Chromosome level assembly reveals a unique immune gene organization and signatures of evolution in the common pheasant.

The common pheasant Phasianus colchicus, belonging to the order Galliformes and family Phasianidae, is the most widespread species. Despite a long history of captivity, the domestication of this bird is still at a preliminary stage. Recently, the demand for accelerating its transformation to poultry for meat and egg production has been increasing. In this study, we assembled high quality, chromosome scale genome of the common pheasant by using PacBio long reads, next-generation short reads, and Hi-C technology. The primary assembly has contig N50 size of 1.33 Mb and scaffold N50 size of 59.46 Mb, with a total size of 0.99 Gb, resolving most macrochromosomes into single scaffolds. A total of 23,058 genes and 10.71 Mb interspersed repeats were identified, constituting 30.31% and 10.71% of the common pheasant genome, respectively. Our phylogenetic analysis revealed that the common pheasant shared common ancestors with turkey about 24.7-34.5 million years ago (Ma). Rapidly evolved gene families, as well as branch-specific positively selected genes, indicate that calcium-related genes are potentially related to the adaptive and evolutionary change of the common pheasant. Interestingly, we found that the common pheasant has a unique major histocompatibility complex B locus (MHC-B) structure: three major inversions occurred in the sequence compared with chicken MHC-B. Furthermore, we detected signals of selection in five breeds of domestic common pheasant, several of which are production-oriented.

Antiviral Activity and Adaptive Evolution of Avian Tetherins.

Tetherin/BST-2 is an antiviral protein that blocks the release of enveloped viral particles by linking them to the membrane of producing cells. At first, BST-2 genes were described only in humans and other mammals. Recent work identified BST-2 orthologs in nonmammalian vertebrates, including birds. Here, we identify the BST-2 sequence in domestic chicken (Gallus gallus) for the first time and demonstrate its activity against avian sarcoma and leukosis virus (ASLV). We generated a BST-2 knockout in chicken cells and showed that BST-2 is a major determinant of an interferon-induced block of ASLV release. Ectopic expression of chicken BST-2 blocks the release of ASLV in chicken cells and of human immunodeficiency virus type 1 (HIV-1) in human cells. Using metabolic labeling and pulse-chase analysis of HIV-1 Gag proteins, we verified that chicken BST-2 blocks the virus at the release stage. Furthermore, we describe BST-2 orthologs in multiple avian species from 12 avian orders. Previously, some of these species were reported to lack BST-2, highlighting the difficulty of identifying sequences of this extremely variable gene. We analyzed BST-2 genes in the avian orders Galliformes and Passeriformes and showed that they evolve under positive selection. This indicates that avian BST-2 is involved in host-virus evolutionary arms races and suggests that BST-2 antagonists exist in some avian viruses. In summary, we show that chicken BST-2 has the potential to act as a restriction factor against ASLV. Characterizing the interaction of avian BST-2 with avian viruses is important in understanding innate antiviral defenses in birds.IMPORTANCE Birds are important hosts of viruses that have the potential to cause zoonotic infections in humans. However, only a few antiviral genes (called viral restriction factors) have been described in birds, mostly because birds lack counterparts of highly studied mammalian restriction factors. Tetherin/BST-2 is a restriction factor, originally described in humans, that blocks the release of newly formed virus particles from infected cells. Recent work identified BST-2 in nonmammalian vertebrate species, including birds. Here, we report the BST-2 sequence in domestic chicken and describe its antiviral activity against a prototypical avian retrovirus, avian sarcoma and leukosis virus (ASLV). We also identify BST-2 genes in multiple avian species and show that they evolve rapidly in birds, which is an important indication of their relevance for antiviral defense. Analysis of avian BST-2 genes will shed light on defense mechanisms against avian viral pathogens.

Molecular cloning and transcriptional regulation of Indian peafowl (Pavo cristatus) IFN-α gene.

