Health monitoring in birds using bio-loggers and whole blood transcriptomics.

Monitoring and early detection of emerging infectious diseases in wild animals is of crucial global importance, yet reliable ways to measure immune status and responses are lacking for animals in the wild. Here we assess the usefulness of bio-loggers for detecting disease outbreaks in free-living birds and confirm detailed responses using leukocyte composition and large-scale transcriptomics. We simulated natural infections by viral and bacterial pathogens in captive mallards (Anas platyrhynchos), an important natural vector for avian influenza virus. We show that body temperature, heart rate and leukocyte composition change reliably during an acute phase immune response. Using genome-wide gene expression profiling of whole blood across time points we confirm that immunostimulants activate pathogen-specific gene regulatory networks. By reporting immune response related changes in physiological and behavioural traits that can be studied in free-ranging populations, we provide baseline information with importance to the global monitoring of zoonotic diseases.

Metal contamination in quail meat: residues, sources, molecular biomarkers, and human health risk assessment.

Quail meat is an emerging source of high-quality animal protein. Quails are exposed to a wide range of xenobiotics such as heavy metals. In this study, residual concentrations of four toxic metals, of significant public health importance, including cadmium (Cd), lead (Pb), arsenic (As), and nickel (Ni), were determined in edible tissues of quails. In addition, metal loads were measured in water, feed, and litter samples collected from same quail farms as possible sources for quail exposure to heavy metals. The possible use of metallothionein (MT) and heat shock protein 70 (Hsp70) as molecular biomarkers of exposure to heavy metals was further investigated. Furthermore, the dietary intake and the potential risk assessment of the examined heavy metals among children and adults were calculated. The edible tissues of quails contained high concentrations of four heavy metals (contents (ppm/ww) ranging from 0.02 to 0.32 in Cd, 0.05 to 1.96 in Pb, 0.002 to 0.32 in As, and 1.17 to 3.94 in Ni), which corresponded to the high contents of these metals in the feeds, water, and litter. MT and Hsp70 mRNA expressions showed positive correlations with the concentrations of heavy metals in tissues indicating the possibility to use these proteins as biomarkers for quail's exposure to toxic metals. Dietary intake of quail meat and risk assessment revealed potential risks especially for children after prolonged exposure to the examined metals. Thus, legislations should be established and continuous screening of metal residues should be adopted in order to reduce the toxic metal concentrations in feeds and drinking water for quails. Reduction of exposure to heavy metals subsequently would lead to minimization of exposure of such toxicants through consumption of quail meat.

Expression and function of microRNA-9 in the mid-hindbrain area of embryonic chick.

BACKGROUND: MiR-9 is a small non-coding RNA that is highly conserved between species and primarily expressed in the central nervous system (CNS). It is known to influence proliferation and neuronal differentiation in the brain and spinal cord of different vertebrates. Different studies have pointed to regional and species-specific differences in the response of neural progenitors to miR-9. METHODS: In ovo and ex ovo electroporation was used to overexpress or reduce miR-9 followed by mRNA in situ hybridisation and immunofluorescent stainings to evaluate miR- expression and the effect of changed miR-9 expression. RESULTS: We have investigated the expression and function of miR-9 during early development of the mid-hindbrain region (MH) in chick. Our analysis reveals a closer relationship of chick miR-9 to mammalian miR-9 than to fish and a dynamic expression pattern in the chick neural tube. Early in development, miR-9 is diffusely expressed in the entire brain, bar the forebrain, and it becomes more restricted to specific areas of the CNS at later stages. MiR-9 overexpression at HH9-10 results in a reduction of FGF8 expression and premature neuronal differentiation in the mid-hindbrain boundary (MHB). Within the midbrain miR-9 does not cause premature neuronal differentiation it rather reduces proliferation in the midbrain. CONCLUSION: Our findings indicate that miR-9 has regional specific effects in the developing mid-hindbrain region with a divergence of response of regional progenitors.

Influence of hatch time and access to feed on intramuscular adipose tissue deposition in broilers.

