<p> In the past two decades or so. microsatellites have become a very widely used genetic tool in many disciplines of biology. Their major downfalL however. is that they often need to be isolated de novo before they can be applied to molecular studies. Traditional shotgun cloning can be successfuL but it is often overly costly and time consuming. Compounding this downfall, isolating microsatellites from some taxa has been shown to be difficult. For example. on average only 0.46% of all clones screened using avian genomes will yield positive clones. This is thought to be a result of a smaller avian genome, a requirement for flight. Several alternative methods have been developed for isolating microsatellites, but the choice as to which isolation method to use is often arbitrary. To address this. four species of birds. the smooth-billed ani (Crotophaga ani). herring gull (Larus argentatus), yellow-bellied elaenia (Elaenia flavogaster), and pukeko (Porphyria porphyria), representing four different orders were used to compare two alternative isolation methods. Enrichment via selective hybridization versus cloning with Lambda Zap phage vector were compared in terms of monetary requirements (total startup cost as well as per isolation attempt cost). and time requirements (total time from start to finish and hands-on experimentation time). No significant difference was detected in terms of number of polymorphic microsatellite loci isolated by each method (p = 0.57), with enrichment yielding more for the anis and elaenias, Lambda Zap yielding more for herring gulls. and both methods isolating equal numbers for pukekos. Nor was any difference found between the methods for dollars spent per sequence with repeat (SWR) using the startup cost (p = 0.30). Enrichment. however. proved to be significantly more effective in terms of dollars per SWR isolated using the per use cost (p = 0.004) as well as hands-on minutes per SWR (p = 0.01) and total minutes per SWR (p < 0.01 ). Based on these tindings. selective hybridization is the better choice for microsatellite isolation. </p> / Thesis / Master of Science (MSc)

Parasitism can be defined as a biological interaction in which one party benefits at the expense of another (Keeton and Gould 1986). However unlike predation, the parasite does not kill its host. In the case of avian brood parasitism, one bird lays its eggs in the nest of a member of its own or another species and then abandons its offspring to the care of its new foster parents. These foster parents or 'hosts' then raise the parasitic young at the expense of their own brood. Brood parasitic birds have reproductive advantages over those which provide parental care for their young because: 1) parental care provided by several fosterers allows a female to produce more successful offspring than she is capable of rearing herself in one season, and 2) parasite offspring are usually distributed among many host nests thereby increasing the probability that at least some offspring will escape predation (Payne 1977). Obligate interspecific brood parasitism, where females only parasitize the nests of other species, is a reproductive strategy adopted by approximately 1% of all bird species and is practiced by members of five families (Anatidae, Cuculidae, Indicatoridae, Icteridae, and Ploceidae). The degree to which interspecific brood parasites reduce host nesting success varies with the reproductive tactics of the parasite. For example, Common Cuckoo chicks (Cuculus canorus) eject eggs and young nest mates from the host nest with the help of an instinctive urge to push out of the nest anything that touches the sensitive shallow depression in the parasitic nestling's back (Lack 1968). Young African Greater Honey-guides (Indicator indicator) stab host nestlings to death with special mandibular hooks that drop off after two weeks of age (Friedmann 1955). In contrast, black-headed duck hatchlings seek only protection and warmth for 1-2 days post hatching and then leave the nest with no further cost to their host (Weller 1968). Finally, Brown-headed Cowbird nestlings intermediately affect host reproductive success by diverting parental resources such as food away from the host's young (Payne 1977). This loss of host fitness results in selective pressure for host defenses against parasitism such as egg ejection (Neudorf and Sealy 1992), clutch abandonment (Burgham and Picman 1989), or increased nest defense early in the nesting cycle (Burgham and Picman 1989, Briskie and Sealy 1989, Neudorfand Sealy 1992). To circumvent these host responses, adaptation in brood parasites has resulted in selective pressure for egg mimicry (Rothstein 1990), egg removal, or shorter incubation periods (Briskie and Sealy 1990, Payne 1977). The Brown-headed Cowbird is the most abundant and widely distributed obligate interspecific brood parasite in North America. Although the breeding behaviour of this bird has been widely studied, most findings are contradictory. The mating system of the Brown-headed cowbird has been described as ranging from monogamous (Laskey 1950, Dufty 1982a, 1982b, Yokel 1986), to promiscuous (Elliot 1980). Most studies also suggest that cowbirds parasitize multiple host species (Friedmann 1929, p 177-188, Jones 1941, McGeen & McGeen 1968, Elliot 1977, Fleischer 1985). However, a few suggest that some individuals may be host specialists (Walkinshaw 1949, McGeen & McGeen 1968). Few of these studies have used genetic techniques to determine the actual mating patterns and to investigate the breeding biology of males and females in a single marked population. The main objective of this study was to use molecular genetic DNA markers as well as behavioural observation to study the mating system and host specificity of a Brown-headed Cowbird population at Delta Marsh, Manitoba. More specifically, my objectives were to: 1) determine whether DNA microsatellite markers are useful for determining parentage in Brown-headed Cowbird populations 2) document the genetic mating system and the patterns of host use by individual females in a population of resident cowbirds. My findings provide the first evidence that microsatellites are useful for high resolution parentage analyses in brood parasitic bird species where there is no A priori information available on male or female parentage. In addition, they are the first to directly quantity the mating system of a Brown-headed Cowbird population and to suggest that individual females are best described as host generalists but may be showing some preference for host nests in one habitat over another. / Thesis / Master of Science (MSc)