Interferon-α (IFN-α) resists viral infections by triggering the transcription of a diverse range of antiviral IFN-stimulated genes (ISGs). However, information about the Indian peafowl (Pavo cristatus) IFN-α (PcIFN-α) has not been reported. In this study, a PcIFN-α gene was amplified, which encoded a protein of 193 amino acids with a 26-amino acid signal peptide sharing 72.16-95.70% identity with other avians in Aves. After expression in prokaryote, PcIFN-α was analyzed for its physicochemical property and antiviral activity. Intriguingly, compared with chicken IFN-α, an effective viral infection therapeutic agent, PcIFN-α showed superior anti-VSV, NDV, and AIV activities, which were then abrogated by rabbit anti-PcIFN-α antibodies in vitro. Moreover, PcIFN-α was shown to be highly sensitive to trypsin; however, it remained stable despite changes in pH and temperature. Additionally, PcIFN-α induced the transcriptional or translational levels of Mx1 and ISG12 genes time-dependently. Overall, the present study revealed that PcIFN-α is a potential novel effective therapeutic agent in antiviral defense responses in peafowl, improving understanding of its involvement in bird antiviral defense.

Absence of protection from West Nile virus disease and adverse effects in red legged partridges after non-structural NS1 protein administration.

The red-legged partridge (Alectoris rufa) is a competent host for West Nile virus (WNV) replication and highly susceptible to WNV disease. With the aim to assess in this species whether the inoculation of non-structural protein NS1 from WNV elicits a protective immune response against WNV infection, groups of partridges were inoculated with recombinant NS1 (NS1 group) or an unrelated recombinant protein (mock group), and challenged with infectious WNV. A third group received no inoculation prior to challenge (challenge group). The NS1 group failed to elicit detectable antibodies to NS1 while in the mock group a specific antibody response was observed. Moreover, no protection against WNV disease was observed in the NS1 group, but rather, it showed significantly higher viral RNA load and delayed neutralizing antibody response, and suffered a more severe clinical disease, which resulted in higher mortality. This adverse effect has not been observed before and warrants further investigations.

Extensive shared polymorphism at non-MHC immune genes in recently diverged North American prairie grouse.

Gene polymorphisms shared between recently diverged species are thought to be widespread and most commonly reflect introgression from hybridization or retention of ancestral polymorphism through incomplete lineage sorting. Shared genetic diversity resulting from incomplete lineage sorting is usually maintained for a relatively short period of time, but under strong balancing selection it may persist for millions of years beyond species divergence (balanced trans-species polymorphism), as in the case of the major histocompatibility complex (MHC) genes. However, balancing selection is much less likely to act on non-MHC immune genes. The aim of this study was to investigate the patterns of shared polymorphism and selection at non-MHC immune genes in five grouse species from Centrocercus and Tympanuchus genera. For this purpose, we genotyped five non-MHC immune genes that do not interact directly with pathogens, but are involved in signaling and regulate immune cell growth. In contrast to previous studies with MHC, we found no evidence for balancing selection or balanced trans-species polymorphism among the non-MHC immune genes. No haplotypes were shared between genera and in most cases more similar allelic variants sorted by genus. Between species within genera, however, we found extensive shared polymorphism, which was most likely attributable to introgression or incomplete lineage sorting following recent divergence and large ancestral effective population size (i.e., weak genetic drift). Our study suggests that North American prairie grouse may have attained relatively low degree of reciprocal monophyly at nuclear loci and reinforces the rarity of balancing selection in non-MHC immune genes.

Serological evidence of exposure to zoonotic tick-borne bacteria in pheasants (Phasianus colchicus).

