How the double spherules of infectious bronchitis virus impact our understanding of RNA virus replicative organelles

Powered by advances in electron tomography, recent studies have extended our understanding of how viruses construct "replication factories" inside infected cells. Their function, however, remains an area of speculation with important implications for human health. It is clear from these studies that whatever their purpose, organelle structure is dynamic (M. Ulasli, M. H. Verheije, C. A. de Haan, and F. Reggiori, Cell. Microbiol. 12:844-861, 2010) and intricate (K. Knoops, M. Kikkert, S. H. Worm, J. C. Zevenhoven-Dobbe, Y. van der Meer, et al., PLOS Biol. 6:e226, 2008). But by concentrating on medically important viruses, these studies have failed to take advantage of the genetic variation inherent in a family of viruses that is as diverse as the archaea, bacteria, and eukaryotes combined (C. Lauber, J. J. Goeman, M. del Carmen Parquet, P. T. Nga, E. J. Snijder, et al., PLOS Pathog. 9:e1003500, 2013). In this climate, Maier et al. (H. J. Maier, P. C. Hawes, E. M. Cottam, J. Mantell, P. Verkade, et al., mBio 4:e00801-13, 2013) explored the replicative structures formed by an avian coronavirus that appears to have diverged at an early point in coronavirus evolution and shed light on controversial aspects of viral biology.

Evidence for systemic spread of the potentially zoonotic intestinal spirochaete Brachyspira pilosicoli in experimentally challenged laying chickens

Brachyspira pilosicoli is a potentially zoonotic anaerobic intestinal spirochaete that is one of several species causing avian intestinal spirochaetosis. The aim of this study was to develop a reproducible model of infection in point-of-lay chickens and compare the virulence of two strains of B. pilosicoli in a model using experimentally challenged laying chickens. Seventeen-week-old commercial laying chickens were experimentally challenged by oral gavage with either B. pilosicoli strain B2904 or CPSp1, following an oral dose of 10 % sodium bicarbonate to neutralize acidity in the crop. Approximately 80 % of the chickens became colonized and exhibited increased faecal moisture content, reduced weight gain and delayed onset of lay. Tissues sampled at post-mortem examination were analysed to produce a quantitative output on the number of spirochaetes present and hence, the extent of colonization. The liver and spleen were colonized, and novel histopathology was observed in these tissues. The infection model we report here has potential use in studies to improve our understanding of the mechanisms by which Brachyspira elicit disease in poultry and in testing novel intervention strategies.

Oral treatment of chickens with Lactobacillus reuteri LM1 reduces Brachyspira pilosicoli-induced pathology

Avian intestinal spirochaetosis (AIS) results from the colonization of the caeca and colon of poultry by pathogenic Brachyspira, notably Brachyspira pilosicoli. Following the ban on the use of antibiotic growth promoters in the European Union in 2006, the number of cases of AIS has increased, which, alongside emerging antimicrobial resistance in Brachyspira, has driven renewed interest in alternative intervention strategies. Lactobacillus-based probiotics have been shown to protect against infection with common enteric pathogens in livestock. Our previous studies have shown that Lactobacillus reuteri LM1 antagonizes aspects of the pathobiology of Brachyspira in vitro. Here, we showed that L. reuteri LM1 mitigates the clinical symptoms of AIS in chickens experimentally challenged with B. pilosicoli. Two groups of 15 commercial laying hens were challenged experimentally by oral gavage with B. pilosicoli B2904 at 18 weeks of age; one group received unsupplemented drinking water and the other received L. reuteri LM1 in drinking water from 1 week prior to challenge with Brachyspira and thereafter for the duration of the study. This treatment regime was protective. Specifically, B. pilosicoli was detected by culture in fewer birds, bird weights were higher, faecal moisture contents were significantly lower (P<0.05) and egg production as assessed by egg weight and faecal staining score was improved (P<0.05). Also, at post-mortem examination, significantly fewer B. pilosicoli were recovered from treated birds (P<0.05), with only mild–moderate histopathological changes observed. These data suggest that L. reuteri LM1 may be a useful tool in the control of AIS.

