Evidence for glutamatergic ganglion cells in the avian retina

This dissertation presents structural, immunochemical and neurochemical evidence for glutamatergic retinotectal synaptic transmission, augmenting and extending previous physiological and anatomical studies. The evidence is especially striking when the laminar patterns of ($\sp3$H) L-glutamate receptor binding, ($\sp3$H) L-glutamate high affinity uptake (HAU) and glutamate immunoreactivity (GLIR) of the dorsal tectum are compared. All show high activity in the tectal SGFS, with a peak in the most superficial laminae of SGFS followed by dip in the b-c region, and a second broad peak in deeper SGFS. Uptake and immunoreactivity bear a stronger resemblance to one another than either does to receptor binding, consistent with the fact that HAU and GLIR are localized in the same structures: glutamatergic terminals, intrinsic cell bodies and their processes. Receptor binding, as attested by the lack of enucleation effects, is a marker of postsynaptic receptors. In summary, these results are consistent with the hypothesis that most of the retinal projection to the optic tectum is glutamatergic: (1) A glutamate/aspartate HAU system exists in the superficial laminae, and it is dependent upon an intact retinal input, as shown developmentally and by retinal ablation; (2) Glutamate-like immunoreactivity appears in retinorecipient tectal regions (partially responsive to enucleation), in cell bodies of retinal ganglion cells and displaced ganglion cells, and in a non-tectal ganglion cell projection, the ectomammilary nucleus; (3) Sodium-independent glutamate receptor binding (which remains unchanged by enucleation) is most intense in the retinorecipient regions of the tectum and the ectomammilary nucleus. This binding is pharmacologically typical of a CNS sensory structure, being dominated by the quisqualate/kainate receptor subclass. Thus, as with other sensory systems, a portion of the retinotectal projection has been shown to include glutamatergic afferents with the distribution and properties expected of the primary projection ^

Avian area vasculosa and CAM as rapid in vivo pro-angiogenic and antiangiogenic models.

Angiogenesis, the development of new blood vessels from preexisting ones, is driven by coordinated signaling pathways governed by specific molecules, hemodynamic forces, and endothelial and periendothelial cells. The processes involve adhesion, migration, and survival machinery within the target endothelial and periendothelial cells. Factors that interfere with any of these processes may therefore influence angiogenesis either positively (pro-angiogenesis) or negatively (antiangiogenesis). The avian area vasculosa (AV) and the avian chorioallantoic membrane (CAM) are two useful tools for studying both angiogenesis and antiangiogenesis since they are amenable to both intravascular and topical administration of target, agents, are relatively rapid assays, and can be adapted very easily to study angiogenesis-dependent processes, such as tumor growth. Both models provide a physiological setting that permits investigation of pro-angiogenic and antiangiogenic agent interactions in vivo.

Competitive fitness in coronaviruses is not correlated with size or number of double-membrane vesicles under reduced-temperature growth conditions.

Positive-stranded viruses synthesize their RNA in membrane-bound organelles, but it is not clear how this benefits the virus or the host. For coronaviruses, these organelles take the form of double-membrane vesicles (DMVs) interconnected by a convoluted membrane network. We used electron microscopy to identify murine coronaviruses with mutations in nsp3 and nsp14 that replicated normally while producing only half the normal amount of DMVs under low-temperature growth conditions. Viruses with mutations in nsp5 and nsp16 produced small DMVs but also replicated normally. Quantitative reverse transcriptase PCR (RT-PCR) confirmed that the most strongly affected of these, the nsp3 mutant, produced more viral RNA than wild-type virus. Competitive growth assays were carried out in both continuous and primary cells to better understand the contribution of DMVs to viral fitness. Surprisingly, several viruses that produced fewer or smaller DMVs showed a higher fitness than wild-type virus at the reduced temperature, suggesting that larger and more numerous DMVs do not necessarily confer a competitive advantage in primary or continuous cell culture. For the first time, this directly demonstrates that replication and organelle formation may be, at least in part, studied separately during infection with positive-stranded RNA virus. IMPORTANCE The viruses that cause severe acute respiratory syndrome (SARS), poliomyelitis, and hepatitis C all replicate in double-membrane vesicles (DMVs). The big question about DMVs is why they exist in the first place. In this study, we looked at thousands of infected cells and identified two coronavirus mutants that made half as many organelles as normal and two others that made typical numbers but smaller organelles. Despite differences in DMV size and number, all four mutants replicated as efficiently as wild-type virus. To better understand the relative importance of replicative organelles, we carried out competitive fitness experiments. None of these viruses was found to be significantly less fit than wild-type, and two were actually fitter in tests in two kinds of cells. This suggests that viruses have evolved to have tremendous plasticity in the ability to form membrane-associated replication complexes and that large and numerous DMVs are not exclusively associated with efficient coronavirus replication.