INTRODUCTION AND OBJECTIVE: Previous studies on tick-borne pathogens in the avian population have focused mainly on the detection of the agents in ticks collected from birds, but data about the presence of tick-borne bacteria in these animals are scant. The aim of the presented study was to verify the exposure to some zoonotic tick-borne bacteria, in particular, Anaplasma phagocytophilum, Borrelia burgdorferi sensu lato, Coxiella burnetii, and the Rickettsia species of the Spotted Fever Group (SFG), in pheasants (Phasianus colchicus) living in a central Italy area, characterized by conditions favourable for the diffusion of the ticks. MATERIALS AND METHOD: Blood serum samples from 276 farm-reared pheasants were examined by indirect immunofluorescence antibody test to detect antibodies against the 4 reported pathogens. RESULTS: A total of 124 (44.92%) birds resulted as seropositive: in particular 3 (1.08%) to C. burnetii, 31 (11.23%) to A. phagocytophilum, 46 (16.67%) to B. burgdorferi s.l., 49 (17.75%) to SFG Rickettsia spp. antigens. Three pheasants resulted positive both to A. phagocytophilum and B. burgdorferi s.l. antigens, whereas 2 - both to B. burgdorferi s.l. and SFG Rickettsia spp. antigens. The antibody titers varied from 1:40-1:320. CONCLUSIONS: The obtained results show that the pheasants had developed antibodies to the investigated tick-borne agents. For this reason, they seem to be involved in the epidemiology of the studied tick-borne bacteria.

Specific alleles at immune genes, rather than genome-wide heterozygosity, are related to immunity and survival in the critically endangered Attwater's prairie-chicken.

The negative effects of inbreeding on fitness are serious concerns for populations of endangered species. Reduced fitness has been associated with lower genome-wide heterozygosity and immune gene diversity in the wild; however, it is rare that both types of genetic measures are included in the same study. Thus, it is often unclear whether the variation in fitness is due to the general effects of inbreeding, immunity-related genes or both. Here, we tested whether genome-wide heterozygosity (20 990 SNPs) and diversity at nine immune genes were better predictors of two measures of fitness (immune response and survival) in the endangered Attwater's prairie-chicken (Tympanuchus cupido attwateri). We found that postrelease survival of captive-bred birds was related to alleles of the innate (Toll-like receptors, TLRs) and adaptive (major histocompatibility complex, MHC) immune systems, but not to genome-wide heterozygosity. Likewise, we found that the immune response at the time of release was related to TLR and MHC alleles, and not to genome-wide heterozygosity. Overall, this study demonstrates that immune genes may serve as important genetic markers when monitoring fitness in inbred populations and that in some populations specific functional genes may be better predictors of fitness than genome-wide heterozygosity.

Protection Afforded by a Recombinant Turkey Herpesvirus-H5 Vaccine Against the 2014 European Highly Pathogenic H5N8 Avian Influenza Strain.

A highly pathogenic avian influenza (HPAI) H5N8 (clade 2.3.4.4) virus, circulating in Asia (South Korea, Japan, and southern China) since the beginning of 2014, reached the European continent in November 2014. Germany, the Netherlands, the United Kingdom, Italy, and Hungary confirmed H5N8 infection of poultry farms of different species and of several wild bird species. Unlike the Asian highly pathogenic (HP) H5N1, this HP H5N8 also went transatlantic and reached the American West Coast by the end of 2014, affecting wild birds as well as backyard and commercial poultry. This strain induces high mortality and morbidity in Galliformes, whereas wild birds seem only moderately affected. A recombinant turkey herpesvirus (rHVT) vector vaccine expressing the H5 gene of a clade 2.2 H5N1 strain (rHVT-H5) previously demonstrated a highly efficient clinical protection and reduced viral excretion against challenge with Asian HP H5N1 strains of various clades (2.2, 2.2.1, 2.2.1.1, 2.1.3, 2.1.3.2, and 2.3.2.1) and was made commercially available in various countries where the disease is endemic. To evaluate the protective efficacy of the rHVT-H5 vaccine against the first German H5N8 turkey isolate (H5N8 GE), a challenge experiment was set up in specific-pathogen-free (SPF) chickens, and the clinical and excretional protection was evaluated. SPF chickens were vaccinated subcutaneously at 1 day old and challenged oculonasally at 4 wk of age with two viral dosages, 10(5) and 10(6) 50% egg infective doses. Morbidity and mortality were monitored daily in unvaccinated and vaccinated groups, whereas viral shedding by oropharyngeal and cloacal routes was evaluated at 2, 5, 9, and 14 days postinoculation (dpi). Serologic monitoring after vaccination and challenge was also carried out. Despite its high antigenic divergence of the challenge H5N8 strain, a single rHVT-H5 vaccine administration at 1 day old resulted in a full clinical protection against challenge and a significant reduction of viral shedding in the vaccinated birds.

