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Abstract The microbiome is a key factor in the health, well-being, and success of vertebrates, contributing to the adaptive capacity of the host. However, the impact of geographic and biotic factors that may affect the microbiome of wild birds in polar environments is not well defined. To address this, we determined the bacterial 16S rRNA gene sequence profiles in faecal samples from pygoscelid penguin populations in the Scotia Arc, focusing on gentoo penguins. This mesopredatory group breeds in defined colonies across a wide geographic range. Since diet could influence microbiome structure, we extracted dietary profiles from a eukaryotic 18S rRNA gene sequence profile. The bacterial microbiome profiles were considered in the context of a diverse set of environmental and ecological measures. Integrating wide geographic sampling with bacterial 16S and eukaryotic 18S rRNA gene sequencing of over 350 faecal samples identified associations between the microbiome profile and a suite of geographic and ecological factors. Microbiome profiles differed according to host species, colony identity, distance between colonies, and diet. Interestingly there was also a relationship between the proportion of host DNA (in relation to total 18S rRNA gene signal) and the microbiome, which may reflect gut passage time. Colony identity provided the strongest association with differences in microbiome profiles indicating that local factors play a key role in the microbiome structure of these polar seabirds. This may reflect the influence of local transfer of microbes either via faecal-oral routes, during chick feeding or other close contact events. Other factors including diet and host species also associate with variation in microbiome profile, and in at least some locations, the microbiome composition varies considerably between individuals. Given the variation in penguin microbiomes associated with diverse factors there is potential for disruption of microbiome associations at a local scale that could influence host health, productivity, and immunological competence. The microbiome represents a sensitive indicator of changing conditions, and the implications of any changes need to be considered in the wider context of environmental change and other stressors.

Avian influenza viruses (AIVs) are a major economic burden to the poultry industry and pose serious zoonotic risks, with human infections being reported every year. To date, the vaccination of birds remains the most important method for the prevention and control of AIV outbreaks. Most national vaccination strategies against AIV infection use whole virus-inactivated vaccines, which predominantly trigger a systemic antibody-mediated immune response. There are currently no studies that have examined the antibody repertoire of birds that were infected with and/or vaccinated against AIV. To this end, we evaluate the changes in the H9N2-specific IgM and IgY repertoires in chickens subjected to vaccination(s) and/or infectious challenge. We show that a large proportion of the IgM and IgY clones were shared across multiple individuals, and these public clonal responses are dependent on both the immunisation status of the birds and the specific tissue that was examined. Furthermore, the analysis revealed specific clonal expansions that are restricted to particular H9N2 immunisation regimes. These results indicate that both the nature and number of immunisations are important drivers of the antibody responses and repertoire profiles in chickens following H9N2 antigenic stimulation. We discuss how the repertoire biology of avian B-cell responses may affect the success of AIV vaccination in chickens, in particular the implications of public versus private clonal selection.

This study examines the tempo and drivers of penguin diversification by combining genomes from all extant and recently extinct penguin lineages, stratigraphic data from fossil penguins and morphological and biogeographic data from all extant and extinct species. Together, these datasets provide new insights into the genetic basis and evolution of adaptations in penguins.

Abstract Malaria parasites such as Plasmodium falciparum have exerted formidable selective pressures on the human genome. Of the human genetic variants associated with malaria protection, beta thalassaemia (a haemoglobinopathy) was the earliest to be associated with malaria prevalence. However, the malaria protective properties of beta thalassaemic erythrocytes remain unclear. Here we studied the mechanics and surface protein expression of beta thalassaemia heterozygous erythrocytes, measured their susceptibility to P. falciparum invasion, and calculated the energy required for merozoites to invade them. We found invasion-relevant differences in beta thalassaemic cells versus matched controls, specifically: elevated membrane tension, reduced bending modulus, and higher levels of expression of the major invasion receptor basigin. However, these differences acted in opposition to each other with respect to their likely impact on invasion, and overall we did not observe beta thalassaemic cells to have lower P. falciparum invasion efficiency for any of the strains tested.