Hepatitis E is an acute, enterically transmitted disease of public health importance. The mechanism of pathogenesis of HEV is poorly understood due to the lack of an in vitro cell culture system and an ideal animal model system. With the discovery of avian HEV and its association with a hepatic disease (Hepatitis-Splenomegaly syndrome), chickens provide an excellent small homologous animal model system to study this important virus. The objectives of this dissertation were to utilize chickens as a model system to study the pathogenesis and replication of avian HEV under the natural route of infection, to identify potential extrahepatic replication sites, to determine and analyze the complete genomic sequence of the avirulent strain of avian HEV, and to study the compartive pathogenesis of the two isolates of avian HEV, the prototype pathogenic and avirulent strains of avian HEV. We attempted to experimentally infect specific-pathogen-free (SPF) adult chickens by the natural fecal-oral route in order to systematically study HEV pathogenesis and replication and to characterize the clinical course and pathological lesions associated with avian HEV infection. Sixty-week-old, specific-pathogen-free (SPF) chickens were inoculated with 5 x104.5 50% chicken infectious dose of avian HEV by oronasal route and IV route. All oronasally- and IV- inoculated chickens had seroconverted to avian HEV antibodies and fecal virus shedding was detected variably from 1 to 20 DPI in the IV group, and from 10 to 56 DPI in the oronasal group. Avian HEV RNA was detected in serum, bile, and liver samples earlier during the course of infection in IV-inoculated chickens than in oronasally-inoculated ones. Gross liver lesions including subcapsular hemorrhages and enlargement of right intermediate lobe and microscopic hepatic lesions in the liver characterized by lymphocytic periphlebitis and phlebitis were observed in inoculated chickens. This is the first report of experimental HEV infection via its natural route in a homologous animal model system. Very little is known about HEV pathogenesis and it has been hypothesized that HEV replicates in tissues other than liver. The replicating negative-strand viral RNA was detected by negative-strand-specific RT-PCR in liver, serum, colon, cecum, jejunum, ileum, duodenum and cecal tonsils,but not in other non-GIT tissues. Immunohistochemistry using an avian HEV capsid protein-specific anti-peptide antibody revealed positive signal in liver and GIT tissues including colon, jejunum, ileum, cecum, cecal tonsils and pancreas. The detection of avian HEV capsid antigen and replicative negative-strand viral RNA in the GIT tissues indicates that HEV replicates in the GI tract following infection by fecal-oral route. The complete genomic sequence of an avirulent strain of avian HEV was determined using primer walking strategy. The full-length genome of the avirulent strain is 6649 nts in length and has a nucleotide sequence identity of 90.1% with the prototype pathogenic strain. Numerous non-silent mutations were observed in ORF1, the region coding for the nonstructural proteins. Six unique non-silent mutations were identified in the capsid-encoding ORF2 region and the ORF3 had four non-silent mutations. Phylogenetic analysis based on full-length genomic sequence revealed that the avirulent strain is clustered together with the pathogenic avian HEV and represents a branch distinct from mammalian HEVs. In order to study the comparative pathogenesis between the pathogenic and avirulent strains of avian HEV, an infectious stock of the avirulent avian HEV was generated and infectivity titer was determined to be 5 x 102.5 CID50 per ml by experimentally infecting young SPF chickens. Six-week-old SPF chickens were inoculated with one of two strains of avian hepatitis E viruses, pathogenic avian HEV recovered from a chicken with HS syndrome and avirulent avian HEV isolated from a healthy chicken to study comparative pathogenesis. Most of the chickens seroconverted by 3 wpi in both pathogenic avian HEV and avirulent avian HEV groups. Avian HEV RNA was detected in feces and serum of the chickens from both the inoculated group from 1 wpi. Microscopic liver lesions included lymphocytic periphlebitis and phlebitis the overall hepatic lesion mean score was higher for the pathogenic avian HEV group compared to the avirulent avian HEV and control groups, suggestive of attenuation In summary, SPF chickens were experimentally infected with avian HEV by natural route to study the systematic pathogenesis and replication. Non-liver replication sites of avian HEV were also identified in a chicken model. The complete genomic sequence of an apparently avirulent strain of avian hepatitis E virus was determined and the comparative pathogenesis of avian hepatitis E virus isolates from a chicken with HS syndrome and from a healthy chicken was also studied by experimental infections in young SPF chickens. The results from this dissertation research have important implications for the understanding of HEV pathogenesis. / Ph. D.

