Perfluoroalkyl compounds (PFCs) are a group of chemical surfactants most notably used in non-stick and stain-resistance applications. Due to their wide-spread use and inherent resistance to degradation, several PFCs have become persistent environmental contaminants. Despite the high concentrations of PFCs reported in wild birds and their eggs, very little is known about the toxicological effects they have on avian species. This thesis investigates the developmental toxicity of PFCs in an avian model species: the domestic chicken (Gallus gallus domesticus). Egg injection experiments were performed to assess the in ovo toxicity of perfluorooctane sulfonate (technical grade, T-PFOS), perfluorooctanoic acid (PFOA), perfluorodecane sulfonate (PFDS) and perfluoroundecanoic acid (PFUdA). Real-time RT-PCR was then used to measure the transcription of candidate biomarker genes in the liver tissue of day 20 embryos. Candidate genes were selected based on their responsiveness to PFC exposure in previously conducted in vitro screening assays. In ovo exposure to PFOS resulted in a dose-dependent decrease in embryo pipping success (a measure of hatching success) with an LD50 of 93 μg/g (3.54 μg/g-672,910 μg/g, 95% confidence interval), however the expression of peroxisome proliferator-activated receptor alpha (PPARα)-regulated genes was not affected in liver tissue as hypothesized. PFOA, PFDS and PFUdA had no effect on the pipping success of chicken embryos. The expression of cytochrome P450 1A4 (CYP1A4) and liver fatty acid binding protein (L-FABP) mRNA increased in embryo liver tissue following in ovo exposure to PFUdA but was only statistically significant at 10 μg/g, which is several orders of magnitude higher than concentrations reported in wild bird eggs. The isomer-specific accumulation of PFOS in chicken embryo livers was also investigated using an in-port derivatization gas-chromatography/mass spectrometry (GC-MS) method. Prior to incubation, chicken eggs were injected with T-PFOS, composed of 63% linear isomer (L-PFOS) and 37.3% branched isomers. The isomer profiles in day-20 embryo liver tissue showed up to 20% enrichment in the proportion of L-PFOS, compared to T-PFOS, with a corresponding decrease in the proportion of branched isomers. This enrichment was inversely proportional to dose. Finally, the transcriptional profiles of cultured chicken embryonic hepatocytes (CEH) exposed to either T-PFOS or L-PFOS were compared using Agilent 4x44k Chicken (V2) Gene Expression microarrays. At equal concentrations (10 μM), T-PFOS altered the expression of significantly more genes (340 genes, >1.5 fold change, false discovery rate adjusted p<0.05) compared to L-PFOS (130 genes). Functional analysis showed that L-PFOS and T-PFOS affected genes involved in lipid metabolism, cellular growth and proliferation, and cell-cell signaling. Pathway and interactome analysis suggested that gene expression may be affected through RXR, oxidative stress response, TP53 signaling, MYC signaling, Wnt/β-catenin signaling and PPARγ and SREBP receptors. In all functional categories and pathways examined, T-PFOS had a more pronounced disruptive effect on transctional regulation than L-PFOS. In summary, egg injection experiments showed that T-PFOS (but not linear PFOA, PFDS or PFUdA) may affect the hatching success of the chicken at environmentally relevant concentrations. It was also demonstrated that the accumulation of PFOS in embryonic liver is isomer specific, and leads to an enrichment of L-PFOS. The increased transcriptional disruption caused by T-PFOS in cultured hepatocytes over L-PFOS suggests that the branched isomers may be largely responsible for the toxicological effects of PFOS. Combined, the results from this thesis demonstrate the importance of considering PFOS isomer burdens during risk assessment. In addition, gene expression analysis identified several candidate mechanisms for PFOS toxicity.

