Wild boars from Sweden, Austria, the Czech Republic and Japan possess intact mannose-binding lectin 2 (MBL2) genes.

The two-nucleotide deletion recently detected in the mannose-binding lectin 2 gene in purebred and crossbred domestic pigs was not found among 68 wild boars representing 4 populations from Europe and Asia. This suggests that the deletion is a result of breeding and/or genetic drift/bottle necks.

Transcription efficiency of different chicken mannose-binding lectin promoter alleles.

The serum collectin mannose-binding lectin (MBL) plays a major role in innate immunity by activation of the lectin complement pathway or by acting as an opsonin. The serum levels of human and animal MBL are associated with susceptibility to a wide range of infections, and the variation of MBL in serum is genetically determined. In the chicken, 14 single nucleotide polymorphisms (SNPs) have so far been found in the MBL promoter region. In this study, the transcription activity of a 670-bp promoter region covering all 14 SNPs from the four MBL promoter alleles A1 to A4 was assessed using a dual-luciferase assay. Of the analysed alleles, A1 showed the highest transcription activity although this allele is frequently found in chickens with low MBL mRNA expression.

Acquisition of resistance after continuous infection with Ascaridia galli in chickens.

SUMMARY Acquired resistance against Ascaridia galli infection was studied in seventy-two 18-week-old white Leghorn chickens allocated to six groups (G1-G6). In order to understand the population dynamics following trickle-infection (100 eggs per chicken twice weekly), chickens of subgroups of G1 were necropsied 3 days after 1, 6 or 12 inoculations (G1A, G1B and G1C respectively), while G2-G4 were inoculated for 6 weeks. G2 was necropsied 4 weeks after the last inoculation. The number of established larvae increased initially (between G1A and G1B) but decreased after repeated inoculations (G1C, G2). G3, G4 and G5 were used to measure the efficacy of anthelminthic treatment and to monitor the acquisition of resistance following a challenge infection. At week 7 G3, G4 and G5 were treated with flubendazole for 7 days in the feed. Two weeks after treatment the chickens in G4 and G5 were challenged with 500 eggs. G6 was left as uninfected control. Necropsy at week 10 after first inoculation revealed a lower establishment rate, an impaired development and a more posterior localization of the larvae in G4 (trickle-infected-treated-challenged) compared with G5 (treated-challenged). IgY level in serum reached noticeable level at 14 dpi in G2 and G4 chickens, and in G4 chickens IgY level further increased after challenge infection. The study provides evidence that acquired resistance against A. galli in chickens leads to a significant yet incomplete protection against re-infection.

MBL1 genotypes in wild boar populations from Sweden, Austria, the Czech Republic, and Japan.

The single nucleotide polymorphism (SNP) G949T in the mannose-binding lectin ( MBL ) 1 gene has been associated with low MBL-A concentration in serum and detected at different frequencies in various European pig populations. However, the origin of this SNP is not known. Part of the MBL1 gene was sequenced in 12 wild boar/Large White crossbred pigs from the second backcross (BC 2 ) generation in a family material originating from two wild boar x Large White intercrosses. Also, MBL-A serum concentration was measured in the entire BC 2 generation (n = 45). Furthermore, the genotypes of 68 wild boars from Sweden, Austria, the Czech Republic, and Japan were determined in regard to five previously described SNPs in MBL1 . The T allele of G949T was present among the BC 2 animals. MBL-A serum concentration in the BC 2 animals showed a bimodal distribution, with one-third of the animals at levels between 0.7 and 1.6 µg mL(-1) and the remaining pigs at levels around 13 µg mL(-1) . There was a co-variation between the presence of the T allele and low MBL-A concentration in serum. The genotyping of the wild boars revealed differences between populations. The T allele of G949T was not detected in the Austrian and Japanese samples and is thus unlikely to be an original feature of wild boars. In contrast, it was present at high frequency (0.35) among the Swedish wild boars, probably representing a founder effect. Five MBL1 haplotypes were resolved. Only two of these were present among the Japanese wild boars compared to four in each of the European populations. This difference may reflect differences in selection pressure and population history.