Supplementary feeding of wild birds indirectly affects ground beetle populations in suburban gardens

Supplementary feeding of wild birds by domestic garden-holders is a globally widespread and popular form of human–wildlife interaction, particularly in urban areas. Vast amounts of energy are thus being added to garden ecosystems. However, the potential indirect effects of this activity on non-avian species have been little studied to date, with the only two previous studies taking place under experimentally manipulated conditions. Here we present the first evidence of a localised depletive effect of wild bird feeding on ground beetles (Coleoptera: Carabidae) in suburban gardens under the usual feeding patterns of the garden-holders. We trapped significantly fewer ground beetles directly under bird-feeding stations than in matched areas of habitat away from feeders. Video analysis also revealed significantly higher activity by ground-foraging birds under the feeding stations than in the control areas. Small mammal trapping revealed no evidence that these species differ in abundance between gardens with and without bird feeders. We therefore suggest that local increases in ground-foraging activity by bird species whose diets encompass arthropods as well as seed material are responsible for the reduction in ground beetle numbers. Our work therefore illustrates that providing food for wild birds can have indirect negative effects on palatable prey species under typical conditions.

Efficacy of a live attenuated Escherichia coli O78:K80 vaccine in chickens and turkeys

A candidate live vaccine for avian pathogenic Escherichia coli (APEC) was constructed from a virulent field APEC O78 strain by mutation of the aroA gene. The mutant was highly similar to the parent wild-type strain in respect of colony morphology, motility, growth in suspension, hemagglutination, Congo Red binding, HEp-2 cell adhesion, and the elaboration of surface antigens type 1 fimbriae and flagella, although production of curli fimbriae was reduced marginally. The mutant proved avirulent when inoculated into 1-day-old chicks by spray application and when presented again in the drinking water at 7 days of age. Chickens and turkeys vaccinated with an O78 aroA mutant were protected against a challenge at 6 wk of age by virulent APEC strains.

Mercury and methylmercury bioaccumulation by polychaete worms is governed by both feeding ecology and mercury bioavailability in coastal mudflats

Polychaete worms are abundant in many mudflats but their importance to coastal food web Hg biomagnification is not known. We sampled sediments and polychaete worms from mudflats in the Bay of Fundy to investigate the bioaccumulation of mercury (Hg) and methylmercury (MeHg) in the coastal invertebrate food web. Hg concentrations in the sediments were low (<20 μg kg−1). Labile Hg (methanol/KOH sediment extraction) in surface sediments (0–1 cm) was positively correlated with Hg bioaccumulation by surface sediment-ingesting polychaetes but, surprisingly, there was a negative correlation between δ15N (i.e. trophic level) and THg bioaccumulation factors in polychaete worms. Worms feeding on deeper sediments contained the greatest MeHg concentrations (69.6 μg kg−1). Polychaetes are an important vector for Hg biomagnification to the coastal avian food web. This research demonstrates that feeding depth and method of feeding are more important than trophic position or sediment Hg concentrations for predicting Hg bioaccumulation.

Cadaver decomposition in terrestrial ecosystems

A dead mammal (i.e. cadaver) is a high quality resource (narrow carbon:nitrogen ratio, high water content) that releases an intense, localised pulse of carbon and nutrients into the soil upon decomposition. Despite the fact that as much as 5,000 kg of cadaver can be introduced to a square kilometre of terrestrial ecosystem each year, cadaver decomposition remains a neglected microsere. Here we review the processes associated with the introduction of cadaver-derived carbon and nutrients into soil from forensic and ecological settings to show that cadaver decomposition can have a greater, albeit localised, effect on belowground ecology than plant and faecal resources. Cadaveric materials are rapidly introduced to belowground floral and faunal communities, which results in the formation of a highly concentrated island of fertility, or cadaver decomposition island (CDI). CDIs are associated with increased soil microbial biomass, microbial activity (C mineralisation) and nematode abundance. Each CDI is an ephemeral natural disturbance that, in addition to releasing energy and nutrients to the wider ecosystem, acts as a hub by receiving these materials in the form of dead insects, exuvia and puparia, faecal matter (from scavengers, grazers and predators) and feathers (from avian scavengers and predators). As such, CDIs contribute to landscape heterogeneity. Furthermore, CDIs are a specialised habitat for a number of flies, beetles and pioneer vegetation, which enhances biodiversity in terrestrial ecosystems.

