Care of laboratory animals and animal experimentation were conducted in accordance with animal ethics guidelines and approved protocols. All animal studies were approved by the Animal Ethics Committee of Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences (CAAS) and the Animal Ethics Committee of Heilongjiang Province (SYXK (H) 2006-032).
NDV vaccine strain LaSota and 10-day-old SPF embryos were provided by Harbin Pharmaceutical Group Bio-vaccine Co. Ltd. Seven-day-old SPF chickens were provided and raised by Harbin Pharmaceutical Group Bio-vaccine Co. Ltd. Commercial NDV strain LaSota live-virus vaccine (L/N: 200805) and inactivated oil emulsion vaccine against ND (L/N: 200805) were purchased from Harbin Pharmaceutical Group Bio-vaccine Co. Ltd. NDV F48E9 strain was provided by State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, CAAS.
Chitosan (with a molecular weight of 71.3 kDa and deacetylation degree of 80%), MTT, RPMI 1640 medium and ConA were purchased from Sigma Ltd. (St. Louis, MO, USA). Sodium tripolyphosphate (TPP) was purchased from Tianjin Institute of Guangfu Refined Chemicals (Tianjin, China), Agarose and SDS from GIBCOBRL Ltd (New Delhi, India), Cell Counting Kit-8 (CCK-8) from Dojindo (Tokyo, Japan), and NDV IgA ELISA Kit from Rapidbio Co. Ltd. (Montgomery, TX, USA).
Purification of NDV
Briefly, the NDV vaccine strain LaSota was propagated in the allantoic cavity of embryonated SPF eggs at 37°C for 120 h post infection. The allantoic fluid was harvested and centrifuged at 3,000 g/min, 5,000 g/min, and 8,000 g/min for 30 min at 4°C, respectively. The upper liquid phase was collected and concentrated at 30,000 g/min for 2.5 h at 4°C. The purified virus was resuspended in phosphate-buffered saline (PBS; pH 7.4) and stored at −20°C until required.
Preparation of Chitosan Solutions and TPP Solutions
According to the principle of ionic crosslinking, nanoparticles can be formed by intra and inter molecular crosslinking between positively charged chitosan and negatively charged TPP. In this study, we used ionic crosslinking method to develop a novel mucosal delivery system based on chitosan nanoparticles. Chitosan solution was prepared by slowly dissolving chitosan with sonication in an aqueous solution of 4% acetic acid until the solution was transparent. Once dissolved, the chitosan solution was diluted with deionized water and filtered through a 0.22 µm filter to produce a final chitosan stock solution of 0.4% (w/v, 4 mg/ml) with a viscosity of 2.54±0.1 centi Poise (cP) as measured using a Model DV-III Plus Programmable Rheometer (Brookfield Engineering Laboratories, Middleboro, MA, USA). The chitosan solution stock was diluted with deionized water to produce chitosan solutions of different concentrations at 0.5 mg/ml, 1.0 mg/ml, and 2.0 mg/ml. TPP was dissolved in deionized water at the concentrations of 0.5 mg/ml, 1.0 mg/ml, and 1.5 mg/ml.
Preparation of Chitosan-NDV Nanoparticles
The chitosan-NDV nanoparticles were prepared using an ionic cross linking method. Two and a half milliliters of NDV solution (with 1.0% sucrose, m/v) were added drop by drop to 5 ml of chitosan solution under magnetic stirring. Subsequently, 2.5 ml of TPP solution was added to the above solution under magnetic stirring at room temperature. The chitosan-NDV nanoparticles were separated by centrifugation at 10,000 g/min for 30 min at 4°C and the supernatant was discarded. The chitosan-NDV nanoparticles were washed with distilled water. After centrifugation the nanoparticles were freeze dried for 24 h using a vacuum freeze-drying machine (BOC Edwards Co. Ltd, UK) and stored at −20°C until required for further analysis. These nanoparticles were named NDV-CS-NPs.
