[Show abstract][Hide abstract] ABSTRACT: Avian pathogenic Escherichia coli (APEC) infections are a serious impediment to sustainable poultry production worldwide. Licensed vaccines are available, but the immunological basis of protection is ill-defined and a need exists to extend cross-serotype efficacy. Here, we analysed innate and adaptive responses induced by commercial vaccines in turkeys. Both a live-attenuated APEC O78 ΔaroA vaccine (Poulvac® E. coli) and a formalin-inactivated APEC O78 bacterin conferred significant protection against homologous intra-airsac challenge in a model of acute colibacillosis. Analysis of expression levels of signature cytokine mRNAs indicated that both vaccines induced a predominantly Th2 response in the spleen. Both vaccines resulted in increased levels of serum O78-specific IgY detected by ELISA and significant splenocyte recall responses to soluble APEC antigens at post-vaccination and post-challenge periods. Supplementing a non-adjuvanted inactivated vaccine with Th2-biasing (Titermax® Gold or aluminium hydroxide) or Th1-biasing (CASAC or CpG motifs) adjuvants, suggested that Th2-biasing adjuvants may give more protection. However, all adjuvants tested augmented humoral responses and protection relative to controls. Our data highlight the importance of both cell-mediated and antibody responses in APEC vaccine-mediated protection toward the control of a key avian endemic disease.
Veterinary Research 12/2015; 46(1):5. DOI:10.1186/s13567-014-0132-5 · 2.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Very virulent infectious bursal disease virus (vvIBDV) induces immunosuppression and inflammation in young birds, which subsequently leads to high mortality. In addition, infectious bursal disease (IBD) is one of the leading causes of vaccine failure on farms. Therefore, understanding the immunopathogenesis of IBDV in both the spleen and the bursae could help effective vaccine development. However, previous studies only profiled the differential expression of a limited number of cytokines, in either the spleen or the bursae of Fabricius of IBDV-infected chickens. Thus, this study aims to evaluate the in vitro and in vivo immunoregulatory effects of vvIBDV infection on macrophage-like cells, spleen and bursae of Fabricius.
The viral load was increased during the progression of the in vitro infection in the HD11 macrophage cell line and in vivo, but no significant difference was observed between the spleen and the bursae tissue. vvIBDV infection induced the expression of pro-inflammatory and Th1 cytokines, and chemokines from HD11 cells in a time- and dosage-dependent manner. Furthermore, alterations in the lymphocyte populations, cytokine and chemokine expression, were observed in the vvIBDV-infected spleens and bursae. A drastic rise was detected in numbers of macrophages and pro-inflammatory cytokine expression in the spleen, as early as 2 days post-infection (dpi). On 4 dpi, macrophage and T lymphocyte infiltration, associated with the peak expression of pro-inflammatory cytokines in the bursae tissues of infected chickens were observed. The majority of the significantly regulated pro-inflammatory cytokines and chemokines, in vvIBDV-infected spleens and bursae, were also detected in vvIBDV-infected HD11 cells. This cellular infiltration subsequently resulted in a sharp rise in nitric oxide (NO) and lipid peroxidation levels.
This study suggests that macrophage may play an important role in regulating the early expression of pro-inflammatory cytokines, first in the spleen and then in the bursae, the latter tissue undergoing macrophage infiltration at 4 dpi.