Targeting membrane-bound viral RNA synthesis reveals potent inhibition of diverse coronaviruses including the middle East respiratory syndrome virus.

Coronaviruses raise serious concerns as emerging zoonotic viruses without specific antiviral drugs available. Here we screened a collection of 16671 diverse compounds for anti-human coronavirus 229E activity and identified an inhibitor, designated K22, that specifically targets membrane-bound coronaviral RNA synthesis. K22 exerts most potent antiviral activity after virus entry during an early step of the viral life cycle. Specifically, the formation of double membrane vesicles (DMVs), a hallmark of coronavirus replication, was greatly impaired upon K22 treatment accompanied by near-complete inhibition of viral RNA synthesis. K22-resistant viruses contained substitutions in non-structural protein 6 (nsp6), a membrane-spanning integral component of the viral replication complex implicated in DMV formation, corroborating that K22 targets membrane bound viral RNA synthesis. Besides K22 resistance, the nsp6 mutants induced a reduced number of DMVs, displayed decreased specific infectivity, while RNA synthesis was not affected. Importantly, K22 inhibits a broad range of coronaviruses, including Middle East respiratory syndrome coronavirus (MERS-CoV), and efficient inhibition was achieved in primary human epithelia cultures representing the entry port of human coronavirus infection. Collectively, this study proposes an evolutionary conserved step in the life cycle of positive-stranded RNA viruses, the recruitment of cellular membranes for viral replication, as vulnerable and, most importantly, druggable target for antiviral intervention. We expect this mode of action to serve as a paradigm for the development of potent antiviral drugs to combat many animal and human virus infections.

Pseudotyping of vesicular stomatitis virus with the envelope glycoproteins of highly pathogenic avian influenza viruses

Pseudotype viruses are useful for studying the envelope proteins of harmful viruses. This work describes the pseudotyping of vesicular stomatitis virus (VSV) with the envelope glycoproteins of highly pathogenic avian influenza viruses. VSV lacking the homotypic glycoprotein (G) gene (VSVΔG) was used to express haemagglutinin (HA), neuraminidase (NA) or the combination of both. Propagation-competent pseudotype viruses were only obtained when HA and NA were expressed from the same vector genome. Pseudotype viruses containing HA from different H5 clades were neutralized specifically by immune sera directed against the corresponding clade. Fast and sensitive reading of test results was achieved by vector-mediated expression of GFP. Pseudotype viruses expressing a mutant VSV matrix protein showed restricted spread in IFN-competent cells. This pseudotype system will facilitate the detection of neutralizing antibodies against virulent influenza viruses, circumventing the need for high-level biosafety containment.

Climate-mediated movement of an avian hybrid zone

The interaction between sibling species that share a zone of contact is a multifaceted relationship affected by climate change [ 1, 2 ]. Between sibling species, interactions may occur at whole-organism (direct or indirect competition) or genomic (hybridization and introgression) levels [ 3–5 ]. Tracking hybrid zone movements can provide insights about influences of environmental change on species interactions [ 1 ]. Here, we explore the extent and mechanism of movement of the contact zone between black-capped chickadees (Poecile atricapillus) and Carolina chickadees (Poecile carolinensis) at whole-organism and genomic levels. We find strong evidence that winter temperatures limit the northern extent of P. carolinensis by demonstrating a current-day association between the range limit of this species and minimum winter temperatures. We further show that this temperature limitation has been consistent over time because we are able to accurately hindcast the previous northern range limit under earlier climate conditions. Using genomic data, we confirm northward movement of this contact zone over the past decade and highlight temporally consistent differential—but limited—geographic introgression of alleles. Our results provide an informative example of the influence of climate change on a contact zone between sibling species.

