Investigation of DNA variation in genes of the immune system in wild populations of Apodemus sylvaticus in relation to infection by Toxoplasma gondii and helminth parasites

The aims of this study are to investigate DNA sequence variation in immune genes from Apodemus sylvaticus in relation to parasite infection. The purpose is to investigate the hypothesis that immune gene variation can influence parasite infection. Evidence from other studies, such as infection of wild voles with Borrelia and cattle with bovine tuberculosis, has demonstrated that the DNA sequence (and therefore the protein sequence) of toll-like receptors (TLRs) is correlated with infection status. Recent laboratory studies on mice showed that the genes for TLR11 or TLR12 are essential for recognition of the parasite Toxoplasma gondii. Furthermore, the NLRP1b gene, a key component in regulating inflammation during infection, her been found in laboratory studies to be responsible for resistance of the mice against toxoplasmosis. Also, it is located at the Toxo1 locus which has been shown to be a key host locus for controlling Toxoplasma parasite proliferation and recent studies on inbred mice confirm that NLRP1b is the main gene that is responsible for this control. But little is known of the role of these innate immune system genes in natural populations. The purpose of this study is to investigate gene diversity in relation to parasite infection in wild rodent populations. A recent study in our laboratory resulted in 126 wild wood mice (Apodemus sylvaticus) being tested for T. gondii infection and other helminth parasites. This provides a topical opportunity to investigate DNA polymorphisms in NLRP1 and TLRs that could be associated with infection. In a study which investigated polymorphisms in relation to TLR11 and TLR 12, several relations were obtained but only one of them is significant which is the relationship between Syphacea infection with H2 and non H2 haplotype. At the start of the project no sequence was available for the NLRP1b gene from A. sylvaticus. Using Clustal alignment of DNA sequences from Mus musculus and other rodents, several combinations of PCR primer pairs were designed and tested for the amplification of parts of this gene from A. sylvaticus. The amplification is complex due to the arrangement of exons and introns, but successful sequences have been obtained for Exon 3 part 2 and Exon 3 part 3 (approximately 450 base pairs) which covers the function region, called NACHT, from 80 mice. Polymorphisms were found at amino acid positions 15 and 22 of exon 3-2 and positions 11 and 36 of exon 3-3. Exon 3-2 amino acid polymorphism is either methionine or leucine at position 15 and glutamic acid or alanine at position 22. Exon 3-3 amino acid polymorphism is either histidine or leucine at position 16 and glutamic acid or lysine at position 36. The polymorphisms found in the NLRP1b gene were examined for any relationship to a broad range of nematode parasites. One of the exon 3-2 haplotypes (H1/H2) shows a significant association with different nematodes and two haplotypes (H2/H3 and H4/H4) have a significant association with Toxoplasma gondii in respect for different helminth parasites. Furthermore, three of the exon 3-3 haplotypes (H1/H4, H4/H4 and H3/H3) show a significant association with different nematodes and one haplotype (H4/H4) has a significant association with Toxoplasma gondii. In addition, one haplotype of exon 3-3 which is H2/H2 showed a significant association with parasitic infection status (negative or multiple parasites). Also, the homozygosity and heterozygosity of the SNPs were investigated. For exon 3-2, the homozygosity of the first SNPs locus showed significant association with Toxoplasma gondii. For exon 3-3, the homozygosity of the first SNPs locus, and the second SNPs locus when analysed separately show significant association with Toxoplasma gondii. But, when analysed together, the heterozygosity of both SNPs locus showed significant association with Toxoplasma gondii in respect for different helminth parasites. In this study, DNA sequence variation was found in the immune genes (TLR11, 12 and NLRP1b) natural populations of wood mice. There was an association between some haplotypes and parasite infection. This provides evidence in support of the hypothesis that variation in immune gene sequences can influence parasite infection. Future studies should be aimed at identifying the detailed interactions between parasites and host immune genes in natural populations of wild animals