Effect of Aloe vera Gel Extract on the Haematological Parameters in White Leghorn Chicks Vaccinated Against Infectious Bursal Disease Virus

1Immunology Section, ICAR-Indian Veterinary Research Institute, 2Avian Diseases Section, Division of Pathology, ICAR-Indian Veterinary Research Institute, 3Division of Biological Standardization, ICAR-Indian Veterinary Research Institute 4Division of Physiology and Reproduction, ICAR-Central Avian Research Institute 5Division of Animal Reproduction, ICARIndian Veterinary Research Institute 6Division of Veterinary Biotechnology, ICARIndian Veterinary Research Institute, Izatnagar, Uttar Pradesh 243 122, India.

Poultry industry is one of the fastest growing enterprises of agriculture sector and has captured large volume of meat and egg market all over the world.Poultry products are the most affordable class of animal protein available.However, this industry faces challenges in protection of occurrence of various diseases that seem to totally wipe out poultry population or severely affect their growth and production rate.Infectious bursal disease (IBD) is one such economically important disease due to its direct (mortality) and indirect effects (immuno suppression, leading to susceptibility to other microbial pathogens) in young chicken of age group 3-6 weeks old 1 .IBD is an acute and highly contagious viral disease which affects both broilers as well as layers caused by Infectious bursal disease virus (IBDV), a double stranded RNA virus belonging to the family Birnaviridae 2 .The IBD virus is a non-enveloped virion with icosahedral symmetry and a diameter ranging from 55-65 nm 3 .Two subtypes of IBDV occur namely, variant and classical forms.Three pathotypes of classical forms are attenuated, virulent and very virulent (vv).Two serotypes of IBDV (based on virulence) occur in chicken viz.virulent serotype 1 and avirulent serotype 2 4 .
The vvIBDV strains causes mortality of about 50-60% in laying hens, 25-30% in broilers and 10-100% in susceptible leghorns 5 and causes depletion of B lymphocytes in bursal follicles, which results in immune-suppression 5 .Vaccination by live (based on classical virulent strains) and inactivated vaccines is used to protect or control this disease.Live vaccines are of three types viz."mild", "intermediate" and "intermediate plus".Mild type vaccine cannot overcome the effect of maternally derived antibodies (MDA) against vvIBDV whereas the other two types are effective even in the presence of MDA but lead to certain degree of bursal atrophy thereby causing immunosuppression 6,7 .In order to overcome such untoward effects and to enhance the effectiveness of vaccine, various herbs and herbal preparations have been evaluated for their immunomodulatory activity and found to be effective 8,9,10,11 .The use of antibiotic growth promoter (AGP) in the poultry ration has been banned in the recent past because it leads to antibiotic resistance in animal and human body 12 .So, the focus is now shifted to find potential alternative to AGP by studying various additives like probiotics, prebiotics and herbs 13,14,15 .In this line, the Aloe vera is a semi-tropical plant that belongs to family Asphodelaceae and has been known since time immemorial for various health benefits 16 .It is a well accepted medicinal plant with proved wide range of properties like anti-bacterial, anti-viral, anti-inflammatory, anti-parasitic, anticancer, wound healing, anti-oxidants and also very good immunomodulating potential 17,18 .The Aloe vera has been explored for its therapeutic property against poultry diseases like Coccidiosis, Fowl Typhoid and Newcastle disease 19 .The gel of Aloe species contains certain immunomodulatory components such as aloctin A and acemannan 20,21 .Immunomodulatory potential of Aloe vera is mainly attributed to the increased leukocyte infiltration at the site of injury, activating macrophages to secrete nitric oxide, stimulating the production of IL-1 in turn boosting Th1/Th2 immune response and other cytokines 22 .Several studies have shown that it can stimulate the immune system of chickens as well 23,24 .Thus, the extract of Aloe vera can be explored to boost the health and immune system of birds vaccinated against infectious agents.Hence, the present study Aloe vera aimed to assess the changes in the haematological parameters and individual cell counts at different period of its administration in white leghorn chicks.

Experimental chickens and housing
Specific pathogen free (SPF) eggs (n=55) for the present study were procured from M/S Venkateshwara Hatcheries Group Limited (VHL), Pune.These eggs were incubated and hatched in Hatchery Unit of ICAR-Central Avian Research Institute, Izatnagar.The hatched out White leghorn chicks (n=48) were housed under standard managemental conditions in the isolation sheds of Avian Disease Section, Division of Pathology, I.V.R.I, Izatnagar.The experiment was carried out as per the approved protocol of the Institute Animal Ethics Committee.