BMC Veterinary Research 12/2015; 11(1). DOI:10.1186/s12917-015-0377-x · 1.78 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Infectious bursal disease (IBD) is caused by infectious bursal disease virus (IBDV), an immunosuppressive virus that targets immune cells such as B cells and macrophage. However, the involvement of dendritic cells (DCs) during IBDV infection is not well understood. In this study the in vitro effects of live and inactivated very virulent IBDV (vvIBDV) UPM0081 on bone marrow derived DCs (BM-DC) were characterized and compared with BM-DC treated with lipopolysaccharide (LPS). Morphologically, BM-DC treated with LPS and vvIBDV showed stellate shape when compared to immature BM-DC. In addition, LPS-treated and both live and inactivated vvIBDV-infected BM-DC expressed high levels of double positive CD86 and MHC class II antigens (>20%). vvIBDV-infected BM-DC showed significantly higher numbers of apoptotic cells compared to LPS. Replication of vvIBDV was detected in the infected BM-DC as evidenced by the increased expression of VP3 and VP4 IBDV antigens based on flow cytometry, real-time PCR and immunofluorescence tests. Levels of different immune-related genes such as IL-1β, CXCLi2 (IL-8), IL-18, IFN-γ, IL-12α, CCR7 and TLR3 were measured after LPS and vvIBDV treatments. However, marked differences were noticed in the onset and intensity of the gene expression between these two treatment groups. LPS was far more potent than live and inactivated vvIBDV in inducing the expression of IL-1β, IL-18 and CCR7 while expression of Th1-like cytokines, IFN-γ and IL-12α were significantly increased in the live vvIBDV treatment group. Meanwhile, the expression of TLR3 was increased in live vvIBDV-infected BM-DC as compared to control. Inactivated vvIBDV-treated BM-DC failed to stimulate IFN-γ, IL-12α and TLR3 expressions. This study suggested that BM-DC may serve as another target cells during IBDV infection which require further confirmation via in vivo studies.
[Show abstract][Hide abstract] ABSTRACT: Chicken Anaemia Virus (CAV) is an economically important virus that targets lymphoid and erythroblastoid progenitor cells leading to immunosuppression. This study aimed to investigate the interplay between viral infection and the host's immune response to better understand the pathways that lead to CAV-induced immunosuppression. To mimic vertical transmission of CAV in the absence of maternally-derived antibody, day-old chicks were infected and their responses measured at various time-points post-infection by qRT-PCR and gene expression microarrays. The kinetics of mRNA expression levels of signature cytokines of innate and adaptive immune responses were determined by qRT-PCR. The global gene expression profiles of mock-infected (control) and CAV-infected chickens at 14 dpi were also compared using a chicken immune-related 5K microarray. Although in the thymus there was evidence of induction of an innate immune response following CAV infection, this was limited in magnitude. There was little evidence of a Th1 adaptive immune response in any lymphoid tissue, as would normally be expected in response to viral infection. Most cytokines associated with Th1, Th2 or Treg subsets were down-regulated, except IL-2, IL-13, IL-10 and IFNγ, which were all up-regulated in thymus and bone marrow. From the microarray studies, genes that exhibited significant (greater than 1.5-fold, false discovery rate <0.05) changes in expression in thymus and bone marrow on CAV infection were mainly associated with T-cell receptor signalling, immune response, transcriptional regulation, intracellular signalling and regulation of apoptosis. Expression levels of a number of adaptor proteins, such as src-like adaptor protein (SLA), a negative regulator of T-cell receptor signalling and the transcription factor Special AT-rich Binding Protein 1 (SATB1), were significantly down-regulated by CAV infection, suggesting potential roles for these genes as regulators of viral infection or cell defence. These results extend our understanding of CAV-induced immunosuppression and suggest a global immune dysregulation following CAV infection.
PLoS ONE 08/2015; 10(8):e0134866. DOI:10.1371/journal.pone.0134866 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background:
Small insertions and deletions (InDels) constitute the second most abundant class of genetic variants and have been found to be associated with many traits and diseases. The present study reports on the detection and characterisation of about 883 K high quality InDels from the whole-genome analysis of several modern layer chicken lines from diverse breeds.
To reduce the error rates seen in InDel detection, this study used the consensus set from two InDel-calling packages: SAMtools and Dindel, as well as stringent post-filtering criteria. By analysing sequence data from 163 chickens from 11 commercial and 5 experimental layer lines, this study detected about 883 K high quality consensus InDels with 93% validation rate and an average density of 0.78 InDels/kb over the genome. Certain chromosomes, viz, GGAZ, 16, 22 and 25 showed very low densities of InDels whereas the highest rate was observed on GGA6. In spite of the higher recombination rates on microchromosomes, the InDel density on these chromosomes was generally lower relative to macrochromosomes possibly due to their higher gene density. About 43-87% of the InDels were found to be fixed within each line. The majority of detected InDels (86%) were 1-5 bases and about 63% were non-repetitive in nature while the rest were tandem repeats of various motif types. Functional annotation identified 613 frameshift, 465 non-frameshift and 10 stop-gain/loss InDels. Apart from the frameshift and stopgain/loss InDels that are expected to affect the translation of protein sequences and their biological activity, 33% of the non-frameshift were predicted as evolutionary intolerant with potential impact on protein functions. Moreover, about 2.5% of the InDels coincided with the most-conserved elements previously mapped on the chicken genome and are likely to define functional elements. InDels potentially affecting protein function were found to be enriched for certain gene-classes e.g. those associated with cell proliferation, chromosome and Golgi organization, spermatogenesis, and muscle contraction.