Optimization of the NDV-CS-NPs Preparation Conditions
Several factors that affected the characteristics of the NDV-CS-NPs including the concentrations of chitosan and TPP solutions, NDV/CS ratio (v/v), the agitating velocity and time were tested. Single factor experiments on the effects of preparation condition of NDV-CS-NPs were conducted. Based on the results obtained in single factor experiments, the key factors were NDV/CS ratio (v/v), TPP concentration, and chitosan concentration. The agitating velocity was 1,000 g/min, and agitating time was 10 min. The orthogonal experiments with three factors and three levels were designed. All the key factors were examined, and morphology, size and zeta potential were used as indicators. The orthogonal experimental scheme is shown in .
Optimization of NDV-CS-NPs preparation conditions.
Morphology, Size and Zeta Potential Measurement of NDV-CS-NPs
A JEM-1200EX transmission electron microscopy (TEM) (Hitachi Ltd, Japan) was used to observe nanoparticle morphology. The colloidal suspension of chitosan nanoparticles was sonicated for 2 min for better dispersion and to prevent particle agglomeration on the copper grid. One drop of colloidal suspension was spread onto a carbon-coated copper grid, which was then dried at room temperature for TEM analysis. The particle sizes and zeta potentials of the NDV-CS-NPs were measured using a Zeta Sizer 2000 from Malvern Instruments (Southborough, MA, USA). Samples were diluted with deionized water, and the measurements were conducted at a scattering angle of 90 degrees and a temperature of 25°C. The diameter was calculated from the autocorrelation function of the intensity of the light scattered from the particles, assuming that the particles had a spherical form.
Evaluation of Encapsulation Efficiency
The encapsulation efficiency of NDV-CS-NPs was determined by separating the nanoparticles from the aqueous medium containing the free virus using centrifugation at 10,000 g/min for 30 min at 4°C. The amount of free NDV in the supernatant was measured spectrophotometrically by a standard Bradford assay using Coomassie Brilliant Blue G-250 with a spectrophotometer at 595 nm. The encapsulation efficiency (EE) of the nanoparticles was calculated as 
is total amount of NDV added, W1
is amount of free NDV. All the measurements were performed in triplicate.
Determination of Viral Titer in the NDV-CS-NPs
The viral titer was calculated by measuring 50% embryo infectious dose (EID50). One hundred micrograms of dried NDV-CS-NPs were added into 10 ml of the PBS buffer (pH 7.2). Subsequently, 2.0 ml of trypsin was added to the above solution and then digested for 72 h at 4°C with a concentration of trypsin solution at 0.25% and centrifuged for 5 min at 1,200 g/min. The obtained supernatant was diluted with sterile saline by a serial of 10-fold dilution, and 10−6, 10−7 and 10−8 were inoculated into the allantoic cavity of five 10-day-old SPF chicken embryonated eggs respectively and incubated at 37°C for 120 h. Dead embryos were discarded at 24 h post inoculation. Eggs were chilled at 4°C and the allantoic fluid was harvested and tested for haemagglutination (HA). The results were considered positive when HA titers were equal to or greater than 7 log2. The EID50 was calculated according to the Reed–Muench method. Five 10-day-old SPF chicken embryonated eggs that were inoculated with trypsin solution served as a control group.
Analysis of NDV Structural Proteins in the NDV-CS-NPs
One hundred micrograms of dried NDV-CS-NPs were added into 1 ml of PBS buffer (pH 7.2) and vortexed for 30 min to ensure that the NDV was fully release. After centrifugation for 10 min at 10,000 g/min, the supernatant containing NDV was collected and stored at −20°C. The NDV structural proteins in the NDV-CS-NPs and the purified NDV were compared for equivalent concentration before and after encapsulation, by Western blotting. Briefly, the supernatant was mixed with 2×SDS buffer and boiled for 5 min. After cooling to room temperature, 20 µl of the samples were loaded onto a 12% SDS-PAGE gel. After electrophoresis, the proteins were transferred to a nitrocellulose membrane using a Bio-Rad semi-dry unit. The membrane was washed with PBS and blocked with 5% skim milk in PBS overnight, and then incubated with NDV-positive chicken serum (HI antibody titer 8.0 log2) at a 1
10 dilution for 1 h. After washing with PBS, HRP-labeled goat-anti-chicken IgY antibody was added at a dilution of 1
10000 for 1 h.
