Immuno-Modulatory Activity of Aqueous Leaf Extract of Moringa Oleifera in Brioler Chickens

This experiment was conducted to investigate the immuno-modulatory activity of aqueous leaf extract of Moringa oleifera on immune response of broiler chickens to Newcastle disease (ND) vaccinations. The performance, blood parameters and serum biochemistry of the birds were also determined. A total of one hundred and twenty (120) day-old commercial broiler chicks were randomly allotted to 3 dietary treatments of 4 replicates each. Each replicate had 10 birds. The treatments: T1 – Control group in which the birds were not given any extract; T2 and T3 – birds in these groups were given the prepared stock solution of Moringa oleifera leaf extract at dose rate of 2500mg/kg and 5000mg/kg of body weight in drinking water. The experimental birds were vaccinated with ND vaccines using a stipulated vaccination regime. The Moringa oleifera leaf extract exhibited significant (p≤0.05) influence on final body weight of the experimental broiler chickens with birds in T3 having an average weight of 1947.43g and birds in T1had 1733.33g. The immune modulating effect of the leaf extract was insignificant (p≥0.05) though it elicited higher antibody titre of Log27 and Log29 in birds in T3 compared to Log26 and Log28 of birds in control group after the first and second ND vaccinations respectively. The leaf extract caused significant (p≤0.05) increase in white blood cells and leucocytes count. The study concluded that the plant extract had slight immune stimulatory effects on response to ND vaccinations and improved the growth performance of broiler chickens. Keywords— Antibody, aqueous, growth, immune response, Moringa oleifera.


INTRODUCTION
Poultry production provides base for the socioeconomic advancement in the majority of developing countries and this has led to increased demand for poultry products especially broiler meat. This is because consumers perceive that it is a healthy product that contains less fat, predominantly unsaturated fatty acids, and particularly polyunsaturated fatty acids, compared to beef or pork products. The continuing survival and growth of the broiler chicken industry in developing countries of the world depends on its ability to compete globally, which is largely dependent on the efficiency of its production system. It is a common practice in the management of poultry to administer antibiotics in drinking water as growth promoters and to prevent or control infectious bacterial diseases. The benefit of such practice is to maintain good health, suppress mortality of birds, and to support maximal growth via improved utilization of nutrients and ultimately improve profit (Zeweil et al., 2006). However, the use of synthetically-produced substances especially antibiotic growth promoters was soon found to have objectionable side-effects (Makanjuola et al., 2014). This has led to antibiotics growth promoter being banned mainly due not only to cross-resistance but also to multiple resistances. Therefore due to the desire for improved economic status in poultry production, researchers revolutionized the application of feed and water additives by focusing on organic or natural supplements instead of using synthetic medicament (Zeweil et al., 2006). The use of medicinal plant either alone or in group (combination) as possible therapeutic measures has become a subject of active scientific investigation (Oyewole, 2012). Some medicinal plant products are known to enhance natural resistance of host to infection due to the presence of bioactive phytochemicals or phyto-nutrients (Soetan and Oyewole, 2009). Moringa (Moringa oleifera Lam.) is a multipurpose tropical tree and it has been dubbed the "miracle tree" or "tree of life" in popular media (Bosch, 2004;Orwa et al., 2009;Radovich, 2013; FAO, 2014) mainly because it is used for food and has numerous industrial, medicinal and agricultural uses, including animal feeding. Moringa leaves have been reported to be a rich source of βcarotene, protein, vitamin C, calcium and potassium and act as a good source of natural antioxidant compounds such as flavonoids, phenolics and carotenoids (Anwar and Bhanger, 2003). It is one of the herbs containing bioceutical agents that could substitute synthetic growth enhancers and supplements in broiler and other livestock production since it possesses important medicinal properties which include antibacterial and antifungal activities (Nickon et al., 2008). ). In Nigeria, leaf preparations of Moringa oleifera is widely used in folklore for the treatment of immune system related disorders (Oyewo et al., 2013). This study was done to investigate the effect of aqueous leaf extraction of Moringa oleifera in drinking water of broiler chickens on their growth performance and immune response to Newcastle disease vaccinations.

Aqueous Leaf Extraction of Moringa oleifera
Fresh leaves of the plant were harvested and air-dryed under normal environmental conditions. The air-dried leaves were ground before extraction and soaked in distilled water for 24 hours using ratio 1:2 (weight/volume). The preparation was then filtered to separate the debris and filtrate using Whatman's filter paper. The filtrate was collected, the solvent was removed using rotary evaporator and the residue obtained after evaporation was weighed. The concentrated stock solution of Moringa leaf extract was prepared by dissolving 500g of the residue in 1 litre of sterile distilled water and stored at 4 o C. The concentrated extract at calculated doses was administered in fresh drinking water which was served to the birds on a daily basis during the period of study.