The effect of hatch time and subsequent access to feed on intramuscular adipose tissue deposition was studied in the pectoralis major muscle of male Ross 308 broiler chickens. Based on their hatch time chicks were classified as early (EH), midterm (MH), or late (LH) hatchers, with an average incubation duration of 497.7 h for EH, 508.8 h for MH, and 514.5 h for LH birds. Chicks were provided access to feed either immediately at hatch, or 24 h after the conclusion of the hatch window. Expression of the adipogenic regulatory genes peroxisome proliferator-activated receptor gamma (PPARγ), and stearoyl-CoA desaturase (SCD), were measured at the time of hatch, and zero, one, 4, 7, 28, and 40 d. Intramuscular adipocyte cell width and visualization of adipose tissue deposition was observed at 28 and 40 d. Expression of PPARγ was increased in the pectoralis major of LH birds at the time of hatch, zero, and one d. The expression of PPARγ at one and 7 d, and SCD at 7 d were increased in all birds that received delayed access to feed. At 28 d, adipocyte cell width was increased in LH birds with delayed access to feed, compared to EH and MH birds with delayed access to feed and LH birds with immediate access to feed. At 40 d, adipocyte cell width was increased in all birds that received delayed access to feed. Also at 40 d, there was a trend (P = 0.078) for more extensive intramuscular adipose tissue deposition in LH than EH birds, and in birds with delayed access to feed (P = 0.075). These data indicate delayed access to feed increases intramuscular adipose tissue deposition in the pectoralis major muscle, and suggest that hatch time influences this regulation.

Effects of breeds and dietary protein levels on the growth performance, energy expenditure and expression of avUCP mRNA in chickens.

The physiological mechanisms of thermogenesis, energy balance and energy expenditure are poorly understood in poultry. The aim of this study was designed to investigate the physiological roles of avian uncoupling protein (avUCP) regulating in energy balance and thermogenesis by using three chicken breeds of existence striking genetic difference and feeding with different dietary protein levels. Three chicken breeds including broilers, hybrid chickens, and non-selection Wuding chickens were used in this study. Total 150 chicks of 1 day of age, with 50 from each breed were reared under standard conditions on starter diets to 30 days. At 30 days of age, forty chicks from each breed chicks were divided into two groups. One group was fed low protein diet (LP, 17.0 %), and the other group was fed high protein diet (HP, 19.5 %) for 60 days. Wuding chickens showed the lowest feed conversion efficiency (FCE) and the highest expressions of avUCP mRNA association with high plasma T3 and insulin concentrations. Hybrid chickens showed the lowest expressions of avUCP mRNA association with high FCE and energy efficiency. Expressions of avUCP mRNA association with diet-induced thermogenesis (DIT) were only observed in broiler and hybrid chickens. The expressions of avUCP mRNA were positive association with plasma insulin, T3 and NEFA concentrations. Age influence on the expression of avUCP mRNA were observed only for hybrid and broiler chickens. It seems that both roles of avUCP regulation thermogenesis and lipid utilisation as fuel were observed in the present study response to variation in dietary protein and breeds.

Evolution of arid zone birds in Australia: leapfrog distribution patterns and mesic-arid connections in quail-thrush (Cinclosoma, Cinclosomatidae).

The quail-thrush, Cinclosoma, include between five and seven species distributed broadly across arid and semi-arid inland Australia, mesic forests of south-eastern Australia and New Guinea. It has been suggested that the arid zone species of quail-thrush arose from forest ancestors as Australia changed from a warm wet climate to a cooler drier climate since the late-Miocene. We generated multilocus (mitochondrial ND2 and eight nuclear loci) gene and species trees with complete taxon sampling of Cinclosoma to investigate evolutionary relationships and species status of some taxa. Topologies reconstructed in congruent, highly-resolved gene trees and species trees that supported the recognition of seven species. Ancestral state reconstruction and divergence time estimates suggest that arid-adapted taxa radiated in parallel with a drying climate and changing habitat. A 'leapfrog' distribution in phenotypes of arid zone taxa was likely a result of ancestral retention of inconspicuous (or camouflaged) plumage patterns. A specimen-based report from 1968 of hybridization between non-sister taxa Cinclosoma castanotum and Cinclosoma marginatum was verified using molecular analysis on specimens collected at the same locality 40 years later. We discuss the implications of hybridization to the evolution of this species group.