In vitro and in vivo Virus Release and Replication of Chitosan Encapsulated NDV
The release of NDV from NDV-CS-NPs was studied using the Coomassie Brilliant Blue G-250 method. The mixture of 0.1 g of dried NDV-CS-NPs and 2 ml of PBS buffer (pH 7.2) was stirred, followed by incubation at 37°C for 100 g/min. Samples were withdrawn at regular time intervals, centrifuged at 10,000 g/min for 30 min at 4°C, and then measured with the spectrophotometer at 595 nm. After the sample was taken each time, the same volume of fresh PBS was added. The experiments were performed in triplicate. An NDV release curve of NDV-CS-NPs was plotted against the release time at the X-axis and the accumulative release amount at the Y-axis 
Eighty 30-day-old SPF chickens were randomly divided into four groups. Chickens in Group 1 were inoculated with 50 µl of the lentogenic live-virus NDV vaccine strain LaSota (106.5 EID50/0.1 ml) by nasal drops. Chickens in Group 2 were inoculated with 50 µl of NDV-CS-NPs (106.5 EID50/0.1 ml) by nasal drops. Chickens in Group 3 were orally treated with 50 µl of NDV-CS-NPs. Chickens in Group 4 were orally treated with 50 µl of PBS.
Samples of the kidneys and Harderian glands of two chickens were randomly taken in each group at 3, 5, 7, 12 and 18 days post immunization and kept at −70°C. Meanwhile, cloacal swab samples were taken at 1, 3, 5, 7, 10, 13, and 16 days post immunization and kept at −20°C for detection of the virus shedding.
Samples of kidneys and Harderian glands with the same weight were ground and the samples of the cloacal swabs were frozen and thawed three times and then centrifuged at 1200 g/min for 5 min. The supernatant was diluted with sterile saline in a 10×dilution series (10−2, 10−3, 10−4, 10−5, and 10−6) for inoculation in the allantoic cavity of 15-day-old embryonated eggs (five eggs per dilution). The inoculated embryonated eggs were observed for the lesions and embryo deaths, and the EID50 was calculated according to the Reed-Muench method.
In vitro Cytotoxicity of NDV-CS-NPs
Chicken embryo kidney (CEK) cells were prepared from the kidneys of 18-day-old chicken embryos and cultured in DMEM, then diluted to 1×106
. Cells were transferred to 96-well plates at 100 µl per well and cultured at 37°C for 5 h. After 100 µl of NDV-CS-NPs (diluted in DMEM culture at 1.5 µg/ml) were added into the wells, the plates were incubated at 37°C for 48 h. Blank chitosan nanoparticles were used as a negative control and original NDV fluid was used as a positive control. Ten microliters of WST-8 reagent was added and incubated for 4 h. The optical density at 570 nm (OD570) was measured to determine survival rate of the cells, which was calculated as 
Where As represents the test wells (containing the cell medium, WST-8 and NDV-CS-NPs); Ac represents the control wells (containing blank chitosan nanoparticles or NDV original fluid); and Ab represents the blank wells (containing the cell medium only). WST-8 is reduced by dehydrogenases in cells to obtain a yellow colored product (formazan), which can be directly used for cytotoxicity assay.
