Immune Regulatory Effects of Enteromorphaclathrata Polysaccharides on Nd Attenuated Vaccine in a Chicken Model Infected with Reticuloendotheliosis Virus

Reticuloendotheliosis virus (REV) infection has frequently affected the poultry industry in recent years. The infection with REV weakens the immune responses of the infected poultry. It is reported that Enteromorphaclathrata polysaccharides are capable of regulating immune function. In order to investigate the immuno regulatory effects of Enteromorphaclathrata polysaccharides (EPS) on the response of REV-infected broilers to a live attenuated Newcastle disease (ND) vaccine. Broilers were intraperitoneally injected with REV at one day of age, subcutaneously infected with EPS at 2 days of age, and vaccinated by nasal drip with a live attenuated ND (Lasota strain) vaccine at 5 days of age. Immune organ index, secretory immunoglobulinA (SIgA), peripheral blood heterophil to lymphocyte ratios (H/L ratio), peripheral blood lymphocyte transformation rates, and interferon-gamma (IFN-γ) and interleukin-2 (IL-2) levels were measured at 3, 7, 14, 21, 28, 35, 42, and 56 days of age. The results showed that EPS increased the immune organ index, and the secretion of small intestine secretory immunoglobulin A, serum ND antibody titers, blood H/L ratio, peripheral blood lymphocyte transformation rates, and IL-2 and IFN-γ levels. These results indicate that EPS are able to enhance the immune responses of chickens both to REV infection and to ND vaccination. Therefore, Enteromorphaclathrata polysaccharides can be considered as an immune regulator in the future.

Reticuloendotheliosis virus (REV) infection has frequently affected the poultry industry in recent years.The infection with REV weakens the immune responses of the infected poultry.It is reported that Enteromorphaclathrata polysaccharides are capable of regulating immune function.In order to investigate the immuno regulatory effects of Enteromorphaclathrata polysaccharides (EPS) on the response of REV-infected broilers to a live attenuated Newcastle disease (ND) vaccine.Broilers were intraperitoneally injected with REV at one day of age, subcutaneously infected with EPS at 2 days of age, and vaccinated by nasal drip with a live attenuated ND (Lasota strain) vaccine at 5 days of age.Immune organ index, secretory immunoglobulinA (SIgA), peripheral blood heterophil to lymphocyte ratios (H/L ratio), peripheral blood lymphocyte transformation rates, and interferon-gamma (IFN-γ) and interleukin-2 (IL-2) levels were measured at 3,7,14,21,28,35,42, and 56 days of age.The results showed that EPS increased the immune organ index, and the secretion of small intestine secretory immunoglobulin A, serum ND antibody titers, blood H/L ratio, peripheral blood lymphocyte transformation rates, and IL-2 and IFN-γ levels.These results indicate that EPS are able to enhance the immune responses of chickens both to REV infection and to ND vaccination.Therefore, Enteromorphaclathrata polysaccharides can be considered as an immune regulator in the future.

IntRoduCtIon
The reticuloendotheliosis virus (REV) is a common immunosuppressive and oncogenic virus (Cui et al, 2006), which may infect several domestic and wild bird species.The infection with REV can cause organ damage, and weaken the immune system of poultry, resulting in poor immune responses to vaccines and immune failure.In addition, the REV increases the sensitivity of poultry to secondary infections.These mixed infections increase mortality and considerably reduce production performance, resulting insignificant damage and losses to the breeding and foodprocessing industries.
Today, antibiotics are widely used in the Chinese poultry industry.However, antibiotics may cause drug resistance and antibiotic residue problems, which not only restrict the exports of animal products, but also threaten human life.Therefore, natural biological agents that have low toxicity and do not result drug residue in animal products have been researched and developed as alternative to the use of antibiotics.
Enteromorpha, commonly known as sea lettuce, are aquatic green algae of the family Ulvaceae.As it is a natural seaweed, Enteromorpha is a low-cost green plant, and has high production potential.In addition, high volumes of Enteromorpha can cause ecological damage to the