The large catalogue of InDels presented in this study along with their associated information such as functional annotation, estimated allele frequency, etc. are expected to serve as a rich resource for application in future research and breeding in the chicken.
[Show abstract][Hide abstract] ABSTRACT: Contents
79 The Chicken Leads the Way in Avian Genomics.
Prepared by J. Smith.
80 The Chicken Genome: Current Status of Genome Assembly and Annotations.
Prepared by D.W. Burt, L. Eöry, A.L. Archibald, B.L. Aken, P. Flicek, K. Howe, W. Chow, M. Dunn, J.M.D. Wood, R. Nag, and W.C. Warren.
83 The Avian RNAseq Consortium: A Community Effort to Annotate the Chicken Genome.
Prepared by J. Smith, D.W. Burt, and the Avian RNAseq Consortium.
89 Noncoding RNAs in the Chicken Genome.
Prepared by J. Hertel, M. Fasold, A. Nitsche, I. Erb, P. Prieto, D. Kedra, C. Notredame, T.E. Steeves, P.P. Gardner, and P.F. Stadler.
91 Genome Sequencing in Birds and Evolutionary Inferences from Avian Genome Sequences.
Prepared by H. Ellegren.
94 The Use of Avian BAC Libraries and Clones.
Prepared by M.N. Romanov and D.K. Griffin.
96 Comparative Genomics.
Prepared by D.M. Larkin, M. Farré, and J. Damas.
100 Avian Cytogenetics Goes Functional.
Prepared by D.K. Griffin, M.N. Romanov, R. O’Connor, K.E. Fowler, and D.M. Larkin.
105 Hypermethylated Chromosome Regions in Chicken and Other Birds.
Prepared by M. Schmid, C. Steinlein, A.-S. Schneider, I. Nanda, and T. Haaf.
109 An Overview of Avian Evolution.
Prepared by S.B. Hedges.
114 An Update on Chicken Sex Determination and Gonadal Sex Differentiation.
Prepared by C.A. Smith.
119 Avian Epigenetics.
Prepared by H. Zhou.
122 Structural Variation and Copy Number Variation in Poultry.
Prepared by R.P.M.A. Crooijmans and M.A.M. Groenen.
124 SNPs and InDels – The Most Abundant Sources of Genetic Variations.
Prepared by A.A. Gheyas, C. Boschiero, and D.W. Burt.
130 Genetic Diversity of Village Chickens.
Prepared by T.T. Desta, R.A. Lawal, and O. Hanotte.
133 Mendelian Traits.
Prepared by D. Wragg.
137 Treasure the Exceptions: Utilizing Chicken Mutant Lines and Advanced Genetic Technologies to Uncover Genes Involved in Developmental Processes.
Prepared by E.A. O’Hare and M.E. Delany.
141 Genomic Landscape of the Chicken DT40 Cell Line.
Prepared by A. Motegi and M. Takata.
145 RNA-seq: Primary Cells, Cell Lines and Heat Stress.
Prepared by C.J. Schmidt, E.M. Pritchett, L. Sun, R.V.N. Davis, A. Hubbard, K.E. Kniel, S.M. Markland, Q. Wang, C. Ashwell, M. Persia, M.F. Rothschild, and S.J. Lamont.
148 Host-Viral Genome Interactions in Marek’s Disease.
Prepared by M.C. McPherson, C.M. Robinson, and M.E. Delany.
154 Transcriptome Variation in Response to Marek’s Disease Virus Acute Infection.
Prepared by L. Preeyanon, C.T. Brown, and H.H. Cheng.