Microbial colonisation is paramount to the normal development of the immune system, particularly at mucosal sites. However, the relationships between the microbiome and the adaptive immune repertoire have mostly been explored in rodents and humans. Here, we report a high-throughput sequencing analysis of the chicken TCRβ repertoire and the influences of microbial colonisation on tissue-resident TCRβ+ cells. The results reveal that the microbiome is an important driver of TCRβ diversity in both intestinal tissues and the bursa of Fabricius, but not in the spleen. Of note, public TCRβ sequences (shared across individuals) make a substantial contribution to the repertoire. Additionally, different tissues exhibit biases in terms of their V family and J gene usage, and these effects were influenced by the gut-associated microbiome. TCRβ clonal expansions were identified in both colonised and germ-free birds, but differences between the groups were indicative of an influence of the microbiota. Together, these findings provide an insight into the avian adaptive immune system and the influence of the microbiota on the TCRβ repertoire.

Abstract Background Despite increasing interest in γδ T cells and their non-classical behaviour, most studies focus on animals with low numbers of circulating γδ T cells, such as mice and humans. Arguably, γδ T cell functions might be more prominent in chickens where these cells form a higher proportion of the circulatory T cell compartment. The TCR repertoire defines different subsets of γδ T cells, and such analysis is facilitated by well-annotated TCR loci. γδ T cells are considered at the cusp of innate and adaptive immunity but most functions have been identified in γδ low species. A deeper understanding of TCR repertoire biology in γδ high and γδ low animals is critical for defining the evolution of the function of γδ T cells. Repertoire dynamics will reveal populations that can be classified as innate-like or adaptive-like as well as those that straddle this definition. Results Here, a recent discrepancy in the structure of the chicken TCR gamma locus is resolved, demonstrating that tandem duplication events have shaped the evolution of this locus. Importantly, repertoire sequencing revealed large differences in the usage of individual TRGV genes, a pattern conserved across multiple tissues, including thymus, spleen and the gut. A single TRGV gene, TRGV3.3, with a highly diverse private CDR3 repertoire dominated every tissue in all birds. TRGV usage patterns were partly explained by the TRGV-associated recombination signal sequences. Public CDR3 clonotypes represented varying proportions of the repertoire of TCRs utilising different TRGVs, with one TRGV dominated by super-public clones present in all birds. Conclusions The application of repertoire analysis enabled functional annotation of the TCRG locus in a species with a high circulating γδ phenotype. This revealed variable usage of TCRGV genes across multiple tissues, a pattern quite different to that found in γδ low species (human and mouse). Defining the repertoire biology of avian γδ T cells will be key to understanding the evolution and functional diversity of these enigmatic lymphocytes in an animal that is numerically more reliant on them. Practically, this will reveal novel ways in which these cells can be exploited to improve health in medical and veterinary contexts.

ABSTRACT: Contaminated chicken meat is a major source of human Campylobacteriosis and rates of infection remain high, despite efforts to limit the colonisation of broiler (meat) chicken flocks on farms. Using conventional testing methods of culture or qPCR, Campylobacter is typically detected amongst broiler flocks from 3 wk of age, leading to the assumption that infection is introduced horizontally into chicken rearing houses at this time. In this study, we use parallel sequencing of a fragment of the Campylobacter outer membrane protein, encoded by the porA gene, to test for presence of Campylobacter DNA amongst fresh fecal samples collected from broiler flocks aged 23 to 28 d. Campylobacter DNA was detected in all of the 290 samples tested using the porA target, and in 48% of samples using 16S bacterial profiling, irrespective of whether or not Campylobacter could be detected using conventional qPCR thresholds. A single porAf2 variant was predominant among flocks that would be determined to be Campylobacter ‘positive’ by conventional means, but a diverse pattern was seen among flocks that were Campylobacter ‘negative’. The ability to routinely detect low levels of Campylobacter amongst broiler flocks at a much earlier age than would conventionally be identified requires a re-examination of how and when biosecurity measures are best applied for live birds. In addition, it may be useful to investigate why single Campylobacter variants proliferate in some broiler flocks and not others.