PCBs are known to cause thyroid disruption in laboratory rats and are thought to be the causal agent in thyroid gland alterations in herring gulls in the Great Lakes. This study examined the regulation of thyroid hormone supply during development in (1) domestic chicken embryos (Gallus domesticus) exposed to a specific dioxin-like PCB congener (PCB-126) and (2) herring gull (Larus argentatus) embryos and pre-fledglings from Great Lakes sites with different chemical pollutant exposures. Specifically, PCB effects on thyroid status were evaluated by measuring plasma thyroid hormone concentrations and brain type II 5'D activity (to determine if PCB exposure was associated with alteration in brain 5'D type II activity that could maintain local T3 supply to the brain). If PCB-126 and PCB mixtures altered thyroid function, we expected to see decreased plasma thyroid hormone concentrations and subsequent increases in 5'D-II activity. Chicken eggs were injected (into the air cell) before incubation with five dose levels (0.0512, 0.128, 0.32, 0.64, 0.8 ng/g) of PCB-126 (3,3, 4,4',5-pentachlorobiphenyl), or vehicle (sunflower oil); sampling was on day 20 of the 21-day incubation period. Studies on PCB-treated embryos included a preliminary study and a larger study encompassing a serious of smaller studies. Herring gull embryos (at pipping, on day 25 of the 26 day incubation), and 28-day pre-fledgling chicks were sampled (for two field seasons) at several Great Lakes sites with different contaminant exposures (with Kent Island being the reference site). In PCB-treated chicken embryos, there were no statistically significant decreases in plasma T4 or T3 concentrations and no significant increases in brain 5'D-II activity in either the preliminary or the larger study. We found no clear pattern of altered thyroid function in herring gulls from polluted Great Lakes' sites. Plasma TH concentrations were not significantly decreased and 5'D-II activity did not significantly increase in birds from more contaminated sites in comparison to birds from Kent Island or sites with less contamination. Although pipped embryos from Strachnan Island had a significant increase in 5'D-II activity when compared to Kent Island, there were no differences in plasma TH concentrations, and brain 5'D-II activity was not significantly increased in birds from sites with greater PCB loads than Strachnan Island. Plasma T4 and T3 concentrations were significantly decreased in prefledglings from West Sister Island and Detroit Edison in comparison to Kent Island, but there was no subsequent increase in brain 5'D-II activity. The present study is the first to evaluate the potential effects of PCBs, alone and in a mixed environmental exposure, on circulating THs and brain 5'D-II activity in developing birds. Although thyroid function was not altered by the specific PCB congener used in my study or by exposure to environmental pollutants, more complete evaluations are needed before determining whether PCBs alter thyroid function in birds. / Master of Science