Assessment of Newcastle disease-specific T cell proliferation in different inbred MHC chicken lines.

In this study, we have described the establishment of an antigen-specific T cell proliferation assay based on recall stimulation with Newcastle disease (ND) antigen; further, we have described the results obtained after recall stimulation of animals containing different major histocompatibility complex (MHC) haplotypes, vaccinated against ND. First optimization of the assay was performed to lower unspecific proliferation and to enhance antigen-specific T cell proliferation. These two issues were achieved using ethylene diamine tetra acetic acid as stabilizing agent in blood samples and autologous immune serum in culture medium. The optimized assay was used to screen chickens with different MHC haplotypes for their ability to perform T cell proliferation. Results showed that the antigen-specific response of CD4(+) and CD8(+) T cells from B12 chickens was generally low, whereas B13, B130 and B201 chickens were medium in CD4(+) or CD8(+) T cell responses. High responses were seen only in few animals of each haplotype and not in general. A polymorphism in the chicken CD8α gene was found in our experimental chicken lines, resulting in incapability to detect CD8α(+) T cells using antibodies from the CT8 clone. Screening chickens with alternative antibodies showed that antibodies from the 2-398 clone were able to discriminate all CD8α(+) cells from CD8α(-) cells, and consequently this antibody was used in a second vaccination experiment performed with chickens of the haplotypes B13 and B130. This experiment showed a significant difference in antigen-specific proliferation of CD4(+) T cells between the two lines, but not in CD8α(+) T cell proliferation.

Feeding activity in groups of newly hatched broiler chicks: effects of strain and hatching time.

The feeding activity of 2 strains of broiler chickens was investigated during their first week of life in relation to their hatching time. Fast (Ross 308) and slow-growing (LB) strains were allocated to 1 of 3 (early, middle, or late hatch) single-strain groups of 80 to 100 as-hatched birds in 4 replicates divided into 2 time-separated blocks. Behavioral observations differed between blocks and were carried out at intervals on d 1 to 6, and the percentage of birds feeding (from trough or paper), drinking, or being otherwise active (block 2 only) were registered. A higher mortality caused by flip-over was seen among the late-hatching birds from the slow-growing strain. The percentage of birds engaged in feeding activity was similar for the 2 strains, but LB birds began to eat from the paper later and were observed eating from the trough less than Ross 308 birds, which in turn were less active than LB, especially in the early and middle hatch groups. Early hatch groups were observed feeding from the paper more than the middle and late hatch groups. Drinking behavior mirrored feeding from the trough, indicating that drinking was prandial. Within strain, no effect of hatch time was found on live weight at hatch, but the feeding behavior of early hatched birds led to a small, transient weight advantage on d 3 after hatch. The transition from feeding on paper to feeding only from the trough may have less effect on birds that feed from the trough sooner, such as the fast-growing strain.

The effects of floor heating on body temperature, water consumption, stress response and immune competence around parturition in loose-housed sows.

The aim of the present study was to study whether floor heating from 12h after onset of nest building until 48 h after birth of the first piglet had any effect on measures related to body temperature, water consumption, stress response and immune competence in loose-housed sows (n=23). In conclusion, the present results indicate that floor heating for a limited period around parturition did not compromise physiological and immunological parameters, water intake and body temperature in loose-housed sows. The water intake peaked the day before parturition and the body temperature peaked on the day of parturition. A cortisol peak at parturition, a transient rise in the number of leucocytes and neutrophils and a transient reduction in the number of lymphocytes, erythrocytes and in the PCV value were observed. Around and after parturition some non-specific immunological variables seemed to be stimulated while others seemed to be compromised.

Influence of chicken serum mannose-binding lectin levels on the immune response towards Escherichia coli.