Intestinal helminth parasites (worms) have afflicted humans throughout history and their eggs are readily detected in archaeological deposits including at locations where intestinal parasites are no longer considered endemic (e.g. the UK). Parasites provide valuable archaeological insights into historical health, sanitation, hygiene, dietary and culinary practices, as well as other factors. Differences in the prevalence of helminths over time may help us understand factors that affected the rate of infection of these parasites in past populations. While communal deposits often contain relatively high numbers of parasite eggs, these cannot be used to calculate prevalence rates, which are a key epidemiological measure of infection. The prevalence of intestinal helminths was investigated through time in England, based on analysis of 464 human burials from 17 sites, dating from the Prehistoric to Industrial periods. Eggs from two faecal-oral transmitted nematodes (Ascaris sp. and Trichuris sp.) and the food-derived cestodes (Taenia spp. and Diphyllobothrium latum syn Dibothriocephalus latus) were identified, although only Ascaris was detected at a high frequency. The changing prevalence of nematode infections can be attributed to changes in effective sanitation or other factors that affect these faecal-oral transmitted parasites and the presence of cestode infections reflect dietary and culinary preferences. These results indicate that the impact of helminth infections on past populations varied over time, and that some locations witnessed a dramatic reduction in parasite prevalence during the industrial era (18th-19th century), whereas other locations continued to experience high prevalence levels. The factors underlying these reductions and the variation in prevalence provide a key historical context for modern anthelmintic programs. Author summary Parasitic worms (helminths) cause many health problems in poorer countries, particularly those in tropical and sub-tropical regions. In modern Europe these infections are very rare and mostly found in those travelling from endemic areas but this wasn’t always the case. Archaeological studies have detected eggs of parasitic helminths in numerous European sites. Key questions include how prevalent these infections were in past communities and whether (or when) these patterns changed over time? This paper addresses both of these questions using a large number of single grave samples from archaeological sites in England dating between Prehistoric and Industrial periods. Helminth infections were detected across all periods but the overall prevalence rates changed over time, being highest in the Roman and Late-Medieval periods. The Industrial period was interesting in that two of the three sites contained very few (or no) parasites whereas the third, London, contained high levels of infection. We discuss factors that may have contributed to the changing parasite landscape and how understanding these factors may influence efforts to control helminth infections in modern endemic areas.

ABSTRACT: Using data on rearing and welfare metrics of multiple commercial broiler flocks, we investigate how welfare measures such as hock burn, mortality, and pododermatitis, among others, impact the likelihood of a flock becoming colonized by Campylobacter. Using both logistic regression and Bayesian networks, we show that, while some welfare metrics were weakly related to Campylobacter colonization, evidence could not be found to suggest that these metrics directly exacerbated Campylobacter colonization, rather that they were both symptoms of the same parent variable – the managing company. Observed dependency on the management of the flock suggested that yet-undiscovered differences in rearing practice were the principal factor explaining both poor bird welfare and increased risk of Campylobacter, suggesting that action can be taken to improve both these factors simultaneously.