Safety Test of NDV-CS-NPs in SPF Chickens
Thirty 4-week-old SPF chickens (Harbin Pharmaceutical Group Bio-vaccine Co. Ltd, Harbin, China) were randomly separated into two groups. Chickens in Group 1 were immunized orally with 0.25 ml of NDV-CS-NPs. Chickens in Group 2 were immunized by eyedrops with 0.25 ml of the ND LaSota lentogenic live-virus vaccine (Harbin Pharmaceutical Group Bio-vaccine Co. Ltd, Harbin, China). Any abnormal changes in the chickens were observed and recorded for three weeks.
Immunization of SPF Chickens
Ninety 30-day-old SPF chickens were randomly divided into six groups. Chickens in Group 1 were immunized intranasally with 50 µl of ND LaSota strain lentogenic live-virus vaccine (106.0 EID50/ml). Chickens in Group 2 were immunized intramuscularly with 50 µl of inactivated NDV vaccine (1010 EID50/ml). Chickens in Group 3 were immunized intranasally with 60 µl of NDV-CS-NPs. Chickens in Group 4 were immunized orally with 60 µl of NDV-CS-NPs. Chickens in Group 5 were immunized orally with 50 µl of blank CS-NPs. Chickens in Group 6 were treated with physiological saline. Blood was collected from the wing vein at 1, 2, 3, 4, 5, 6, 7, 8, 10 and 12 weeks post immunization.
Lymphocyte Proliferation Test
Lymphocyte proliferation of the immunized chickens was conducted using MTT (3-(4, 5)-dimethylthiahiazo (-z-y1)-3, 5-diphenytetrazoliumromide) colorimetric assay as previously described 
. The spleens of the immunized chickens were removed aseptically at 6 and 8 weeks post the immunization. The spleens were filtered through 200 micron copper mesh, and single cell suspensions were prepared from the filtrate. Erythrocytes were lysed using 0.75% Tris-NH4
Cl (pH 7.4). Spleen cells were suspended in RPMI 1640 medium containing 10% fetal serum and then diluted to 4×106
ml. Cell suspensions were transferred to 96-well plates at 100 µl per well. Twenty microliters of purified and inactivated NDV were used as a specific stimulating antigen. Wells containing 20 µg/ml of ConA were used as positive controls, and those without stimulating antigen were used as negative controls. All of the cells were cultured at 5% CO2
and 37°C for 44 h, and then 20 µl of MTT was added into each well, followed by culturing for another 5 h. One hundred microliters of DMSO were added into each well. All experiments were repeated three times and each was measured in triplicate. The OD570
was measured to determine the stimulation index using the following formula:
average OD value in the test group divided by average OD value in the control group.
HI Antibodies in Serum
Half a milliliter of blood samples was centrifuged at 2,200 g/min for 10 min at 4°C to collect serum. Each serum sample was transferred to 1.5 ml of prelabeled eppendorf tube and preserved at 4°C until tested individually by hemagglutination inhibition test.
IgA Antibody in Intestinal Mucus
To evaluate the mucosal immune response, intestinal mucus was collected from two euthanized chickens once a week. To collect the intestinal mucus, 4 ml of PBS was gently instilled into the duodenum lumen, allowing it to pass through the length of the duodenum before being collected in prelabeled tubes 
. The ND IgA antibody in the intestinal mucus was detected by an NDV IgA ELISA Kit from Rapidbio Co. Ltd (West Hills, CA, USA).
An experiment was conducted to evaluate the protective efficacy after inoculation with different vaccines. When the level of ND serum antibodies in every immune group increased to 6.0 log2, five chickens were selected at random from the six groups and infected intramuscularly with the highly virulent NDV strain F48E9 for challenge studies with a viral titer of 108 EID50/0.1 ml. Clinical signs of disease and mortality were monitored on a daily basis for seven days.
All experiments were repeated three times and each measured in triplicate. Data were presented as mean values ± standard deviation (SD). Mean values were analyzed using the one-sided Student’s t-test. Differences were considered to be statistically significant at p<0.05.