This study aimed to investigate the effect of mannose-binding lectin (MBL) on infections with Escherichia coli in chickens. Initially, the basic levels of MBL in 4 different lines of layer chickens, namely ISA Brown, Lohmann Selected Leghorn, Lohmann Braun, and Hellevad, were investigated. This investigation revealed a 2-to 3-fold difference in the basic levels of MBL in serum between some of these commercial lines. Furthermore, the ontogeny of the basic level of MBL in serum of an experimental chicken line was investigated. The level of MBL was very stabile for long periods, with an elevation at 5 to 7 wk of age. Another elevation in MBL level started around 18 to 19 wk of age and stayed elevated at least until 38 wk of age. In this study, it was hypothesized that chickens with high levels of MBL (H-type) may be less prone to disease caused by E. coli infection than chickens with low levels of MBL (L-type) after attempts were made to immunosuppress the chickens by immunization with a live attenuated infectious bursal disease virus (IBDV) vaccine strain. The H-type and L-type chickens were divided into 4 groups receiving either no treatment (I-E-), E. coli alone (I-E+), IBDV alone (I+E-), or IBDV and E. coli (I+E+). Body weight gain was depressed by IBDV immunization as well as E. coli inoculation. The depression of BW gain was significantly larger in L-type chickens compared with H-type chickens. The antibody response to E. coli was significantly depressed by IBDV vaccination and antibody titers to E. coli were elevated by experimental E. coli inoculation, but only in the group not given IBDV (I-E- vs. I-E+). On d 28, T-cell responses in L-type chickens showed a lower percentage of proliferating CD4+ and CD8+ T cells compared with the H-type, regardless of treatment. In conclusion, immune reactions toward infections with E. coli differed between chickens having different basal serum MBL levels, and as such, MBL may be of importance for future selection of more robust chickens for outdoor or organic farming.

Effect of early life thermal conditioning and immune challenge on thermotolerance and humoral immune competence in adult laying hens.

Effects of early life experience with climatic (heat) and hygienic [lipopolysaccharide (LPS)] stress on adaptability to the same stressors in later life were studied in laying hens. Chicks were exposed to 37 degrees C for 24 h at d 5 of age (n = 12) or were i.v.-administered once with 1 mg/kg of BW of LPS at 6 wk of age (n = 12), whereas a control group was reared under standard conditions receiving a placebo treatment of PBS (n = 36). At 24 wk of age, hens treated in early life were reexposed to the same stressor. Early life control hens were exposed to heat stress (n = 12), i.v.-administered with LPS (n = 12), or not exposed (n = 12). To evaluate improvement of adaptability, effects of climatic and hygienic stress on performance, humoral immune competence, and endocrine responsiveness were investigated in hens with early life experience to the stressors and hens only exposed to the stressors in later life. Early life heat exposure did not affect performance, immune, and endocrine parameters. Treatment x time interactions were found for level of antibody (Ab) binding to LPS and keyhole limpet hemocyanin (KLH) after LPS administration, indicating that hens with early life LPS experience differed in response level (Ab binding to LPS) and response pattern (Ab binding to LPS and KLH) compared with hens administered with LPS only at adult age. Our data suggest that early life heat stress exposure did not affect adaptability of laying hens to heat stress in later life. However, early life LPS exposure affected kinetics and magnitude of Ab levels binding to LPS and KLH, indicating that early life LPS exposure can enhance the status of immune reactivity or induce a higher sensitivity to LPS.

An assay for measuring the mannan-binding lectin pathway of complement activation in chickens.

To investigate the ability of chicken mannan-binding lectin (cMBL) to work as a complement activator, a heterogeneous ELISA test was developed, in which the deposition of human complement factor 4 (C4) was used as a measure of complement activation ability. Serum from different experimental chicken lines was tested. The correlation between serum cMBL concentrations and human C4 deposition was high (correlation = 0.8549, P < 0.0001). There was no difference in C4 deposition among sera from the chicken lines when calculated as C4 deposition relative to the cMBL concentration.

Comparison of growth performance and immune parameters of three commercial chicken lines used in organic production.