Helminth infections are among the World Health Organization's top neglected diseases with significant impact in many Less Economically Developed Countries. Despite no longer being endemic in Europe, the widespread presence of helminth eggs in archaeological deposits indicates that helminths represented a considerable burden in past European populations. Prevalence of infection is a key epidemiological feature that would influence the elimination of endemic intestinal helminths, for example, low prevalence rates may have made it easier to eliminate these infections in Europe without the use of modern anthelminthic drugs. To determine historical prevalence rates we analysed 589 grave samples from 7 European sites dated between 680 and 1700 CE, identifying two soil transmitted nematodes (Ascaris spp. and Trichuris trichiura) at all locations, and two food derived cestodes (Diphyllobothrium latum and Taenia spp.) at 4 sites. The rates of nematode infection in the medieval populations (1.5 to 25.6% for T. trichiura; 9.3-42.9% for Ascaris spp.) were comparable to those reported within modern endemically infected populations. There was some evidence of higher levels of nematode infection in younger individuals but not at all sites. The genetic diversity of T. trichiura ITS-1 in single graves was variable but much lower than with communal medieval latrine deposits. The prevalence of food derived cestodes was much lower (1.0-9.9%) than the prevalence of nematodes. Interestingly, sites that contained Taenia spp. eggs also contained D. latum which may reflect local culinary practices. These data demonstrate the importance of helminth infections in Medieval Europe and provide a baseline for studies on the epidemiology of infection in historical and modern contexts. Since the prevalence of medieval STH infections mirror those in modern endemic countries the factors affecting STH decline in Europe may also inform modern intervention campaigns.

Human endogenous retroviruses (HERVs) are under genomic and epigenetic control but can be expressed in normal tissues, producing RNA transcripts some of which are translated. While it has not been demonstrated experimentally in modern humans, cDNA copies from HERV RNA (namely HERV-K HML-2 or HK2) were produced after the human-chimp split and until at least 250,000 years ago. We were interested in determining if such cDNA could be a ligand for pattern recognition receptors (PRRs) of the innate immune response. The AIM-2-like receptors for DNA, interferon-γ-inducible protein 16 (IFI16) and Cyclic GMP-AMP synthase (cGAS) were candidate PRRs. IFI16 can detect cDNA produced during HIV-1 replication, causing increased T cell death. While HIV-1 has emerged relatively recently as a human pathogen, the cDNA functionality of IFI16 could have been selected for during the course of human evolution. Here we present a novel hypothesis that the products of reverse transcription of HK2, which has been proliferating in the genome of human ancestors for 30 million years, could interact with IFI16. In support of our hypothesis, we provide preliminary data showing that IFI16 (but not cGAS) interacts with synthetic single-stranded HK2 oligos corresponding to the first product of reverse transcription. Further, we show that ssDNA detection by IFI16 has variability with respect to sequence features but is not dependent on strong secondary structures mimicking dsDNA. Among the HK2 oligos, IFI16 interacts more intensely with those derived from LTRs, suggesting these oligos have undetermined structural features that allow IFI16 to bind with greater affinity. Further, cells with stem cell features that naturally allow HK2 expression were found to express many components of the innate immune system including cGAS but not IFI16. Based on the presented preliminary data we further postulate another hypothesis: that the IFI16 functionality in human cells has been acting as “second-line” defense to control abnormal HK2 replication in somatic tissues. The absence of this protein in stem cells and a stem cell line could permit these cells to express HERVs which contribute to stem cell identity. Finally, we also comment on potential studies that could support or refute our hypothesis.

There is growing interest in uncovering the viral diversity present in wild animal species. The remote Antarctic region is home to a wealth of uncovered microbial diversity, some of which is associated with its megafauna, including penguin species, the dominant avian biota. Penguins interface with a number of other biota in their roles as marine mesopredators and several species overlap in their ranges and habitats. To characterize the circular single-stranded viruses related to those in the phylum Cressdnaviricota from these environmental sentinel species, cloacal swabs (n = 95) were obtained from King Penguins in South Georgia, and congeneric Adélie Penguins, Chinstrap Penguins, and Gentoo Penguins across the South Shetland Islands and Antarctic Peninsula. Using a combination of high-throughput sequencing, abutting primers-based PCR recovery of circular genomic elements, cloning, and Sanger sequencing, we detected 97 novel sequences comprising 40 ssDNA viral genomes and 57 viral-like circular molecules from 45 individual penguins. We present their detection patterns, with Chinstrap Penguins harboring the highest number of new sequences. The novel Antarctic viruses identified appear to be host-specific, while one circular molecule was shared between sympatric Chinstrap and Gentoo Penguins. We also report viral genotype sharing between three adult-chick pairs, one in each Pygoscelid species. Sequence similarity network approaches coupled with Maximum likelihood phylogenies of the clusters indicate the 40 novel viral genomes do not fall within any known viral families and likely fall within the recently established phylum Cressdnaviricota based on their replication-associated protein sequences. Similarly, 83 capsid protein sequences encoded by the viruses or viral-like circular molecules identified in this study do not cluster with any of those encoded by classified viral groups. Further research is warranted to expand knowledge of the Antarctic virome and would help elucidate the importance of viral-like molecules in vertebrate host evolution.