Aloe vera plant
The Aloe vera plants were procured from local Nursery at Gandhi Puram, Izatnagar.The leaves were washed thoroughly with warm water, spines removed by trimming and the gel from leaves was separated by filleting the skin.Then, the separated gel was homogenised in homogenizer.The homogenized gel was purified by centrifugation at 10,000 rpm at 4 °C for 30 minutes 25 to separate the fibre and pure gel layer.The top pure and clear gel was collected without disturbing fibre content and stored at 4°C until further use.

Experimental design
Forty-eight SPF day-old Babcock chicks were randomly divided into four groups (n= 12 chicks/group).The chicks were reared under uniform managemental conditions for a period of 45 days.The chicks were divided as into four experimental groups: group A (control-only drinking water), group B (Aloe vera treated only), group C (vaccinated with IBDV vaccine), and group D (IBDV vaccinated and Aloe vera treated).IBD Vaccine (Live intermediate plus strain) was administered through intra ocular route at second week of age and Aloe vera gel was administrated @ 3% through drinking water from day old to the entire study period (45 days).

Sample collection and analysis
Blood samples (~1.5 ml) were collected in EDTA anticoagulant from different groups of experimental chicks on 7, 14, 21 and 28 day post IBDV vaccination.The hematological parameters comprising of white blood corpuscles (WBC) count, red blood cells (RBC) count, total leukocyte (TLC) count (Lymphocytes, Monocytes, Heterophils), Hemoglobin (Hb), packed cell volume (PCV), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) were analyzed using Automatic method (automatic cell counter) in Vet Hematology analyzer (Abacus junior, Radim, Italy) after putting the samples on electric mixer.

Statistical analysis
All the data obtained were analyzed using the statistical software SPSS®™ 20.0 (IBM, Corp. USA).Two-way analysis of variance (ANOVA) was used to determine the statistically significant differences in mean values between the groups.The means were subjected to Duncan Multiple range test and values with p<0.05 were considered significant.

