Bats and their endoparasites : characterising pipistrelle infections and toll-like receptor (TLR2 and TLR4) gene variations

Bats are unique mammals since they are able to fly and due to their crucial ecosystem roles, they are designated as keystone species. However, in many parts of the world, it is difficult to study bats due to the existence of protective legislation caused by their threatened status. Consequently, bat endoparasite studies are limited and even less is known about the bat immune system. To address this paucity of knowledge, this study was conducted using 99 pipistrelle bats (Pipistrellus pipistrellus, n=93 and P. pygmaeus, n=6 bats) that were obtained opportunistically from the Greater Manchester and Lancashire region between September 2005 and September 2008. These bats were infected with several species of helminths and protozoan parasites as previously described (Lord, 2010; Dodd et al., 2014). The data within this thesis describes further characterisation of the protozoan infections in this pipistrelle population through development of PCR-based molecular typing tools. This approach has allowed the molecular differentiation between Trypanosoma dionisii and T. vespertilionis infections, confirmed that all eimerian infections were caused by Eimeria rioarribaensis and also confirmed that Bartonella sp. infection is most likely to be non-zoonotic. In addition, Cryptosporidium sp. and Borrelia sp. infection data is presented; the former being the first report in a UK bat. Analysis of the infection profiles with respect to bat genotyping data (Dodd et al., 2014) shows that the parasites are randomly distributed with the exception of the E. rioarribaensis infections which appear to cluster in a sub-population of pipistrelles that are genetically more homogeneous. Since Toll-like receptors (TLRs) are an important element of the mammalian innate immune system, a PCR strategy was developed to isolate TLR4 and TLR2 genes from the pipistrelle bats (n=59). The TLR4 sequences were highly variable at the amino acid level (haplotypes, n=42), and a phylogenetic analysis of the protein sequences showed that they clustered into 7 major groups. Analysis of infection profiles in these bats showed that two TLR4 clusters appeared to correlate with susceptibility to trypanosomes (cluster 6) and Toxoplasma gondii (cluster 3). In addition, bats in TLR4 cluster 6 had a significantly reduced helminth burden. The TLR2 sequences were more conserved at the amino acid level (haplotypes, n=5); however, 7 bats were heterozygous at the TLR2 locus and interestingly, these correlated with a significantly reduced helminth burden. Overall, this thesis highlights the difficulty of studying bat endoparasites and this is often confounded by the lack, or absence, of parasitic material to assist developing molecular-based tools. Despite this difficulty, interesting data have been generated with respect to the pipistrelle genetics, including Toll-like receptor variations, and eimerian, trypanosome, T. gondii and helminth infection profiles, and this is worthy of further detailed investigations