Circoviruses infect a variety of animal species and have small (~1.8–2.2 kb) circular single-stranded DNA genomes. Recently a penguin circovirus (PenCV) was identified associated with an Adélie Penguin (Pygoscelis adeliae) with feather disorder and in the cloacal swabs of three asymptomatic Adélie Penguins at Cape Crozier, Antarctica. A total of 75 cloacal swab samples obtained from adults and chicks of three species of penguin (genus: Pygoscelis) from seven Antarctic breeding colonies (South Shetland Islands and Western Antarctic Peninsula) in the 2015−2016 breeding season were screened for PenCV. We identified new variants of PenCV in one Adélie Penguin and one Chinstrap Penguin (Pygoscelis antarcticus) from Port Charcot, Booth Island, Western Antarctic Peninsula, a site home to all three species of Pygoscelid penguins. These two PenCV genomes (length of 1986 nucleotides) share > 99% genome-wide nucleotide identity with each other and share ~87% genome-wide nucleotide identity with the PenCV sequences described from Adélie Penguins at Cape Crozier ~4400 km away in East Antarctica. We did not find any evidence of recombination among PenCV sequences. This is the first report of PenCV in Chinstrap Penguins and the first detection outside of Ross Island, East Antarctica. Given the limited knowledge on Antarctic animal viral diversity, future samples from Antarctic wildlife should be screened for these and other viruses to determine the prevalence and potential impact of viral infections.

Coccidiosis in poultry, caused by protozoan parasites of the genus Eimeria, is an intestinal disease with substantial economic impact. With the use of anticoccidial drugs under public and political pressure, and the comparatively higher cost of live-attenuated vaccines, an attractive complementary strategy for control is to breed chickens with increased resistance to Eimeria parasitism. Prior infection with Eimeria maxima leads to complete immunity against challenge with homologous strains, but only partial resistance to challenge with antigenically diverse heterologous strains. We investigate the genetic architecture of avian resistance to E. maxima primary infection and heterologous strain secondary challenge using White Leghorn populations of derived inbred lines, C.B12 and 15I, known to differ in susceptibility to the parasite. An intercross population was infected with E. maxima Houghton (H) strain, followed 3 weeks later by E. maxima Weybridge (W) strain challenge, while a backcross population received a single E. maxima W infection. The phenotypes measured were parasite replication (counting fecal oocyst output or qPCR for parasite numbers in intestinal tissue), intestinal lesion score (gross pathology, scale 0–4), and for the backcross only, serum interleukin-10 (IL-10) levels. Birds were genotyped using a high density genome-wide DNA array (600K, Affymetrix). Genome-wide association study located associations on chromosomes 1, 2, 3, and 5 following primary infection in the backcross population, and a suggestive association on chromosome 1 following heterologous E. maxima W challenge in the intercross population. This mapped several megabases away from the quantitative trait locus (QTL) linked to the backcross primary W strain infection, suggesting different underlying mechanisms for the primary- and heterologous secondary- responses. Underlying pathways for those genes located in the respective QTL for resistance to primary infection and protection against heterologous challenge were related mainly to immune response, with IL-10 signaling in the backcross primary infection being the most significant. Additionally, the identified markers associated with IL-10 levels exhibited significant additive genetic variance. We suggest this is a phenotype of interest to the outcome of challenge, being scalable in live birds and negating the requirement for single-bird cages, fecal oocyst counts, or slaughter for sampling (qPCR).

