No description available.

No description available.

Orientador: Hélio José Montassier / Banca: Manoel Victor Franco Lemos / Banca: Ricardo Luiz Moro de Souza / Resumo: A nucleoproteína (NP) do Vírus da Influenza Aviária (VIA) é um importante alvo antigênico no imunodiagnóstico desta doença, devido à sua baixa variabilidade entre as diferentes estirpes do VIA, resultando em uma elevada reatividade cruzada, e por ser também uma proteína altamente imunogênica para hospedeiros vertebrados. Neste estudo, o gene codificador da NP do VIA foi parcialmente clonado e expresso em Escherichia coli como uma proteína recombinante fusionada ao polipeptídeo SUMO e uma etiqueta de poli-histidina para seu uso no desenvolvimento de um ensaio de ELISA indireto para a detecção de anticorpos específicos contra o VIA. A NP recombinante foi expressada na fração solúvel e foi mais facilmente purificada. Após análise em relação aos seus principais sítios de antigenicidade e caracterização por meio de Western blotting, a NP recombinante foi utilizada como uma preparação antigênica no ELISA indireto para detecção de anticorpos contra o VIA presentes em amostras de soro de galinha. A análise comparativa do teste desenvolvido no presente estudo com um ELISA comercial apresentou valores de 95%, 97% e 96,7% de sensibilidade, especificidade e acurácia, respectivamente e um índice κappa de 0,88. Os resultados permitem concluir que a NP recombinante do VIA desenvolvida neste estudo possui características favoráveis para ser aplicada como antígeno no ELISA indireto, constituindo-se em um método sensível e específico para o imunodiagnóstico da Influenza Aviária em galinhas / Abstract: The nucleoprotein (NP) of Avian Influenza Virus (AIV) is an important antigenic target for immunodiagnosis of this disease, due to its low variability among different AIV strains, resulting in high cross-reactivity, and the also highly immunogenic for vertebrate hosts. In this study, the gene enconding NP of AIV was cloned and expressed in Escherichia coli as a recombinant protein fused to SUMO polypeptide with a polyhistidine tag and used to develop an indirect ELISA for the detection of AIV-specific antibodies. The recombinant NP was expressed in the soluble fraction and easily purified. After Analysis of the main sites of antigenicity and characterization in Western-Blotting, the recombinant NP was optimized as an antigen preparation for indirect ELISA to detect anti-AIV antibodies in chicken serum samples. The comparative analysis of this ELISA with a commercial ELISA showed values of 95%, 97%, 96.7% of sensitivity, specificity and accuracy, respectively, and an agreement of k=0.88. In conclusion, the results indicated that the recombinant NP of AIV produced in this study is a good source of antigen for indirect ELISA and provides a sensitive and specific method for the immunodiagnosis of Avian Influenza in chickens / Mestre

published_or_final_version / abstract / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Pathology / Master / Master of Philosophy

published_or_final_version / Community Medicine / Master / Master of Public Health

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

I investigated parasitic infection and immunocompetence in populations of introduced bird species in New Zealand (NZ) that had experienced a range of population bottlenecks (11-808 individuals), and compared these parameters to non-bottlenecked conspecifics in the United Kingdom (UK). My aims were two-fold; firstly to assess if population bottlenecks are linked to increased parasite loads and/or decreased immunocompetence, and secondly, to assess at what severity of bottleneck these effects become evident. I found that ectoparasite load (chewing lice, Order: Phthiraptera, Sub-Orders: Amblycera & Ischnocera) was significantly higher in the more severely bottlenecked species in NZ than in the UK, whilst this difference became non-significant at more moderate bottlenecks. The difference was mainly driven by the Sub-Order Amblycera. The prevalence of avian malaria (Plasmodium spp.) was significantly negatively correlated to bottleneck size within NZ, after controlling for body mass. Total leucocyte and differential lymphocyte counts were elevated in the less bottlenecked species that were infected with malaria, whilst the populations at the more severe end of the bottleneck spectrum did not exhibit such a response. Furthermore, heterophil/lymphocyte (HL) ratio (a parameter used as an indicator of environmental and/or immunological stress), was significantly raised in the more bottlenecked species when compared to their UK counterparts, and this difference was correlated with the size of the bottleneck. Immunocompetence was further assessed by the experimental challenge of six introduced birds species in NZ with the mitogen phytohaemagglutinin (PHA). Immune response to PHA was significantly correlated to bottleneck size, but in the opposite direction to that predicted; immune response was greater in the more bottlenecked species. However, this may be an indication of increased investment in immunity, due to increased parasite and pathogen pressure or differential investment in varying components of the immune system. Finally, the immune response to PHA was compared in nestlings of two species that had experienced very different bottlenecks (70 vs. 653). After controlling for ectoparasitic infestation, I found no difference between the two species; however, this finding may be confounded by interspecific competition. Overall, my findings suggest that more severe population bottlenecks may result in increased susceptibility to pathogens, and impact on the immune system. This has a number of implications for the development of conservation protocols, and future avenues of research are suggested.

