A Wider and Deeper Peptide-Binding Groove for the Class I Molecules from B15 Compared with B19 Chickens Correlates with Relative Resistance to Marek's Disease.

The chicken MHC is known to confer decisive resistance or susceptibility to various economically important pathogens, including the iconic oncogenic herpesvirus that causes Marek's disease (MD). Only one classical class I gene, BF2, is expressed at a high level in chickens, so it was relatively easy to discern a hierarchy from well-expressed thermostable fastidious specialist alleles to promiscuous generalist alleles that are less stable and expressed less on the cell surface. The class I molecule BF2*1901 is better expressed and more thermostable than the closely related BF2*1501, but the peptide motif was not simpler as expected. In this study, we confirm for newly developed chicken lines that the chicken MHC haplotype B15 confers resistance to MD compared with B19. Using gas phase sequencing and immunopeptidomics, we find that BF2*1901 binds a greater variety of amino acids in some anchor positions than does BF2*1501. However, by x-ray crystallography, we find that the peptide-binding groove of BF2*1901 is narrower and shallower. Although the self-peptides that bound to BF2*1901 may appear more various than those of BF2*1501, the structures show that the wider and deeper peptide-binding groove of BF2*1501 allows stronger binding and thus more peptides overall, correlating with the expected hierarchies for expression level, thermostability, and MD resistance. Our study provides a reasonable explanation for greater promiscuity for BF2*1501 compared with BF2*1901, corresponding to the difference in resistance to MD.

Correction: Protective porcine influenza virus-specific monoclonal antibodies recognize similar haemagglutinin epitopes as humans.

[This corrects the article DOI: 10.1371/journal.ppat.1009330.].

Protective porcine influenza virus-specific monoclonal antibodies recognize similar haemagglutinin epitopes as humans.

Pigs are natural hosts for the same subtypes of influenza A viruses as humans and integrally involved in virus evolution with frequent interspecies transmissions in both directions. The emergence of the 2009 pandemic H1N1 virus illustrates the importance of pigs in evolution of zoonotic strains. Here we generated pig influenza-specific monoclonal antibodies (mAbs) from H1N1pdm09 infected pigs. The mAbs recognized the same two major immunodominant haemagglutinin (HA) epitopes targeted by humans, one of which is not recognized by post-infection ferret antisera that are commonly used to monitor virus evolution. Neutralizing activity of the pig mAbs was comparable to that of potent human anti-HA mAbs. Further, prophylactic administration of a selected porcine mAb to pigs abolished lung viral load and greatly reduced lung pathology but did not eliminate nasal shedding of virus after H1N1pdm09 challenge. Hence mAbs from pigs, which target HA can significantly reduce disease severity. These results, together with the comparable sizes of pigs and humans, indicate that the pig is a valuable model for understanding how best to apply mAbs as therapy in humans and for monitoring antigenic drift of influenza viruses in humans, thereby providing information highly relevant to making influenza vaccine recommendations.

OMIP-089: Cattle T-cell phenotyping by an 8-color panel.

This multiplex staining panel was developed to differentiate cattle T cells into conventional (CD4 and CD8) and unconventional (γδ-TCR) subsets as well as their stage of differentiation and activation. The combination of CD45RO and CD62L allows the identification of naïve (TNaïve ), central memory (TCM ), effector memory (TEM ) and terminal effector (TTE ) T cells. Activated cattle T cells (TAV ) can be identified by the cell surface expression of CD25. This panel was developed using cryopreserved cattle peripheral blood mononuclear cells (PBMCs) and tested on fresh as well as stimulated PBMCs. Therefore, this 8-color, 10-parameter flow cytometry panel simultaneously identifies cattle TNaïve , TAV , TCM , TEM , TTE and γδ-TCR cells. This panel will improve our ability to examine T-cell response to pathogens and vaccines in cattle including the potential to identify previously undescribed subpopulations. Furthermore, this panel can be readily optimized for other bovid species as many of these reagents are likely to cross react.

OMIP-085: Cattle B-cell phenotyping by an 8-color panel.

