Dissecting the Genomic Architecture of Resistance to Eimeria maxima Parasitism in the Chicken.

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).

Re-evaluation of the chicken MIP family of chemokines and their receptors suggests that CCL5 is the prototypic MIP family chemokine, and that different species have developed different repertoires of both the CC chemokines and their receptors.

Analysis of the chicken genome has shown that the chicken has a different repertoire of chemokines and chemokine receptors to those of mammals and other species. In this study, we report the sequencing and analysis of a bacterial artificial chromosome containing the entire chicken MIP family CC chemokine cluster. The gene duplication and divergence events that have taken place in mammals do not appear to have occurred as extensively in the avian lineage, as chickens possess fewer MIP family chemokine genes than humans or mice. We previously proposed that the four chicken MIP family members be named chicken (ch) CCLi1-4, according to their position on chicken chromosome 19, until such time as further analysis could determine if any of them were direct orthologues of mammalian MIP family members. Our analysis herein, combined with that of others, suggests that chCCLi4 is the orthologue of mammalian CCL5, and that chCCLi3 (K203) may be an orthologue of human CCL16. The other two chemokines do not have obvious orthologues, and thus we propose that they should still be called chCCLi1 and chCCLi2, until their biological function is further characterised. A similar pattern applies to the MIP family chemokine receptors, with only three receptor genes present at the relevant locus in the chicken genome, compared to four in man and mouse (CCR1, CCR2, CCR3 and CCR5). Of the three chicken receptor genes, only two look likely to be receptors for the MIP family chemokines, the third grouping with human, mouse and chicken CCR8 in phylogenetic analysis. The two chicken MIP CC receptors (CCRs) are not direct orthologues of the mammalian MIP CCRs.

A genomic analysis of chicken cytokines and chemokines.

As most mechanisms of adaptive immunity evolved during the divergence of vertebrates, the immune systems of extant vertebrates represent different successful variations on the themes initiated in their earliest common ancestors. The genes involved in elaborating these mechanisms have been subject to exceptional selective pressures in an arms race with highly adaptable pathogens, resulting in highly divergent sequences of orthologous genes and the gain and loss of members of gene families as different species find different solutions to the challenge of infection. Consequently, it has been difficult to transfer to the chicken detailed knowledge of the molecular mechanisms of the mammalian immune system and, thus, to enhance the already significant contribution of chickens toward understanding the evolution of immunity. The availability of the chicken genome sequence provides the opportunity to resolve outstanding questions concerning which molecular components of the immune system are shared between mammals and birds and which represent their unique evolutionary solutions. We have integrated genome data with existing knowledge to make a new comparative census of members of cytokine and chemokine gene families, distinguishing the core set of molecules likely to be common to all higher vertebrates from those particular to these 300 million-year-old lineages. Some differences can be explained by the different architectures of the mammalian and avian immune systems. Chickens lack lymph nodes and also the genes for the lymphotoxins and lymphotoxin receptors. The lack of functional eosinophils correlates with the absence of the eotaxin genes and our previously reported observation that interleukin- 5 (IL-5) is a pseudogene. To summarize, in the chicken genome, we can identify the genes for 23 ILs, 8 type I interferons (IFNs), IFN-gamma, 1 colony-stimulating factor (GM-CSF), 2 of the 3 known transforming growth factors (TGFs), 24 chemokines (1 XCL, 14 CCL, 8 CXCL, and 1 CX3CL), and 10 tumor necrosis factor superfamily (TNFSF) members. Receptor genes present in the genome suggest the likely presence of 2 other ILs, 1 other CSF, and 2 other TNFSF members.

In vivo and in vitro studies of genetic resistance to systemic salmonellosis in the chicken encoded by the SAL1 locus.