Experimental design and Animal Management
A total of one hundred and twenty (120) day-old broiler chicks of the Abor acre breed purchased from a reputable hatchery in Akure, Ondo State, Nigeria were used for the study which lasted for a period of 6 weeks. The birds were reared on deep litter using routine management procedures as outlined by the Teaching and Research Farm of FUTA and feed and water provided ad libitum. The chicks were divided into three treatment groups (T1, T2 and T3) with four replicates of 10 birds each using a completely randomized design. Birds in control group (T1) were not given any extract while birds in T2 and T3 received the prepared stock solution of Moringa oleifera leaf extract at dose rate of 2500mg/kg and 5000mg/kg of body weight respectively in their drinking water. The experimental chickens were vaccinated with Newcastle disease vaccines (NDV) -NDV intra-ocular (Hithner B1 strain) at 3 days old and NDV LaSota via the oral route at 28 days old. The study was conducted for a period of 6 weeks.

Performance criteria measurement:
Initial weight of birds was measured at day old, then on a weekly basis for the final weight. Thereafter weight gain for each week over the trial period was measured as the difference between the initial weight and the final weight. The feed consumption was recorded per replicate and the feed conversion ratio calculated as a ratio of feed consumed to weight gain of birds per replicate.

Blood and Sera collection:
Samples of blood for the purpose of serum analysis were collected from 3 birds per replicate in each treatment group before the trial commenced via the heart to determine baseline maternal antibody titre levels against Newcastle disease. The birds were sedated using chloroform before the bleeding exercise. Thereafter, in each treatment 12 birds (3 per replicate) were randomly selected and blood was collected 14 days after administering each of the ND vaccines through the jugular vein for serological analysis to determine the antibody titre values. At the end of the 6 weeks experimental period blood was also collected for haematological and serum protein biochemistry analysis from 12 birds in each treatment.

Laboratory Analysis 2.6.1 Haematological Analysis
Erythrocyte sedimentation rate (ESR), packed cell volume (PCV), red blood cell count (RBC), haemoglobin concentration (HB) and white blood cell differentials were analysed as described by (Lamb, 1981). The Mean Corpuscular Haemoglobin Concentration (MCHC), Mean Corpuscular Haemoglobin (MCH) and the Mean Corpuscular Volume (MCV) were also calculated accordingly.

Haemagglutination and Haemagglutination Inhibition Test (HA/HI Test)
Serum samples taken from the experimental broiler chickens were analysed using beta (β) micro haemagglutination inhibition technique as described by Thayer and Beard, (1998) to determine the antibody titre levels. The antibody titre is a measure of the humoral immune response elicited in the experimental birds to Newcastle disease vaccinations.

Serum Biochemical Analysis
Diagnostic kits (Randox Laboratories, UK Test Kits) was used to analyse serum biochemical parameters (Total protein, Globulin, Albumin, Alanine transferase, Alaninie phosphatise, Aspartate transaminase ) of the experimental broiler chickens.

Statistical Analysis
Data obtained were subjected to one-way analysis of variance (ANOVA). Where significant differences were observed, the mean were separated using the SAS statistical package (2012).  Table 1 presents the performance characteristics of the experimental broiler chickens. The administration of the Moringa oleifera leaf extract resulted in significant (p≤0.05) increase in the FBW and WG of experimental birds. The FBW for birds in treatment 3 (1947.43g) was the highest and significantly (p≤0.05) different from that of birds in treatment 1 (1733.33g). The WG result followed a similar trend with birds in T3 gaining an average weight of 1911.43g and birds in T1 having 1733.33g. The table also shows that the FBW and WG of birds given the leaf extract (birds in T1 and T2) were not significantly (p≥0.05) different regardless of the dose. The FI and FCR though not significantly (p≥0.05) influenced by administration of leaf extract were seen to be highest as the dose increased.

Immunological Responses
In Table 2 the antibody titre values of the experimental birds in response to ND vaccinations are shown. The table revealed that the birds had a uniform maternal antibody titre value of Log24 across the treatments. However after the ND vaccinations, administration of the leaf extracts elicited higher titre values with no significant (p≥0.05) effect with increasing dose. The birds in T2 and T3 had similar antibody titre values of Log27 and Log29 while those in T1 had Log26 and Log28 after the first and second ND vaccinations respectively.