This 8-color panel has been optimized to distinguish between functionally distinct subsets of cattle B cells in both fresh and cryopreserved peripheral blood mononuclear cells (PBMCs). Existing characterized antibodies against cell surface molecules (immunoglobulin light chain (S-Ig[L]), CD20, CD21, CD40, CD71, and CD138) enabled the discrimination of 24 unique populations within the B-cell population. This allows the identification of five putative functionally distinct B-cell subsets critical to infection and vaccination responses: (1) naïve B cells (BNaïve ), (2) regulatory B cells (BReg ), (3) memory B cells (BMem ), (4) plasmablasts (PB), and (5) plasma cells (PC). Although CD3 and CD8α can be included as an additional dump channel, it does not significantly improve the panel's ability to separate "classical" B cells. This panel will promote better characterization and tracking of B-cell responses in cattle as well as other bovid species as the reagents are likely to cross react.

Pickering emulsion-templated ionotropic gelation of tocotrienol microcapsules: effects of alginate and chitosan concentrations and gelation process parameters.

BACKGROUND: Throughout the past decade, Pickering emulsion has been increasingly utilized for the encapsulation of bioactive compounds due to its high stability and biocompatibility. In the present work, palm tocotrienols were initially encapsulated in a calcium carbonate Pickering emulsion, which was then subjected to alginate gelation and subsequent chitosan coating. The effects of wall material (alginate and chitosan) concentrations, gelation pH and time, and chitosan coating time on the encapsulation efficiency of palm tocotrienols were explored. RESULTS: Our findings revealed that uncoated alginate microcapsules ruptured upon drying and exhibited low encapsulation efficiency (13.81 ± 2.76%). However, the addition of chitosan successfully provided a more complex and rigid external wall structure to enhance the stability of the microcapsules. By prolonging the crosslinking time from 5 to 30 min and increasing the chitosan concentration from 0.1% to 0.5%, the oil encapsulation efficiency was increased by 28%. Under the right gelation pH (pH 4), the extension of gelation time from 1 to 12 h resulted in an increase in alginate-Ca2+ crosslinkings, thus strengthening the microcapsules. CONCLUSION: With the optimum formulation and process parameters, a high encapsulation efficiency (81.49 ± 1.75%) with an elevated oil loading efficiency (63.58 ± 2.96%) were achieved. The final product is biocompatible and can potentially be used for the delivery of palm tocotrienols. © 2021 Society of Chemical Industry.

The UK Veterinary Immunological Toolbox Website: promoting vaccine research by facilitating communication and removing reagent barriers.

Using the best animal models to study immune responses against specific pathogens or vaccines can dramatically accelerate our understanding. Veterinary species are well studied, particularly livestock, to reduce their disease burden. They have also proven to be powerful models, especially for zoonotic pathogens and novel vaccination strategies. A prerequisite for any model selection is having the right quality and range of species-specific immunological reagents. To help promote the widest possible use of veterinary species, an open access website (https://www.immunologicaltoolbox.co.uk) has been created as a central community annotated hub for veterinary immunological reagents. The website is also the portal into services offered by the UK Immunological Toolbox project that includes antibody generation, sequencing and recombinant expression. The funding for this effort is linked into sustainable sources, but ultimate success relies on community engagement to continually increase the quality and quantity of information. It is hoped that as more users and reagent owners engage, it will become an essential resource for researchers, veterinarians and clinicians alike by removing barriers that prevent the use of the most informative animal models.

Differentially expressed genes during spontaneous lytic switch of Marek's disease virus in lymphoblastoid cell lines determined by global gene expression profiling.

Marek's disease virus (MDV), an alphaherpesvirus of poultry, causes Marek's disease and is characterized by visceral CD4+TCRαß+ T-cell lymphomas in susceptible hosts. Immortal cell lines harbouring the viral genome have been generated from ex vivo cultures of MD tumours. As readily available sources of large numbers of cells, MDV-transformed lymphoblastoid cell lines (LCLs) are extremely valuable for studies of virus-host interaction. While the viral genome in most cells is held in a latent state, minor populations of cells display spontaneous reactivation identifiable by the expression of lytic viral genes. Spontaneous reactivation in these cells presents an opportunity to investigate the biological processes involved in the virus reactivation. For detailed characterization of the molecular events associated with reactivation, we used two lymphoblastoid cell lines derived from lymphomas induced by pRB1B-UL47eGFP, a recombinant MDV engineered to express enhanced green fluorescent protein (EGFP) fused with the UL47. We used fluorescence-activated cell sorting to purify the low-frequency EGFP-positive cells with a spontaneously activating viral genome from the majority EGFP-negative cells and analysed their gene expression profiles by RNA-seq using Illumina HiSeq2500. Ingenuity pathway analysis on more than 2000 differentially expressed genes between the lytically infected (EGFP-positive) and latently infected (EGFP-negative) cell populations identified the biological pathways involved in the reactivation. Virus-reactivating cells exhibited differential expression of a significant number of viral genes, with hierarchical differences in expression levels. Downregulation of a number of host genes including those directly involved in T-cell activation, such as CD3, CD28, ICOS and phospholipase C, was also noticed in the LCL undergoing lytic switch.