Immune Regulatory Effects of Enteromorphaclathrata Polysaccharides on Nd Attenuated Vaccine in a Chicken Model Infected with Reticuloendotheliosis Virus
marine environment.Polysaccharides derived from Enteromorpha have a wide range of pharmacological properties (Jianteng et al. 2014), including antitumoral activity, reduction of blood lipids and glucose, as well as antioxidant, antibacterial, antiviral, and immuno regulatory functions (Wang et al. 2012;Shao et al. 2014;Teng et al. 2013;Shihong et al. 2010;Mezghani et al. 2013;Lü et al. 2014;Wei et al. 2014).Therefore, Enteromorphaclathrata polysaccharides have a higher potential application value compared with antibiotics.However, there are no studies on the immunoregulatory effects of Enteromorphaclathrata polysaccharides on the response of REV-infected chickens to ND vaccine.
This study aimed at investigating the immunoregulatory effects of Enteromorphaclathrata polysaccharides on the response of REV-infected chickens to an attenuated live ND vaccine.The REV infected chicken model was established, the immune organ index, secretory immunoglobulin A (SIgA), peripheral blood H/L ratios, peripheral blood lymphocyte transformation rates, and iInterferon-gamma (IFN-γ) and interleukin-2 (IL-2) levels were measured to evaluate immuno regulatory effects of EPS, which laid the foundation for the further development of Enteromorphaclathrata polysaccharides as a new immune adjuvant and vaccine immune enhancer in the poultry industry.

Virus strains
The REV (SNV-ori-p4 strain) was donated by Professor Cui, Z (College of Veterinary Medicine, Shandong Agricultural University).The tissue culture infectious dose (TCID50) of REV-SNV titer was calculated by the Reed-Muench method, using indirect immunofluorescence to identify REV-positive virus cultures in DF-1 chicken fibroblasts (Liang et al. 2013), the TCID50 was 10-4.58/0.1 mL.

Isolation of Enteromorphaclathrata polysaccharides
Fresh Enteromorpha were collected from Qingdao (China), and the polysaccharides were obtained by hot water extraction and ethanol precipitation after cleaning, incubation in an oven at 37ºC, and pulverization in a Ultra-Micro Pulverizer.The details of this method are described as follows (Fuchao et al. 2010).Flesh Enteromorpha were precisely weighed, and then deionized water was added in the proportion of 1:100 (quality ratio).The mixture was stirred for 3 hours under 100ºC.The solution was filtered using filter paper while still hot, the residue was washed three times with deionized water, 95% ethanol was three times to the filtrate, which was stored at 4ºC overnight, and then centrifuged (3,000 rpm for 20 min).After drying in oven at 37ºC, the precipitate was washed with ether for fat removal.The precipitate was completely dissolved in deionized water (1 g:50 mL), 0.1% trypsin was added and the precipitate was digested for 2 h at 37ºC to remove proteins using Sevage reagent, and the supernatant was removed after stirring (3,000 rpm for 10 min) at room temperature.The Sevage solvent was added three times to the supernatant.Anhydrous ethanol was added to the supernatant fluid at 1:3 (volume ratio), precipitated overnight at 4ºC, and then centrifuged at 3,000 rpm for 20 min.After centrifuging (3,000 rpm for 20 min), the precipitate consisted of raw Enteromorpha polysaccharides.Enteromorpha polysaccharides were purified in C18 solid-phase extraction column to determine polysaccharide yield (Zuo et al. 2013).Finally, PMP precolumn derivatization ultra-high performance liquid chromatographytandem mass spectrometry was used to determine the composition and yield of the Enteromorphaclathrata polysaccharides.

Experimental birds
This experiment was approved by the Committee on the Ethics of Animal of Shandong (Permit No.: 20143782).In total, 175 one-d-old broiler chicks were randomly divided into five groups.Groups I and group II were intraperitoneally injected at 1 d of age with 0.1 mL of REV at 10 4.58 TCID 50 .Groups I and III were subcutaneously injected at 2d of age with 0.1 mL of

Immune Regulatory Effects of Enteromorphaclathrata Polysaccharides on Nd Attenuated Vaccine in a Chicken Model Infected with Reticuloendotheliosis Virus
Enteromorphaclathrata polysaccharides at 5 mg/L for three days.Group IV was intraperitoneally injected at 1 d of age with 0.1 mL physiological saline solution.Birds of Groups I to IV were vaccinated with an attenuated live NDV LaSota strain vaccine at 5d of age by nasal drip.Group V was set as the blank control.Five chicks per group were randomly sacrificed after feed and water fasting for 6 h on days 3, 7, 14, 21, 28, 35, 42, and 56 for the measurement of the evaluated indices.

Immune organ index
Five chicks per group were randomly selected to determine their weight and immune organ indexes.Birds were euthanized and dissected in a UV radiationsterilized isolation chamber.The spleen, thymus, and bursa of Fabricius were surgically excised and weighed after the removal of surface moisture with a sterile filter paper.Immune organ index was expressed as their weight organs relative to live bodyweight (McCarty et al. 2004).