A number of inbred lines of chickens have been shown to be resistant or susceptible to systemic salmonellosis caused by Salmonella enterica serovar Gallinarum in adult birds, or by S. enterica serovar Enteritidis and S. enterica serovar Typhimurium in young chicks. Resistant lines show only moderate pathology and low mortality rates, whereas susceptible lines display extensive pathological changes and higher levels of mortality following Salmonella infection. Genetic resistance to salmonellosis is dominant and not linked to sex, MHC or Slc11a1 (formerly known as Nramp1), which leads to resistance in mice and other species. A novel locus encoding resistance to salmonellosis has been identified on chicken chromosome 5, and designated SAL1. The nature of the differences in pathology found between resistant and susceptible chicken lines in vivo indicates that resistance is expressed at the level of the mononuclear phagocyte system. Macrophages from adult resistant line birds cleared Salmonella serovar Gallinarum from infected macrophages within 24 h, whereas Salmonella bacteria persisted within macrophages from susceptible line birds for at least 48 h. Clearance of Salmonella by macrophages was accompanied by a strong and reproducible respiratory burst response in resistant lines, but little or no response in susceptible lines. Macrophages from an outbred chicken line showed variable responses. No differences were seen in macrophage nitric oxide production in cells from resistant or susceptible lines. These differences suggest that increased macrophage antimicrobial activity correlates with resistance and that macrophage activity plays an important role in genetic resistance to systemic salmonellosis in the chicken.

Salmonella carrier state in chicken: comparison of expression of immune response genes between susceptible and resistant animals.

Asymptomatic Salmonella enterica serovar Enteritidis carrier state in poultry has serious consequences on food safety and public health due to the risks of food poisoning following consumption of contaminated products. An understanding the mechanisms of persistence of Salmonella in the digestive tract of chicken can be achieved by a better knowledge of the defects in the control of infection in susceptible versus resistant animals. The gene expression of innate immune response factors including anti-microbial molecules, inflammatory and anti-infectious cytokines was studied in the caecal lymphoid tissue associated with the carrier state. Expression levels of these genes were assessed by real-time PCR and were compared in two inbred lines of chickens differing in resistance to the carrier state following oral inoculation of S. enterica serovar Enteritidis at 1 week of age. No correlation was observed between resistance/susceptibility to caecal carrier state and level of interleukin (IL)-1beta, IL-8, IL-18, inducible NO synthase (iNOS) and natural resistance associated macrophage protein 1 (NRAMP1). A high baseline level of defensin gene expression was recorded in young animals from the susceptible line. In contrast, a significantly low expression of interferon-gamma (IFN-gamma) gene was observed in these susceptible infected animals in comparison to resistant ones and healthy counterparts. IFN-gamma expression level represents a valuable indication of immunodeficiency associated with persistence of Salmonella in the chicken digestive tract, and IFN-gamma thus represents a factor to consider in the development of prophylactic measures for the reduction of Salmonella carrier state.

Re-evaluation of chicken CXCR1 determines the true gene structure: CXCLi1 (K60) and CXCLi2 (CAF/interleukin-8) are ligands for this receptor.

The original report of chicken CXCR1 (Li, Q. J., Lu, S., Ye, R. D., and Martins-Green, M. (2000) Gene (Amst.) 257, 307-317) described it as a single exon gene, with two isoforms (differing in their start codon). In comparison with mammalian CXCR1, the reported chicken CXCR1 was longer at both the NH(2) and COOH termini, and it lacked the conserved (C/S)CXNP motif present in the last transmembrane region of all known chemokine receptors. A re-evaluation of chicken CXCR1, comparing known expressed sequence tags with the chicken genome sequence, suggested that the gene contains two exons. We isolated a cDNA corresponding to our prediction, which was significantly different in sequence to the reported CXCR1. In particular, there were three frameshifts in our sequence, compared with the reported sequence, that restored higher identity in the COOH-terminal half of the protein to mammalian CXCR1 (61% total amino acid identity compared with 52% for the reported CXCR1), restored the (C/S)CXNP motif, and gave a predicted protein of the same length as mammalian CXCR1. In human, CXCR1 is the receptor for CXCL8. In the chicken, there are two syntenic genes, CXCLi1 and CXCLi2, which look equally like orthologues of human CXCL8. We demonstrate that both of these chemokines are ligands for chicken CXCR1. We also demonstrate that heterophils express chicken CXCR1 and that the receptor is Galpha(i) protein-linked.

