The effects of different levels of Aloe vera gel on ileum microflora population and immune response in broilers: a comparison to antibiotic effects

Abstract

The purpose of the present research is to study the effects of different levels of Aloe vera gel on microflora population, humoral and cellular immune response, and relative weight of lymphoid organs compared to the effects brought about by antibiotic. The groups included the control group (basal diet) as well as three groups with different levels of Aloe vera gel (added to basal diet at 1.5%, 2% and 2.5%), and finally a group with basal diet supplemented by 15 ppm virginiamycin antibiotic. Regarding ileum microflora, the increase in Aloe vera gel in the feed resulted in significant reduction in the number of E. coli colonies and significant increase in the number of Lactobacillus colonies compared to the control and antibiotic groups. As far as humoral immunity is concerned, Newcastle disease virus antibody titre on the 24th and 38th day was the highest in 2.5% and 2% Aloe vera gel groups, respectively. In addition, sheep red blood cells antibody titre reached its highest level in the 2.5% Aloe vera gel group on the days 24, 28, 34 and 38. The differences among the groups regarding sheep red blood cells antibody titre were significant on the days 24, 28 and 34, while no significant differences were observed on the 38th day. Regarding the cellular immunity, the highest response was obtained by injecting phytohemagglutinin-P and the highest relative weight of spleen and bursa of Fabricius was found in the 2.5% Aloe vera gel group. The findings of the study suggest reduction in number of E. coli colonies and increase in number of Lactobacillus colonies as a result of increased Aloe vera gel in the feed. Furthermore, the 2.5% Aloe vera gel group resulted in significantly higher levels of humoral and cellular immunity, as well as relatively greater weight of lymphoid organs, which suggests that Aloe vera gel can be a proper replacement for the virginiamycin antibiotic.

Keywords:

• Aloe vera
• microflora
• immune response
• antibiotic
• broiler

1. Introduction

In poultry industry, birds are maintained in confinement systems and large flocks in order to achieve higher economic returns. In such a situation, birds are exposed to stress and inducing factors such as high population, vaccination and temperature changes. Intestinal microflora is influenced, and sometimes imbalanced, by these factors, leading to deteriorated health conditions for chickens. Therefore, the imbalance in intestinal microflora may result in weakened immune system and failure of growth performance in chickens. Additives, such as antibiotics, may assist in dealing with such problems. Further more long-term inclusion of antibiotics in animals and poultry diets can result in increased microorganism resistance to such groups in human body. Thus, antibiotics are banned from being added to feed in EU and some other countries.

This has led researchers to attempt finding alternatives to antibiotics. They have found different replacements for growth-stimulating antibiotics such as probiotics, prebiotics, organic acids and herbal medicines during recent years (Griggs and Jacob 2005). Depending on the composition of mixtures, herbal medicines have a variety of properties. The active compounds of herbs can be found in the form of glycosides, alkaloids, volatile oils and organic acids, and more importantly in polysaccharides (Li 2000).

Yuan et al. (1993) suggested that polysaccharides found in herbal and fungal medicines have antibacterial effects. Polysaccharides act as immunomodulators and even perform antimicrobial activities (Xia and Cheng 1988). It has been reported that polysaccharides can be used as feed supplements or as booster in vaccination. Antibacterial (Yuan et al. 1993), antiviral (Wei et al. 1997; Liu et al. 1999) and antiparasitic (Pang et al. 2000) effects of polysaccharides on broilers have also been reported.

Aloe vera is one of the most well-known herbal medicines with various properties such as healing, anti-inflammatory, anti-oxidative, anti-diabetic and immunomodulatory effects as well as antibacterial, antifungal and antiparasitic properties (Waihenya et al. 2002; Boudreau and Beland 2006). Studies suggest that chemical composition of Aloe plant, to a large extent, depends on the species analysed. High water content (98.5–99.5% of fresh matter) is an important characteristic of Aloe vera filet. In addition, polysaccharide constitutes over 60% of its remaining solid (McAnalley 1993). Aloe vera contains different types of polysaccharides most significantly acemannan. Several studies confirmed the immunomodulatory effects of acemannan. As a polymer formed by mannose, acemannan can attach to mannose receptors in macrophages which tend to bind with carbohydrate structures containing mannose (Tietze et al. 1982; Lee 1988). Such bonds activate microphages and in turn release cytokines. Recent studies on acemannan extracted from Aloe vera gel suggested that acemannan can enhance intestinal microflora population, including the number of Lactobacillus colonies, in broilers (Lin et al. 2005).