163 The National Avian Research Facility.
Prepared by A. Hart, R. Kuo, L. Eöry, P. Kaiser, and D.W. Burt.
Cytogenetic and Genome Research 07/2015; 145(2):78-179. DOI:10.1159/000430927 · 1.56 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Eimeria are recognised as highly pathogenic parasites of chickens. Presently, research aimed at reducing their impact is hindered by the lack of non-subjective medium/high throughput diagnostic tools. Here, we tested a pre-existing real-time PCR (qPCR) to quantify Eimeria tenella from chicken tissue and faecal samples. Chickens were inoculated with 500, 1500 or 4500 E. tenella oocysts; then, parasite burdens were quantified employing i) qPCR analysis of DNA extracted from caecal tissues collected at five and eight days post-infection (dpi) and ii) faecal oocyst counts (FOC) on samples taken six to eight dpi. Real-time PCR test results indicated a significant dose-dependent increase in parasite numbers among study groups (p=0.0002 and p=0.0019) for samples collected 5 dpi (i.e. prior to gametogony) but not in those from day eight (after most oocyst shedding). In contrast, no significant dose-dependent increase in FOC was observed in samples from the three groups collected 6-8 dpi. Further evaluation of this technique, via the quantification of E. tenella in naturally infected broilers from across the United Kingdom, indicated a concordant relationship (r2 = 0.8894) between qPCR test results and FOC. This method overcomes the limitations of coproscopic quantification and allows reproducible medium to high-throughput examination of a variety of biological samples, thus representing a valuable diagnostic tool for determining the impact of Eimeria infections on chicken farms. Importantly, qPCR also has significant implications for animal welfare via improved statistical power and reduced group sizes in experimental studies.
[Show abstract][Hide abstract] ABSTRACT: type A is the main etiological factor for necrotic enteritis, a multifactorial enteric disease that penalizes performance, health, and welfare of poultry. Lack of knowledge of host responses and disease pathogenesis is slowing down progress on developing therapies for disease control. A combined genomewide and targeted gene approach was used to investigate pathways and biological functions affected by the infusion of culture supernatant in the duodenum of broilers in 2 experiments. An in situ isolated loop of duodenum was prepared in anesthetized broilers of 3 wk of age (Exp. 1) and was infused either with crude culture supernatant ( = 7; treated), positive for necrotic enteritis B-like toxin (NetB) as determined by a cytotoxicity assay, or with a control preparation ( = 6; control). Birds were maintained alive for 1 h and then euthanized for tissue recovery. The use of the Affymetrix chicken genome array on RNA samples from loop tissue showed top biological functions affected by culture supernatant infusion included cell morphology, immune cell trafficking, and cell death; pathways affected included death receptor signaling, inflammatory response, and nuclear factor (NF)-κB signaling. In a second in situ study (Exp. 2), broilers were maintained alive for 4 h to monitor temporal expression patterns of targeted genes. Duodenal tissue was removed at 0.5, 1, 2, and 4 h post-infusion with culture supernatant ( = 9) or a control preparation ( = 5) for histology and gene expression analysis. Genes encoding proinflammatory cytokines, such as interferon γ (γ), cell trafficking, such as neuroblastoma 1 () and B cell CLL/Lymphoma 6 (), and cell death, such as Fas cell surface death receptor () and GTPase IMAP family member 8 (), were differentially expressed in the duodenum of treated and control broilers ( < 0.05). We have demonstrated that culture supernatant (NetB positive) infusion resulted in histological and gene expression changes consistent with necrotic enteritis in the duodenum of broilers. In the absence of live bacteria, crude culture supernatant resulted in early immunomodulation, inflammation, and cell death in the duodenum. The pathways identified here can be targeted for the development of new drugs, vaccines, and novel therapies for necrotic enteritis in broilers.