The Effect of Heavy Metal Stress on Avian Proximal Tubule Urate Secretion

In both humans and birds, urate is an important antioxidant when maintained at normal plasma concentrations. Though human kidneys primarily reabsorb filtered urate, while those of birds perform mostly secretion, both maintain urate levels at ~300microM. The importance of maintaining urate levels within the homeostatic range was observed when the study of several prominent diseases revealed an association with hyperuricemia. This study examined the effect of elevated zinc concentration on avian urate secretion. Here, acute exposure of chicken proximal tubule epithelial cells (cPTCs) to zinc stress had no effect on urate secretion, but prolonged zinc-induced cellular stress inhibited active transepithelial urate secretion with no change in Mrp4 expression, glucose transport, or transepithelial resistance. Moreover, zinc had no effect on urate transport by isolated brush border membrane vesicles, suggesting involvement of a more complex cellular stress adaptation. Previous work has demonstrated that AMP-activated protein kinase (AMPK), a critical metabolic regulator, conserves energy during cellular stress by shutting down ATP-utilizing processes and activating ATP-generating processes. Pharmacological activation of AMPK by AICAR produced decreased urate secretion by cPTCs similar to the effect seen with prolonged exposure to zinc, while the AMPK inhibitor Compound C prevented both AICAR and zinc inhibition of urate secretion, suggesting a stress induced mechanism of regulation. Supported by NSF. IACUC #A08-046.

AVIAN ROTAVIRUSES

The prevalence of antirotavirus antibodies in chickens and turkeys in the Gonzales, Texas and Llano, Texas areas was studied. Caged layer chicken flocks were found to have a prevalence of 64% when samples were taken randomly. This compares to 45% in chicken broiler breeder flocks and 92% in turkey breeding flocks. The natural occurrence of turkey rotavirus infection in two separate field studies showed an increase in mortality varying from 9% to 45% above expected death losses. Clinically, pasted vents, lacitude, and general malaise were noted in affected poults. Lesions noted on post mortem examination were; slight ballooning of the small intestine, excessively large ceca, and mild hyperemia of the small and large intestines.^ The use of maternal antibody from simian rotavirus immunized chickens' eggs for preventing murine rotavirus infection in infant mice was investigated. There was a reduction from 91% to 15% incidence when infant mice were treated twice daily with egg yolk immunoglobulin.^ The need for a convenient, easily grown and rapidly reproducing model for avian and mammalian rotaviruses led to the use of coturnix chicks. The turkey rotavirus was adapted to the quail chicks be serial passage. Transmission and scanning electron microscopy as well as micropathological methods were used in the study of the pathogenesis of rotavirus infection in quail and infant mice. ^

The development of poultry farms risk assessment tool for avian influenza in Imo State, Nigeria

Background: Nigeria was one of the 13 countries where avian influenza outbreak in poultry farms was reported during the 2006 avian influenza pandemic threat and was also the first country in Africa to report the presence of H5N1influenza among its poultry population. There are multiple hypotheses on how the avian influenza outbreak of 2006 was introduced to Nigeria, but the consensus is that once introduced, poultry farms and their workers were responsible for 70% of the spread of avian influenza virus to other poultry farms and the population. ^ The spread of avian influenza has been attributed to lack of compliance by poultry farms and their workers with poultry farm biosecurity measures. When poultry farms fail to adhere to biosecurity measures and there is an outbreak of infectious diseases like in 2006, epidemiological investigations usually assess poultry farm biosecurity—often with the aid of a questionnaire. Despite the importance of questionnaires in determining farm compliance with biosecurity measures, there have been few efforts to determine the validity of questionnaires designed to assess poultry farms risk factors. Hence, this study developed and validated a tool (questionnaire) that can be used for poultry farm risk stratification in Imo State, Nigeria. ^ Methods: Risk domains were generated using literature and recommendations from agricultural organizations and the Nigeria government for poultry farms. The risk domains were then used to develop a questionnaire. Both the risk domain and questionnaire were verified and modified by a group of five experts with a research interest in Nigeria's poultry industry and/or avian influenza prevention. Once a consensus was reached by the experts, the questionnaire was distributed to 30 selected poultry farms in Imo State, Nigeria that participated in this study. Survey responses were received for all the 30 poultry farms that were selected. The same poultry farms were visited one week after they completed the questionnaires for on-site observation. Agreement among survey and observation results were analyzed using a kappa test and rated as poor, fair, moderate, substantial, or nearly perfect; and internal consistency of the survey was also computed. ^ Result: Out of the 43 items on the questionnaire, 32 items were validated by this study. The agreement between the survey result and onsite observation was analyzed using kappa test and ranged from poor to nearly perfect. Most poultry farms had their best agreements in the contact section of the survey. The least agreement was noted in the farm management section of the survey. Thirty-two questions on the survey had a coefficient alpha > 0.70, which is a robust internal consistency for the survey. ^ Conclusion: This study developed 14 risk domains for poultry farms in Nigeria and validated 32 items from the original questionnaire that contained 43 items. The validated items can be used to determine the risk of introduction and spread of avian influenza virus in poultry farms in Imo State, Nigeria. After further validations in other states, regions and poultry farm sectors in Nigeria; this risk assessment tool can then be used to determine the risk profile of poultry farms across Nigeria.^