Owing to the higher demands for avoiding medication and antibiotics, health status of the production animals plays an important role in the poultry industry, especially in organic poultry systems. Immunity plays a major role in keeping the host free from disease, and it is evident that the host's genetic make-up influences immunity and disease resistance/susceptibility in chickens. Previously, breeding strategies aimed at selection for resistance against specific diseases with the risk of creating less disease resistance against other pathogens. Changing breeding strategies towards selection of chickens with a more general and broad disease resistance or robustness may therefore improve the overall health status, animal welfare, and food security in the poultry production. The aim of this study was therefore to compare the immunocompetence of the presumed "robust" Hellevad chickens with two chicken lines widely used in organic production, Bovans Brown (Bovans) and Hisex White (Hisex). The chickens were subjected to a routine vaccination program comprising one parasite and four viral vaccines. The current study indicates that considerable differences in immunocompetence may exist between commercial layer lines used in organic production. The Hellevad chickens were found to have higher body weight at the end of the experiment (17 weeks of age) than the other two lines. Furthermore, Hellevad and Hisex chickens were found to have higher levels of humoral innate immunity with regard to sample to positive ratio of natural antibodies in serum and concentration of mannose-binding lectin in serum as compared to Bovans. Moreover, indications of an inflammatory response were observed in the Bovans at week 5, corresponding to 1 week after vaccination with live infectious bursal disease virus. With regard to adaptive immune parameters such as IgY concentration in blood and infectious bursal disease virus (IBDV)-specific antibody titres, the Hellevad and Hisex chickens had lower levels than the Bovans. How the differences observed in growth and immune parameters in the three chicken lines influence the immune protection against infection needs to be studied further.

Quantification and phenotypic characterisation of peripheral IFN-γ producing leucocytes in chickens vaccinated against Newcastle disease.

The aim of this study was to optimise and evaluate an intracellular cytokine staining (ICS) assay for assessment of T cell IFN-γ responses in chickens vaccinated against Newcastle disease (ND). We aimed to validate currently available antibodies to chicken IFN-γ using transfected CHO cells. Moreover, this ICS assay was evaluated for use to detect mitogen and antigen induced IFN-γ production in chicken peripheral blood leucocytes. Chickens from an inbred white leghorn line containing two MHC haplotypes, B19 and B21, were divided into three experimental groups; one group was kept as naive controls, one group was vaccinated intramuscularly twice with a commercial inactivated ND virus (NDV) vaccine, and the last group was vaccinated orally twice with a commercial live attenuated NDV vaccine. PBMC were ex vivo stimulated with ConA or with NDV antigen. The ICS assay was used to determine the phenotype and frequency of IFN-γ positive cells. ConA stimulation induced extensive IFN-γ production in both CD3+TCRγδ+ (γδ T cells) cells and CD3+TCRγδ- cells (αß T cells), but no significant differences were observed between the experimental groups. Furthermore, a large proportion of the IFN-γ producing cells were CD3- indicating that other cells than classic T cells, secreted this cytokine. NDV antigen stimulation induced IFN-γ production but to a lower extent than ConA and with a large variation between individuals. The CD3+TCR1γδ-CD8α+ (CTL) population produced the highest NDV specific IFN-γ responses, with significantly elevated levels of IFN-γ producing cells in the B19 chickens vaccinated orally with live attenuated NDV vaccine. This was not the case in the B21 animals, indicating a haplotype restricted variation. In contrast, the CD3+TCR1γδ-CD4+ (Th) population did not show a significant increase in IFN-γ production in NDV stimulated samples which was in part due to a high number of IFN-γ producing cells after incubation with medium alone. In conclusion, an ICS assay for phenotyping of IFN-γ producing chicken leukocytes was set up that proved useful in identifying cytokine producing cells upon either mitogen or antigen-specific stimulation.

Altered circulating levels of serotonin and immunological changes in laying hens divergently selected for feather pecking behavior.