Detection of small intestine secretory immunoglobulin A (SIgA)
A 10-cm segment of the small intestine between the duodenum and the jejunum was sectioned, rinsed with sterile saline until clean.TwomL PBS (pH 7.4), containing0.1% bovine serum albumin and protease inhibitors were added, and the solution was homogenized.The homogenate was incubated at room temperature for 4 h, centrifuged for 10 min at 4,000 rpm, and the supernatant was collected to determine sIgA levels, using a chicken secretory IgA (sIgA) ELISA kit.

Serum ND-HI antibody assay
Cardiac blood (1.5 mL/bird) was collected and into a 1.5 mL Eppendorf tube under sterile conditions, stored at 37ºC for 2 h, and then the supernatant was collected.ND antibody titers were determined by hemagglutination inhibition (HI) using two-fold dilution series of the sera.The titers were expressed as log2 values of the highest dilution showing the complete inhibition of agglutination.

Blood heterophil to lymphocyte ratio detection
Approximately 1 mL blood was aseptically collected from the heart of each bird and placed in EDTA-Na anticoagulant tubes.Blood H/L ratios were determined using an automatic hematology analyzer (model XT 1800i, SYSMEX, Japan).

Peripheral blood lymphocyte transformation assay
A volume of 2 mL blood/bird was aseptically collected from the heart and placed into EDTA-Na anticoagulant tubes.Blood was diluted with 2 mL of serum-free RPMI-1640, gently shaken, and then 4 mL of lymphocyte separation medium were added.The solution was centrifuged at 2000 rpm for 20 min.The resulting fluid was clearly separated into four layers.The second layer was a yellowish-white membrane, consisting of lymphocytes, which was removed and suspended in RPMI-1640 culture medium, centrifuged at 2000 rpm for 20 min, after which the supernatant was discarded.This procedure was repeated twice.The precipitated cells were then suspended in RPMI-1640 culture medium containing 10% fetal bovine serum.Cell density was adjusted to 2×10 6 /mL using RPMI-1640 media, and incubated in 96-well culture plates (0.1mL/well, and four wells/sample).Con A (30 mg/mL) was added to three of the four wells.The plates were incubated at 37ºC in humid atmosphere of 5% CO 2 until a monolayer of lymphocytes grew.Subsequently, 30 mL of MTT stain (5 mg/mL) were added to each well, and the plates were re-incubated for 4 h.Absorbance was read at 490 nm, and the values were used to calculate lymphocyte transformation rates (LTR) (Guo et al. 2014).

Detection of IL-2 and IFN-γ in the serum
A volume of 1.5 mL aseptic cardio blood was pipetted into Eppendorf tubes and allowed to clot at 37ºC for 1h.The serum was then separated by centrifugation (4,000 rpm for 15 min) and stored at −20ºC for IFN-γ and IL-2 detection.The levels of IL-2 and IFN-γ were determined using a chicken IL-2 ELISA kit (Life Span BioSciences, Inc., USA) and an IFN-γ ELISA kit (Aviva Systems Biology, Inc., USA).

Statistical analysis
All data were analyzed by SPSS Statistics 19.0 software and expressed as mean±SD.One-way analysis of variance was applied to perform multiple comparison analysis.P values of <0.05 were used to define statistical significance.

The isolation of Enteromorphaclathrata polysaccharides
The yield rate of Enteromorphaclathrata polysaccharides (EPS) was 23.5%, and their purity rate was 69.2%.PMP precolumn derivatization in ultra-high

Immune Regulatory Effects of Enteromorphaclathrata Polysaccharides on Nd Attenuated Vaccine in a Chicken Model Infected with Reticuloendotheliosis Virus
performance liquid chromatography in tandem mass spectrometry was used to obtain EPS composition and yield, as shown in Table 1.The purified EPS consisted of six compounds, out of which the most abundant were the monosaccharidesxylose (32.3%) and rhamnose (28.7%).

Immune organ index
The immune organ indexes obtained in the experimental birds on different days of evaluation are shown in Figure 1.The immune organ index of all five groups peaked on day 21 post-injection and then decreased.The immune organ index of group III was significantly higher compared with other four groups (p<0.05).Groups I and V presented similar immune organ indexes (p>0.05).The immune organ index of group II was significantlylower compared with those of groups I and III (p<0.01).

Small intestine secretory immunoglobulin A (sIgA) titers
As shown in Figure 2, the small intestine sIgA titers of groups I, III, and IV peaked on day 21 post-injection, then gradually decreased, whereas in groups II and V, the peak was reached on day 7 post-injection and then began to decrease.In addition, slgA secretion in group III was significant higher than that of other four groups (p<0.01), and in group II, it was lower compared with other four groups (p<0.05) at any time point.