Periodic introductions of influenza A viruses (IAVs) from wild birds contribute to emergence of novel strains that infect domestic poultry, lower mammals, and humans, but the mechanisms of emergence are unclear. The objectives of this dissertation research are to infer the genesis of two emerging IAVs, low pathogenic avian influenza (LPAI) H10N8 and highly pathogenic avian influenza (HPAI) H7N8 viruses, and to characterize the antigenic diversity and genetic evolution of contemporary H7 avian influenza viruses (AIVs) from North America. First, AIVs that are genetically close to the human H10N8 isolate were recovered at the live poultry market (LPM) visited by the first H10N8 patient. High seroprevalence of H10 virus was observed in ducks and chickens from five LPMs in the region. These findings suggested that LPM was the most probable source of human infection with the H10N8 virus, and this virus appeared to be present throughout the LPM system in the city. Second, the novel H7N8 virus most likely circulated among diving ducks in the Mississippi flyway during autumn 2015 and was subsequently introduced to Indiana turkey, in which it evolved from LPAI into HPAI. H4N8 IAVs from diving ducks possess a gene constellation comprising five H7N8–like gene segments. These findings suggest that viral gene constellations circulating among diving ducks could contribute towards the emergence of IAVs that can affect poultry. Diving ducks may serve as a unique reservoir, contributing to the maintenance, diversification, and transmission of IAVs in wild birds. Third, antigenic and genetic characterization of 93 H7 AIVs from North America showed limited antigenic diversity. Gradual accumulation of nucleotide and amino acid substitutions in the H7 gene of AIVs from wild and domestic birds caused a wide genetic diversity. These findings suggested that continuous genetic evolution has not led to significant antigenic diversity for contemporary H7 AIVs isolated from wild and domestic birds in North America. In summary, these findings not only improve our understanding of the ecology and evolution of IAVs but also provide information for formulation of effective disease prevention and control strategies.

No description available.

M. Tech. (Department of Biotechnology, Faculty of Applied and Computer Science), Vaal University of Technology. / Influenza-A virus is a single stranded negative sense RNA virus that is a member of a Orthomyxoviridae group. The virus is diverse and consists of 16 haemagglutinin (H) and 9 neuraminidase (N) glycoproteins subtypes that form a serotype. Avian influenza virus (AIV) has been detected in more than 100 bird species from 26 different families, although Anseriformes and Charadriiformes are considered the natural hosts of the virus. A 12-month study was conducted at the African Pride Irene Country Club lodge in Pretoria where the prevalence of AIV was monitored in a community of wild birds. The African Pride Irene Country Club lodge houses a population of wild bird species such as Egyptian geese (Alopochen aegytptiaca), Yellow-billed duck (Anas undulata), Red knobbed coot (Fulica cristata), African sacred ibis (Threskiornis aethiopicus) and Hadeda ibis (Bostrycha hagedash). A total of 3674 faecal samples were collected over a period of 12 months and screened for AIV group using the Matrix gene (M-gene) real time reverse-transcriptase PCR (rRT-PCR). Positive samples were submitted for virus isolation in embryonated chicken eggs. In addition, the RNAs were screened using H5 and H7 subtype specific rRT-PCR and a conventional universal RCR assay that targets the HA gene was also used. Polymerase Chain Reaction (PCR) products were requenced using Sanger sequencing and the viral isolates were subjected to Next Generation sequencing (NGS). Twenty percent of the samples tested positive for the AIV group and four virus subtypes were identified. One virus isolate was identified through NGS as H3N6; two through conventional PCR and Sanger sequencing as H9Nx and H6Nx. Of the twenty percent samples that tested positive for AIV 98% were identified as H7Nx by subtype specific through rRT-PCR. The highest frequency of AIV positive samples was detected between the months of January and February 2017 (20%), with smaller peaks detected in february and March 2016 (0.3%). Lower peaks were also detected between the months July and November 2016 (0.1%), respectively. A high prevalence of AIV was detected in the late summer months with a frequency of 65% positive, although a low prevalence was also detected in the autumn (0.6%) winter (0.6%) and spring 0.08%). Thus, the study provides a valuable insight into the seasonal prevalence of AIV in a heterogeneous wild duck population in Gauteng Province.

Call number: LD2668 .T4 1958 M32

published_or_final_version / abstract / toc / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

published_or_final_version / abstract / Microbiology / Doctoral / Doctor of Philosophy