The evolution of the Indian Ocean parrots (Psittaciformes): extinction, adaptive radiation and eustacy.

Parrots are among the most recognisable and widely distributed of all bird groups occupying major parts of the tropics. The evolution of the genera that are found in and around the Indian Ocean region is particularly interesting as they show a high degree of heterogeneity in distribution and levels of speciation. Here we present a molecular phylogenetic analysis of Indian Ocean parrots, identifying the possible geological and geographical factors that influenced their evolution. We hypothesise that the Indian Ocean islands acted as stepping stones in the radiation of the Old-World parrots, and that sea-level changes may have been an important determinant of current distributions and differences in speciation. A multi-locus phylogeny showing the evolutionary relationships among genera highlights the interesting position of the monotypic Psittrichas, which shares a common ancestor with the geographically distant Coracopsis. An extensive species-level molecular phylogeny indicates a complex pattern of radiation including evidence for colonisation of Africa, Asia and the Indian Ocean islands from Australasia via multiple routes, and of island populations 'seeding' continents. Moreover, comparison of estimated divergence dates and sea-level changes points to the latter as a factor in parrot speciation. This is the first study to include the extinct parrot taxa, Mascarinus mascarinus and Psittacula wardi which, respectively, appear closely related to Coracopsis nigra and Psittacula eupatria.

Assessment of bioactivity of a recombinant chicken interferon-gamma expressed using a baculovirus expression system.

The full-length coding sequence of chicken interferon-γ (ChIFN-γ) was cloned into a baculovirus nonfusion vector, pFastBacDual, and expressed in Sf21 insect cells. Recombinant ChIFN-γ (rChIFN-γ) protein was found to be expressed both intracellularly as well as in the culture supernatants. The affinity-purified rChIFN-γ contained 14, 17, and 28 kDa proteins, possibly representing both glycosylated and nonglycosylated protein forms of ChIFN-γ. The bioactivity of rChIFN-γ was confirmed in vitro by production of nitric oxide in a chicken macrophage cell line (HD11) and antiviral activity against vesicular stomatitis virus in primary chicken embryonic fibroblast cells. Further, HD11 cells stimulated with rChIFN-γ showed significant upregulation of inducible nitric oxide synthases, IFN-γ, interleukin-1ß, interleukin-12p35, signal transducers and activators of transcription 1, class II, major histocompatibility complex, transactivator, and major histocompatibility complex II-ß chain (BL-B) transcripts. In conclusion, the present study provides information on the ability of functionally active rChIFN-γ expressed in a baculovirus system in inducing significant transcriptional upregulation of various immune system-related genes, including those that encode cytokines, antigen-presenting molecules, and transcription factors involved in the major histocompatibility complex and IFN-signaling pathway.

Bioinformatic discovery and initial characterisation of nine novel antimicrobial peptide genes in the chicken.

Antimicrobial peptides (AMPs) are essential components of innate immunity in a range of species from Drosophila to humans and are generally thought to act by disrupting the membrane integrity of microbes. In order to discover novel AMPs in the chicken, we have implemented a bioinformatic approach that involves the clustering of more than 420,000 chicken expressed sequence tags (ESTs). Similarity searching of proteins-predicted to be encoded by these EST clusters-for homology to known AMPs has resulted in the in silico identification of full-length sequences for seven novel gallinacins (Gal-4 to Gal-10), a novel cathelicidin and a novel liver-expressed antimicrobial peptide 2 (LEAP-2) in the chicken. Differential gene expression of these novel genes has been demonstrated across a panel of chicken tissues. An evolutionary analysis of the gallinacin family has detected sites-primarily in the mature AMP-that are under positive selection in these molecules. The functional implications of these results are discussed.