This study investigated factors associated with two mortality events: avian botulism in waterfowl and mortality associated with hatching asynchrony in prefledgling Franklins gulls (Larus pipixcan). The initial focus of my research was on the spatiotemporal relationship between mortality of Franklins gulls and the onset of botulism outbreaks in waterfowl, and the suitability of gull carcasses for proliferation and toxigenesis of Clostridium botulinum. From 1999 to 2001, dead hatch-year Franklins gulls were by far the most abundant carcasses, and the only source of toxin-laden maggots found on transects prior to the occurrence of avian botulism in waterfowl. Nest density was a significant predictor of hatch-year gull carcass density. High density of toxic material from gull carcasses prior to the onset of botulism in waterfowl coincided with high densities of susceptible birds; hence, mortality of Franklins gulls has the potential to be a major initiating factor for botulism outbreaks at Eyebrow Lake, Saskatchewan. The causes of gull mortality were conditions or diseases associated with starvation, stress, or immunosuppression, and most mortality occurred in third-hatched chicks. To separate effects of laying order from effects of hatching asynchrony on prefledgling survival, a cross-fostering experiment was conducted to create clutches containing asynchronously hatching eggs of the same laying order, and of similar egg mass, egg volume, and female quality. Hatching order, independent of laying order, significantly affected survival to fledging, whereas laying order had no observable effect, indicating that intraclutch variation in egg quality does not predetermine the fate of prefledglings, and may be less important than hatching asynchrony for survival of prefledgling Franklins gulls. Relationships among hatching asynchrony, laying order, mass, corticosterone, immune function, growth, and survival at two stages of development were complex. Hatching asynchrony significantly affected early and late prefledgling survival, and was directly or indirectly associated with mass, corticosterone level, and cell-mediated immune responses at early and later stages of development. Both hatching asynchrony and mass appeared to play key roles in mediating life history trade-offs among cell-mediated immune function, growth, and survival. In contrast to cell-mediated immune responses, primary humoral immune response was not directly affected by hatching order or mass, nor was it associated with survival to fledging. Rather, it was associated with laying order, neonatal testosterone, corticosterone at 2 weeks, growth of leg length, and clutch initiation date, illustrating the importance of examining more than one branch of the immune system in studies of life history trade-offs. This study is a step toward using a multipronged and multidisciplinary approach to demonstrate interactions and trade-offs among life history traits, the physiological mechanisms that produce these relationships, and how these relationships may change depending on stage of development.

Perfluoroalkyl compounds (PFCs) are a group of chemical surfactants most notably used in non-stick and stain-resistance applications. Due to their wide-spread use and inherent resistance to degradation, several PFCs have become persistent environmental contaminants. Despite the high concentrations of PFCs reported in wild birds and their eggs, very little is known about the toxicological effects they have on avian species. This thesis investigates the developmental toxicity of PFCs in an avian model species: the domestic chicken (Gallus gallus domesticus). Egg injection experiments were performed to assess the in ovo toxicity of perfluorooctane sulfonate (technical grade, T-PFOS), perfluorooctanoic acid (PFOA), perfluorodecane sulfonate (PFDS) and perfluoroundecanoic acid (PFUdA). Real-time RT-PCR was then used to measure the transcription of candidate biomarker genes in the liver tissue of day 20 embryos. Candidate genes were selected based on their responsiveness to PFC exposure in previously conducted in vitro screening assays. In ovo exposure to PFOS resulted in a dose-dependent decrease in embryo pipping success (a measure of hatching success) with an LD50 of 93 μg/g (3.54 μg/g-672,910 μg/g, 95% confidence interval), however the expression of peroxisome proliferator-activated receptor alpha (PPARα)-regulated genes was not affected in liver tissue as hypothesized. PFOA, PFDS and PFUdA had no effect on the pipping success of chicken embryos. The expression of cytochrome P450 1A4 (CYP1A4) and liver fatty acid binding protein (L-FABP) mRNA increased in embryo liver tissue following in ovo exposure to PFUdA but was only statistically significant at 10 μg/g, which is several orders of magnitude higher than concentrations reported in wild bird eggs. The isomer-specific accumulation of PFOS in chicken embryo livers was also investigated using an in-port derivatization gas-chromatography/mass spectrometry (GC-MS) method. Prior to incubation, chicken eggs were injected with T-PFOS, composed of 63% linear isomer (L-PFOS) and 37.3% branched isomers. The isomer profiles in day-20 embryo liver tissue showed up to 20% enrichment in the proportion of L-PFOS, compared to T-PFOS, with a corresponding decrease in the proportion of branched isomers. This enrichment was inversely proportional to dose. Finally, the transcriptional profiles of cultured chicken embryonic hepatocytes (CEH) exposed to either T-PFOS or L-PFOS were compared using Agilent 4x44k Chicken (V2) Gene Expression microarrays. At equal concentrations (10 μM), T-PFOS altered the expression of significantly more genes (340 genes, >1.5 fold change, false discovery rate adjusted p<0.05) compared to L-PFOS (130 genes). Functional analysis showed that L-PFOS and T-PFOS affected genes involved in lipid metabolism, cellular growth and proliferation, and cell-cell signaling. Pathway and interactome analysis suggested that gene expression may be affected through RXR, oxidative stress response, TP53 signaling, MYC signaling, Wnt/β-catenin signaling and PPARγ and SREBP receptors. In all functional categories and pathways examined, T-PFOS had a more pronounced disruptive effect on transctional regulation than L-PFOS. In summary, egg injection experiments showed that T-PFOS (but not linear PFOA, PFDS or PFUdA) may affect the hatching success of the chicken at environmentally relevant concentrations. It was also demonstrated that the accumulation of PFOS in embryonic liver is isomer specific, and leads to an enrichment of L-PFOS. The increased transcriptional disruption caused by T-PFOS in cultured hepatocytes over L-PFOS suggests that the branched isomers may be largely responsible for the toxicological effects of PFOS. Combined, the results from this thesis demonstrate the importance of considering PFOS isomer burdens during risk assessment. In addition, gene expression analysis identified several candidate mechanisms for PFOS toxicity.

