The Thermostable live oral pellet Newcastle disease vaccine (OPNDV) developed at the Department of Veterinary microbiology using D58 isolate of NDV was given to chicks at the age of 10th day through oral route. Each oral pellet vaccine grain had 106.5 EID50 / grain.
B1 Broiler Chicks with no history of vaccination from day old were housed in animal house at the Madras Veterinary College. They were provided with unmedicated broiler starter mash and water ad libidum and maintained as per the guidelines provided by the CPCSEA (Committee for the purpose of control and supervision on experiments on animals, Chennai, India). The trial on the birds was approved by CPCSEA (Proposal number: 2491/DR/01).
Harderian glands and lachrymal fluid was collected as per the procedure described by . Tracheal fluid was collected by swabbing and the swabs were placed in Tryptose phosphate broth (TPB). Intestinal washings were derived by lavage with TPB. Bile was collected from gall bladder using 22 gauge needle .
Experimental design and sample collection
The experimental group comprised of 40 chicks. The birds were divided into 2 groups (20 birds each). The first group was vaccinated with oral pellet Newcastle disease vaccine at a dose of one grain per bird. Control group was not vaccinated. Samples such as Harderian gland, lachrymal fluid, tracheal fluid, intestinal washings, bile and serum were collected from both the groups on 7, 14 and 21 days to assess mucosal immune response.
Indirect ELISA for IgA
The optimum dilutions of test antigen (infected allantoic fluid and tissue homogenates), samples and conjugate for the test system were determined by checkerboard titration .
The reaction volume for the entire assay was 100 μl / well. After each step, the ELISA plate was washed six times in an automatic plate washer (Bio-Rad, Model # 1575, USA) utilizing 400 μl of wash solution. The microplate (Immunoplate) was adsorbed with NDV protein in coating buffer to all the wells overnight at 4OC or at 37OC for 60 minutes. After washing, the plate was blocked with blocking buffer and incubated for 60 min at 37OC. After another washing, 1:1000 dilutions of samples were added in duplicate for optimum results and the plate was incubated for 45 min at 37OC. After another washing, goat anti chicken IgA peroxidase conjugate at 1:5000 dilution in blocking buffer was added to the washed plate and incubated for 30 min at 37OC. The enzyme substrate solution (ABTS) was added to the microplate and incubated for 10 min in the dark. The reaction was stopped by adding 1% SDS. Optical density values were measured at 405 nm in an automatic ELISA reader (Bio-Rad, Model # 550, CA, USA). Cut-off between positive and negative was kept as 2 times the value of negative.
Twelve birds were used for challenge studies. Virulent Newcastle disease strain was used for challenge testing.
The data were analysed using Minitab statistical software package. The duplicate optical density (OD) values of IgA ELISA were checked for measure of dispersion by calculating the standard deviation (SD) and coefficient of variation (COV) between OD values. If the COV was more than 20 per cent, the readings were not included for further calculation.