A B-cell targeting virus disrupts potentially protective genomic methylation patterns in lymphoid tissue by increasing global 5-hydroxymethylcytosine levels.

The mechanisms by which viruses modulate the immune system include changes in host genomic methylation. 5-hydroxymethylcytosine (5hmC) is the catalytic product of the Tet (Ten-11 translocation) family of enzymes and may serve as an intermediate of DNA demethylation. Recent reports suggest that 5hmC may confer consequences on cellular events including the pathogenesis of disease; in order to explore this possibility further we investigated both 5-methylcytosine (5mC) and 5hmC levels in healthy and diseased chicken bursas of Fabricius. We discovered that embryonic B-cells have high 5mC content while 5hmC decreases during bursa development. We propose that a high 5mC level protects from the mutagenic activity of the B-cell antibody diversifying enzyme activation induced deaminase (AID). In support of this view, AID mRNA increases significantly within the developing bursa from embryonic to post hatch stages while mRNAs that encode Tet family members 1 and 2 reduce over the same period. Moreover, our data revealed that infectious bursal disease virus (IBDV) disrupts this genomic methylation pattern causing a global increase in 5hmC levels in a mechanism that may involve increased Tet 1 and 2 mRNAs. To our knowledge this is the first time that a viral infection has been observed to cause global increases in genomic 5hmC within infected host tissues, underlining a mechanism that may involve the induction of B-cell genomic instability and cell death to facilitate viral egress.

Clonal structure of rapid-onset MDV-driven CD4+ lymphomas and responding CD8+ T cells.

Lymphoid oncogenesis is a life threatening complication associated with a number of persistent viral infections (e.g. EBV and HTLV-1 in humans). With many of these infections it is difficult to study their natural history and the dynamics of tumor formation. Marek's Disease Virus (MDV) is a prevalent α-herpesvirus of poultry, inducing CD4+ TCRαß+ T cell tumors in susceptible hosts. The high penetrance and temporal predictability of tumor induction raises issues related to the clonal structure of these lymphomas. Similarly, the clonality of responding CD8 T cells that infiltrate the tumor sites is unknown. Using TCRß repertoire analysis tools, we demonstrated that MDV driven CD4+ T cell tumors were dominated by one to three large clones within an oligoclonal framework of smaller clones of CD4+ T cells. Individual birds had multiple tumor sites, some the result of metastasis (i.e. shared dominant clones) and others derived from distinct clones of transformed cells. The smaller oligoclonal CD4+ cells may represent an anti-tumor response, although on one occasion a low frequency clone was transformed and expanded after culture. Metastatic tumor clones were detected in the blood early during infection and dominated the circulating T cell repertoire, leading to MDV associated immune suppression. We also demonstrated that the tumor-infiltrating CD8+ T cell response was dominated by large oligoclonal expansions containing both "public" and "private" CDR3 sequences. The frequency of CD8+ T cell CDR3 sequences suggests initial stimulation during the early phases of infection. Collectively, our results indicate that MDV driven tumors are dominated by a highly restricted number of CD4+ clones. Moreover, the responding CD8+ T cell infiltrate is oligoclonal indicating recognition of a limited number of MDV antigens. These studies improve our understanding of the biology of MDV, an important poultry pathogen and a natural infection model of virus-induced tumor formation.

Distinct effector functions mediated by Fc regions of bovine IgG subclasses and their interaction with Fc gamma receptors.

