No description available.

The major histocompatibility complex of the rat (RT1 complex) encodes two sets of class II molecules referred to as RT1 B and RT1 D. The RT1 Bα gene was isolated from a Sprague-Dawley (RT1b) rat genomic library using a rat RT1 Bα chain cDNA as a hybridization probe. The coding and the majority of the intron DNA sequence was determined. The structure of the RT1 Bα gene is equivalent to that of H-2 and HLA a chain genes. Comparison of the nucleotide and predicted amino acid sequences of the RT1 Bα gene to those of the H-2 and HLA genes revealed a high degree of overall sequence conservation. However, two regions of the first external domain (a1), residues 19-23 and 45-78, exhibit marked sequence variation. Two blocks of conserved nucleotide sequence were identified in the 5' promoter region of the RT1 Bα gene that have been described in all MHC class II genes sequenced to date. These conserved sequences may be involved in the co-ordinate regulation of expression of class II genes. The cloned RT1 Bα gene was efficiently transcribed when transfected into mouse L cells. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

Parasitism is one of the most common ways of living and it has arised in many taxa. Parasites feed and live inside or on their hosts resulting in both long and short term consequences for the host. This thesis is exploring the phenotypic and genotypic effects of animals living with parasitic infections. I have been studying three different parasite groups and their associated host species: the great snipe, a lekking freshwater wader bird that migrates between Africa and Northern Europe; the tree sparrow, a stationary passerine found close to human settlements and lastly the water vole, a large rodent living in riparian habitats. Avian malaria is one of the most commonly studied parasites affecting birds. Atoxoplasma, an intestinal protozoan parasite is less studied but is thought to be endemic in free-ranging birds. Given the freshwater habitat great snipes inhabit, a prevalence of 30% avian malaria infections is not high and that the prevalence fluctuated among years. Sequencing of the avian malaria cytochrome b gene revealed that parasites are similar to avian malaria parasites found in African birds suggesting that they were infected on the wintering grounds in Africa. Tree sparrows had few malaria infected individuals, a result that is consistent with other studies of stationary birds at high latitudes. Atoxoplasma infections were common in tree sparrows and capture-recapture analyses show decreased survival in infected compared to uninfected birds and signs of lower mating success among infected. Genetic analyses comparing the transcriptome between mated and unmated great snipe males revealed that the genotype is important for mating success and health status for some of the expressed genes. That variations in some of these genes are involved in maintaining a good health status and mating success supports handicap models for sexual selection in this lek mating system. The major histocompatibility complex (MHC) is a part of the immune system and it contains genes involved in immune response. In water voles, a number of new MHC alleles were identified. Based on their in silico phenotype they were grouped into supertypes to facilitate studies on how helminth infections affect the MHC diversity in the water voles. Some of these MHC supertypes provided resistance to one helminth species, but the same supertype caused the opposite effect for other helminth parasites. Overall, parasites are a driving force for maintaining genetic diversity and parasite infections lowers survival rate, which would lead to a lower lifetime breeding success.

The Major Histocompatibility Complex class II B (MHC II B) gene encodes a protein that is part of the adaptive immune system and critical for the non-self recognition ability of immune cells. This gene has been characterized in the Bald Eagle, ten unique alleles were found in two subpopulations at the geographic extremes of the range margins. Geographic genetic variation is suggested by the presence of population specific alleles. The results showed considerable divergence of groups of Bald Eagle alleles when compared to alleles from other birds of prey. Particular codons within the exon II show signs of balancing selection driving the evolution of the MHC II B. Transcription data showed statistically significant differential expression of alleles. This can be interpreted as meaning a particular locus is being preferentially expressed in blood. The analysis of the polymorphism of this adaptive marker may aid managers of wildlife during this age of global climate change and the biodiversity crisis. / by Andrew Smith. / Thesis (M.S.)--Florida Atlantic University, 2010. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2010. Mode of access: World Wide Web.

Infectious disease is a major issue facing the livestock industry. Further understanding of the role of genetic factors in the observed phenotypic variability of the immune response to pathogens and vaccination could assist in designing improved preventative measures such as more efficacious vaccines and targeted breeding strategies to select for disease resistance. Major candidate genes for controlling immune responsiveness are located within the major histocompatibility complex (MHC). The highly polymorphic classical MHC genes are key determinants in the strength and type of immune response. However, it has proved difficult to establish genotyping approaches to define functionally relevant allelic variations for outbred species such as cattle, not least because the peptide binding clefts (PBC) of classical MHC molecules are highly polymorphic, and the genes within the MHC complex are closely linked. The overall aim of this project was to investigate the role of MHC genes in immune responsiveness in approximately 200 F2 and backcross Holstein-Charolais cattle. These animals were generated as part of the Roslin Bovine Genome (RoBoGen) herd, established through a quantitative trait loci (QTL) project, in which a number of phenotypic traits including immune traits were measured. The immune traits included responses to a Foot-and-mouth disease virus (FMDV) peptide, and vaccines against bovine respiratory syncytial virus (BRSV), para-influenza virus 3 (PIV-3) and bovine herpes virus-1 (BHV-1), as well as T cell response to Staphylococcus aureus. The immune phenotypes measured included IgG and interferon- (IFN- ) levels and T cell proliferation. The cattle MHC region, known as bovine leukocyte antigens (BoLA), resides on bovine chromosome 23. The BoLA region contains approximately 200 genes most of which are immune-related. Class II gene polymorphisms were considered to be the most likely to influence the immune traits measured, and the project primarily focused on the best defined gene, BoLA-DRB3. A sequence-based typing technique was successfully improved to facilitate genotyping of the PBC of BoLA-DRB3 in all generations of the RoBoGen herd (approximately 400 animals) and identified 24 distinct alleles. The sequence information obtained also enabled further analysis of the role of defined ‘pockets’ within the PBC, which directly determine peptide binding affinity. All datasets were statistically analysed using a residual maximum likelihood (REML) model and it was shown that several of the DRB3 alleles within the RoBoGen herd had highly significant (p<0.05) associations with the immune response to the FMDV peptide. In addition DRB3 alleles were identified which had significant associations with the response to the respiratory pathogen vaccinations and exposure to S. aureus. The pocket analysis also enabled the identification of several amino acid positions within the PBC which were significantly associated with the immune response traits. In order to explore whether DQ Class II gene polymorphisms also played a role in the variability of responses and whether BoLA Class I-Class II haplotypes could be discerned, microarrays which utilized allele specific oligonucleotides for BoLA Class I and Class II DQ genes were employed. In addition, to investigate whether the number of DQ gene pairs per chromosome influenced the responses, a quantitative polymerase chain reaction (qPCR) assay to determine DQA gene dosage was developed. However, due to the extremely complex nature of the BoLA region both, techniques would require improving to be used for large-scale studies. Nonetheless, information about haplotypes was determined from the microarray results and the qPCR technique lays the foundations for future development to investigate DQ gene dosage. The MHC region in cattle is very complex due the high level of polymorphisms and gene duplications. It is likely that many genes play a role in the immune response to both pathogens and vaccines. However, from the evidence presented here, polymorphisms in the PBC of BoLA-DRB3, particularly within the pockets, are significantly associated with variation in immune response to many different antigens and this information could be exploited in the design of vaccines or breeding cattle for improved disease resistance.