The YfgL lipoprotein is essential for type III secretion system expression and virulence of Salmonella enterica Serovar Enteritidis.

Salmonella enterica, like many gram-negative pathogens, uses type three secretion systems (TTSS) to infect its hosts. The three TTSS of Salmonella, namely, TTSS-1, TTSS-2, and flagella, play a major role in the virulence of this bacterium, allowing it to cross the intestinal barrier and to disseminate systemically. Previous data from our laboratory have demonstrated the involvement of the chromosomal region harboring the yfgL, engA, and yfgJ open reading frames in S. enterica serovar Enteritidis virulence. Using microarray analysis and real-time reverse transcription-PCR after growth of bacterial cultures favorable for either TTSS-1 or TTSS-2 expression, we show in this study that the deletion in S. enterica serovar Enteritidis of yfgL, encoding an outer membrane lipoprotein, led to the transcriptional down-regulation of most Salmonella pathogenicity island 1 (SPI-1), SPI-2, and flagellar genes encoding the TTSS structural proteins and effector proteins secreted by these TTSS. In line with these results, the virulence of the DeltayfgL mutant was greatly attenuated in mice. Moreover, even if YfgL is involved in the assembly of outer membrane proteins, the regulation of TTSS expression observed was not due to an inability of the Delta yfgL mutant to assemble TTSS in its membrane. Indeed, when we forced the transcription of SPI-1 genes by constitutively expressing HilA, the secretion of the TTSS-1 effector protein SipA was restored in the culture supernatant of the mutant. These results highlight the crucial role of the outer membrane lipoprotein YfgL in the expression of all Salmonella TTSS and, thus, in the virulence of Salmonella. Therefore, this outer membrane protein seems to be a privileged target for fighting Salmonella.

Macrophages isolated from chickens genetically resistant or susceptible to systemic salmonellosis show magnitudinal and temporal differential expression of cytokines and chemokines following Salmonella enterica challenge.

Macrophages from inbred chickens that are resistant to salmonellosis show greater and more rapid expression of proinflammatory chemokines and cytokines, including the key Th1-inducing cytokine interleukin-18, upon Salmonella challenge than those from susceptible birds. This suggests the possibility that salmonellosis resistant-line macrophages signal more effectively and rapidly and are more able to induce protective Th1 adaptive responses.

Identification and characterization of a functional, alternatively spliced Toll-like receptor 7 (TLR7) and genomic disruption of TLR8 in chickens.

Based upon the recognition of antiviral compounds and single stranded viral RNA the Toll-like receptors TLR7 and TLR8 are suggested to play a significant role in initiating antiviral immune responses. Here we report the molecular characterization of the chicken TLR7/8 loci which revealed an intact TLR7 gene and fragments of a TLR8-like gene with a 6-kilobase insertion containing chicken repeat 1 (CR1) retroviral-like insertion elements. The chicken TLR7 gene encodes a 1047-amino-acid protein with 62% identity to human TLR7 and a conserved pattern of predicted leucine-rich repeats. Highest levels of chicken TLR7 mRNA were detected in immune-related tissues and cells, especially the spleen, caecal, tonsil and splenic B cells. Alternative spliced forms of TLR7 mRNA were identified in chicken, mouse and human and expressed in similar tissues and cell types to the major form of chicken TLR7. The chicken TLR7+ HD11 cell line and fresh splenocytes produced elevated levels of interleukin-1beta (IL-1beta) mRNA after exposure to the agonists R848 and loxoribine. Interestingly, none of the TLR7 agonists stimulated increased type I interferon (IFN) mRNA whereas poly(I:C) (a TLR3 agonist) up-regulated both chicken IFN-alpha and chicken IFN-beta mRNA. In contrast, TLR7 agonists, particularly R848 and poly(U) stimulated up-regulation of chicken IL-1beta, and chicken IL-8 mRNAs more effectively than poly(I:C). Stimulation of chicken TLR7 with R848 was chloroquine sensitive, suggesting signalling within an endosomal compartment, as for mammalian TLR7. The deletion of TLR8 in galliforms, accompanied with the differential response after exposure to TLR7 agonists, offers insight into the evolution of vertebrate TLR function.