Journal of Animal Science 06/2015; DOI:10.2527/jas.2014-8597 · 2.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The Eimeria species are highly pathogenic parasites of chickens. Research aimed at reducing their impact is hindered by a lack of non-subjective, quantitative, tools to measure parasite replication in the host. The time-consuming, and often time-sensitive, nature of existing approaches precludes their use in large-scale genetic, epidemiological, and evolutionary analyses. We have used quantitative real-time PCR (qPCR) to accurately quantify Eimeria tenella in chicken tissue and shown this to be more efficient and sensitive than traditional methodologies. We tested four chicken-specific reference qPCR assays and found beta-actin (actb) to be optimal for sample normalisation. In an experimental setting, chickens were inoculated with 500, 1500, or 4500 E. tenella oocysts and parasite replication and the impact of infection measured by i) qPCR analysis of DNA extracted from caecal tissues collected at five and eight days post-infection (dpi), ii) faecal oocyst counts (FOCs) on samples taken from six to eight dpi, and iii) lesion scoring on caeca collected post-mortem at five and eight dpi. Quantitative real-time PCR test results indicated a significant dose-dependent increase in parasite numbers among study groups for samples collected five dpi (i.e., prior to gametogony) (R2 = 0.994) (p < 0.002) but not in those from day eight (after most oocyst shedding) (R2 = 0.006) (p > 0.379). A strong dose-dependent increase in parasite replication and severity of infection was also revealed by FOC (R2 = 0.997) and lesion scoring. Importantly, qPCR offers substantial improvements for animal welfare via improved statistical power and reduced group sizes in experimental studies. The described qPCR method overcomes subjective limitations of coproscopic quantification, allows reproducible medium- to high-throughput examination of tissues, faeces, and oocysts, and is a valuable tool for determining the impact of Eimeria infections in both experimental and field settings.
Parasitology International 06/2015; 64(5). DOI:10.1016/j.parint.2015.06.010 · 1.86 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chickens raised under village production systems are exposed to a wide variety of pathogens, and current or previous infections may affect their susceptibility to further infections with another parasite, and/or can alter the manifestation of each infection. It is possible that co-infections may be as important as environmental risk factors. However, in cross-sectional studies, where the timing of infection is unknown, apparent associations between infections may be observed due to parasites sharing common risk factors. This study measured antibody titres to 3 viral (Newcastle disease, Marek's disease and infectious bursal disease) and 2 bacterial (Pasteurella multocida and Salmonella) diseases, and the infection prevalence of 3 families of endo- and ecto-parasites (Ascaridida, Eimeria and lice) in 1056 village chickens from two geographically distinct populations in Ethiopia. Samples were collected during 4 cross-sectional surveys, each approximately 6 months apart. Constrained ordination, a technique for analysis of ecological community data, was used to explore this complex dataset and enabled potential relationships to be uncovered and tested despite the different measurements used for the different parasites. It was found that only a small proportion of variation in the data could be explained by the risk factors measured. Very few birds (9/1280) were found to be seropositive to Newcastle disease. Positive relationships were identified between Pasteurella and Salmonella titres; and between Marek's disease and parasitic infections, and these two groups of diseases were correlated with females and males respectively. This may suggest differences in the way that the immune systems of male and female chickens interact with these parasites. In conclusion, we find that a number of infectious pathogens and their interactions are likely to impact village chicken health and production. Control of these infections is likely to be of importance in future development planning.
Preventive Veterinary Medicine 11/2014; 117(2). DOI:10.1016/j.prevetmed.2014.07.002 · 2.17 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In the poultry production industry, chickens with access to outdoor areas are exposed to a wide range of parasites e.g. the helminth Ascaridia galli. By real-time quantitative RT-PCR, the relative gene expression of the T helper 1 (Th1) cytokine IFN-γ, the T helper 2 (Th2) cytokine IL-13, the anti-inflammatory cytokines IL-10 and TGF-β4 and the pro-inflammatory cytokine IL-17F were determined over a period of 3 weeks in A. galli and non-A. galli-infected chickens. A characteristic Th2 response was observed in the jejunum of A. galli-infected chickens with increased expression of IL-13 and decreased expression of IFN-γ from day 14 post infection. At the putative time of larvae invasion into the intestinal mucosa (day 7), an increased expression of IFN-γ, IL-10, and TGF-β4 was observed in the spleen. At the putative onset of the innate immune response (day 10), a decreased expression of jejunal IFN-γ and IL-13 was observed. Finally, at the expected period of an adaptive immune response (days 14–21) a general decreased expression of IFN-γ and TGF-β4 in spleen and IFN-γ in jejunum was followed by a decreased expression of IFN-γ and IL-10 at day 21 in caecal tonsils.