Drinking water application of Denargard Tiamulin for control of Brachyspira pilosicoli infection of laying poultry

Avian intestinal spirochaetosis (AIS) caused by Brachyspira spp., and notably Brachyspira pilosicoli, is common in layer flocks and reportedly of increasing incidence in broilers and broiler breeders. Disease manifests as diar- rhoea, increased feed consumption, reduced growth rates and occasional mortality in broilers and these signs are shown in layers also associated with a delayed onset of lay, reduced egg weights, faecal staining of eggshells and non-productive ovaries. Treatment with Denagard® Tiamulin has been used to protect against B. pilosicoli colonisation, persistence and clinical presentation of AIS in commercial layers, but to date there has been no de- finitive study validating efficacy. Here, we used a poultry model of B. pilosicoli infection of layers to compare the impact of three doses of Denagard® Tiamulin. Four groups of thirty 17 week old commercial pre-lay birds were all challenged with B. pilosicoli strain B2904 with three oral doses two days apart. All birds were colonised within 2 days after the final oral challenge and mild onset of clinical signs were observed thereafter. A fifth group that was unchallenged and untreated was also included for comparison as healthy birds. Five days after the final oral Brachypira challenge three groups were given Denagard® Tiamulin in drinking water made up following the manufacturer's recommendations with doses verified as 58.7 ppm, 113 ppm and 225 ppm. Weight gain body condition and the level of diarrhoea of birds infected with B. pilosicoli were improved and shedding of the organism reduced significantly (p = 0.001) following treatment with Denagard® Tiamulin irrespective of dose given. The level and duration of colonisation of organs of birds infected with B. pilosicoli was also reduced. Confirming previous findings we showed that the ileum, caeca, colon, and both liver and spleen were colonised and here we demonstrated that treatment with Denagard® Tiamulin resulted in significant reduction in the numbers of Brachyspira found in each of these sites and dramatic reduction in faecal shedding (p b 0.001) to ap- proaching zero as assessed by culture of cloacal swabs. Although the number of eggs produced per bird and the level of eggshell staining appeared unaffected, egg weights of treated birds were greater than those of untreated birds for a period of approximately two weeks following treatment. These data conclusively demonstrate the ef- fectiveness of Denagard® Tiamulin in reducing B. pilosicoli infection in laying hens.

Drinking water application of Denagard® Tiamulin for control of Brachyspira pilosicoli infection of laying poultry

Avian intestinal spirochaetosis (AIS) caused by Brachyspira spp., and notably Brachyspira pilosicoli, is common in layer flocks and reportedly of increasing incidence in broilers and broiler breeders. Disease manifests as diarrhoea,increased feed consumption, reduced growth rates and occasional mortality in broilers and these signs are shown in layers also associated with a delayed onset of lay, reduced egg weights, faecal staining of eggshells and non-productive ovaries. Treatment with Denagard® Tiamulin has been used to protect against B. pilosicoli colonisation, persistence and clinical presentation of AIS in commercial layers, but to date there has been no definitive study validating efficacy. Here, we used a poultry model of B. pilosicoli infection of layers to compare the impact of three doses of Denagard® Tiamulin. Four groups of thirty 17 week old commercial pre-lay birds were all challengedwith B. pilosicoli strain B2904with three oral doses two days apart. All birdswere colonised within 2 days after the final oral challenge and mild onset of clinical signs were observed thereafter. A fifth group that was unchallenged and untreated was also included for comparison as healthy birds. Five days after the final oral Brachypira challenge three groups were given Denagard® Tiamulin in drinking water made up following the manufacturer's recommendations with doses verified as 58.7 ppm, 113 ppm and 225 ppm. Weight gain body condition and the level of diarrhoea of birds infected with B. pilosicoli were improved and shedding of the organism reduced significantly (p = 0.001) following treatment with Denagard® Tiamulin irrespective of dose given. The level and duration of colonisation of organs of birds infected with B. pilosicoli was also reduced. Confirming previous findings we showed that the ileum, caeca, colon, and both liver and spleen were colonised and here we demonstrated that treatment with Denagard® Tiamulin resulted in significant reduction in the numbers of Brachyspira found in each of these sites and dramatic reduction in faecal shedding (p b 0.001) to approaching zero as assessed by culture of cloacal swabs. Although the number of eggs produced per bird and the level of eggshell staining appeared unaffected, egg weights of treated birds were greater than those of untreated birds for a period of approximately two weeks following treatment. These data conclusively demonstrate the effectiveness of Denagard® Tiamulin in reducing B. pilosicoli infection in laying hens.