No description available.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 90-119).

An intestinal cell line from rainbow trout, Oncorhynchus mykiss, was developed and challenged against several bioactive components. Primary cultures initiated from the distal segment produced the cell line, RTgutGC. RTgutGC showed optimal growth in L15 supplemented with 10-20% fetal bovine serum (FBS) at room temperature. RTgutGC has undergone over 100 passages and stained minimally for β-galactosidase, suggesting this to be an immortal cell line. Late passage cultures gave a consistent polygonal morphology with distinct borders. RTgutGC stained positive for alkaline phosphatase (AP) under certain culture conditions, hence may produce intestinal-specific alkaline phosphatase (IAP). Lipopolysaccharide (LPS) was used as a model microbial endotoxin for determining the sensitivity of the cells to a natural ligand in the gastrointestinal tract (GIT). Exposure of LPS was compared between RTgutGC and two mammalian intestinal cell lines (HT-29 and Caco-2). LPS induced cell death in RTgutGC, potentially through an alternative pathway seen in higher vertebrate response. Cytotoxicity of LPS against RTgutGC, seeded at normal density, was reduced in the presence of glutamine compared to L15 alone (t test, p≤ 0.05). RTgutGC seeded at a super density, where AP was strongly expressed, also showed less toxicity towards LPS. Two isoforms of tumor necrosis factor alpha (TNF-α) transcripts were up-regulated after LPS treatment in RTgutGC. Six rainbow trout cell lines, including RTgutGC, showed constitutive transcript expression of several immune-related genes: Major Histocompatibility (MH) class II α and ß. When MH activity was examined at the protein level, the cell lines showed constitutive expression of MH class I proteins, but not for MH class II molecules. RTS11, a rainbow trout spleen monocyte/ macrophage-like cell line, was the only line to express all MH transcripts and proteins. The utility of the anti-rainbow trout MH protein sera was demonstrated by exposing RTgutGC to poly IC. After a 3 day treatment, RTgutGC showed up-regulation of β2m protein expression. Thus, the cellular and immunological responses in fish intestinal cells can be modeled using the methods presented in this study.

An intestinal cell line from rainbow trout, Oncorhynchus mykiss, was developed and challenged against several bioactive components. Primary cultures initiated from the distal segment produced the cell line, RTgutGC. RTgutGC showed optimal growth in L15 supplemented with 10-20% fetal bovine serum (FBS) at room temperature. RTgutGC has undergone over 100 passages and stained minimally for β-galactosidase, suggesting this to be an immortal cell line. Late passage cultures gave a consistent polygonal morphology with distinct borders. RTgutGC stained positive for alkaline phosphatase (AP) under certain culture conditions, hence may produce intestinal-specific alkaline phosphatase (IAP). Lipopolysaccharide (LPS) was used as a model microbial endotoxin for determining the sensitivity of the cells to a natural ligand in the gastrointestinal tract (GIT). Exposure of LPS was compared between RTgutGC and two mammalian intestinal cell lines (HT-29 and Caco-2). LPS induced cell death in RTgutGC, potentially through an alternative pathway seen in higher vertebrate response. Cytotoxicity of LPS against RTgutGC, seeded at normal density, was reduced in the presence of glutamine compared to L15 alone (t test, p≤ 0.05). RTgutGC seeded at a super density, where AP was strongly expressed, also showed less toxicity towards LPS. Two isoforms of tumor necrosis factor alpha (TNF-α) transcripts were up-regulated after LPS treatment in RTgutGC. Six rainbow trout cell lines, including RTgutGC, showed constitutive transcript expression of several immune-related genes: Major Histocompatibility (MH) class II α and ß. When MH activity was examined at the protein level, the cell lines showed constitutive expression of MH class I proteins, but not for MH class II molecules. RTS11, a rainbow trout spleen monocyte/ macrophage-like cell line, was the only line to express all MH transcripts and proteins. The utility of the anti-rainbow trout MH protein sera was demonstrated by exposing RTgutGC to poly IC. After a 3 day treatment, RTgutGC showed up-regulation of β2m protein expression. Thus, the cellular and immunological responses in fish intestinal cells can be modeled using the methods presented in this study.