This study investigated factors associated with two mortality events: avian botulism in waterfowl and mortality associated with hatching asynchrony in prefledgling Franklins gulls (Larus pipixcan). The initial focus of my research was on the spatiotemporal relationship between mortality of Franklins gulls and the onset of botulism outbreaks in waterfowl, and the suitability of gull carcasses for proliferation and toxigenesis of Clostridium botulinum. From 1999 to 2001, dead hatch-year Franklins gulls were by far the most abundant carcasses, and the only source of toxin-laden maggots found on transects prior to the occurrence of avian botulism in waterfowl. Nest density was a significant predictor of hatch-year gull carcass density. High density of toxic material from gull carcasses prior to the onset of botulism in waterfowl coincided with high densities of susceptible birds; hence, mortality of Franklins gulls has the potential to be a major initiating factor for botulism outbreaks at Eyebrow Lake, Saskatchewan. The causes of gull mortality were conditions or diseases associated with starvation, stress, or immunosuppression, and most mortality occurred in third-hatched chicks. To separate effects of laying order from effects of hatching asynchrony on prefledgling survival, a cross-fostering experiment was conducted to create clutches containing asynchronously hatching eggs of the same laying order, and of similar egg mass, egg volume, and female quality. Hatching order, independent of laying order, significantly affected survival to fledging, whereas laying order had no observable effect, indicating that intraclutch variation in egg quality does not predetermine the fate of prefledglings, and may be less important than hatching asynchrony for survival of prefledgling Franklins gulls. Relationships among hatching asynchrony, laying order, mass, corticosterone, immune function, growth, and survival at two stages of development were complex. Hatching asynchrony significantly affected early and late prefledgling survival, and was directly or indirectly associated with mass, corticosterone level, and cell-mediated immune responses at early and later stages of development. Both hatching asynchrony and mass appeared to play key roles in mediating life history trade-offs among cell-mediated immune function, growth, and survival. In contrast to cell-mediated immune responses, primary humoral immune response was not directly affected by hatching order or mass, nor was it associated with survival to fledging. Rather, it was associated with laying order, neonatal testosterone, corticosterone at 2 weeks, growth of leg length, and clutch initiation date, illustrating the importance of examining more than one branch of the immune system in studies of life history trade-offs. This study is a step toward using a multipronged and multidisciplinary approach to demonstrate interactions and trade-offs among life history traits, the physiological mechanisms that produce these relationships, and how these relationships may change depending on stage of development.

The study objectives of this thesis were to evaluate the effects of delayed feeding and specific aspects of the Programmed Nutrition (PN) feeding strategy (Alltech, Inc.) on growth performance, carcass characteristics, and skeletal characteristics of commercial broiler chicks through market age, as well as investigate the effects of breed and the PN feeding strategy on early growth and development. When commercial broiler chicks were fed reduced nutrient diets, delayed feeding decreased early growth performance and carcass yield (P<0.05), whereas post-hatch PN conditioning for 72 hours improved early growth performance and alleviated the negative effects of delayed feeding on carcass yield (P<0.05). Through market age, delayed feeding improved Gain: Feed (P<0.05), while PN had the opposite effect. Interactive effects and main effects of delayed feeding and PN were observed for tissue mineral concentration (P<0.05). PN lowered bone ash % (P<0.05) and increased meat oxidation of broiler chicks during storage (P<0.05). PN also had negative effects on early growth performance and bone breaking strength (P<0.05) of various meat-type breeds, but especially for non-commercial, moderate-growing or fast-growing breeds. In conclusion, PN may be suitable for commercial broiler chicks that experience delayed feeding and are fed reduced nutrient diets.

