Immunomodulatory Potential of Microencapsulated Multispecies Probiotic Consortium in Newcastle Disease Virus Vaccinated Chicken

In poultry industry, prevention and control of diseases have led to a substantial increase in use of antibiotics in recent years. The search for alternative to antibiotics in poultry feed and addition of probiotics has been proposed due to increasing reports of drug resistance developing microorganisms led to severe diseases and its residues presence in meat which is potential health hazard to consumers (Fuller, 2001). According to the FAO and WHO, probiotics are “live microorganisms which when administered in adequate amounts confer a health benefit on the host” (Lee and Salminen, 2009). It has been reported that certain Lactobacilli can induce an increase in the cellular or humoral systemic immune response (Perdigon et al.,1988) and can also influences the cells such as macrophage involved in the inflammatory immune response (Kato et al., 1983; Perdigon et al., 1988). The mRNA expression levels of Toll like receptors (TLRs) TLR2 and TLR4 was increased (P<0.05) when graded levels of the probiotic and prebiotic were supplemented, while, TLR7 did not show any significant change (Sheoran et al., 2018). There are also reports about the effect of LAB (Lactic Acid Bacteria) as immunostimulator and as inducer of cytokine release (De Simone et al., 1995). Several studies has been carried over to assess the effect of probiotics on humoral (Landy and Kavyani, 2013; Talebi et al., 2015) and cell mediated immune response (Brisbin et al., 2011; Dalgaard et al., 2010) in NDV vaccinated chicken.
        
Timmerman et al., (2006) provided evidence that multispecies probiotics are more effective than monospecies probiotics and also species-specific probiotics elicit different health effects than do probiotics derived from another host species. It has been shown that probiotics stimulate different subsets of immune system cells to produce cytokines, which in turn play a role in the induction and regulation of the immune response. Ghafoor et al., (2005) studied effects of probiotics (Protexin) on the hemagglutination inhibition HI titer of antibodies against avian influenza virus (AIV) and post field AIV challenge. The findings were compared with the cyclophosphamide treated AIV-vaccinated; untreated and AIV-vaccinated and unvaccinated and control birds. This investigation revealed that protexin treated chicks have higher AIV-HI antibody and no AIV post challenge mortality compared to the cyclophosphamide treated and untreated chicks. The overall finding of this study clearly demonstrate that the use of this multi strain probiotics has good effect on immune response of broilers.
        
Khalifa et al., (2014) evaluated the effect of probiotic and/or Escherichia coli infection on antibody responses to routine vaccination against New Castle Disease Virus (NDV) and Infectious Bursal Disease Virus (IBDV) in broiler chicks. They concluded that using of probiotic routinely in ration of broiler chicks enhances immune status and improves humoral immune response against NDV and IBDV as well as treatment of E. coli infection in chicks before vaccination program improve the antibody response against NDV and IBDV vaccines. Keeping in view of the above facts, a study has been undertaken to evaluate the effect of microencapsulated multispecies probiotic supplementation on immune response against Newcastle disease vaccinated broiler chicken.

Probiotic consortium preparation, Experimental chicken and management
 
Probiotic consortia containing Lactobacillus plantarum, Enterococcus faecium, Enterococcus hirae, Pediococcus acidilactici and Weissella paramesenteroides was made based on their immunomodulatory potential using in vitro cytokine expression studies and T and B cell receptors profiling (Divya et al., 2019) was used in this study.
        
Commercial day-old VenCobb broiler chicks (n=40) were wing banded, weighed individually and assigned to four groups on the basis of initial body weight. Each trial group had 10 broiler chicks. Group-1 were fed with microencapsulated probiotic formulation and vaccinated against NDV B1 and Lasota vaccine, Group-2 were fed with commercial probiotic organisms and vaccinated against NDV B1 and Lasota vaccine, Group-3 were used as Positive Control group without probiotic treatment and vaccinated against NDV B1 and Lasota vaccine, Group-4 were used as Negative  Control Group – unvaccinated group without probiotic treatment Chicks were fed with chick mash during 1^st two weeks of age, followed by fed with grower mash. The broilers were reared for a period of five weeks under cage system.
        
Microencapsulated probiotic consortium in a beads form (2×10⁸ CFU/g) containing Lactobacillus plantarum, Enterococcus faecium, Enterococcus hirae, Pediococcus acidilactici and Weissella paramesenteroides. commercial probiotic consists of Saccharomyces cerevisiae SC-47, Saccharomyces boulardii, Lactobacillus acidophilus, Propionibacterium freudenreichii and Sea weed powder. All the birds were kept under uniform management conditions throughout the experimental period.
 