Influence of dietary protein on serum metabolites and antioxidant status: A study in Chrysolophus amherstiae.

This experiment was conducted to study the effect of feeding graded levels of dietary crude protein (CP) on serum biochemical profile of Lady Amherst's pheasants (LAP). Eighteen male LAP were randomly distributed into three groups of six each in an experiment based on completely randomized design. The CP content of the diets was 13.4%, 16.5%, and 19.1%, in groups I, II, and III, respectively. Serum concentrations of uric acid was lowest (P < 0.05) in group I. Relationship between serum concentration of uric acid and nitrogen intake was linear (R(2) = 0.39, P < 0.01). Concentrations of other serum metabolites and enzymes were similar among the groups. Serum concentration of triiodothyronine (T3 ) was highest (P < 0.05) in group I, followed by groups II and III. Total antioxidant capacity (TAC) of serum was lower (P < 0.007), whereas serum concentration of malondialdehyde (MDA) was higher (P < 0.001) in group I as compared to groups II and III. Regression of serum concentration of TAC (R(2) = 0.74, P < 0.01) and MDA (R(2) = 0.39, P < 0.05) was polynomial. Heterophil to lymphocyte ratio was higher (P < 0.007) in group I as compared to groups II and III. Relationship between H/L ratio and nitrogen intake was polynomial (R(2) = 0.69, P < 0.05). Cell mediated immune response measured as foot web index was similar among the groups. Based upon the results, it was concluded that a diet containing 16.5% crude protein would be optimum for improving antioxidant defense and the ability of Lady Amherst's pheasant to combat stress. Zoo Biol. 35:346-354, 2016. © 2016 Wiley Periodicals, Inc.

Molecular characterization of classical and nonclassical MHC class I genes from the golden pheasant (Chrysolophus pictus).

Classical major histocompatibility complex (MHC) class I allelic polymorphism is essential for competent antigen presentation. To improve the genotyping efforts in the golden pheasant, it is necessary to differentiate more accurately between classical and nonclassical class I molecules. In our study, all MHC class I genes were isolated from one golden pheasant based on two overlapping PCR amplifications. In total, six full-length class I nucleotide sequences (A-F) were identified, and four were novel. Two (A and C) belonged to the IA1 gene, two (B and D) were alleles derived from the IA2 gene through transgene amplification, and two (E and F) comprised a third novel locus, IA3 that was excluded from the core region of the golden pheasant MHC-B. IA1 and IA2 exhibited the broad expression profiles characteristic of classical loci, while IA3 showed no expression in multiple tissues and was therefore defined as a nonclassical gene. Phylogenetic analysis indicated that the three IA genes in the golden pheasant share a much closer evolutionary relationship than the corresponding sequences in other galliform species. This observation was consistent with high sequence similarity among them, which likely arises from the homogenizing effect of recombination. Our careful distinction between the classical and nonclassical MHC class I genes in the golden pheasant lays the foundation for developing locus-specific genotyping and establishing a good molecular marker system of classical MHC I loci.

Contrasting patterns of selection and drift between two categories of immune genes in prairie-chickens.

Immune-receptor genes of the adaptive immune system, such as the major histocompatibility complex (MHC), are involved in recognizing specific pathogens and are known to have high rates of adaptive evolution, presumably as a consequence of rapid co-evolution between hosts and pathogens. In contrast, many 'mediating' genes of the immune system do not interact directly with specific pathogens and are involved in signalling (e.g. cytokines) or controlling immune cell growth. As a consequence, we might expect stronger selection at immune-receptor than mediating genes, but these two types of genes have not been compared directly in wild populations. Here, we tested the hypothesis that selection differs between MHC (class I and II) and mediating genes by comparing levels of population differentiation across the range of greater prairie-chickens (Tympanuchus cupido). As predicted, there was stronger population differentiation and isolation by distance at immune receptor (MHC) than at either mediating genes or neutral microsatellites, suggesting a stronger role of local adaptation at the MHC. In contrast, mediating genes displayed weaker differentiation between populations than neutral microsatellites, consistent with selection favouring similar alleles across populations for mediating genes. In addition to selection, drift also had a stronger effect on immune receptor (MHC) than mediating genes as indicated by the stronger decline of MHC variation in relation to population size. This is the first study in the wild to show that the effects of selection and drift on immune genes vary across populations depending on their functional role.