Haematological Parameters
The blood parameters of the experimental broiler chickens are shown in Table 3. In all the indices measured only the WBC and lymphocyte counts were significantly (p≤0.05) influenced by administration of the leaf extracts.
The WBC values of birds in T2 (2.61×10 6 mm 3 ) and T3 (2.66×10 6 mm 3 ) were similar but significantly (p≤0.05) different from that of birds in T1 (2.51×10 6 mm 3 ). The lymphocyte counts also followed similar trend. The PCV and MCV values though not significantly (p≥0.05) different amongst treatments decreased with higher doses of the leaf extracts. It was observed that the ESR, RBC, Hb, MCH, MCHC values increased in experimental birds with no significant (p≥0.05) differences among the doses.

Serum Biochemistry
The trend of the serum biochemical parameters of the broiler chickens measured following administration of the leaf extract are presented in Table 4. The leaf extracts did not have any significant (p≥0.05) influence on serum indices of the experimental birds. An increase was observed in protein indices (Total protein, albumin and globulin) of the birds administered the leaf extract which was not dose dependent.

IV. DISCUSSION
The immuno-modulatory effects of aqueous leaf extracts of Moringa oleifera in broiler chickens in response to Newcastle disease vaccinations as investigated in this present study is negligible. However, the birds given the In this study, inclusion of Moringa oleifera leaf extract in water of broiler chickens influenced the performance characteristics. The leaf extracts caused improved productive performance of the experimental broiler chickens judging from the trend of the growth performance traits. This was made obvious, in that birds given the highest dose of Moringa oleifera extract exhibited the best performance characteristics which reflected in their body weights. This could be attributed to complete amino acids, considerable amount of vitamins, and mineral content; antioxidant, immunostimulant and antibacterial properties of Moringa oleifera leaf (Makkar and Becker, 1997;Fahey, 2005;Anwar et al., 2007). It could also be related to the effect of the available nutritional components and some growth stimulating constituents of Moringa oleifera which probably resulted in the improvement of live body weight of the chickens (Kakengi et al., 2007). In addition, the minute quantity of anti-nutritional factors that affect palatability of feeds were not implicated to compromise the bioavailability of nutrients and growth stimulating compounds present in Moringa oleifera leaves (Foidl et  al., 2001). It has also being reported that the crude extract of Moringa oleifera like other herbal drug may contain digestion enhancing properties which stimulates favorable growth of good bacteria while decreasing harmful microorganisms (Hernandez et al., 2004). However, the mechanism by which this herbal product influences the growth performance and gut micro flora of poultry are poorly understood (Hernandez et al., 2004). The aqueous leaf extract of Moringa oleifera led to a boost in the total white blood cell and lymphocytes counts in experimental broiler chickens. This further reiterates it's immunomodulation capability, since white blood cells are involved in fighting infection and clearing off injured or dead cells and tissues in body (Jeremy et al., 2001; Oyewo et al., 2013). The trend in antibodies produced in response to ND vaccinations in experimental birds also supports the result of the total white blood cell and lymphocyte counts. The other haematological parameters were not influenced by the leaf extract and values obtained fell within the normal range for healthy chickens as described by Animashahun et al (2006). This indicates that aqueous extract of Moringa oleifera leaves did not have any detrimental effect on physiological indices measured. This is however different from the study of Oyewo et al., (2013) where it was reported that the administration of aqueous leaf extract of Moringa oleifera induced anaemia in wistar rats which might have been caused by haemolysis of erythrocytes, due to the levels of saponins and heavy metals (lead) in the aqueous leaf extract. A note of caution was therefore suggested in the use of the crude extract of Moringa oleifera in high doses because of the levels of heavy metals in the aqueous leaf extract. Evaluation of serum biochemical parameters may provide useful information in the assessment of health status of birds and serves to reflect many metabolic alterations of organs and tissues when feeding unconventional feed sources (Kudair and Al-Hussary 2010). In this study, the serum biochemical parameters of the experimental chickens was not influenced by leaf extracts of Moringa oliefera. This result of the serum biochemical profile is not in line with the previous work of Adedapo et al (2009) who reported significant effect of Moringa oleifera extracts on serum ALT, AST and ALP which led him to conclude that high doses of the aqueous extract could lead to liver damage. It is however supported by the work of (Kudair and Al-Hussary 2010) that reported that Moringa oleifera leaf has no significant effect on the serum biochemical indices of broiler chickens.

V.
CONCLUSION It can be concluded in this study that Moringa oleifera leaf extracts served to moderately boost immunological responses to ND vaccinations, though increasing the dose did not enhance the suggested immunomodulatory activity. It also achieved improved productive performance in the broiler chickens. However, there is need for further investigation to reiterate the capability of Moringa oleifera aqueous leaf extract as a substitute to synthetic supplements such as antibiotics in improving broiler production.