A genome scan for quantitative trait loci affecting the Salmonella carrier-state in the chicken.

Selection for increased resistance to Salmonella colonisation and excretion could reduce the risk of foodborne Salmonella infection. In order to identify potential loci affecting resistance, differences in resistance were identified between the N and 6(1) inbred lines and two QTL research performed. In an F2 cross, the animals were inoculated at one week of age with Salmonella enteritidis and cloacal swabs were carried out 4 and 5 wk post inoculation (thereafter called CSW4F2 and CSW4F2) and caecal contamination (CAECF2) was assessed 1 week later. The animals from the (N x 6(1)) x N backcross were inoculated at six weeks of age with Salmonella typhimurium and cloacal swabs were studied from wk 1 to 4 (thereafter called CSW1BC to CSW4BC). A total of 33 F2 and 46 backcross progeny were selectively genotyped for 103 and 135 microsatellite markers respectively. The analysis used least-squares-based and non-parametric interval mapping. Two genome-wise significant QTL were observed on Chromosome 1 for CSW2BC and on Chromosome 2 for CSW4F2, and four suggestive QTL for CSW5F2 on Chromosome 2, for CSW5F2 and CSW2BC on chromosome 5 and for CAECF2 on chromosome 16. These results suggest new regions of interest and the putative role of SAL1.

Identification and functional characterization of chicken toll-like receptor 5 reveals a fundamental role in the biology of infection with Salmonella enterica serovar typhimurium.

Toll-like receptors (TLRs) are a major component of the pattern recognition receptor repertoire that detect invading microorganisms and direct the vertebrate immune system to eliminate infection. In chickens, the differential biology of Salmonella serovars (systemic versus gut-restricted localization) correlates with the presence or absence of flagella, a known TLR5 agonist. Chicken TLR5 (chTLR5) exhibits conserved sequence and structural similarity with mammalian TLR5 and is expressed in tissues and cell populations of immunological and stromal origin. Exposure of chTLR5+ cells to flagellin induced elevated levels of chicken interleukin-1beta (chIL-1beta) but little upregulation of chIL-6 mRNA. Consistent with the flagellin-TLR5 hypothesis, an aflagellar Salmonella enterica serovar Typhimurium fliM mutant exhibited an enhanced ability to establish systemic infection. During the early stages of infection, the fliM mutant induced less IL-1beta mRNA and polymorphonuclear cell infiltration of the gut. Collectively, the data represent the identification and functional characterization of a nonmammalian TLR5 and indicate a role in restricting the entry of flagellated Salmonella into systemic sites of the chicken.

Heritability of susceptibility to Salmonella enteritidis infection in fowls and test of the role of the chromosome carrying the NRAMP1 gene.

373 thirteen-week-old chicks issued from a commercial cross and 312 chickens from the L2 line were intravenously inoculated with 10 (6) Salmonella enteritidis and the numbers of Salmonella in the spleen, liver and genital organs were assessed 3 days later. Heritabilities of the number of Salmonella were estimated at 0.02 +/-0.04 and 0.05 +/-0.05 in the liver; at 0.29 +/-0.07 and 0.10 +/-0.06 in the spleen; and at 0.16 +/- 0.05 and 0.11 +/-0.08 in the genital organs, in the first and second experiments, respectively. The difference between the two experiments could result from sampling variations and from differences in the genetic structure of the two populations possibly including both heterosis and additive effects as well as their interaction in the first experiment. Genetic correlations between the number of bacteria in the genital organs and liver (0.56 +/-0.58 and 0.76 +/- 0.32 in the first and second experiments, respectively) and spleen (0.37 +/-0.24 and 0.79 +/-0.23) were positive. Moreover a significant within-sire effect of VIL1, a marker gene for NRAMP1, was observed in 117 progeny resulting from 25 informative matings. These results indicate that there are genetic differences in the resistance to visceral infection by S. enteritidis in these commercial egg-laying flocks, and suggest that these differences are at least partly due to genetic polymorphism in the NRAMP1 region.