Transcriptomic Characterization of Innate and Acquired Immune Responses in Red-Legged Partridges (Alectoris rufa): A Resource for Immunoecology and Robustness Selection.

Present and future challenges for wild partridge populations include the resistance against possible disease transmission after restocking with captive-reared individuals, and the need to cope with the stress prompted by new dynamic and challenging scenarios. Selection of individuals with the best immune ability may be a good strategy to improve general immunity, and hence adaptation to stress. In this study, non-infectious challenges with phytohemagglutinin (PHA) and sheep red blood cells allowed the classification of red-legged partridges (Alectoris rufa) according to their overall immune responses (IR). Skin from the area of injection of PHA and spleen, both from animals showing extreme high and low IR, were selected to investigate the transcriptional profiles underlying the different ability to cope with pathogens and external aggressions. RNA-seq yielded 97 million raw reads from eight sequencing libraries and approximately 84% of the processed reads were mapped to the reference chicken genome. Differential expression analysis identified 1488 up- and 107 down-regulated loci in individuals with high IR versus low IR. Partridges displaying higher innate IR show an enhanced activation of host defence gene pathways complemented with a tightly controlled desensitization that facilitates the return to cellular homeostasis. These findings indicate that the immune system ability to respond to aggressions (either diseases or stress produced by environmental changes) involves extensive transcriptional and post-transcriptional regulations, and expand our understanding on the molecular mechanisms of the avian immune system, opening the possibility of improving disease resistance or robustness using genome assisted selection (GAS) approaches for increased IR in partridges by using genes such as AVN or BF2 as markers. This study provides the first transcriptome sequencing data of the Alectoris genus, a resource for molecular ecology that enables integration of genomic tools in further studies.

Detecção de resistência às fluoroquinolonas em Campylobacter isolados de frangos de criação orgânica / Detection of fluoroquinolone resistance in Campylobacter strains from organic poultry

Estudos têm revelado que a resistência às quinolonas em cepas de Campylobacter está relacionada à presença da mutação Treonina-86 para Isoleucina. Com o objetivo de investigar a presença dessa mutação em cepas de Campylobacter sensíveis e resistentes à ciprofloxacina e enrofloxacina, o conteúdo cecal de 80 frangos de corte de criação orgânica, abatidos sob Serviço de Inspeção Estadual (S.I.E.) do Estado do Rio de Janeiro, foram coletados e investigados para a presença de Campylobacter. A determinação da resistência à ciprofloxacina e enrofloxacina foi feita pela técnica de difusão em disco e de diluição em ágar para determinação da Concentração Inibitória Mínima (CIM). A detecção da mutação na Região Determinante de Resistencia às Quinolonas (RDRQ) no gene gyrA foi realizada através de sequenciamento. Campylobacter foi isolado a partir de 100% das amostras avaliadas, sendo 68,75% correspondente à C. jejuni e 31,25% à C. coli. No teste de difusão em disco, 100% das cepas foram resistentes à ciprofloxacina e 56,25% das cepas foram resistentes à enrofloxacina. No teste de diluição em ágar, todas as cepas foram resistentes à ciprofloxacina apresentando CIM variando de ≥ 16-64μg/mL, e resistência ou resistência intermediaria à enrofloxacina foi detectada em 42,50% (CIM ≥ 4-32μg/mL) e 38,75% (CIM = 2μg/mL) das cepas, respectivamente. A mutação Tre-86-Ile, foi observada em 100% das cepas analisadas. Além dessa mutação, foram observadas outras mutações não silenciosas (Val-73-Glu, Ser-114-Leu, Val-88-Asp, Ala-75-Asp, Ser-119-Gli, Arg-79-Lis) e mutações silenciosas (His-81-His, Ser-119-Ser, Ala-120-Ala, Fen-99-Fen, Ala-122-Ala, Gli-74-Gli, Ile-77-Ile, Ala-91-Ala, Leu-92-Leu, Val-93-Val, Ile-106-Ile, Tre-107-Tre, Gli-113-Gli, Ile-115-Ile, Gli-110-Gli). A observação de que cepas sensíveis à enrofloxacina pelos testes fenotípicos apresentavam a substituição Tre-86 para Ile sugere que outros mecanismos podem contribuir para a resistência à enrofloxacina em Campylobacter...