The aim of this study was to investigate the changes in immunological parameters as well as changes with respect to plasma levels of serotonin and tryptophan in lines selected for and against feather pecking (FP) behavior [high FP (HP) line and low FP (LP) line] for 5 generations. The hens from the HP line had a higher plasma serotonin level than those from the LP line (0.059 vs. 0.037 micromol/L, F(2,27) = 0.031, P < 0.05). The plasma level of tryptophan was, on average, 67.30 micromol/L and did not differ between the lines (68.3 vs. 66.3 micromol/L, F(2,28) = 0.36, P < 0.05). The HP line had a higher response to infectious bursal disease virus vaccination after 1 wk post-vaccination compared with the control and LP lines. The number of white blood cells (P < 0.0001) and the expression of MHC class I molecules on CD4 (P < 0.02), CD8beta (P < 0.006) and on B cells (P < 0.03) were highest in the LP line compared with the control and HP lines. Selection for or against FP, therefore, changes the number of white blood cells and the expression of MHC class I molecules on T and B cells, which may influence the health status of the birds.

Immune response to a killed infectious bursal disease virus vaccine in inbred chicken lines with different major histocompatibility complex haplotypes.

The influence of MHC on antibody responses to killed infectious bursal disease virus (IBDV) vaccine was investigated in several MHC inbred chicken lines. We found a notable MHC haplotype effect on the specific antibody response against IBDV as measured by ELISA. Some MHC haplotypes were high responders (B201, B4, and BR5), whereas other MHC haplotypes were low responders (B19, B12 and BW3). The humoral response of 1 pair of recombinants isolated from a Red Jungle Fowl (BW3 and BW4) being identical on BF and BG, but different on BL, indicated that part of the primary vaccine response was an MHC II restricted T-cell dependent response. The humoral response in another pair of recombinant haplotypes originating in 2 different White Leghorn chickens being BF21, BL21, BG15 (BR4) and BF15, BL15, BG21 (BR5) on the MHC locus indicated that the BG locus may perform an adjuvant effect on the antibody response as well. Vaccination of chickens at different ages and in lines with different origin indicated that age and background genes also influence the specific antibody response against inactivated IBDV vaccine.

Discrepancy between transcriptional products and cell surface expression of MHC class I antigens in metastatic and non-metastatic Lewis lung tumor cells.

Major histocompatibility complex class I transcriptional products and cell surface expression of their corresponding proteins were measured in tumorigenic Lewis lung carcinoma cells with either high metastatic activity (G4 cells) or with no metastatic activity (G2 cells). The transcriptional products were measured by hybridization to gene-specific oligonucleotide probes for H-2Kb and H-2Db respectively. The cell surface density of the corresponding H-2 glycoproteins was determined by FACS cell sorter analysis and by radioimmunoassay using anti-H-2Kb and anti-H-2Db specific monoclonal antibodies. The analyses revealed that the cell surface density of both Kb and Db was reduced 4-9 fold in G4 cells compared to G2 cells. However, this reduction of G4 cell surface expression of MHC class I molecules was not reflected at the mRNA level since both subclones had similar low levels of detectable Kb and Db specific mRNA. beta 2-microglobulin was analysed at the mRNA and protein level and found not to be the rate-limiting factor in the MHC class I expression of the metastatic G4 cells. Thus, the cell surface expression of H-2Kb and H-2Db by the two Lewis lung carcinoma subclones did not correlate with the amount of specific mRNA. Other regulatory mechanisms of gene expression acting at the levels between transcription and the appearance of the gene product at the cell surface must therefore account for the observed difference in the cell surface expression of MHC class I molecules of the two Lewis lung carcinoma cells. The potential importance of MHC class I expression in the metastatic capacity of the tumor cells is discussed.

Major histocompatibility complex-linked immune response of young chickens vaccinated with an attenuated live infectious bursal disease virus vaccine followed by an infection.

