In this study, the aim was to establish and optimize a method for the detection of NDV-specific memory T cells in the chicken. The assay was then used to determine differences in the

development of NDV-specific T cells Selleck BI-2536 upon ND vaccination in chickens differing in the major histocompatibility complex (MHC). Two animal experiments were performed. Experiment 1 was performed to determine the proliferative capacity of four different MHC haplotypes, while experiment 2 was performed to determine recall proliferation after experimental vaccination in two MHC haplotypes. Experimental chickens for optimization of a method for recall proliferation and for experiment 1.  Offspring from different inbred chicken lines were used: line 2 (B12), line 133 (B13), line 130 (B130) and line 201 (B201), the MHC haplotypes are shown in parentheses. All lines are bred at Aarhus University [11]. The birds were vaccinated through drinking water at 3 and 8 weeks of age with a live attenuated Newcastle disease vaccine (Poulvac NDW; Fort Dodge Animal Health Ltd. Southhampton, UK) and once at 16 weeks of age intramuscularly (IM) with inactivated ND vaccine (Poulvac I-ND; Fort Dodge Animal Health Ltd.), according to Danish legislation. Blood samples were taken in the jugular vein and stabilized with either EDTA or heparin for optimization C646 concentration purposes and with EDTA

only for the MHC screening. Birds for optimization and MHC screening were tested up to 2 years after vaccination. Experimental chickens experiment 2.  For this purpose, animals from two inbred chicken lines that differ immunologically with respect to their peripheral blood CD4/CD8 ratios were chosen. These were line 133 (B13) and

line 130 (B130), the MHC haplotypes are shown in parentheses [11]. Ten birds from each line were vaccinated orally at 4 and 8 weeks of age with 1 dose of live attenuated Newcastle disease vaccine (Poulvac NDW; Fort Dodge Animal Health Ltd.). Recall proliferation was performed 3 weeks after the last vaccination. Blood samples were taken from the jugular vein and stabilized with EDTA. MHC genotyping of chickens for experimental vaccination.  All chickens used in the experiment were produced from MHC-characterized parents. The MHC haplotypes Suplatast tosilate of the offspring were confirmed by genotyping the LEI0258 microsatellite locus [12] by a PCR-based fragment analysis [13]. Genomic DNA was isolated from peripheral blood using the ArchivePure™DNA Blood Kit (5 PRIME GmbH, Hamburg, Germany) according to the manufacturer’s instructions. Amplification by PCR and gel documentation were performed as earlier described [14]. PBMC isolation.  PBMC were purified from heparinized or EDTA-stabilized peripheral blood density gradient centrifugation. One millilitre of blood was diluted with 1 ml of phosphate-buffered saline (PBS) and layered onto an equal volume of Ficoll-Paque™ PLUS (Amersham Biosciences, Uppsala, Sweden) before centrifugation at 400 g for 35 min at 20 °C.