The chicken CD4 gene has remained conserved in evolution.

CD4 has a central role in thymocyte differentiation and cell-mediated immunity. We isolated and analyzed chicken CD4. The gene spans 11.5 kb and is composed of ten exons. The promoter is TATA-less and similar to the mouse and human CD4 promoters, with two transcription start sites as determined by 5'RACE analysis. In general the introns are short, although the 5'untranslated region includes a large intron of 5.6 kb containing binding sites of the putative CD4 silencer. The single-strand conformation polymorphism technique was used to identify a polymorphism to map the gene, which lies on chicken Chromosome 1 in a position showing conserved synteny to mouse and human. This is the first report describing the organization of CD4 from a non-mammalian species. The structure and localization of chicken CD4 and many sequence motifs important in its regulation have remained conserved during evolution.

Allelic variation in TLR4 is linked to susceptibility to Salmonella enterica serovar Typhimurium infection in chickens.

Toll-like receptor 4 (TLR4) is part of a group of evolutionarily conserved pattern recognition receptors involved in the activation of the immune system in response to various pathogens and in the innate defense against infection. We describe here the cloning and characterization of the avian orthologue of mammalian TLR4. Chicken TLR4 encodes a 843-amino-acid protein that contains a leucine-rich repeat extracellular domain, a short transmembrane domain typical of type I transmembrane proteins, and a Toll-interleukin-1R signaling domain characteristic of all TLR proteins. The chicken TLR4 protein shows 46% identity (64% similarity) to human TLR4 and 41% similarity to other TLR family members. Northern blot analysis reveals that TLR4 is expressed at approximately the same level in all tissues tested, including brain, thymus, kidney, intestine, muscle, liver, lung, bursa of Fabricius, heart, and spleen. The probe detected only one transcript of ca. 4.4 kb in length for all tissues except muscle where the size of TLR4 mRNA was ca. 9.6 kb. We have mapped TLR4 to microchromosome E41W17 in a region harboring the gene for tenascin C and known to be well conserved between the chicken and mammalian genomes. This region of the chicken genome was shown previously to harbor a Salmonella susceptibility locus. By using linkage analysis, TLR4 was shown to be linked to resistance to infection with Salmonella enterica serovar Typhimurium in chickens (likelihood ratio test of 10.2, P = 0.00138), suggesting a role of TLR4 in the host response of chickens to Salmonella infection.

The properties of the single chicken MHC classical class II alpha chain ( B-LA) gene indicate an ancient origin for the DR/E-like isotype of class II molecules.

In mammals, there are MHC class II molecules with distinctive sequence features, such as the classical isotypes DR, DQ and DP. These particular isotypes have not been reported in non-mammalian vertebrates. We have isolated the class II (B-L) alpha chain from outbred chickens as the basis for the cloning and sequencing of the cDNA. We found only one class II alpha chain transcript, which bears the major features of a classical class II alpha sequence, including the critical peptide-binding residues. The chicken sequence is more similar to human DR than to the DQ, DP, DO or DM isotypes, most significantly in the peptide-binding alpha(1) domain. The cDNA and genomic DNA sequences from chickens of diverse origins show few alleles, which differ in only four nucleotides and one amino acid. In contrast, significant restriction fragment length polymorphism is detected by Southern blot analysis of genomic DNA, suggesting considerable diversity around the gene. Analysis of a large back-cross family indicates that the class II alpha chain locus ( B-LA) is located roughly 5.6 cM from the MHC locus, which encodes the classical class II beta chains. Thus the chicken class II alpha chain gene is like the mammalian DR and E isotypes in three properties: the presence of the critical peptide-binding residues, the low level of polymorphism and sequence diversity, and the recombinational separation from the class II beta chain genes. These results indicate that the sequence features of this lineage are both functionally important and at least 300 million years old.