Antimicrobial properties of herbs, therefore, have significant effects on improvement of immune response and growth performance through increasing the number of useful bacteria (e.g. Lactobacillus and Bifidobacteria). Thus, the present study attempts to study the effects of different levels of Aloe vera gel on ileum microflora population, humoral immune response, cellular immunity and relative weight of lymphoid organs in broilers compared to those effects of antibiotic.

2. Material and methods

2.1. Experimental animals and design, and feed preparation

The study was conducted at Poultry Farming Research Hall of Agriculture College, Islamic Azad University – Karaj Branch. Three hundred one-day old Ross 308 male broilers were studied here. Experimental diets were fed from 1 to 42 days of age. Feed and water were available ad libitum.

Diets were determined according to Ross requirements manual and using UFFDA software. The starter diet with 3334 kcal ME/kg and 25.12% crude protein (CP) was fed for the 1 to 10 days. A grower diet with 3150 kcal ME/kg and 22.10% CP was fed from 11 to 24 days and a finisher diet with 3200 kcal ME/kg and 19.76% CP was fed from 25 to 42 days. The birds were randomly distributed into five groups with four replicates (each consisting of 15 broilers). The control group was fed by basal diet with no additive; the feed for the next three groups were treated by 1.5%, 2% and 2.5% Aloe vera gel respectively, and the fifth group was fed by a diet containing 15 ppm antibiotic (virginiamycin).

Aloe vera gel was purchased from Barij Essence Company. The pH of Aloe vera gel was 4.49. It was colourless with negative microbial load. It should be mentioned that the feeds were mixed with Aloe vera gel, then they were fed to the chickens at the beginning of each farming period (starter, grower and finisher). First, Aloe vera gel was mixed with soybean meal, later on, corn and other additives were added.

2.2. Microbial enumeration

On the day 42, two male broilers were randomly selected from each replicate. The birds were sacrificed and 1 g of their ileum content was immediately mixed with 9 ml phosphate buffer saline (PBS). Samples were serially diluted from 10⁻¹ to 10⁻⁷. Lactobacillus count was performed in MRS (de Man, Rogosa and Sharpe) agar after incubation in anaerobic chamber at 37°C for 48 h. The E. coli count was performed in MacConkey agar after aerobic incubation at 37°C for 24 h. The numbers of colony forming unit (CFU) were expressed as log₁₀ CFU per gram.

2.3. Humoral immune response

Vaccination against Newcastle disease virus (NDV) was performed on day 9 using eye drop (Live B strain) and subcutaneous injection was done to all chicks. As a booster, Newcastle vaccine (Lasota) was fed to broilers on the 16th day using drinking water. Eight to 22 days after vaccination, two male broilers were picked out randomly from each replicate and the samples were taken from the vein under their right wing. The immune response was assessed by hemagglutination inhibition (HI) test. On the days 21 and 31, 1 ml sheep red blood cells (SRBC) (3%) washed in sterile PBS was injected to two male broilers from each replicate through the vein under the left wing. Three and 7 days after the injection, 1 ml blood sample was taken from the same broilers through the vein under the right wing. To determine SRBC antibody titre, micro-titre hemagglutination was used (Wegmann and Smithies 1966). The SRBC and NDV antibody titres were expressed as log₂.

2.4. Cell-mediated immunity

The cellular immune response was assessed following cutaneous basophilic hypersensitivity test in vivo by injecting phytohemagglutinin-P (PHA-P). On the 38th day, two birds were randomly selected from each replicate. The toe thickness between the third and fourth interdigital space of the left and right feet was measured using a micrometer. Immediately after the measurement, 100 µg of PHA-P, suspended in 0.1 ml of PBS, was injected into the right toe and 0.1 ml of PBS into the left foot (control). The web swelling of both feet was measured after 24 h of injection. The response was determined by subtracting the skin thickness of the first measurement from the second, and the values of the left foot (control) from the right foot (Corrier and DeLoach 1990). On the day 42, two birds per replicate from each group were sacrificed and lymphoid organs (spleen and bursa) were collected, weighed and expressed as percent live weight.

2.5. Statistics

The data were analysed by General Linear Model (GLM) procedure for completely randomised design with five groups and four replicates using the SAS software (1996) and the means were compared with Duncan's Multiple Range test at the level of P<0.05.