Recent observed changes in bird distributions provide an unprecedented opportunity to gain a deeper understanding of the processes that influence species' persistence. By modelling presence-absence data from the North American Breeding Bird Survey, we found evidence that the breeding range of the western burrowing owl has contracted at its northern, western, and eastern boundaries since 1967. We suggest that the species' breeding distribution is also expanding southwards to former wintering grounds into northern Mexico, facilitated by the appearance of new breeding habitat created by irrigated agriculture in the arid areas of southwestern United States and northwestern Mexico. This dissertation explores the hypothesis that burrowing owls from northern migratory populations have become resident breeders in areas of northwestern Mexico that were formerly used only by migratory owls during winter, contributing to both population declines near the northern extent of the species' breeding range and population increases in the southern half of the species' range. We used novel DNA microsatellite markers to test patterns of gene flow predicted by this migration-mediated range-shift hypothesis. We genotyped 1,560 owls from 36 study locations in Canada, Mexico, and the United States. Analyses of molecular variance provided evidence that burrowing owl populations in both northwestern Mexico and Canada are genetically different from the rest of the populations in the breeding range, lending some support to the migration-mediated range-shift hypothesis. We found evidence of subtle genetic differentiation associated with subtropical irrigated agricultural areas in southern Sonora and Sinaloa, demonstrating that land use can produce location-specific population dynamics leading to genetic structure even in the absence of dispersal barriers. We also used stable isotopes 2/H, 13/C, and 15/N in feathers to test philopatry and breeding dispersal patterns predicted by this migration-mediated range-shift hypothesis. Burrowing owl populations near the northern edge of the species' breeding range had a high proportion of immigrants compared to interior populations, while other populations had high levels of philopatry. Stable isotopes also provided evidence of breeding dispersal events from Canadian populations to northwestern Mexico in support of the migration-mediated range-shift hypothesis, but similar isotope signatures in nestling feathers between these two regions prevent stronger inferences.

I evaluated the spatial and temporal patterns of the avian communities in the Colorado River delta, Mexico, and their relationship with vegetation type and surface water. I also developed plausible conservation and restoration guidelines for riparian areas and native birds in the region. The study included monthly point counts at 30 transects (240 points) from May 2002 to July 2003, breeding counts at 175 sites (3 times per year) during 2002 and 2003, and habitat measurements at the survey points.The most common species were Mourning Doves, Red-winged Blackbirds, and Brown-headed Cowbirds, but another 64 species were commonly found, including Verdins, Song Sparrows, Yellow-breasted Chats and Abert's Towhees. Surface water was the most important habitat feature related to avian richness and density regardless of vegetation type or land cover (P < 0.005). During summer, species richness was explained by variations in water and the cover of cottonwoods (r2 = 0.56, P < 0.001), and the variation in bird densities was explained by variations in water and the cover of willows (r2 = 0.35, P = 0.003).When comparing native versus saltcedar dominated sites, both with the presence (wet) or absence (dry) of surface water, the diversity of birds was more influenced by the presence of water than by vegetation type. Bird abundance was more influenced by vegetation type, but water also had an important effect, as wet sites had higher bird abundance than dry sites with the same vegetation type, and saltcedar wet areas had similar avian abundance to native dry sites. On all cases, the presence of water was an important factor determining the ecological value, in terms of avian richness, abundance, and diversity, of both native riparian and saltcedar areas. Saltcedar areas with surface water had avian characteristics similar to native riparian sites.The dedication of instream flows and pulse floods, the maintenance of vegetation cover and structural diversity, and an increase of older riparian stands will secure the viability of existing bird populations and will increase the probability of recovery of the species that are still extirpated from the floodplain of the Colorado River in Mexico.