In this study we determined protein and gene expression in the caeca of newly hatched chickens inoculated with cecal contents sourced from hens of different ages. Over 250 proteins exhibited modified expression levels in response to microbiota inoculation. The most significant inductions were observed for ISG12-2, OASL, ES1, LYG2, DMBT1-L, CDD, ANGPTL6, B2M, CUZD1, IgM and Ig lambda chain. Of these, ISG12-2, ES1 and both immunoglobulins were expressed at lower levels in germ-free chickens compared to conventional chickens. In contrast, CELA2A, BRT-2, ALDH1A1, ADH1C, AKR1B1L, HEXB, ALDH2, ALDOB, CALB1 and TTR were expressed at lower levels following inoculation of microbiota. When chicks were given microbiota preparations from different age donors, the recipients mounted differential responses to the inoculation which also differed from the response profile in naturally colonised birds. For example, B2M, CUZD1 and CELA2A responded differently to the inoculation with microbiota of 4- or 40-week-old hens. The increased or decreased gene expression could be recorded 6 weeks after the inoculation of newly hatched chickens. To characterise the proteins that may directly interact with the microbiota we characterised chicken proteins that co-purified with the microbiota and identified a range of host proteins including CDD, ANGPTL6, DMBT1-L, MEP1A and Ig lambda. We propose that induction of ISG12-2 results in reduced apoptosis of host cells exposed to the colonizing commensal microbiota and that CDD, ANGPTL6, DMBT1-L, MEP1A and Ig lambda reduce contact of luminal microbiota with the gut epithelium thereby reducing the inflammatory response.

Many genes important in immunity are found as multigene families. The butyrophilin genes are members of the B7 family, playing diverse roles in co-regulation and perhaps in antigen presentation. In humans, a fixed number of butyrophilin genes are found in and around the major histocompatibility complex (MHC), and show striking association with particular autoimmune diseases. In chickens, BG genes encode homologues with somewhat different domain organisation. Only a few BG genes have been characterised, one involved in actin-myosin interaction in the intestinal brush border, and another implicated in resistance to viral diseases. We characterise all BG genes in B12 chickens, finding a multigene family organised as tandem repeats in the BG region outside the MHC, a single gene in the MHC (the BF-BL region), and another single gene on a different chromosome. There is a precise cell and tissue expression for each gene, but overall there are two kinds, those expressed by haemopoietic cells and those expressed in tissues (presumably non-haemopoietic cells), correlating with two different kinds of promoters and 5' untranslated regions (5'UTR). However, the multigene family in the BG region contains many hybrid genes, suggesting recombination and/or deletion as major evolutionary forces. We identify BG genes in the chicken whole genome shotgun sequence, as well as by comparison to other haplotypes by fibre fluorescence in situ hybridisation, confirming dynamic expansion and contraction within the BG region. Thus, the BG genes in chickens are undergoing much more rapid evolution compared to their homologues in mammals, for reasons yet to be understood.