Cattle possess three IgG subclasses. However, the key immune functions, including complement and NK cell activation, and enhancement of phagocytosis, are not fully described for bovine IgG1, 2 and 3. We produced chimeric monoclonal antibodies (mAbs) consisting of a defined variable region linked to the constant regions of bovine IgG1, 2 and 3, and expressed His-tagged soluble recombinant bovine Fc gamma receptors (FcγRs) IA (CD64), IIA (CD32A), III (CD16) and Fcγ2R. Functional assays using bovinized mAbs were developed. IgG1 and IgG3, but not IgG2, activated complement-dependent cytotoxicity. Only IgG1 could activate cattle NK cells to mobilize CD107a after antigen crosslinking, a surrogate assay for antibody-dependent cell cytotoxicity. Both IgG1 and IgG2 could trigger monocyte-derived macrophages to phagocytose fluorescently labelled antigen-expressing target cells. IgG3 induced only weak antibody-dependent cellular phagocytosis (ADCP). By contrast, monocytes only exhibited strong ADCP when triggered by IgG2. IgG1 bound most strongly to recombinant FcγRs IA, IIA and III, with weaker binding by IgG3 and none by IgG2, which bound exclusively to Fcγ2R. Immune complexes containing IgG1, 2 and 3 bound differentially to leukocyte subsets, with IgG2 binding strongly to neutrophils and monocytes and all subclasses binding platelets. Differential expression of the FcγRs on leukocyte subsets was demonstrated by surface staining and/or RT-qPCR of sorted cells, e.g., Fcγ2R mRNA was expressed in monocytes/macrophages, neutrophils, and platelets, potentially explaining their strong interactions with IgG2, and FcγRIII was expressed on NK cells, presumably mediating IgG1-dependent NK cell activation. These data reveal differences in bovine IgG subclass functionality, which do not correspond to those described in humans, mice or pigs, which is relevant to the study of these IgG subclasses in vaccine and therapeutic antibody development.

Diversification of immunoglobulin genes by gene conversion in the domestic chicken (Gallus gallus domesticus).

Sustainable modern poultry production depends on effective protection against infectious diseases and a diverse range of antibodies is key for an effective immune response. In the domestic chicken, somatic gene conversion is the dominant process in which the antibody immunoglobulin genes are diversified. Affinity maturation by somatic hypermutation (SHM) also occurs, but the relative contribution of gene conversion versus somatic hypermutation to immunoglobulin (Ig) gene diversity is poorly understood. In this study, we use high throughput long-read sequencing to study immunoglobulin diversity in multiple immune-associated tissues in Rhode Island Red chickens. To better understand the impact of genetic diversification in the chicken, a novel gene conversion identification software was developed (BrepConvert). In this study, BrepConvert enabled the identification of over 1 million gene conversion events. Mapping the occurrence of putative somatic gene conversion (SGC) events throughout the variable gene region revealed repetitive and highly restricted patterns of genetic insertions in both the antibody heavy and light chains. These patterns coincided with the locations of genetic variability in available pseudogenes and align with antigen binding sites, predominately the complementary determining regions (CDRs). We found biased usage of pseudogenes during gene conversion, as well as immunoglobulin heavy chain diversity gene (IGHD) preferences during V(D)J gene rearrangement, suggesting that antibody diversification in chickens is more focused than the genetic potential for diversity would suggest.

A Customizable Suite of Methods to Sequence and Annotate Cattle Antibodies.

Studying the antibody response to infection or vaccination is essential for developing more effective vaccines and therapeutics. Advances in high-throughput antibody sequencing technologies and immunoinformatic tools now allow the fast and comprehensive analysis of antibody repertoires at high resolution in any species. Here, we detail a flexible and customizable suite of methods from flow cytometry, single cell sorting, heavy and light chain amplification to antibody sequencing in cattle. These methods were used successfully, including adaptation to the 10x Genomics platform, to isolate native heavy-light chain pairs. When combined with the Ig-Sequence Multi-Species Annotation Tool, this suite represents a powerful toolkit for studying the cattle antibody response with high resolution and precision. Using three workflows, we processed 84, 96, and 8313 cattle B cells from which we sequenced 24, 31, and 4756 antibody heavy-light chain pairs, respectively. Each method has strengths and limitations in terms of the throughput, timeline, specialist equipment, and cost that are each discussed. Moreover, the principles outlined here can be applied to study antibody responses in other mammalian species.

Induction of lymphomas by inoculation of Marek's disease virus-derived lymphoblastoid cell lines: prevention by CVI988 vaccination.