L’évaluation de la douleur chez les oiseaux est difficile, puisque la plupart se comportent comme des proies et ont tendance à masquer tout signe extérieur de douleur. Les doses et les drogues utilisées pour traiter la douleur des oiseaux sont la plupart du temps basées sur une extrapolation d’autres espèces, ainsi que sur l’expérience clinique. Peu d’études de pharmacocinétique, d’efficacité et de toxicité sont disponibles dans la littérature. La plupart des études rapportées utilisent des stimuli nociceptifs éloignés des douleurs cliniques, comme les stimuli électriques ou thermiques, qui sont difficilement extrapolables à des situations rencontrées en pratique. L’objectif de notre projet était d’évaluer les effets analgésiques de deux doses de meloxicam chez le pigeon à l’aide d‘un modèle de fracture du fémur. La douleur post-opératoire a été évaluée pendant les quatre premiers jours suivant la chirurgie par trois méthodes : le suivi du poids porté sur la patte opérée comparativement à l’autre patte, quatre différentes échelles descriptives de douleur et la réalisation d’éthogrammes à l’aide d’enregistrements vidéo. L’administration de 0,5 mg/kg PO q12h de meloxicam n’a pas permis de réduire significativement les indicateurs de douleur mesurés comparativement à un groupe témoin recevant de la saline. Les pigeons ayant reçu 2 mg/kg PO q12h de meloxicam ont montré une réduction significative des indicateurs de douleur mesurés par les différentes méthodes. Nos résultats suggèrent que l’administration de 2 mg/kg PO q12h aux pigeons suite à une chirurgie orthopédique procure une analgésie supérieure aux doses actuellement recommandées dans la littérature. / Pain assessment is especially difficult in avian species, since many birds behave as prey and do not show any external signs of distress. Choice of drugs and dosages used in clinical practice are most of the time based on extrapolation from other species and clinical experience. Few pharmacokinetic, efficacy or toxicity research studies are available in the literature. Most of these studies used noxious stimuli, such as electric or thermal stimuli, and results are therefore difficult to extrapolate to clinical pain. The objective of our project was to study the analgesic efficacy of two dosages of meloxicam in pigeons, using a femoral fracture pain model. Postoperative pain was assessed during the first four postoperative days by three different methods: weight bearing load on the fractured limb versus the controlateral limb, four different descriptive pain scales and ethogram realization based on video recordings. Administration of 0.5 mg/kg PO q12h of meloxicam did not show any reduction in the measured level of pain compared to the control group that received saline. However, pigeons that received 2 mg/kg PO q12h showed a significant decrease in their pain levels, with the three different pain assessment methods. Our results suggest that 2 mg/kg can provide a superior level of analgesia compared to the dosages recommended in the current literature, for pigeons that have undergone an orthopedic surgery.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Rokshana Parvin Molecular epidemiology and biological properties of avian influenza viruses of subtype H5N1 and H9N2 Institute of Virology Submitted in November 2014 Pages 106, Figures 7, Table 1, References 339, Publications 4 Keywords: Avian Influenza Virus, H5N1, H9N2, Reassortment, Mutation, Replication and Growth kinetics Introduction Avian influenza viruses (AIVs) are the major cause of significant disease outbreaks with high morbidity and mortality worldwide in domestic birds resulting in great economic losses. Especially the subtypes of highly pathogenic avian influenza viruses (HPAIV) H5N1 and low pathogenic avian influenza viruses (LPAIV) H9N2 became the most prevalent AIVs in poultry causing regular disease outbreaks in many countries of Asia, the Middle East and Europe and are still ongoing events. Therefore, continues monitoring, surveillance and characterization of the circulating viruses are of high priority. Objectives The current study was designed for three main objectives; i) Molecular epidemiology of the HPAIV H5N1 in migratory birds in Bangladesh, ii) Molecular characterization of the AIV subtype H9N2 and iii) Biological properties of the AIV subtype H9N2. Materials and methods In first the part of the investigations, two HPAIV H5N1 strains were confirmed from 205 pools of fecal surveillance samples in Bangladesh. The two isolated H5N1 viruses were characterized by genome amplification and sequence analysis of the all eight genome segments. In the second part