Serum ND antibody titers
The changes in serum ND antibody titers are presented in Figure 3.The ND antibody titer in group V was zero, as expected.The ND antibody titers of the infected groups (I, II, III, IV) reached a maximum value on day 21.The ND antibody titers of Group III were significantly higher than those of other groups (p<0.01) and remained high for until 42 days.The ND antibody titers of Group II were significantly lower than those of Groups I, III and IV (p<0.05).No significant difference was observed between Groups I and IV (p>0.05).

Blood heterophil to lymphocyte ratio
The blood H/L ratios of all groups are listed in Table 2.The H/L ratio of groups I, III and IV reached a maximum value on day 21.The H/L ratios of Group III were significantly higher than those of other groups (p<0.05) at all time points.No significant difference was observed between Groups I and IV (p>0.05).The H/L ratios of Group II were significantly lower than those of Groups I, III and IV (p<0.01) at all time points.

Immune Regulatory Effects of Enteromorphaclathrata Polysaccharides on Nd Attenuated Vaccine in a Chicken Model Infected with Reticuloendotheliosis Virus
Changes in peripheral blood lymphocyte transformation rates The blood lymphocyte transformation rates of all experimental groups obtained on the different days post-injection are listed in Table 3.The lymphocyte transformation rates of groups I, III, and IV reached maximum values on day 28 post-injection.The lymphocyte transformation rates of Group III were significantly higher than those of other groups (p<0.05) at all time points.Significant differences were observed between Groups I and IV (p<0.05).The lymphocyte transformation rates of Group I were lower that Group IV at all time points.The lymphocyte transformation rates of Group II was significantly lower than that of other groups (p<0.05) and reached peak value on day 7 post-injection.

Peripheral blood IL-2 levels
The IL-2 levels in the peripheral blood of all groups obtained on the different days post-injection are presented in Figure 4.The IL-2 levels of group I, III, and IV gradually increased.In group II, IL-2 values peaked on day 21 post-inoculation and then decreased, while in group III, peak values observed on day 14 and then a decrease until day 21.The IL-2 levels of Group III were significantly higher than those of other groups (p<0.01) at all time points.The IL-2 levels of Groups I and IV were similar until day 21 post-inoculation (p>0.05),but were significantly different thereafter (p<0.05).In comparison with other groups, the IL-2 levels of Group II were significantly lower (p<0.05)at all time points.

Changes of IFN-γ levels in the peripheral blood
The IFN-γ level changes in the peripheral blood of the experimental groups on the different days post-injection are shown in Figure 5.The IFN-γ levels of Groups I, III and IV were gradually increased.In Group II, peak IFN-γ values were observed on day 21 postinoculation, then sharply decreased to levels lower than those of the control group.The IFN-γ levels of

Immune Regulatory Effects of Enteromorphaclathrata Polysaccharides on Nd Attenuated Vaccine in a Chicken Model Infected with Reticuloendotheliosis Virus
polysaccharides are able to strengthen vaccinal response by increasing the production of IFN-γ in chickens.

ConCluSIon
The purified Enteromorphaclathrata polysaccharides contains six components, out of which xylose and rhamnose are the most abundant.Enteromorphaclathrata polysaccharides can not only strengthen the immune responses of chicken infected with reticuloendotheliosis virus, but also significantly enhance the response to an ND vaccine.Therefore, Enteromorphaclathrata polysaccharides can be used as an immune regulator.

Figure 2 -
Figure 2 -The changes of sIgA in small intestine according to experimental group and days post-inoculation.(REV: reticuloendotheliosis virus, EPS: Enteromorphaclathrata polysaccharides, NDV: ND vaccine).

Figure 4 -
Figure 4 -Changes in IL-2 levels in the peripheral blood of chickens, according to experimental group and days post-inoculation.(REV: reticuloendotheliosis virus, EPS: Enteromorphaclathrata polysaccharides, NDV: ND vaccine).

Table 1 -
Composition and monosaccharide contents of in EPS

Table 2 -
Effect of EPS on the blood H/L ratio (%), according to experimental group and days post-inoculation.indicates significant difference in the results of group II compared with the other groups at p<0.05. *

Table 3 -
Effect of EPS on blood lymphocyte transformation ratio in chickens (%), according to experimental group and days post-inoculation.
*indicates significant difference in the results of group II compared with the other groups at p<0.05.