Studies have shown that resistance to quinolones in Campylobacter strains is related with Threonine-86-Isoleucine mutation. In order to investigate the presence of this mutation in sensitive and resistant Campylobacter strains to ciprofloxacin and enrofloxacin, the cecal contents of 80 broilers from organic raising chickens, slaughtered under State Inspection Service (S.I.S) of the State of Rio de Janeiro, were collected and tested for the presence of Campylobacter. The determination of ciprofloxacin and enrofloxacin susceptibility was done by disk diffusion and agar dilution methods for determining the Minimum Inhibitory Concentration (MIC). The detection of mutation in Quinolone Resistance Determinant Region (QRDR) in gyrA gene was done by sequencing. Campylobacter was isolated from 100% of the samples, being 68.75% C. jejuni and 31.25% C. coli. By the disk diffusion method, resistance to ciprofloxacin was observed in all isolates and 56.25% of the strains were resistant to enrofloxacin. By agar dilution method, all strains were resistant to ciprofloxacin (MIC ≥ 16μg/mL to ≥ 64μg/mL) and full and intermediate resistance to enrofloxacin was detected in 42.50% (MIC ≥ 4-32μg/mL) and 38.75% (MIC =2μg/mL) of the strains, respectively. Mutation Thr-86-Ile was observed in 100% of the isolates investigated. In addition to this mutation, others no silent mutations (Val-73-Glu, Ser-114-Leu, Val-88-Asp, Ala-75-Asp, Gly-119-Ser, Arg-79-Lys) and silent mutations (His-81-His, Ser-119-Ser, Ala-120-Ala, Phe-99-Phe, Ala-122-Ala, Gly-74-Gly, Ile-77-Ile, Ala-91-Ala, Leu-92-Leu, Val-93-Val, Ile-106-Ile, Thr-107-Thr, Gly-113-Gly, Ile-115-Ile, Gly-110-Gly) were detected. All the enrofloxacin-sensitive strains by the phenotypic methods had the Thr-86 to Ile substitution, which suggests other mechanisms contributing to enrofloxacin resistance in Campylobacter...

Molecular tools to support metabolic and immune function research in the Guinea Fowl (Numida meleagris).

BACKGROUND: Guinea fowl (Numidia meleagris) production as an alternative source of meat and poultry has shown potential for economic viability. However, there has been little progress in characterizing the transcriptome of the guinea fowl. In this study RNA-sequencing and de novo transcriptome assembly of several Guinea fowl tissues (pancreas, hypothalamus, liver, bone marrow and bursa) which play key roles in regulating feed intake, satiety, and immune function was performed using Illumina's Hi-Seq 2000. RESULTS: 74 million sequences were generated and assembled into 96,492 contigs using the Trinity software suite. Over 39,000 of these transcripts were found to have in silico translated protein sequences that are homologous to chicken protein sequences. Gene ontology analysis uncovered 416 transcripts with metabolic functions and 703 with immune function. CONCLUSION: The transcriptome information presented here will support the development of molecular approaches to improve production efficiency of the guinea fowl and other avian species.

Sublethal Pb exposure produces season-dependent effects on immune response, oxidative balance and investment in carotenoid-based coloration in red-legged partridges.