of the investigations, a confirmed AIV H9N2 from a retrospective analysis derived from a poultry farm in Bangladesh was characterized. Furthermore, three AI-H9N2 viruses were isolated and characterized from a commercial broiler and broiler breeder flock with clinical respiratory manifestations in Egypt. Full length genome amplification, cloning, sequencing and comprehensive phylogenetic analyses were performed for all eight genome segments. In the final part of the study, four selected Eurasian lineage H9N2 viruses - three G1 sub-lineages H9N2 and one European wild bird H9N2 virus - were propagated in embryonated chicken eggs (ECE) and Madin-Darby canine kidney epithelial cell culture systems. The ECE-grown and cell culture-grown viruses were monitored for replication kinetics based on tissue culture infectious dose (TCID50), hemagglutination assay (HA) and quantitative real time RT-PCR (qRT-PCR). The cellular morphology after infections was analyzed by immunofluorescence assay and cellular ELISA was performed to screen the sensitivity of the viruses to amantadine. Results The two newly isolated HPAIV H5N1 strains from migratory birds belonged to clade 2.3.2.1 and clustered together with other recently isolated viruses in Bangladesh derived from ducks, chickens, quails and crow. The amino acid sequences were also genetically similar although, some unique amino acid substitutions were observed. These substitutions were not related to the known conserved region of the molecular determinants of the virus. The phylogenetic analyses of the isolated AIV H9N2 from Bangladesh and Egypt revealed their close relationship with their respective contemporary isolates and maintained ancestor relation with A/Quail/HK/G1/1997 confirming that all studied H9N2 belonged to G1 sub-lineage. All six internal gene segments of the Bangladeshi AIV H9N2 showed high sequence homology with the HPAIV subtype H7N3 from Pakistan. In addition, also the PB1 internal gene showed high nucleotide homologies with a recently circulating HPAI-H5N1 virus from Bangladesh. Thus, the Bangladeshi AIV H9N2 is genetically a unique strain which shares internal gene segments with different HPAI viruses and takes part in reassortment events. On the other hand, the internal gene segments of the Egyptian H9N2 viruses were similar to the other members of the G1 sub-lineage with no evidence of reassortment events. In this virus rather point mutations within their respective gene segments are observed. With regard to the biological characterization, the three G1-H9N2 viruses produced comparatively higher titer than the Eurasian wild type-AIV H9N2. Overall, the ECE-grown viruses yielded higher titers than cell culture-grown viruses. Following a single passage in cell culture, individual nucleotide substitutions were noticed in HA, NA and NS gene sequences but none of them are related to the conserved region that can alter virus pathogenesis or virulence. All of the studied H9N2 viruses were sensitive to amantadine. Conclusion The present study demonstrated for the first time the presence of HPAI H5N1 in the wild migratory bird population in Bangladesh and determine as one of the major cause to introduce the new clade of HPAIV H5N1 into the Bangladeshi poultry flocks. The Bangladeshi AIV H9N2 strain has exhibited two independent reassortment events with HPAIV of subtype H7N3 and H5N1.The Egyptian AIV H9N2 strains were limited to regular point mutations which is very common for AIVs. The G1-H9N2 viruses showed a higher replication profile when compared to European wild bird-AIV H9N2. Both the ECE and MDCK cell system allowed efficient replication but the ECE system is considered as the better cultivation system for H9N2 viruses in order to get maximum amounts of virus within a short time period. In this study new strains of AIV H9N2 and H5N1 with significant genetic constitutions were described. Thus, continuous monitoring of the field samples, rapid reporting soon after outbreaks, molecular characterization to confirm the emergence of new reassortant strains and the biological properties to know its impact on the virulence are recommended. / Rokshana Parvin Molekulare Epidemiologie und biologische Charakterisierung von aviären Influenzaviren der Subtypen H5N1 und H9N2 Institut für Virologie Eingereicht im November 2014 Seiten 106, Abbildungen 7, Tabelle 1, Literaturangaben 339 , Publikationen 4 Schlüsselwörter: Aviäres Influenza Virus, H5N1, H9N2, Reassortment, Mutation, Replikation und Wachstumskinetik Einleitung Weltweit kommt es in der Geflügelproduktion durch Infektionen mit aviären Influenzaviren (AIV) zu hohen Morbiditäts- und