Avian species identity and predation success in tropical cacao agroforestry of the Napu Valley in Central Sulawesi, Indonesia

Avian ecosystem services such as the suppression of pests are considered being of high ecological and economic importance in a range of ecosystems, especially in tropical agroforestry. But how bird predation success is related to the diversity and composition of the bird community, as well as local and landscape factors, is poorly understood. The author quantified arthropod predation in relation to the identity and diversity of insectivorous birds, using experimental exposure of artificial, caterpillar-like prey on smallholder cacao agroforestry systems, differing in local shade management and distance to primary forest. The bird community was assessed using both mist netting (targeting on active understory insectivores) and point count (higher completeness of species inventories) sampling. The study was conducted in a land use dominated area in Central Sulawesi, Indonesia, adjacent to the Lore Lindu National Park. We selected 15 smallholder cacao plantations as sites for bird and bat exclosure experiments in March 2010. Until July 2011, we recorded several data in this study area, including the bird community data, cacao tree data and bird predation experiments that are presented here. We found that avian predation success can be driven by single and abundant insectivorous species, rather than by overall bird species richness. Forest proximity was important for enhancing the density of this key species, but did also promote bird species richness. The availability of local shade trees had no effects on the local bird community or avian predation success. Our findings are both of economical as well as ecological interest because the conservation of nearby forest remnants will likely benefit human needs and biodiversity conservation alike.

Avian occupancy probabilities, vegetation structure, and presence of Northern Bobwhite (Colinus virginianus) in eastern and central Oklahoma (2009-2011)

Changes in land use and land cover throughout the eastern half of North America have caused substantial declines in populations of birds that rely on grassland and shrubland vegetation types, including socially and economically important game birds such as the Northern Bobwhite (Colinus virginianus; hereafter bobwhites). As much attention is focused on habitat management and restoration for bobwhites, they may act as an umbrella species for other bird species with similar habitat requirements. We quantified the relationship of bobwhites to the overall bird community and evaluated the potential for bobwhites to act as an umbrella species for grassland and shrubland birds. We monitored bobwhite presence and bird community composition within 31 sample units on selected private lands in the south-central United States from 2009 to 2011. Bobwhites were strongly associated with other grassland and shrubland birds and were a significant positive predictor for 9 species. Seven of these, including Bell's Vireo (Vireo bellii), Dicksissel (Spiza americana), and Grasshopper Sparrow (Ammodramus savannarum), are listed as species of conservation concern. Species richness and occupancy probability of grassland and shrubland birds were higher relative to the overall bird community in sample units occupied by bobwhites. Our results show that bobwhites can act as an umbrella species for grassland and shrubland birds, although the specific species in any given situation will depend on region and management objectives. These results suggest that efficiency in conservation funding can be increased by using public interest in popular game species to leverage resources to meet multiple conservation objectives.

Nueua relacion, en que se declara la feliz victoria, que han conseguido dos navios malteses de siete javeques de moros, que avian saqueado la villa de Azuaga llevandose muchos cautivos y vasos sagrados : de los que quemaron quatro, llevando presos tres ... : sucedio el dia 12 de agosto del presente año 1779 : [primera y segunda parte]

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