Vaccination schedule
 
The birds were vaccinated against Newcastle Disease according to the routine vaccination schedule. Chicks were vaccinated against B1 Strain (live, lentogenic) of NDV at 7^th day of age (ocular), then by Lasota strain (live, lentogenic) at 21^st day of age (oral) based on the routine vaccination schedule and dose.
 
Feed supplementation
 
Experimental birds were supplemented with 1gram of microencapsulated probiotic consortium per chick for 1-7 days age, then 3 grams per chick for 7-14 days age, followed by 6 grams per chick for 14-35 days age. The encapsulated beads containing probiotic organisms were mixed in feed and fed to chicken. The commercial probiotic powder was fed to chicken as per recommended level mentioned in product details. Commercial feed was fed to chicken as per recommended level mentioned in broiler performance goals-venCobb400 feed chart.
 
Assessment of humoral immune response by ELISA
Serum collection: Sera collected (n=24) from 6 birds each in all four trial groups on 7^th, 14^th and 28^th after primary and booster vaccination against NDV vaccine.
 
Serum antibody titres assessment: Enzyme linked immuno sorbent assay (ELISA) was performed to assess the antibody titres against NDV. The absorbance was measured at 405nm by ELISA plate Reader. The optical density was converted into titre values based on the calculation recommended by kit manufacturer (Silva et al., 2009).
 
Assessment of chicken Th 1 and Th 2 cytokine profile by real time PCR
Preparation of peripheral blood mononuclear cells (PBMC): Blood samples (n=24) from 6 birds each in all four trial groups after 7 days of primary and booster vaccination. Peripheral blood mononuclear cells were prepared (Brisbin et al., 2010).
 
RNA extraction and quantification: RNA was extracted from peripheral mononuclear cell suspension (n=24) by using Nucleospin™ RNA extraction kit. The extracted RNA was quantified using Picodrop Spectrophotometer (Nanodrop 2000, Thermo Scientific Inc).
 
cDNA synthesis: cDNA was synthesized from extracted RNA using iScript cDNA synthesis Kit (Bio Rad Inc) as per the manufacturer’s instructions by the addition of the following reagents of total 20 µl mix in 0.2 ml PCR tubes viz., RNA (2 µg) - 10 µl, iScript Reaction Mix (5X)- 4 µl, iScript reverse transcriptase - 1 µl and nuclease free water - 5 µl.
        
The tubes were then incubated at 25°C for 5 minutes, subsequently at 42°C for 30 minutes and finally at 85°C for 5 minutes. The resultant synthesized cDNA was stored at -20°C until further use.
 
Cytokine profiling by Real time PCR: Real time quantification of cytokines expression with respective to in vivo in different groups were fed with microencapsulated probiotic consortium, commercial probiotic powder and control group were performed in a CFX96 Touch Real-Time PCR Detection System (Bio-Rad) using the SyBr green dye. Further protocol was followed as per Brisbin et al., (2010). The lists of primers used in the real time PCR were given in the following Table 1.
 

 
Assessment of cell mediated immune response by flow cytometry
Preparation of splenic leucocytes: Spleen tissues were collected (n=24) after 21 days of 1^st vaccine against NDV in sample collection containers. Further process was followed as per protocol mentioned in the Brisbin et al., (2010).
 
Assessment of B cell receptor expression using FITC conjugated Bu1A antibody by flow cytometry: Samples were prepared for analysis of B cell expression by flow cytometry as per protocol mentioned by Dong et al., (2011). Bu1A FITC conjugate was used to analyze the B cell expression in this study.
 
Analysis of T cell receptor expression using FITC conjugated CD3 antibody by flow cytometry: The same protocol was followed (as mentioned in 2.6.2) for flow cytometry studies using B cell receptor and instead of Bu1A FITC conjugate, 0.15µg of rat anti human CD3: FITC antibody (Bio-Rad Laboratories India Pvt. Ltd) targeted against T cell was used. Phenotypical results were expressed as both the percentage of Bu1A and CD3 positive cells or mean fluorescence intensity (MFI) with in a particular cell subset. Data was analyzed by using BD FACS software (Dong et al., 2011).
 
Assessment of innate immune response through macrophage activity by immunohistochemistry method: To assess the innate immune response, immunohisto- chemistry was performed and enumerated macrophage population in spleen tissues by using CD163 receptor after 21 days of post vaccination. Macrophage count was done by electron microscopy in 6 fields at 10x magnification.