Mortalitätsraten und damit verbunden zu hohen wirtschaftlichen Verlusten. Zu den bedeutenden AIV in der Geflügelwirtschaft werden die hoch pathogenen aviären Influenzaviren (HPAIV) des Subtyps H5N1 sowie AIV des Subtyps H9N2 gezählt. Letztere besitzen die Charakteristika von niedrigpathogenen aviären Influenzaviren. Durch diese Subtypen kommt es regelmäßig in vielen Ländern in Asien, im Nahen Osten und Europa zu wiederholten Krankheitsgeschehen. Dies bedingt die dringende Notwendigkeit von andauerndem Monitoring, Überwachung und Charakterisierung der zirkulierenden Viren. Ziele der Untersuchungen Die vorliegende Studie soll folgende drei Hauptfragestellungen beantworten: i) Molekulare Epidemiologie des HPAIV H5N1 bei Zugvögeln in Bangladesch, ii) Molekulare Charakterisierung von AIV des Subtyps H9N2 und iii) Biologische Eigenschaften von AIV des Subtyps H9N2. Materialien und Methoden Der erste Teil der Arbeit befasst sich mit zwei HPAIV Stämmen des Subtyps H5N1, welche im Monitoring Programm in Bangladesch von insgesamt 205 gepolten Kotproben, isoliert wurden. Die Charakterisierung der beiden Isolate erfolgte durch Vervielfältigung der acht Genomsegmente und nachfolgende phylogenetische Analysen. Der zweite Teil der Arbeit beschreibt die retrospektive Analyse eines AIV des Subtyps H9N2, welches von einer Geflügelproduktionsanlage in Bangladesch eingesandt wurde. Weiterhin wurden aus einer Geflügelmast- und Legehennenhaltung mit respiratorischer Symptomatik drei AIV des Subtyps H9N2 isoliert und charakterisiert. Auch hier wurde das gesamte Genom amplifiziert, kloniert und nachfolgend phylogenetisch analysiert. Im letzten Teil der Studie wurden vier europäische AIV H9N2 Isolate, von welchen 3 Isolate zur H9N2 Sublinie G1 gehören und ein Isolat von einem Wildvogel selektiert und in embryonierten Hühnereiern (EHE) und auf Madin-Darby canine kidney (MDCK) Zellen passagiert. Mittels 50% tissue culture infectious dose (TCID50), Hämagglutinationstest (HA) und RT-real-time-PCR (qRT-PCR) wurden von diesen so passagierten Viren die Vermehrungskinetik bestimmt. Die Morphologie der infizierten Zellen nach Infektion wurde mittels Immunfluoreszenztest analysiert. Eine Bestimmung der Amantadin Empfindlichkeit dieser Viren erfolgte mit einem ELISA. Ergebnisse Die beiden neuen HPAIV des Subtyps H5N1 von Zugvögeln können in die Clade 2.3.2.1 eingeordnet werden und clustern mit kürzlich aus Enten, Hühnern, Wachteln und Krähen isolierten AIV aus Bangladesch. Eine Verwandtschaft der Viren konnte auch auf Ebene der Aminosäure Sequenz gezeigt werden, obwohl einige einzigartige Aminosäure Austausche nachgewiesen wurden. Diese Austausche zeigen keine Verbindung mit bekannten konservierten Regionen der molekularen Determinanten der Viren. Die phylogenetische Analyse der AIV aus Bangladesch und Ägypten zeigt eine deutliche Verbindung mit den derzeit zirkulierenden AIV auf diesem geographischen Gebiet sowie die Verwandtschaft zu dem Isolat A/Quail/HK/G1/1997. Dies bestätigt, dass die in dieser Studie analysierten AIV zu der Subline G1 gehören. Alle sechs internen Gensegmente des AIV H9N2 aus Bangladesch zeigen eine hohe Sequenz Homologie mit einem HPAIV des Subtyps H7N3 aus Pakistan. Zusätzlich zeigt das interne Gene PB1 eine hohe Homologie auf Nukleinsäureebene zu einem derzeit in Bangladesch zirkulierenden HPAIV des Subtyps H5N1. Somit ist das AIV H9N2 aus Bangladesch als ein einzigartiges Isolat anzusehen, welches durch Reassortierung interne Gensegmente mit hochpathogenen AIV teilt. Im Gegensatz dazu, sind die internen Gene des AIV H9N2 aus Ägypten sehr ähnlich zu anderen Mitgliedern der Sublinie G1, welche keine Hinweise auf Reassorierung zeigen. Nur einzelne Punktmutationen konnten in den entsprechenden Gensegmenten nachgewiesen werden. In Hinblick auf die biologische Charakterisierung, konnte in den drei AIV H9N2 der Sublinie G1 vergleichsweise höhere Titer nachgewiesen werden als in einem europäischen AIV H9N2 Wildtypisolat. Insgesamt zeigten die in EHE passagierten Viren höhere Titer als die MDCK-Zell passagierten Viren. Schon nach einer Passage auf Zellkultur konnten einzelne Nukleotidaustausche in den HA, NA und NS kodierenden Gensegmenten nachgewiesen werden, wobei keine dieser Veränderungen einen Einfluss auf konservierte Regionen haben, die die Pathogenese oder Virulenz der Viren beeinflussen. Alle untersuchten H9N2 Viren sind sensitiv gegenüber Amantadin. Schlussfolgerungen Die vorliegende Studie zeigt erstmalig das Vorkommen von HPAIV H5N1 bei Zugvögeln in Bangladesch, welches als Haupteintragsquelle der neuen HPAIV H5N1 in der dortigen Geflügelhaltung angesehen wird. Das AIV H9N2 aus Bangladesch