[Show abstract][Hide abstract] ABSTRACT: Abstract Text:
Salmonella Enteritidis is a threat to human health due to the consumption of contaminated meat or eggs. A genome wide association study was performed using a 60 K SNP chip to identify genomic regions associated with resistance to salmonella carrier-state. We genotyped 209 chicks issued from an F8 advanced intercross line between two experimental inbred lines and experimentally infected with S. Enteritidis. Their carrier-state was assessed by measuring the fecal count of S. Enteritidis 4 weeks after infection. After quality controls, 22,557 markers could be used for a genome wide association analysis performed with the plink software. Half of the markers associated with the highest probabilities were located in genomic regions previously identified for their association with resistance to S. Enteritidis carrier-state or resistance to S. Typhimurium in chicken. This study therefore confirms and refines the location of several regions of interest.
Chicken; Salmonella Enteritidis; GWAS
10th World Congress on Genetics Applied to Livestock Production; 08/2014
[Show abstract][Hide abstract] ABSTRACT: Serological data for Salmonella and Infec-tious Bursal Disease Virus (IBDV) were recorded for 760 indigenous Ethiopian village chickens raised in two distinct geographical regions, Horro and Jarso. Chickens were gen-otyped with a 620K SNP array. A multidimensional scaling analysis showed that the two populations were genetically distinct. In Horro chickens, genome-wide scans revealed nine SNP with chromosome-wide significant association with Salmonella resistance and seven SNP with genome-wide significant association with IBDV resistance. In Jarso chickens, these scans revealed one SNP with genome-wide and two SNP with chromosome-wide significant associa-tion with Salmonella resistance, and one SNP with genome-wide and three SNP with chromosome-wide significant association with IBDV resistance. All significant SNP for each region for either disease were located on different chromosomes. Most of these SNP had a significant additive effect and were located close to annotated genes that are known to impact the immune response in chickens.
10 th World Congress of Genetics Applied to Livestock Production; 08/2014
[Show abstract][Hide abstract] ABSTRACT: This study aimed to evaluate the response of Harderian gland (HG) cells after in vitro stimulation with class B synthetic oligodeoxyribonucleotides (ODN) containing CpG motifs. This knowledge is of importance for the development of mucosal vaccines for poultry, such as eye-drop or spray vaccines, to determine if class B CpG ODN can act as an vaccine adjuvant or as a prophylactic treatment mainly against respiratory disease viruses. The relative expression of Toll-like receptor 21 (TLR21), interferon (IFN)-γ, interleukin (IL)-1β and IL-10 genes were quantified at 1, 3, 6 and 18 h post-stimulation of HG cells from 5-week-old birds. In addition, it was also investigated if expression of these genes was affected by the age of the birds (differences between 5- and 12-week-old birds), concentrations of ODN or cell preparation method used. Class B CpG ODN induced upregulation of TLR21 and IFN-γ mRNA expression levels at 1 h post-stimulation depending on concentration of ODN used but only in HG cells isolated from young birds.
[Show abstract][Hide abstract] ABSTRACT: The observed genetic divergence likely emanated from limited gene flow, variation in demographic history and route and time of introduction. Moreover, the two populations might be descended from different ancestral origins and they have been established and developed under different management histories. Ecological variability and demographic structure of the community may have also contributed to this divergence. Horro chickens are genetically more homogenous than Jarso chickens (Figure 4). The optimal K was 2, however biologically meaningful pattern was observed at K = 4 following the marketsheds sampled. Table 2. Indicators of genetic diversity (mean (std)). Study site Horro Jarso Location W. Ethiopia E. Ethiopia Agro-ecology Sub-humid Semi-arid Topography Undulating Rugged Religion 98% Christians 99% Muslims Rainfall 1685mm 700mm Temperature 19°C 21°C Flock size 8 birds 4 birds Table 1. Description of the study sites. Figure 1. The study sites. Figure 2. The binned MAF.
34th International Society for Animal Genetics, Xi'an, China; 07/2014