Multispecies probiotics effect on innate immune response
Macrophage activity in probiotic treated chicken: The immunocytochemistry showed  increase in number of macrophages  in probiotc treared group which provided the evidence for involvement of probiotic towards innate immunity. Approximatly, 438, 359 and 119 macrophages were observed in microencapsulated probiotic treated group, commercial probiotic treated group and control group, respectively. Results were shown in Fig 1. Similar results were observed by Lin and Karin (2007). They concluded that macrophage activity was increased in probiotic fed group chicken and phagocytic activity of macrophages also  increased due to probiotic feeding (L. plantarum), subsequently cytokine expression also increased upon macrophage activation. Kristeen et al., (2017) stated that macrophage population increased upon infection with both genotypes of NDV but NDV AF2240 resulted in a higher peak in macrophage cell number at 2.86 × 10⁵ ± 16 cells compared to NDV IBS002 at 1.70 × 10⁵ ± 15 cells on the fourth day after infection.
 

 
Multispecies Probiotic effect on humoral immune response: Antibody titre values to find out the humoral immune response against NDV were measured by ELISA and the results presented in Fig 2. Microencapsulated probiotic treated group has shown higher NDV antibody titres compared to commercial probiotic and control groups. These results were in agreement with the findings of Landy and Kavyani (2013), Nikpiran et al., (2013) and Khammas and Saigh (2014) where multi-strain probiotic (primalac®) supplementation  had shown significantly higher ELISA antibody titres against NDV vaccine compared to control groups. Similarly, Khaksefidi and Ghoorchi (2006) also reported that antibody production against NDV in broiler chicks treated with probiotic was significantly higher at 10 days post immunization than that in untreated group. Talebi et al., (2015) reported that supplementation of single species of probiotic bacterium (Enterococcus faecium) had the highest antibody titre against NDV and significantly (p=0.0049) differ when compared with those of only vaccinated chickens. The health benefits and immunomodulatory properties offered by probiotic bacteria are strain specific and there is no universal strain or species that can provide all proposed benefits. Hence, multispecies probiotic is comparatively better than single species probiotic bacteria to exhibit beneficial effects in chicken (Izquierdo et al., 2009).
 

 
 
Multispecies Probiotic effect on cell mediated immunity: Microencapsulated probiotic fed group chicken had shown higher IL- 2 and IL-12 cytokine expression which are responsible for driving the Th1 immune response and also higher IL-4 and IL-10 cytokine expression which are responsible for driving the Th2 immune response compared to commercial probiotic fed group chicken ( Figs 3 and 4). Similar results were found in study of Brisbin et al., (2011) who reported that chickens  fed with mixture of Lactobacillus cultures (lactobacillus salivaris, L. reuteri, L. acidophillus) had shown higher IL-12p40 cytokine expression compared to chicken fed with only PBS. The chicken IL 2 is an active growth factor for different immune cell types including T cells. The chicken IL4 stimulates the Th2 cells which is responsible for humoral immunity. Avian IL -12 is a pro-inflammatory cytokine produced by phagocytic cells and antigen-presenting cells (APC) within a few hours after bacteria or intracellular parasites infection and it also activates NK cells, and stimulate the production of Interferon-g (IFN-gamma). The role of IL-10 in chicken on many cell types such as non killer cell and also T and B lymphocytes has been observed (Giansanti et al., 2006). There was also increased lymphocyte count due to the effect of supplementing probiotics to feed (Hanamanta et al., 2010).
 

 

 
Multispecies probiotics effect on T-cell and B-cell mediated immune responses: Flow cytometry analysis was carried out for all groups chicken and staining percentages were determined from 10⁴ cells per sample by analysis on a fluorescence activated cell sorter (FACScan; Becton Dickinson). The gated stained and unstained populations of cells were determined from a mean value for the negative control samples of each group of chicken. Higher proportions of Bu1A and CD3 positive cells were observed in chicken fed with probiotic when compared to control group chicken.(Figs 5 and 6). Huang et al., (2013) reported that probiotics enhanced the formation of pool of CD8⁺ T cells in the intestinal mucosa and also correlated with the results of Haghighi et al., (2008) who reported that the elevation of CD8⁺ T cells density by probiotics treatment. Similarly, in study of Dalgaard et al., (2010) reported that the proliferative capacity of peripheral CD4⁺ and CD8⁺ cells specific for NDV was addressed 3 weeks after vaccination and found to be significantly higher in L133 bred line than in L130 bred line chicken.
 

 

Microencapsulated Probiotic consortium containing Lactobacillus plantarum, Enterococcus faecium, Enterococcus hirae, Pediococcus acidilactici and Weissella paramesenteroides used in this study has improved the immune response in terms of both innate and adaptive immunity as exemplified in in vivo trial conducted in commercial chicken.