Ingestion of lead (Pb) shot pellets constitutes the main cause of Pb poisoning in avifauna. We studied the effects of sublethal Pb exposure on immunity, carotenoid-based coloration, oxidative stress and trade-offs among these types of responses during spring and autumn in red-legged partridges (Alectoris rufa). We evaluated constitutive immunity testing lysozyme and natural antibody levels, and blood bactericidal and phagocytic activities. We studied induced immunity by testing PHA and humoral responses. We analyzed fecal parasite and bacterial abundance and oxidative stress biomarkers. Pb exposure in spring reduced natural antibody levels, whereas in autumn, it reduced lysozyme levels and increased phagocytic activity. Pb exposure increased PHA response in both seasons, and decreased T-independent humoral response in autumn. Pb exposure also increased noncoliform and decreased coliform Gram-negative gut bacteria. In spring, Pb exposure decreased antioxidant levels and increased coloration in males, whereas in autumn, it increased retinol levels but reduced coloration in both genders. Our results suggest that in spring, Pb-exposed females used antioxidants to cope with oxidative stress at the expense of coloration, whereas Pb-exposed males increased coloration, which may reflect an increased breeding investment. In autumn, both genders prioritized oxidative balance maintenance at the expense of coloration.

Adverse effects of thiram-treated seed ingestion on the reproductive performance and the offspring immune function of the red-legged partridge.

Pesticide research traditionally has focused on compounds with high acute toxicity or persistence, but the adverse sublethal effects of pesticides with different properties also may have important consequences on exposed wildlife. The authors studied the effects of thiram, a fungicide used for seed coating with known effects as endocrine disruptor. Red-legged partridges (Alectoris rufa; n = 15 pairs per treatment group) were fed wheat treated with 0%, 20%, or 100% of the thiram application rate used in autumn (25 d) and late winter (10 d) to mimic cereal sowing periods. The authors studied the effects on reproductive performance, carotenoid-based ornamentation and cellular immune responsiveness of adult partridges, and their relationship with changes in oxidative stress biomarkers and plasma biochemistry. The authors also studied the effect of parental exposure on egg antioxidant content and on the survival, growth, and cellular immune response of offspring. Exposure to thiram-coated seeds delayed egg laying, reduced clutch size, and affected egg size and eggshell thickness. Partridges exposed to the 20% thiram dose exhibited reduced egg fertility and brood size (55% and 28% of controls, respectively). Chick survival was unaffected by parental exposure to treated seeds, but adverse effects on their growth rate and cellular immune response were apparent. These effects on reproduction and immune function may have important demographic consequences on farmland bird populations.

Changes in selected biochemical indices, leukocyte profile, and pterins as biomarkers of immune system activity due to antipecking measures in pheasants.

The physiological changes in response to beak trimming and spectacle usage as antipecking measures were monitored in 10-mo-old common pheasants (Phasianus colchicus). Short-term analysis conducted before the beginning of the laying period showed immediate increases of plasma corticosterone (P < 0.05) and lactate dehydrogenase (P < 0.001) concentrations and decrease of plasma triglycerides (P < 0.01) levels in response to both beak trimming and the application of spectacles. Beak-trimmed pheasants exhibited higher plasma corticosterone concentrations than pheasants fitted with spectacles (P < 0.001). To assess long-term changes, blood samples for biochemical (neopterin and biopterin determination) and hematological (leukocyte profile determination) examinations were taken from beak-trimmed, spectacles-fitted, and control pheasant hens housed in cages during their laying period. At the end of the laying period, hens fitted with spectacles exhibited lower concentrations of plasma neopterin (P = 0.005) and biopterin (P = 0.005) than beak-trimmed pheasant hens. Our findings suggest that the immune system was suppressed in spectacles-fitted pheasant hens as a result of chronic stress, as also indicated by the higher heterophil-to-lymphocyte ratio (P = 0.001) compared with beak-trimmed hens. Our study found a negative correlation (r = -0.31, P = 0.019) between the heterophil-to-lymphocyte ratio and plasma neopterin concentration. This study demonstrated that both beak trimming and use of spectacles are not only stressful procedures for pheasants, but long-term effects may also include a negative impact on the immune system.

Transcriptome sequencing of black grouse (Tetrao tetrix) for immune gene discovery and microsatellite development.