The influence of the MHC on infectious bursal disease virus (IBDV) vaccine response in chickens was investigated in three different chicken lines containing four different MHC haplotypes. Two MHC haplotypes were present in all three lines with one haplotype (B19) shared between the lines. Line 1 further contains the BW1 haplotype isolated from a Red Jungle Fowl. Line 131 further contains the B131 haplotype isolated from a meat-type chicken. Finally, Line 21 further contains the international B21 haplotype. The chickens were vaccinated with live attenuated commercial IBDV vaccine at 3 wk of age, followed by a challenge with virulent IBDV at 6 wk of age. In this study, we found a notable MHC haplotype effect on the specific antibody response against IBDV, as measured by ELISA. The BW1 haplotype was found to have a significantly higher serum antibody titer against IBDV (7,872) than haplotypes B19 (mean 5,243), B21 (5,570), and B131 (5,333) at 8 d postinfection. However, a virus-neutralizing antibody test did not reflect this result. Nevertheless, the MHC haplotype-associated protective immunity was further supported by the bursa of Fabricius (bursa) recovery from the disease, as measured by histological scorings of the bursa. Chickens carrying the BW1 haplotype had a significantly lower bursa lesion score (1.7) than the haplotypes B19 (mean 3.8), B21 (3.6), and B131 (4.3) 8 d postinfection. Furthermore, multiple line effects were found in other variables when comparing Day 6 with Day 8. Body weight, relative weights of the bursa and the spleen, percentage and relative number of MHC II molecules on MHC II-positive lymphocytes, percentage and relative number of CD4 molecules on CD4-positive lymphocytes, and the specific antibody response all differed significantly among lines. Line 1, with Red Jungle Fowl genes, was clearly differentiated from the other two investigated lines. These results suggest an MHC II restricted T-cell dependent secondary antibody response against IBDV.

Serum levels of mannan-binding lectin in chickens prior to and during experimental infection with avian infectious bronchitis virus.

Mannan-binding lectin (MBL) is a glycoprotein and a member of the C-type lectin super family, the collectin family, and the acute phase protein family. The MBL exerts its function by directly binding to microbial surfaces through its carbohydrate recognition domains, followed by direct opsonization or complement activation via MBL-associated serine proteases (MASP)-1 and -2. Thus, MBL plays a major role in the first-line innate defense against pathogens. We investigated the MBL concentrations in serum during experimental infectious bronchitis virus (IBV) infections in chickens. The results showed that the acute phase MBL response to infection with IBV was, to a degree (P < 0.0068), dependent on whether the chickens were inoculated after 12 h of rest (dark) or after 12 h of activity (light). The acute phase response in chickens challenged after 12 h of activity peaked after 4.6 d with an increase of 24%, whereas the acute phase response in chickens challenged after 12 h of rest peaked after 3.1 d with an increase of 51%. The specific antibody titer against IBV was also tested, and a difference (P < 0.0091) between the two experimental groups was found with peak titer values of 6,816 and 4,349. However, the highest value was found in chickens inoculated after 12 h of activity. Thus, an inverse relation exists between the MBL response and the IBV specific antibody response. The ability of MBL to activate the complement cascade was tested in a heterologous system by deposition of human C4 on the chicken MBL/MASP complex. The complement activation was directly associated with the concentration of MBL in serum, indicating neutralization of the virus before the humoral antibody response took over.

Effects of different antimicrobial treatments on serum acute phase responses and leucocyte counts in pigs after a primary and a secondary challenge infection with Actinobacillus pleuropneumoniae.