Lymphoblastoid cell lines 265(L) and 990(O) are monoclonal lymphomas, derived respectively from liver and ovarian tumours, generated in inbred P-line (MHC B(19)/B(19)) chickens infected with RB-1B strain of Marek's disease virus (MDV) and pRB-1B5 BAC clone respectively. These were inoculated into inbred, MDV-susceptible, P-line chickens by intra-venous or intra-abdominal routes. Additional groups of birds were vaccinated using 1000 plaque-forming units of CVI988 vaccine 8 days prior to inoculation of the cell lines. Non-vaccinated birds developed visceral Marek's disease tumours with an increased rate 30 to 60 days post inoculation. Vaccination prevented tumour and disease development in challenged birds. TCRß repertoire analysis by spectratyping and sequencing of the inoculum was used to track tumour identity in primary tumours and tumour cell lines derived from inoculated birds. These data revealed that the tumours were a consequence of de novo virus infection and not metastasis and expansion of the inoculated tumour cells. Moreover, the data showed that the two MDV-derived cell lines were not transplantable even in syngeneic P-line birds. The data also demonstrated the application of spectratyping as a tool to track tumour identity in lymphoma transplantation studies.

Fc-Mediated Functions of Porcine IgG Subclasses.

The pig is an important agricultural species and powerful biomedical model. We have established the pig, a large natural host animal for influenza with many physiological similarities to humans, as a robust model for testing the therapeutic potential of monoclonal antibodies. Antibodies provide protection through neutralization and recruitment of innate effector functions through the Fc domain. However very little is known about the Fc-mediated functions of porcine IgG subclasses. We have generated 8 subclasses of two porcine monoclonal anti influenza hemagglutinin antibodies. We characterized their ability to activate complement, trigger cytotoxicity and phagocytosis by immune cells and assayed their binding to monocytes, macrophages, and natural killer cells. We show that IgG1, IgG2a, IgG2b, IgG2c and IgG4 bind well to targeted cell types and mediate complement mediated cellular cytotoxicity (CDCC), antibody dependent cellular cytotoxicity (ADCC) and antibody mediated cell phagocytosis (ADCP). IgG5b and IgG5c exhibited weak binding and variable and poor functional activity. Immune complexes of porcine IgG3 did not show any Fc-mediated functions except for binding to monocytes and macrophages and weak binding to NK cells. Interestingly, functionally similar porcine IgG subclasses clustered together in the genome. These novel findings will enhance the utility of the pig model for investigation of therapeutic antibodies.

The Veterinary Immunological Toolbox: Past, Present, and Future.

It is well-recognized that research capability in veterinary species is restricted by a lack of immunological reagents relative to the extensive toolboxes for small rodent biomedical model species and humans. This creates a barrier to the strategic development of disease control solutions for livestock, companion animals and wildlife that not only affects animal health but can affect human health by increasing the risk of transmission of zoonotic pathogens. There have been a number of projects aimed at reducing the capability gaps in the veterinary immunological toolbox, the majority of these focusing on livestock species. Various approaches have been taken to veterinary immunological reagent development across the globe and technological advances in molecular biology and protein biochemistry have accelerated toolbox development. While short-term funding initiatives can address specific gaps in capability, they do not account for long-term sustainability of reagents and databases that requires a different funding model. We review the past, present and future of the veterinary immunological toolbox with specific reference to recent developments discussed at the International Union of Immunological Societies (IUIS) Veterinary Immunology Committee (VIC) Immune Toolkit Workshop at the 12th International Veterinary Immunology Symposium (IVIS) in Seattle, USA, 16-19 August 2019. The future availability of these reagents is critical to research for improving animal health, responses to infectious pathogens and vaccine design as well as for important analyses of zoonotic pathogens and the animal /human interface for One Health initiatives.

In vitro Interactions of Chicken Programmed Cell Death 1 (PD-1) and PD-1 Ligand-1 (PD-L1).

In the present study, we determined the in vitro characteristics and binding interactions of chicken PD-1 (chPD-1) and PD-L1 (chPD-L1) and developed a panel of specific monoclonal antibodies against the two proteins. ChPD-1 and chPD-L1 sequence identities and similarities were lower compared with those of humans and other mammalian species. Furthermore, in phylogenetic analysis, chPD-1 and chPD-L1 were grouped separately from the mammalian PD-1 and PD-L1 sequences. As in other species, chPD-1 and chPD-L1 sequences showed signal peptide, extracellular domain, a transmembrane domain and intracellular domain. Based on the three dimensional (3D) structural homology, chPD-1, and chPD-L1 were similar to 3D structures of mammalian PD-1 and PD-L1. Further, Ig V domain of chPD-1 and the Ig V and Ig C domains of chPD-L1 were highly conserved with the mammalian counterparts. In vitro binding interaction studies using Superparamagnetic Dynabeads® confirmed that recombinant soluble chPD-1/PD-L1 fusion proteins and surface chPD-1/PD-L1 proteins interacted with each other on COS cells. Two monoclonal antibodies specific against chPD-1 and five antibodies against chPD-L1 were developed and their specific binding characteristics confirmed by immunofluorescence staining and Western blotting.