Consequences of Simulated Loss of Open Cerrado Areas to Bird Functional Diversity

Over the past 35 years, more than two thirds of the Cerrado`s original expanse has been taken by agriculture. Even if some attempts have been made to conserve closed cerrado physiognomies, open cerrado physiognomies, richer in species and more fragile, have been systematically ignored. These open physiognomies are used by almost half of the Cerrado bird species, many of which being endemics. Using data from 11 surveys carried out in Cerrado landscapes, we asked what would happen to bird functional diversity if open cerrado species became extinct. Open cerrado birds would be able to keep on average 59% of the functional diversity. If they became extinct, on average 27% of the functional diversity would be lost. In this case, the remaining functional diversity would be lower than what would be expected by chance in five sites. Although many functions were shared by both open cerrado and forest species, there was some degree of complementarity between them, highlighted by the decrease in functional diversity when the former became extinct. Destruction of open cerrado physiognomies would lead to a habitat simplification, decrease in bird functional diversity, and, ultimately, to a considerable impact on community functioning. Thus, open cerrado physiognomies must receive much more conservation attention than they are currently receiving, because they maintain a high bird functional diversity that would otherwise be considerably diminished Were open cerrado species to become extinct.

Clock Genes, Melanopsins, Melatonin, and Dopamine Key Enzymes and Their Modulation by Light and Glutamate in Chicken Embryonic Retinal Cells

The avian circadian system is composed of the retina, the mammalian homolog region of the suprachiasmatic nucleus (SNC), and the pineal gland. The retina, itself, displays many rhythmic physiological events, such as movements of photoreceptor cells, opsin expression, retinal reisomerization, and melatonin and dopamine production and secretion. Altogether, these rhythmic events are coordinated to predict environmental changes in light conditions during the day, optimizing retina function. The authors investigated the expression pattern of the melanopsin genes Opn4x and Opn4m, the clock genes Clock and Per2, and the genes for the key enzymes N-Acetyltransferase and Tyrosine Hidroxylase in chicken embryo dispersed retinal cells. Primary cultures of chicken retina from 8-day-old embryos were kept in constant dark (DD), in 12-h light/12-h dark (12L:12D), in 12L:12D followed by DD, or in DD in the absence or presence of 100 mu M glutamate for 12 h. Total RNA was extracted throughout a 24-h span, every 3 h starting at zeitgeber time 0 (ZT0) of the 6th day, and submitted to reverse transcriptase-polymerase chain reaction (RT-PCR) followed by quantitative PCR (qPCR) for mRNA quantification. The data showed no rhythmic pattern of transcription for any gene in cells kept in DD. However under a light-dark cycle, Clock, Per2, Opn4m, N-Acetyltransferase, and Tyrosine Hydroxylase exhibited rhythmic patterns of transcription. In DD, 100 mu M glutamate was able to induce rhythmic expression of Clock, strongly inhibited the expression of Tyrosine Hydroxylase, and, only at some ZTs, of Opn4x and Opn4m. The neurotransmitter had no effect on Per2 and N-Acetyltransferase transcription. The authors confirmed the expression of the protein OPN4x by immunocytochemistry. These results suggest that chicken embryonic retinal cells contain a functional circadian clock, whose synchronization requires light-dark cycle or glutamate stimuli. (Author correspondence: amdlcast@ib.usp.br).

The timing of Neotropical speciation dynamics: A reconstruction of Myiopagis flycatcher diversification using phylogenetic and paleogeographic data

Neotropical forests have brought forth a large proportion of the world`s terrestrial biodiversity, but the underlying evolutionary mechanisms and their timing require further elucidation. Despite insights gained from phylogenetic studies, uncertainties about molecular clock rates have hindered efforts to determine the timing of diversification processes. Moreover, most molecular research has been detached from the extensive body of data on Neotropical geology and paleogeography. We here examine phylogenetic relationships and the timing of speciation events in a Neotropical flycatcher genus (Myiopagis) by using calibrations from modern geologic data in conjunction with a number of recently developed DNA sequence dating algorithms and by comparing these estimates with those based on a range of previously proposed molecular clock rates. We present a well-supported hypothesis of systematic relationships within the genus. Our age estimates of Myiopagis speciation events based on paleogeographic data are in close agreement with nodal ages derived from a ""traditional"" avian mitochondrial 2%/My clock, while contradicting other clock rates. Our comparative approach corroborates the consistency of the traditional avian mitochondrial clock rate of 2%/My for tyrant-flycatchers. Nevertheless, our results argue against the indiscriminate use of molecular clock rates in evolutionary research and advocate the verification of the appropriateness of the traditional clock rate by means of independent calibrations in individual studies. (C) 2009 Elsevier Inc. All rights reserved.