3. Results and discussion

Table 1 shows the effect of groups on ileum microflora population. The increase in Aloe vera gel contained in the feed results in significant linear reduction in number of E. coli colonies as well as significant linear increase in number of Lactobacillus colonies compared to the antibiotic and control groups (P<0.05). The increase in the number of Lactobacillus bacteria in Aloe vera gel groups confirms antibacterial effects of Aloe vera, which stimulates the growth of useful intestinal flora and reduces the presence of gram-negative bacteria.

Table 1. The ileum microflora population of broilers fed with Aloe vera gel and antibiotic.

Groups	Counts of E. coli (log10 CFU/g)	Counts of Lactobacillus (log10 CFU/g)
CON	7.18^a	4.63^b
1.5% A.V	4.36^bc	5.82^a
2% A.V	4.26^c	5.88^a
2.5% A.V	4.19^c	6.02^a
15 ppm VM	4.44^b	4.69^b
SEM	0.32	0.35
Note: Means with different superscripts within the column are significantly different (P<0.05).
CON, control; A.V, Aloe vera gel; VM, virginiamycin.

Guo et al. (2004) reported that polysaccharides contained in mushrooms and herbal medicines can improve intestinal microflora population, reduce harmful bacteria count (e.g. E. coli) and increase the number of beneficial bacteria (e.g. Lactobacillus). On the other hand, Willis et al. (2009) found that herb polysaccharides had effects similar to those of prebiotic.

It can be concluded that polysaccharides contained in Aloe vera (particularly acemannan) have effects similar to those of prebiotics; that is, they increase the number of Lactobacillus colonies and reduce gram-negative bacteria. Previous studies show that acemannan added to the broiler diet decreased the number of intestinal E. coli colonies (Lin et al. 2005). In fact, short-chain fatty acids, as the final product of Lactobacillus fermentation, can lower intestinal pH and make the environment unfavourable for gram-negative bacteria.

The effects of different groups on humoral immune response are presented in Table 2. The highest SRBC antibody titre during the first period (24th day) was observed in the 2.5% Aloe vera gel group showing no significant difference from the antibiotic group (P>0.05). However, significant increase was observed and compared to the control group (P<0.05). In addition, no significant difference was observed between different levels of Aloe vera gel groups (P>0.05). On the 28th and 34th days, the highest antibody titre for SRBC was that of the 2.5% Aloe vera gel group showing a significant difference from the control and antibiotic groups (P<0.05). No significant difference was observed among the Aloe vera gel groups on the 28th and 34th days regarding the antibody titre for SRBC (P>0.05). On the 38th day, there was no significant difference between the groups (P>0.05), but the highest antibody titre was that of the 2.5% Aloe vera gel group.

Table 2. The humoral immune response of broilers fed with Aloe vera gel and antibiotic.

	Antibody response to SRBC (log2)				Antibody response to NDV (log2)
Groups	24 days	28 days	34 days	38 days	24 days	38 days
CON	1.75^b	4.75^b	6.62^bc	11.25	1.75^c	1.62^c
1.5% A.V	3.25^a	5.50^ab	7.37^a	11.12	2.12^bc	2.25^bc
2% A.V	3.25^a	6.12^a	7.25^ab	11.50	2.5^b	3.62^a
2.5% A.V	3.37^a	6.25^a	7.50^a	11.87	3.12^a	3.25^a
15 ppm VM	3.12^a	4.87^b	6.50^b	10.62	2.62^ab	2.37^b
SEM	0.159	0.245	0.200	0.364	0.182	0.222
Note: Means with different superscripts within the column are significantly different (P<0.05).
CON, control; A.V, Aloe vera gel; VM, virginiamycin; SRBC, sheep red blood cells; NDV, Newcastle disease virus.

Antibody titre for NDV on the 24th day was the highest in the 2.5% Aloe vera gel group showing no significant difference from the antibiotic group (P>0.05), while a significant difference was observed between the control group and the 1.5 and 2% Aloe vera gel groups (P<0.05). On the 38th day, the highest antibody titre for NDV was observed in the 2% Aloe vera gel group showing significant difference compared to the control and antibiotic groups (P<0.05). Studies suggest significant increase in NDV antibody titre in broilers treated with 2% Aloe vera (solved in their drinking water) compared to control group (Valle-paraso et al. 2005). In addition, it has been shown that adding acemannan to Newcastle vaccine significantly increases antibody titre on the 21st day after vaccination (Chinnah et al. 1992).