RESULTS AND DISCUSSION
The results of hematological parameters at 7, 14, 21 and 28 day post IBDV vaccination for all the four experimental groups are summarized in Table .1 and 2. The blood indices are a fundamental tool used widely to monitor the effects of nutrition, therapy and environment in veterinary medicine 26 .The groups (B, C, and D) showed significant (P<0.05)difference in WBC count on 7, 14, 21 and 28 th day post vaccination (DPV).Especially, the group D (IBDV vaccinated plus Aloe vera) showed significantly higher (Pd"0.01)WBC count (21.55,  19.34, 26.79 and 28.38 X 10 3 cell/µl) than the control group (16.99, 12.35, 13.29 and 12.21 X 10 3 cell/µl) during the study period.The reason for higher WBC count in group B, C, and D might be due to the stimulation of immune response cells by both vaccine as well as by Aloe vera gel.Lymphocyte count was found to be highest in the group D ( 23.43 and 21.74 X 10 3 cell/µl) on 21 and 28 DPV, respectively and it was significantly higher (p<0.05)than all other group.Although, the group B (16.84,12.64X 10 3 cells/µl) failed to show any significant difference with group A (15.06, 11.81 X 10 3 /µl) on 7 and 21 DPV in terms of lymphocyte count, but it revealed its additive effects on hematological cells when combined with vaccine during entire study period.Our results are consistent with the findings of Valle et al. 27 who reported that inclusion of 2% Aloe vera gel in drinking water showed an increase in total WBC and lymphocyte counts in broilers vaccinated against Newcastle disease.Similarly, Darabighane et al. 28 observed that inclusion of Aloe vera gel in feed of broiler ration showed increased the WBC counts.In another study, the extract of Aloe vera gel @ 300 mg/ml administered intra-peritoneally in Swiss albino mice showed significantly (P<0.01)higher lymphoproliferative response than control group 29 .In addition, the turkey poults receiving 20 ml/l of Aloe vera gel have also been documented with significantly higher WBC and Lymphocytes counts than control group 30 .Recently, Darabighane et al. 31 reported that the boilers fed with either Aloe vera gel or peppermint @ 10g/kg or Vitamin E @ 100 mg/kg showed significantly (p<0.05)higher WBC count than control group.
Moreover, the group B showed significant higher monocyte count on 14 (0.93 X 10 3 cell/µl) and 21 (1.17 X 10 3 cell/µl) DPV than group A and C. The reason for higher monocyte count may be attributed to the presence of mannose polysaccharide (acemannan) which binds with mannose receptor on monocyte and stimulates production of various cytokines like IL-1, IL-6, IL-12 and TNF-±.These secreted cytokines further acts on the B and T lymphocytes to increase their growth and proliferation 32,33 .The acemannan compound also exhibits anti-tumor, anti-microbial and immunomodulatory effects as reported earlier 34 .
As far as the heterophil counts are concerned, the group D showed 1.89, 3.24 and 5.85 X 10 3 cell/µl on 14, 21 and 28 DPV, respectively, and group C showed 1.35, 1.89 and 1.45 X 10 3 cell /  µl on 14, 21 and 28 DPV, respectively, which are significantly higher than the group A. The increased heterophils count imparts its phagocytic activity against vaccine antigen.However, at 7 DPV the heterophil count did not differ significantly among the groups.
The RBC, Hb and PCV values were significantly higher in all the groups than the control group, which might be due the presence of erythropoietic factors in the extract of Aloe vera leaf 35 .Similarly, Mahdavi et al. 36 reported that the broiler fed with 1% Aloe vera gel powder in ration showed highest WBC, RBC and hemoglobin count.In another study, the broiler chickens fed with Aloe vera leaf juice @ 20 g/L in drinking water, daily for 42 days showed significantly (P<0.05)higher value of Hb concentration, PCV percentage and total leukocyte count than the control group 37 .Similarly, the broiler chicken fed with 7.5g/kg Aloe vera powder in ration revealed increased RBC count and Hb concentration 38 .The parameters like the MCV, MCH and MCHC measurements showed no significance difference (P d" 0.05) amongst the groups.
In the present study, the chicks fed with Aloe vera gel extract @ 3% in the drinking water did not show any adverse effects on their health and immune status rather it was found to stimulate the immune system as observed for the results for lymphocytes and other parameters/immune cells.This finding is consistent with that of Khan et al. 39 who reported that Fayoumi's chick fed with Aloe vera leaf @ 2.0% in ration did not show any sign of adverse effects on their health.In an earlier study by Pittman 40 , the whole Aloe vera leaf was recommended for the control of immune deficiency disorders and Bolu et al. 30 stated that the extract would serve as a better alternative to antibiotic in poultry industry.
Seeing the importance of various herbs and their products gaining importance in animal and health production systems 41,42,15 , the present findings with the use of Aloe vera with IBDV vaccinated chicks as a model, with addition to earlier beneficial reports of this herb 19,17,23,18,16,24 could pave way for further experimental and clinical trials with this valuable herb against virulent IBDV and other infectious pathogens of poultry as well as to be exploited as a promising immune-enhancer and health beneficial element.
In summary, the extract of Aloe vera gel increased the leukocytes count in IBDV vaccinated and unvaccinated white leghorn chicks thereby stimulating non-specific immune response without affecting their normal haematological parameters.It can be used as an additive in poultry ration to achieve better FCR and to boost health and immune status of poultry against IBDV, for which purpose further explorative studies carried out could reflect its beneficial potential to be use as an additive in poultry ration to achieve better production and health along with boosting the immune status of poultry birds against infectious diseases.

Table 1 .
Hematological changes in IBDV vaccinated and unvaccinated groups of chicks treated with Aloe vera gel extract abcd Means in row and columns bearing different superscripts differ significantly (P<0.05).ABCD Means in columns wise bearing different superscripts differ significantly (P<0.05).PQRS Means in row wise bearing different superscripts differ significantly (P<0.05).Values (Mean+SE) RBC -Red Blood Cells; WBC-White Blood Cells; MCV -Mean Cell Volume; MCH -Mean Cell; Hb-Haemoglobin; MCHC -Mean Cell Haemoglobin Concentration; Hb -Haemoglobin, PCV -Packed Cell Volume

Table 2 .
TLC changes in IBDV vaccinated and unvaccinated groups of chicks treated with Aloe vera gel extract