Development of new nuclear markers and characterization of single nucleotide polymorphisms in kelp gull (Larus dominicanus)

The present study seeks to develop nuclear markers for the kelp gull (Larus dominicanus). We hereby report the characterization of 12 independent nuclear introns, where 104 single nucleotide polymorphisms (SNPs) in 8138 sequenced base pairs were observed. These SNP markers are the first to be designed for genotyping a gull species. The markers will provide useful tools for understanding which processes act or acted upon kelp gulls to cause their low genetic variability in mitochondrial DNA. In addition, these markers open a new opportunity for population genetic and evolutionary studies in the Laridae group.

Evolutionary history of Ramphastos toucans: Molecular phylogenetics, temporal diversification, and biogeography

The toucan genus Ramphastos (Piciformes: Ramphastidae) has been a model in the formulation of Neotropical paleobiogeographic hypotheses. Weckstein (2005) reported on the phylogenetic history of this genus based on three mitochondrial genes, but some relationships were weakly supported and one of the subspecies of R. vitellinus (citreolaemus) was unsampled. This study expands on Weckstein (2005) by adding more DNA sequence data (including a nuclear marker) and more samples, including R v. citreolaemus. Maximum parsimony, maximum likelihood, and Bayesian methods recovered similar trees, with nodes showing high support. A monophyletic R. vitellinus complex was strongly supported as the sister-group to R. brevis. The results also confirmed that the southeastern and northern populations of R. vitellinus ariel are paraphyletic. X v. citreolaemus is sister to the Amazonian subspecies of the vitellinus complex. Using three protein-coding genes (COI, cytochrome-b and ND2) and interval-calibrated nodes under a Bayesian relaxed-clock framework, we infer that ramphastid genera originated in the middle Miocene to early Pliocene, Ramphastos species originated between late Miocene and early Pleistocene, and intra-specific divergences took place throughout the Pleistocene. Parsimony-based reconstruction of ancestral areas indicated that evolution of the four trans-Andean Ramphastos taxa (R. v. citreolaemus, R. a. swainsonii, R. brevis and R. sulfuratus) was associated with four independent dispersals from the cis-Andean region. The last pulse of Andean uplift may have been important for the evolution of R. sulfuratus, whereas the origin of the other trans-Andean Ramphastos taxa is consistent with vicariance due to drying events in the lowland forests north of the Andes. Estimated rates of molecular evolution were higher than the ""standard"" bird rate of 2% substitutions/site/million years for two of the three genes analyzed (cytochrome-b and ND2). (C) 2009 Elsevier Inc. All rights reserved.

Morphometric analysis of the AMPA-type neurons in the Deiters vestibular complex of the chick brain

Chicken (Gallus gallus) brains were used to investigate the typology and the immunolabel pattern for the subunits composing the AMPA-type glutamate receptors (GluR) of hindbrain neurons of the dorsal (dND) and ventral nuclei (vND) of the Deiter`s vestibular complex (CD), which is the avian correspondent of the lateral vestibular nucleus (LVN) of mammals. Our results revealed that neurons of both divisions were poor in GluR1. The vND, the GluR2/3+ and GluR4+ label presented no area or neuronal size preference, although most neurons were around 75%. The dND neurons expressing GluR2/3 are primarily around 85%, medium to large-sized 85%, and predominantly 60% located in the medial portion of the rostral pole and in the lateral portion of the caudal pole. The majority of dND neurons containing GluR4 are also around 75%, larger (70% are large and giant), exhibiting a distribution that seems to be complementary to that of GluR2/3+ neurons. This distinct arrangement indicates functional differences into and between the DC nuclei, also signaling that such variation could be attributed to the diverse nature of the subunit composition of the GluRs. Discussion addresses the morphological and functional correlation of the avian DC with the LVN of mammals in addition to the high morphological correspondence, To include these data into the modern comparative approach we propose to adopt a similar nomenclature for the avian divisions dND and vND that could be referred as dLVN and vLVN. (C) 2008 Elsevier B.V. All rights reserved.