zeigt zwei unabhängige Reassortierungen mit HPAIV des Subtyps H7N3 und H5N1. Hingegen zeigt das ägyptische AIV H9N2 Punktmutationen, welche sehr typisch für diese Viren sind. Die hier untersuchten AIV H9N2 der Sublinie G1 zeigen im Vergleich zu einem europäischen AIV H9N2 eine höhere Replikationsrate. Eine Replikation der Viren konnte in EHE und MDCK-Zellen gezeigt werden, jedoch wird das EHE als das geeignetere System für die Kultivierung von H9N2 Viren betrachtet, da hier in einer kürzeren Zeitspanne mehr Virus produziert werden kann. Des Weiteren konnten in dieser Studie neue Isolate von AIV des Subtyps H9N2 und H5N1mit einem bedeutenden genetischen Aufbau beschrieben werden. Daher wird ein kontinuierliches Monitoring von Feldproben, unverzügliche Meldung von Ausbruchsgeschehen, die molekulare Charakterisierung zur Dokumentation eventuell auftretender neuer Reassortanten sowie Untersuchungen der biologischer Eigenschaften zur Virulenzbestimmung empfohlen.

Widespread anthropogenic invasions have prompted concerns that naturalized organisms could threaten biodiversity. In particular, invasive weeds can negatively affect native biota through a variety of means, including disrupting mutualisms. This thesis was designed to observe and test dispersal mutualisms in a native forest during autumn when the majority of plant species are fruiting. In this thesis I examined whether the invasive plant barberry (Berberis glaucocarpa) was influencing the behaviour of a native frugivore bellbird (Anthornis melanura) and a range of dispersal related services in a native forest, Kowhai Bush near Kaikoura. To test these 18 banded bellbirds were followed through autumn 2011. These observe bellbirds were split between control and test bird. Barberry fruit was removed from the test bird territories. I recorded whether bellbirds changed their territory sizes, foraging and daily behaviours. During 52 hours of observations, bellbirds were never observed feeding on barberry fruit. No significant changes to bellbird behaviour or territories were observed after the removal of barberry fruit. Bellbird diet overall was dominated by invertebrates (83% of foraging observations), with smaller contributions from fruit (16%, nearly all on Coprosma robusta), nectar and honeydew. Since bellbirds did not eat barberry fruit, removal of this weed is unlikely to negatively affect bellbirds during autumn. Which other bird species were dispersing barberry was recorded. I recorded 242 hours of videotape footage on 24 fruiting plants. A total of 101 foraging events were recorded of 4 different bird species: silvereyes (Zosterops lateralis) 42 visits, blackbirds (Turdus merula) 27 visits, song thrush (Turdus philomelos) 29, and starlings (Sturnus vulgaris) 3 visits. The species differed in the mean length of time they spent in plants, so the overall contribution to barberry fruit removal was 32.6% silvereyes, 24.3% blackbirds, 42.9% song thrush and 0.1% starlings. To find out the relative contribution of exotic and native birds to dispersal of fruits in Kowhai Bush, I mist-netted 221 birds of 10 species and identified any seeds in the 183 faeces they deposited. A total of 21 plant species were observed fruiting in Kowhai Bush during this time. A total of 11 different plant species were identified from 1092 seeds. Birds were further observed feeding on 3 other plant species which were not observed in faecal samples. This left 7 plants with unobserved dispersal vectors. There were likely four main dispersers, bellbirds, silvereyes, song thrush and blackbirds and five minor, brown creeper (Mohoua novaeseelandiae), tui (Prosthemadera novaeseelandiae), fantails (Rhipidura fuliginosa), dunnock (Prunella modularis) and starlings. However there was considerable variability between these bird species dispersal abilities. Introduced birds’ song thrush and blackbirds were observed dispersing naturalized plant seeds at higher than expected rates in comparison to native frugivores bellbirds and silvereyes. I also measured the gape sizes on mist netted birds and on samples of fruit from Kowhai Bush. Both silvereyes and bellbirds were found to be eating fruit larger than their gape, but despite this two native (Hedycarya arborea and Ripogonum scandens) and three exotic plants (Vitis vinifera, Taxus baccata and Crataegus monogyna) had large fruit that were probably mainly dispersed by song thrush and blackbirds. Hence, introduced birds were important seed dispersers for large fleshy fruited seeds in Kowhai Bush. Demonstrating that interactions among native and exotic flesh fruited plants and frugivores is important within forest communities.