Toll-like Receptors (TLR) are phylogenetically conserved transmembrane proteins responsible for detection of pathogens and activation of immune responses in diverse animal species. The stimulation of TLR by pathogen-derived molecules leads to the production of pro-inflammatory mediators including cytokines and nitric oxide. Although TLR-induced events are critical for immune induction, uncontrolled inflammation can be life threatening and regulation is a critical feature of TLR biology. We used an avian macrophage cell line (HD11) to determine the relationship between TLR agonist-induced activation of inflammatory responses and the transcriptional regulation of TLR. Exposure of macrophages to specific TLR agonists induced upregulation of cytokine and nitric oxide pathways that were inhibited by blocking various components of the TLR signalling pathways. TLR activation also led to changes in the levels of mRNA encoding the TLR responsible for recognising the inducing agonist (cognate regulation) and cross-regulation of other TLR (non-cognate regulation). Interestingly, in most cases, regulation of TLR mRNA was independent of NFκB activity but dependent on one or more of the MAPK pathway components. Moreover, the relative importance of ERK, JNK and p38 was dependent upon both the stimulating agonist and the target TLR. These results provide a framework for understanding the complex pathways involved in transcriptional regulation of TLR, immune induction and inflammation. Manipulation of these pathways during vaccination or management of acute inflammatory disease may lead to improved clinical outcome or enhanced vaccine efficacy.

Cell division is an essential cellular process that requires an array of known and unknown proteins for its spatial and temporal regulation. Here we develop a novel, high-throughput screening method for the identification of bacterial cell division genes and regulators. The method combines the over-expression of a shotgun genomic expression library to perturb the cell division process with high-throughput flow cytometry sorting to screen many thousands of clones. Using this approach, we recovered clones with a filamentous morphology for the model bacterium, Escherichia coli. Genetic analysis revealed that our screen identified both known cell division genes, and genes that have not previously been identified to be involved in cell division. This novel screening strategy is applicable to a wide range of organisms, including pathogenic bacteria, where cell division genes and regulators are attractive drug targets for antibiotic development.

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+ TCRαβ+ 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 TCRβ 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.

Apicomplexan parasites are responsible for a myriad of diseases in humans and livestock; yet despite intensive effort, development of effective sub-unit vaccines remains a long-term goal. Antigenic complexity and our inability to identify protective antigens from the pool that induce response are serious challenges in the development of new vaccines. Using a combination of parasite genetics and selective barriers with population-based genetic fingerprinting, we have identified that immunity against the most important apicomplexan parasite of livestock (Eimeria spp.) was targeted against a few discrete regions of the genome. Herein we report the identification of six genomic regions and, within two of those loci, the identification of true protective antigens that confer immunity as sub-unit vaccines. The first of these is an Eimeria maxima homologue of apical membrane antigen-1 (AMA-1) and the second is a previously uncharacterised gene that we have termed 'immune mapped protein-1' (IMP-1). Significantly, homologues of the AMA-1 antigen are protective with a range of apicomplexan parasites including Plasmodium spp., which suggest that there may be some characteristic(s) of protective antigens shared across this diverse group of parasites. Interestingly, homologues of the IMP-1 antigen, which is protective against E. maxima infection, can be identified in Toxoplasma gondii and Neospora caninum. Overall, this study documents the discovery of novel protective antigens using a population-based genetic mapping approach allied with a protection-based screen of candidate genes. The identification of AMA-1 and IMP-1 represents a substantial step towards development of an effective anti-eimerian sub-unit vaccine and raises the possibility of identification of novel antigens for other apicomplexan parasites. Moreover, validation of the parasite genetics approach to identify effective antigens supports its adoption in other parasite systems where legitimate protective antigen identification is difficult.

Neurogenesis continues through the adult life of mice in the subgranular zone of the dentate gyrus in the hippocampus, but its function remains unclear. Measuring cellular proliferation in the hippocampus of 719 outbred heterogeneous stock mice revealed a highly significant correlation with the proportions of CD8+ versus CD4+ T lymphocyte subsets. This correlation reflected shared genetic loci, with the exception of the H-2Ea locus that had a dominant influence on T cell subsets but no impact on neurogenesis. Analysis of knockouts and repopulation of TCRα-deficient mice by subsets of T cells confirmed the influence of T cells on adult neurogenesis, indicating that CD4+ T cells or subpopulations thereof mediate the effect. Our results reveal an organismal impact, broader than hitherto suspected, of the natural genetic variation that controls T cell development and homeostasis.