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Title: The Genetics of Susceptibility to Toxoplasma gondii and Related Phenotypes
Author: Darlay, Rebecca
ISNI:       0000 0001 3404 6494
Awarding Body: The University of Leeds
Current Institution: University of Leeds
Date of Award: 2007
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The intracellular protozoan Toxoplasma gondii causes a range of pathologies in its hosts, including abortion and ocular diseases, and can lead to mortality in immunocompromised individuals. The role of candidate immuneresponse genes in the variation of immune responsiveness .to T.gondii-related phenotypes were investigated: abortion and antibody responses in sheep and antibody responses in man. In the sheep, losses due to abortion are of major economic significance. Two separate ovine phenotypes were analysed: whether or not a lamb is aborted, and whether or not a ewe has had an aborted lamb. Study of the· epidemiology of abortion in two pedigree Charollais flocks (total flock sizes = 8192 and 1541) showed that abortion was associated with productivity as ewes which have many lambs have a high risk of aborting at least one of those lambs. Abortion was also associated with parity, as the chance of a ewe aborting reduced with successive pregnancies. Abortion was a repeatable event since 16 % of ewes that had an abortion were likely to abort in a future pregnancy. Any ewe which has an unexplained abortion should therefore be removed from the flock for future breeding purposes. There is a genetic component to the variation in abortion in sheep, with the aborted lamb phenotype having a heritability of 41.0 % and the aborting ewe phenotype· having a heritability of 12.2 %. Novel polymorphisms were detected in candidate immune response genes in order to investigate this component. Sheep InRNA sequences for 1FNG, 1L1B, 1L4 and TNF were compared to published bovine and human sequences, in order to design primers to amplify genomic DNA from these genes. Arnplicons were then analysed by DHPLC to detect potential novel polymorphisms, and interesting amplicons were sequenced and polymorphic sites characterised. A previously reported polymorphism from the literature 1L2647 was also investigated. RFLP assays were designed for 1FNGI68, 1FNG285, 1L1B679 and 1L2647. A Taqrrian assay was also designed for 1L4285. Ewes and lambs from the flocks (total n = 750) were genotyped at these polymorphic sites and association with the two abortion phenotypes and antibody responsiveness was tested by transmission disequilibrium testing, case-control analysis and GLM. Aborting ewes were more likely to carry the 1FNGI68T allele than the 1FNGI6sC allele, while lambs that had been aborted were more likely to have carried an 1FNG285G than 1FNG285A. The induction of an immune response in the placenta during pregnancy, where the immune environment is skewed in favour of tolerance of the semi-allogenic foetus, results in a pro-inflammatory response that is detrimental to the survival of the foetus. We speculate that these 1FNG alleles are in linkage disequilibrium with a polymorphism that results in hyperinduction· of IFN-y during the immune response to an infectious pathogen (such as T. gondii), and that the excess pro-inflammatory response results in untenable damage to the placenta and developing fo~tus. Human infection with T. gondii is also common, causing miscarriage and congenital infection, as well as causing extensive pathology in immunocompromised individuals. In order to investigate seroprevalence in human populations, antibody responses to T. gondii were assessed in a random sample of the UK. population (n = 249), and in thr~e South American populations from ~uanuco (n = 320) and Pilcopata (n = 104) in Peru and Isiboro-Secure (n = 111) in· Bolivia. We determined from IgG reactivity that ser!Jprevalence of T. gondii infection in the UK is - 17 %, in accordance with previous studies. The seroprevalence was much higher in three South American populations, with 77 % ofBolivian samples showing anti-To gondii IgG activity. Interestingly, the seroprevalence in different populations in Peru were markedly different. Samples from Pilcopata, which shares a. similar climate to Bolivia, also showed 77 % seroprevalence, but only 41 % of Huanuco samples showed IgG reactivity to T. gondii antigens. The Huanuco group, however, have the most variable IgG titres, and concentrations were on average much higher than all the other populations. . Investigation ofpolymorphisms in the immune response genes that might contribute to or control antibody responsiveness shows associations with a number of polymorphisms. The genes tested were ILl 0, 1LIB, 1LIRN, CD14, 1LIB, 1L4, 1L12B, LTA, TNF, HLA-DRBI, HLA-DQAI, HLA-DQBI, 1FNGRI, 1FNG, NOS2, CCLII, TGFB and 1FNGR2. CDI4_J59C (situated in the promoter) is associated (p = 0.004) with anti-To gondii IgG antibody concentrations in the Huanuco population, with those individuals with the lowest IgG responses to i. gondii more likely to carry this allele. Also within the Huanuco population were associations between lQ.-A-DRBI *4 (p = 0.003) and HLA-DQAI*0102 (p < 0.001) and high IgG levels; as well as between HLA-DRBI *4 and high IgM concentrations. These HLA Class II genes are involved in presentation of exogenous antibody. Finally, 1FNG2/o9A was associated with low levels ofIgG in the Huanuco population, while allele 1 of a polymorphism in the IFN-y receptor gene, 1FNGRlintron, is associated with high levels ofIgG in the same population. No other associations were found in the· South American population, but allele 2 (two 70 bp repeats) of IL4VNTR was also found to be associated with lower levels ofIgM in the UK population. These associations found between antibody responsiveness to T. gondii and human polymorphisms may be mirrored by similar mechanisms in the sheep, and hence all hosts.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available