazanfari et al. (2002) injected mice intraperitoneally with different doses of garlic extracts and found a significant increase of delayed type hypersensitivity response, but not of antibody response, to sheep Red Blood Cell (RBC). The literature is very limited regarding the effect of garlic on immune system in broilers. Gabor et al. (1998) reported that a liquid product containing feed acidifiers, garlic and microbial cell extracts augmented the serological response to the vaccine prepared from inactivated NDV. The literature shows that studies made on garlic have been accompanied with different results in some cases. For example, Birrenkott et al. (2000) reported that 3% garlic powder did not have any significant effect on serum and yolk cholesterol when laying hens were fed diets for 8 month, whereas Chowdhury et al. (2002) observed a significantly lower serum and yolk cholesterol when laying hens were fed for 6 week on diets supplemented with 2-10% sun-dried garlic paste. Although the reason for this is unknown, it likely relates to preparation methods, the stability of chemical components and the duration of the study (Amagase et al., 2001). Therefore, the first objective of this study was to further study the effect of garlic, in powder form, on serological response of broilers to live NDV vaccine. The second objective was to examine the quantitative changes of blood leukocytes as functional parameters in immune responses.
MATERIALS AND METHODS
Experimental design: From October to November 2007, two hundred and forty, two-day-old, male Ross x Ross broiler chicks were housed in poultry research section of Shahid Chamran University (Iran) and randomly divided into 4 groups of 60 birds each. Chicks in groups 1 and 2 were given control mash diet (based on corn and soybean meal), but those in groups 3 and 4 were fed on control diet supplemented with 1 and 3% garlic powder, respectively. All chicks were fed for 6 week on rations formulated to meet the nutrient requirements of broilers (National Research Council, 1994). Feed and water were provided ad libitum. All birds except those in group 1 were vaccinated by eye-drop with B1 strain (Pestikal, Croatia) at 9 and 18 day of age. Ten blood samples were taken via wing vein from each group on days 0, 14, 24 and 34 after first vaccination. The sera were stored at -20°C until the end of the experiment. The assessment of NDV-specific antibody levels were made by conventional hemagglutination-inhibition test (4 HA unit of Ag) as per Thayer and Beard (1998) and enzyme-linked immunosorbent assay (Synbiotic kit, USA). EDTA-mixed blood samples were examined for total and differential leukocyte count using Natt and Herrick solution and Wright staining according to Dein (1986). The data were analyzed as one-way design by analysis of variance (Petrie and Watson, 2006). The significance of data was set at p<0.05.
Preparation method for dietary garlic: High quality garlic bulbs were purchased from local markets, peeled and cut into smaller pieces. Then, they were dried in oven at 50 to 60°C to produce powder. Diets were prepared the following day and were stored at room temperature for a maximum of two weeks.
RESULTS AND DISCUSSION
The results of Table 1 and 2 show that maternal antibody titer to NDV significantly decreased with age in the non-vaccinated control chicks (group 1) and reached to a very slight level at the age of 23 days, whereas it had a remarkable increase (p<0.05) in all vaccinated groups until 33 days of age. This finding suggested that humoral immune response to NDV was elicited after vaccination.
| Table 1: | Effect of dietary garlic on serum HI titer1 (Log2) in broiler chicks vaccinated against ND virus |
| a-cValues in columns/rows with no common superscript differ significantly (p<0.05), 1Values represent means±SE for each treatment; n = 10. 2Non-vaccinated control group, *First vaccination at 9 days of age | |
| Table 2: | Effect of dietary garlic on serum ELISA titer1 in broiler chicks vaccinated against ND virus |
| a-dValues in columns/rows with no common superscript differ significantly (p<0.05). 1Values represent means±SE for each treatment; n = 10. 2Non-vaccinated control group. *First vaccination at 9 days of age | |
It was observed in the current study that there were no difference (p>0.05) among vaccinated groups in anti-NDV titers (Table 1, 2). Ghazanfari et al. (2002) injected mice intraperitoneally using two sources of garlic (freshly-prepared and commercial tablet extracts) for 5 days at doses of 1-300 mg kg-1, but did not find any increase in anti-SRBC antibody level in comparison to control group. However a liquid product, developed using feed acidifiers, garlic and microbial cell extracts, made a significant rise in serological response of broilers to inactivated NDV vaccine when it was applied in a concentration of 1 mg L-1 in drinking water beginning 2-3 days before parenteral vaccination and continued for 17-20 days (Gabor et al., 1998). This discrepancy likely relates to the difference in the type of vaccine (live vs. killed), the type of garlic preparation or the presence of other components in the product applied by Gabor et al. (1998). Also, the strain of bird may affect the response to a given garlic preparation. Chowdhury et al. (2002) studied the effect of sun-dried garlic paste on reproduction parameters in different strains of laying hens and found significantly different responses in some traits among the strains. Garlic powder represent the composition of garlic cloves better than any other type of processed garlic, however some changes do occur during processing. Therefore, the other possible reason for the negative results is that the active constituents may not have been sufficient to stimulate humoral immunity when garlic powder is added to diet up to level of 3%.
A significant increase of total leukocyte and percentage of lymphocytes was observed in vaccinated chicks 14 days after vaccination (Table 3, 4), suggesting that immune response was activated by vaccine, but there were no difference (p>0.05) among vaccinated groups, being in agreement with serological finding. To the best of our knowledge, the effect of dietary garlic on hematological parameters in broilers has not been previously reported in the literature, although Dorhoi et al. (2006) reported that the standardized ethanol extract of garlic did not stimulate the proliferation of lymphocytes taken from laying hens and even impaired the phagocytic capacity of monocyte-derived macrophage culture at 200 mg L-1.
| Table 3: | Effect of dietary garlic on total leukocyte count1 in broiler chicks vaccinated against ND virus |
| a-cValues in columns with no common superscript differ significantly (p<0.05). 1Values are number of WBC/μL and represent means±SE; n = 10. 2Non-vaccinated control group. *First vaccination at 9 days of age | |
| Table 4: | Effect of dietary garlic on differential leukocyte count1 in broiler chicks vaccinated against ND virus |
| a-bValues in rows with no common superscript differ significantly (p<0.05). 1Values represent means±SE for each treatment; n = 10. *Non-vaccinated control group. **First vaccination at 9 days of age | |
Considering the results obtained from the current study, it is concluded that inclusion of garlic powder to the diet of broilers does not have any beneficial effect on serological response against NDV.
ACKNOWLEDGMENT
The authors wish to express their gratitude to research council of Shahid Chamran University for the financial support.