Enhanced humoral immunity can be attributed to polysaccharides (acemannan) contained in Aloe vera gel. Several studies confirm the role of polysaccharides contained in herbs and mushrooms in stimulation of immune system. Polysaccharides can increase cytokines and antibodies, and enhance the performance of natural killers and B- and T- lymphocytes (Nie and Zhang 1999). In general, polysaccharides can affect the humoral immune response and cellular immunity (Lien and Gao 1990).

The effects of acemannan on immunity system have been studied in several researches. As a polymer formed by mannose, acemannan can attach to mannose receptors in macrophages which tend to bind with carbohydrate structures containing mannose (Tietze et al. 1982; Lee 1988). Such bonds activate microphages and release growth factors such as Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) that controls the growth and divisions of cells which act as myeloid precursor. In addition, acemannan contained in Aloe vera can release cytokines such as IL-1, IL-6, IL-12 and TNF-α (Tumour Necrosis Factor) from macrophages (Wombel and Helderman 1988; Ramamoorthy et al. 1996; Zhang and Tizard 1996). This can stimulate the growth of B lymphocytes and increase the number of T lymphocytes.

The highest response to PHA-P was observed in the 2.5% Aloe vera gel group with a significant difference compared to the antibiotic and control groups (P<0.05). The lowest response was that of the antibiotic group. Considering the role of acemannan in increased activity of macrophages and lymphocytes, the improved response to PHA-P Aloe vera gel groups was expectable.

In general, the increased relative weight of lymphoid organs in Aloe vera gel groups may reflect the improved immunity (humoral and cellular) against antigens. As seen in Table 3, the greatest relative weight of lymphoid organs was that of the 2.5% Aloe vera gel group which can be the result of increased T- and B-lymphocytes.

Table 3. The cellular immune response and relative weight of lymphoid organs of broilers fed with Aloe vera gel and antibiotic.

		Relative lymphoid organ weights (g/100 g body weight)
	Increase in skin thickness (mm) to PHA-P	Spleen	Bursa
Groups	38 days	42 days	42 days
CON	0.50^c	0.072^a	0.094^b
1.5% A.V	0.70^ab	0.102^a	0.110^ab
2% A.V	0.63^b	0.101^a	0.122^a
2.5% A.V	0.78^a	0.109^a	0.128^a
15 ppm VM	0.44^c	0.077^b	0.097^b
SEM	0.036	0.0043	0.0297
Note: Means with different superscripts within the column are significantly different (P<0.05). CON, control; A.V, Aloe vera gel; VM, virginiamycin; PHA-P, phytohemagglutinin-P.

Spleen is the secondary lymphoid organ responding to antigens present. Spleen plays an important role in antibody generation and cellular immunity (Cyster 2005). Therefore, the increased weight of spleen in Aloe vera gel groups, particularly in the 2.5% Aloe vera gel group, shows the increased amount of lymphocytes in this organ which can be a sign of enhanced humoral and cellular immune response in the 2.5% Aloe vera gel group. Lin et al. (2005) reported that 0.1% polysaccharide added to basal diet can increase relative weight of thymus and bursa of Fabricius compared to the control group.

Cook and Samman (1996) reported that indirect positive effects of herbal medicines on immune system. Since Lactobacillus can stimulate immune system, herbs are expected to enhance the performance of immune system by increasing Lactobacillus. Numerous studies have been carried out on the antibacterial properties of Aloe vera. This study have revealed the role of acemannan in increasing intestinal Lactobacillus colonies and reduction in E. coli colonies in broilers (Lin et al. 2005). As already mentioned, the higher amount of Aloe vera gel contained in diets resulted in increased Lactobacillus colonies while decreasing the number of E. coli colonies; Since short-chain fatty acids, as the final product of fermentation, are generated by Lactobacillus, they can lower the intestinal pH and make the environment unfavourable for gram-negative bacteria. In effect, Aloe vera can indirectly enhance immune response by removing gram-negative bacteria.

4. Conclusion

The findings of this study suggest that increased level of Aloe vera gel lowers E. coli colonies and increases the number of Lactobacillus colonies; a process that may improve intestinal microflora and indirectly enhance the performance immune system through elimination of pathogens. In addition, an improved performance of immune system was observed in Aloe vera gel groups compared to the control and antibiotic groups. Moreover, the 2.5% Aloe vera gel group represented significantly heavier lymphoid organs, and improved both humoral and cellular immune responses. Therefore, the 2.5% Aloe vera gel group can be suggested as the best replacement for the antibiotic virginiamycin.

Acknowledgements

The authors wish to thank Manager and Personnel of Barij Essence Pharmaceutical Co. for providing them with Aloe vera gel required in this study.