The black grouse (Tetrao tetrix) is a galliform bird species that is important for both ecological studies and conservation genetics. Here, we report the sequencing of the spleen transcriptome of black grouse using 454 GS FLX Titanium sequencing. We performed a large-scale gene discovery analysis with a focus on genes that might be related to fitness in this species and also identified a large set of microsatellites. In total, we obtained 182 179 quality-filtered sequencing reads that we assembled into 9035 contigs. Using these contigs and 15 794 length-filtered (greater than 200 bp) singletons, we identified 7762 transcripts that appear to be homologues of chicken genes. A specific BLAST search with an emphasis on immune genes found 308 homologous chicken genes that have immune function, including ten major histocompatibility complex-related genes located on chicken chromosome 16. We also identified 1300 expressed sequence tag microsatellites and were able to design suitable flanking primers for 526 of these. A preliminary test of the polymorphism of the microsatellites found 10 polymorphic microsatellites of the 102 tested. Genomic resources generated in this study should greatly benefit future ecological, evolutionary and conservation genetic studies on this species.

Contrasted effects of an oxidative challenge and α-melanocyte-stimulating hormone on cellular immune responsiveness: an experiment with red-legged partridges Alectoris rufa.

Oxidative stress is increasingly recognized as a key selective force shaping evolutionary trade-offs. One such trade-off involves investing in immunity versus combating oxidative stress. While there is broad evidence that mounting an immune response causes increased oxidative stress, the effect that increased oxidative stress during development has at a later stage on immune responsiveness remains little known. The production of melanin-based coloration in vertebrates is influenced by oxidative stress and by hormones, such as the alpha-melanocyte-stimulating hormone (α-MSH). Oxidative stress could impair immunity, and this might be a cost associated with the production of melanin traits. α-MSH has immunomodulatory effects, with most evidence pointing towards an improvement of immunity (improved pro-inflammatory activity). Here, we investigated the effects of an oxidative challenge (exposure to a pro-oxidant compound, diquat) and of experimentally elevated α-MSH on the cell-mediated immune responses (CMIR) of growing young (1 month old) red-legged partridges Alectoris rufa in captivity. CMIR were assessed in response to primary and secondary challenges with phytohemagglutinin (PHA). We specifically tested whether an oxidative challenge during growth and development had a delayed effect (4 months after exposure) on immunity. We found that the diquat treatment did not affect primary CMIR, but significantly reduced secondary CMIR. Elevated α-MSH increased primary CMIR in males, but not in females. Our experimental results are consistent with a trade-off between investing in activities that generate oxidative stress (e.g., growth, reproduction, production of ornaments) versus investing in immunity, and shed new lights onto the inter-relationships between immunity, oxidative stress and the expression of melanin-based coloration in vertebrates, revealing a novel, delayed physiological cost that can contribute to ensuring honest signaling.

Drift and selection influence geographic variation at immune loci of prairie-chickens.

Previous studies of immunity in wild populations have focused primarily on genes of the major histocompatibility complex (MHC); however, studies of model species have identified additional immune-related genes that also affect fitness. In this study, we sequenced five non-MHC immune genes in six greater prairie-chicken (Tympanuchus cupido) populations that have experienced varying degrees of genetic drift as a consequence of population bottlenecks and fragmentation. We compared patterns of geographic variation at the immune genes with six neutral microsatellite markers to investigate the relative effects of selection and genetic drift. Global F(ST) outlier tests identified positive selection on just one of five immune genes (IAP-1) in one population. In contrast, at other immune genes, standardized G'(ST) values were lower than those at microsatellites for a majority of pairwise population comparisons, consistent with balancing selection or with species-wide positive or purifying selection resulting in similar haplotype frequencies across populations. The effects of genetic drift were also evident as summary statistics (e.g., Tajima's D) did not differ from neutrality for the majority of cases, and immune gene diversity (number of haplotypes per gene) was correlated positively with population size. In summary, we found that both genetic drift and selection shaped variation at the five immune genes, and the strength and type of selection varied among genes. Our results caution that neutral forces, such as drift, can make it difficult to detect current selection on genes.