The present study was performed at the Department of Pathology and Parasitology, Faculty of Animal Science and Veterinary Medicine, Patuakhali Science and Technology University (PSTU) during the period from February 2018 to February 2019. The present investigation was carried at Barishal sadar, Babugonj and Gournadi Upazilas of Barishal district in Bangladesh.
Experimental design and treatments:
Nine (09) commercial farms (Broiler 05 and layer 04) were randomly selected from three different Upazila of Barisal district. A total of 240 serum samples were collected randomly from broiler (135) and layer (105) chickens. After two weeks of immunization with commercial ND vaccines (ND virus strain Clone 30), blood samples were collected for sera. In case of broiler, 1-2 weeks, 3-4 weeks and > 4 weeks of age group and in case of layer, 15-24 weeks, 25-34 weeks, 35-44 weeks, 45-54 weeks and > 55 weeks age groups were selected for determination of age-wise HI antibody titers. HI test was performed using 4 HA unit of Newcastle disease virus (NDV) antigen to detect ND-specific HI antibodies in vaccinated chickens.
Blood collection and serum preparation:
A total of 240 blood samples were collected aseptically from the wing vein of birds (from the commercial layer and broiler). Then the samples were kept at room temperature for two hours to clot blood inside the syringe. After clotting, fluid portions of blood were placed in graduated centrifuge tubes and centrifuged at 2500 rpm for 5 minutes. The clear sera samples were poured in an Eppendorf tube which was labeled and stored at -200 C in the freezer of the Laboratory. All the serum samples were analyzed for NDV antibodies by HI test.
Preparation of chicken red blood cell (RBC) suspension (1% v/v):
To prepare RBC suspension in PBS, a total of 3 ml of blood were collected from the wing vein of a healthy unvaccinated chicken aseptically in a disposable syringe containing 3 ml of Alsever’s solution as an anticoagulant. The blood was placed in a centrifuge tube from the syringe and was centrifuged at 1500 rpm for 15 minutes. The plasma and buffy coat were removed carefully with a pipette. After washing three times with phosphate-buffered saline (PBS), 1% suspension of RBC in PBS (1ml suspension+ 99 ml PBS) was prepared for HI test.
Hemagglutination test and determination of 4HA unit virus for HI test:
The HA test is used to standardize the antigens for the HI test. 50 μl PBS was dispensed into a row of 12 wells in V-bottomed microtiter plates for each antigen used in the test. One additional row of wells was included for a positive HA control. 50 μl undiluted antigen was added to the 1st well of each corresponding row. The antigen (first through 11th well) was serially diluted with a multichannel micropipette set to deliver 50 μl. The resulting dilutions was range from 1:2 in the first well, to 1:2048 in the 11th well; the 12th row, containing only PBS, was serve as a cell control. 50 μl 1% erythrocyte suspension was added to each well and shake/agitate the plate to thoroughly mix reactants.
Hemagglutination inhibition (HI) test:
HI test was done according to the procedure of OIE (2012). For HI test, a two-fold serial dilution of 25μl serum was made with PBS in V-bottomed microtiter plates (Nunc) up to 10th well with a multichannel pipette. 25 μl of 4 hemagglutinating (HA) units of Newcastle disease virus or antigen was added up to 11th well. To facilitate antigen-antibody reaction the plates were kept at room temperature for more than 30 minutes. Then 25 μl of 1% (v/v) chicken RBC suspension was added to each well. The 11th well-considered as a positive control that contains antigen and RBCs and the 12th well considered as the negative control that contains only RBCs. After gentle mixing, the RBCs were allowed to settle at room temperature for 40 minutes. Agglutination was assessed by tilting the plates.
Histopathological examination:
Postmortem examination and diagnosis of the Newcastle diseases of in vaccinated chickens below protective level was done by following the standard procedures. After dissecting the dead birds, samples were incised with sterile scissors. All samples (liver, heart, spleen, lungs, trachea, intestine, proventriculus, cecal tonsil etc.) were collected after postmortem examination of infected birds below the protective level from nine commercial poultry farms. After collection of samples, it was preserved in 10% neutral buffered formalin solution for further histopathological examination. Formalin-fixed tissue samples were processed and stained as per the standard method (Titford, 2009).
Statistical analysis:
All data were summarized with the help of MS Excel and analyzed by means of the GraphPad Prism 5.0 statistical software (GraphPad Software, San Diego, USA). Differences with a P value <0.05 were regarded as statistically significant.