Perfluoroalkyl compounds (PFCs) are a group of chemical surfactants most notably used in non-stick and stain-resistance applications. Due to their wide-spread use and inherent resistance to degradation, several PFCs have become persistent environmental contaminants. Despite the high concentrations of PFCs reported in wild birds and their eggs, very little is known about the toxicological effects they have on avian species. This thesis investigates the developmental toxicity of PFCs in an avian model species: the domestic chicken (Gallus gallus domesticus). Egg injection experiments were performed to assess the in ovo toxicity of perfluorooctane sulfonate (technical grade, T-PFOS), perfluorooctanoic acid (PFOA), perfluorodecane sulfonate (PFDS) and perfluoroundecanoic acid (PFUdA). Real-time RT-PCR was then used to measure the transcription of candidate biomarker genes in the liver tissue of day 20 embryos. Candidate genes were selected based on their responsiveness to PFC exposure in previously conducted in vitro screening assays. In ovo exposure to PFOS resulted in a dose-dependent decrease in embryo pipping success (a measure of hatching success) with an LD50 of 93 μg/g (3.54 μg/g-672,910 μg/g, 95% confidence interval), however the expression of peroxisome proliferator-activated receptor alpha (PPARα)-regulated genes was not affected in liver tissue as hypothesized. PFOA, PFDS and PFUdA had no effect on the pipping success of chicken embryos. The expression of cytochrome P450 1A4 (CYP1A4) and liver fatty acid binding protein (L-FABP) mRNA increased in embryo liver tissue following in ovo exposure to PFUdA but was only statistically significant at 10 μg/g, which is several orders of magnitude higher than concentrations reported in wild bird eggs. The isomer-specific accumulation of PFOS in chicken embryo livers was also investigated using an in-port derivatization gas-chromatography/mass spectrometry (GC-MS) method. Prior to incubation, chicken eggs were injected with T-PFOS, composed of 63% linear isomer (L-PFOS) and 37.3% branched isomers. The isomer profiles in day-20 embryo liver tissue showed up to 20% enrichment in the proportion of L-PFOS, compared to T-PFOS, with a corresponding decrease in the proportion of branched isomers. This enrichment was inversely proportional to dose. Finally, the transcriptional profiles of cultured chicken embryonic hepatocytes (CEH) exposed to either T-PFOS or L-PFOS were compared using Agilent 4x44k Chicken (V2) Gene Expression microarrays. At equal concentrations (10 μM), T-PFOS altered the expression of significantly more genes (340 genes, >1.5 fold change, false discovery rate adjusted p<0.05) compared to L-PFOS (130 genes). Functional analysis showed that L-PFOS and T-PFOS affected genes involved in lipid metabolism, cellular growth and proliferation, and cell-cell signaling. Pathway and interactome analysis suggested that gene expression may be affected through RXR, oxidative stress response, TP53 signaling, MYC signaling, Wnt/β-catenin signaling and PPARγ and SREBP receptors. In all functional categories and pathways examined, T-PFOS had a more pronounced disruptive effect on transctional regulation than L-PFOS. In summary, egg injection experiments showed that T-PFOS (but not linear PFOA, PFDS or PFUdA) may affect the hatching success of the chicken at environmentally relevant concentrations. It was also demonstrated that the accumulation of PFOS in embryonic liver is isomer specific, and leads to an enrichment of L-PFOS. The increased transcriptional disruption caused by T-PFOS in cultured hepatocytes over L-PFOS suggests that the branched isomers may be largely responsible for the toxicological effects of PFOS. Combined, the results from this thesis demonstrate the importance of considering PFOS isomer burdens during risk assessment. In addition, gene expression analysis identified several candidate mechanisms for PFOS toxicity.