The role of the light chain in the structure and binding activity of two cattle antibodies that neutralize bovine respiratory syncytial virus.

Cattle antibodies have unusually long CDR3 loops in their heavy chains (HCs), and limited light chain (LC) diversity, raising the question of whether these mask the effect of LC variation on antigen recognition. We have investigated the role of the LC in the structure and activity of two neutralizing cattle antibodies (B4 and B13) that bind the F protein of bovine respiratory syncytial virus (bRSV). Recombinant Fab fragments of B4 and B13 bound bRSV infected cells and showed similar affinities for purified bRSV F protein. Exchanging the LCs between the Fab fragments produced hybrid Fabs: B13* (B13 HC/B4 LC) and B4* (B4 HC/B13 LC). The affinity of B13* to the F protein was found to be two-fold lower than B13 whilst the binding affinity of B4* was reduced at least a hundred-fold compared to B4 such that it no longer bound to bRSV infected cells. Comparison of the structures of B4 and B13 with their LC exchanged counterparts B4* and B13* showed that paratope of the HC variable domain (VH) of B4 was disrupted on pairing with the B13 LC, consistent with the loss of binding activity. By contrast, B13 H3 adopts a similar conformation when paired with either B13 or B4 LCs. These observations confirm the expected key role of the extended H3 loop in antigen-binding by cattle antibodies but also show that the quaternary LC/HC subunit interaction can be crucial for its presentation and thus the LC variable domain (VL) is also important for antigen recognition.

Early pathogenesis during infectious bursal disease in susceptible chickens is associated with changes in B cell genomic methylation and loss of genome integrity.

We propose a model by which an increase in the genomic modification, 5-hydroxymethylcytosine (5hmC), contributes to B cell death within the chicken bursa of Fabricus (BF) infected with infectious bursal disease virus (IBDV). Our findings indicate that, following an IBDV infection, Rhode Island Red (RIR) chickens have fewer surviving B cells and higher levels of 5hmC in the BF than the more resistant 15l line of birds. Elevated genomic 5hmC levels within the RIR BF are associated with markers of immune responses: infiltrating T cells and increased expression of CD40L, FasL and iNOS. Such changes correlate with genomic fragmentation and the presence of IBDV capsid protein, VP2. To explore the effects of CD40L, the immature B cell line, DT40, was exposed to recombinant chicken CD40L that resulted in changes in nuclear 5hmC distribution. Collectively, our observations suggest that T cell infiltration exacerbates early immunopathology within the BF during an IBDV infection contributing to B cell genomic instability and death to facilitate viral egress and immunosuppression.

A Versatile Panel of Reference Gene Assays for the Measurement of Chicken mRNA by Quantitative PCR.

Quantitative real-time PCR assays are widely used for the quantification of mRNA within avian experimental samples. Multiple stably-expressed reference genes, selected for the lowest variation in representative samples, can be used to control random technical variation. Reference gene assays must be reliable, have high amplification specificity and efficiency, and not produce signals from contaminating DNA. Whilst recent research papers identify specific genes that are stable in particular tissues and experimental treatments, here we describe a panel of ten avian gene primer and probe sets that can be used to identify suitable reference genes in many experimental contexts. The panel was tested with TaqMan and SYBR Green systems in two experimental scenarios: a tissue collection and virus infection of cultured fibroblasts. GeNorm and NormFinder algorithms were able to select appropriate reference gene sets in each case. We show the effects of using the selected genes on the detection of statistically significant differences in expression. The results are compared with those obtained using 28s ribosomal RNA, the present most widely accepted reference gene in chicken work, identifying circumstances where its use might provide misleading results. Methods for eliminating DNA contamination of RNA reduced, but did not completely remove, detectable DNA. We therefore attached special importance to testing each qPCR assay for absence of signal using DNA template. The assays and analyses developed here provide a useful resource for selecting reference genes for investigations of avian biology.