The susceptibility to an initial challenge and a re-challenge inoculation with Actinobacillus pleuropneumoniae was analysed in pigs that were treated with antimicrobials of different efficacies following the first exposure to A pleuropneumoniae. In brief, 30 nine-week-old specific pathogen-free pigs were allocated to five groups of six. After acclimatisation, four groups were inoculated with A pleuropneumoniae serotype 2. At the onset of clinical signs, three of the groups of pigs were treated with enrofloxacin, tetracycline or penicillin. A fourth group served as the inoculated control and the fifth group as a control group that had not been inoculated. On day 28, all five groups were re-challenged with the same strain of A pleuropneumoniae serotype 2 as had been used in the first inoculation. No treatments were carried out at this time. The acute phase responses and differential leucocyte counts were monitored in detail after both inoculations. Leucocytosis and acute phase responses in the forms of serum amyloid A, pig-major acute phase protein and haptoglobin were recorded in all of the inoculated groups after the onset of clinical signs following the first inoculation. A porcine mannan-binding lectin-A response was less evident in the pigs. Acute phase responses resembling those of the first inoculation were observed in the pigs that had not previously been inoculated and in the pigs treated with enrofloxacin. Acute phase responses were not recorded in the other three groups, where the pigs had seroconverted to A pleuropneumoniae serotype 2 following the first inoculation.

Flow cytometric assessment of antigen-specific proliferation in peripheral chicken T cells by CFSE dilution.

Carboxyfluorescein succinimidyl ester (CFSE) dilution is a well established method for analysis of dividing cells by flow cytometry. In other species the method has been extensively used in the study of antigen-specific T cells. The purpose of this study was to apply the method to chicken peripheral mononuclear blood cells (PBMC) and to evaluate and optimize its performance in relation to detection of vaccine-induced chicken T cells specific for Newcastle disease virus (NDV). The method was based on analysis of CFSE dilution upon ex vivo recall stimulation with whole vaccine antigen. Analysis of proliferation was combined with the use of monoclonal antibodies directed against the lymphocyte surface markers CD4 and CD8 in order to phenotype the responding cells. Problems with nonspecific background proliferation especially in the CD8 compartment were significantly reduced by replacing medium containing fetal calf serum with serum-free medium. It was rendered probable that antigen-specific cellular immunity can be assessed by this method as NDV-vaccinated chickens showed a significantly higher proliferative capacity than age-matched naïve controls. Furthermore it was shown that the recall stimulation lead to a proliferative response in T cells expressing αß-type TCRs but also those expressing the γδ-type. In summary, the method was found challenging but nevertheless useful to quantify the proliferative response of chicken antigen-specific T cells. Further investigations though, are needed in order to prove what cell subsets are true antigen-specific responders and what cells are bystander activated. Nevertheless, the method is expected to be a valuable tool to evaluate and quantify vaccine responses to current and new chicken vaccines in the future.

Flow cytometric assessment of chicken T cell-mediated immune responses after Newcastle disease virus vaccination and challenge.

The objective of this study was to use flow cytometry to assess chicken T cell-mediated immune responses. In this study two inbred genetic chicken lines (L130 and L133) were subjected to two times vaccination against Newcastle disease (ND) and a subsequent challenge by ND virus (NDV) infection. Despite a delayed NDV-specific antibody response to vaccination, L133 appeared to be better protected than L130 in the subsequent infection challenge as determined by the presence of viral genomes. Peripheral blood was analyzed by flow cytometry and responses in vaccinated/challenged birds were studied by 5-color immunophenotyping as well as by measuring the proliferative capacity of NDV-specific T cells after recall stimulation. Immunophenotyping identified L133 as having a significantly lower CD4/CD8 ratio and a lower frequency of gammadelta T cells than L130 in the peripheral T cell compartment. Furthermore, peripheral lymphocytes from L133 exhibited a significantly higher expression of CD44 and CD45 throughout the experiment. Interestingly, also vaccine-induced differences were observed in L133 as immune chickens had a significantly higher CD45 expression on their lymphocytes than the naïve controls. Immune chickens from both lines had a significantly higher frequency of circulating gammadelta T cells than the naïve controls both after vaccination and challenge. Finally, the proliferative capacity of peripheral CD4+ and CD8+ cells specific for NDV was addressed 3 weeks after vaccination and 1 week after infection and found to be significantly higher in L133 than in L130 at both sampling times. In conclusion, we found the applied flow cytometric methods very useful for the study of chicken T cell biology.