Characterisation of selected innate immunity genes in felids

CEUS Central European Science Partnership Project: Austria Czech Republic Pamela Burger (Vetmeduni Vienna, Research Institute of Wildlife Ecology) Petr Horin (University of Veterinary & Pharmaceutical Sciences Brno, Central Institute of Technology) Abstract Each genome is under immense pressure due to different environmental influences. One of the strongest natural selection pressures on the genome are pathogens, which vehemently challenge the immune system. In response, usually highly polymorphic multi-gene families, e.g., the Major Histocompatibility Complex (MHC), encode key receptors that recognize and present foreign molecules to specialized immune cells, as part of the adaptive (acquired) immunity. If the variability in adaptive immune genes is reduced, like in some wild felid species, other components of the immune system should be selected to provide an adequate immune response. Such complementary mechanisms have been described for example in cheetahs, which show a stronger innate than adaptive immune response than leopards. In this project we follow an ecological comparative approach to investigate the evolution of the innate immune response in domestic and wild felids by characterizing genes, which are part of the innate immunity but closely communicate with adaptive immunity genes. We will characterize and comparatively analyse innate and adaptive immunity genes in wild endangered and domestic felids, including a cat population with a disease (feline coronavirus). This project fills a knowledge gap on the existence, function and (co- )evolution of innate immunity genes in the family Felidae and beyond, in mammalian species. Novel information on immunegenetic diversity and selection will enable effective genetic monitoring in wild- ranging felids and facilitate selective breeding strategies in managed populations. The project is performed within the framework of the International CEUS (Central European Science) Partnership Austria Czech Republic.

One of the strongest natural selection pressures on the genome are pathogens, which vehemently challenge the immune system. In response, usually highly polymorphic multigene families encode key receptor molecules that recognize and present foreign peptides to phagocytic immune cells, as part of the adaptive immunity. If the variability in adaptive immune genes is reduced, like in some wild felid species, other components of the immune system should be selected to provide an adequate immune response. As part of the innate immunity, natural killer (NK) cells are a highly heterogeneous population due to differential expression of NK receptors (NKRs). These are functionally related to adaptive immune genes. Despite their importance, the capacities and genetic heterogeneity of NK cells in felids are largely unknown. This project aimed to study the evolution of innate immunity genes in domestic and wild felid species with a special focus on natural killer cell receptors (NKRs). We followed an ecological comparative approach to investigate the evolution of the innate immune response in felids by characterizing genes, which are part of the innate immunity but closely communicate with adaptive immunity genes. As part of the characterisation and diversity of immune response genes it is important to have, first, a chromosome-level reference genome, and second, in-depth knowledge about the population structure in the investigated wild feline species. Therefore, we generated a de novo reference genome for cheetahs and investigated population structure and subspecies boundaries in African and Asiatic cheetahs using single nucleotide polymorphisms (SNPs) and mitochondrial DNA markers. Further genome resources were developed for the Pallas Cat and the snow leopard. The Toll-like receptor genes in cheetahs from South Africa, Namibia and Botswana showed lower nucleotide diversity, but similar amino acid haplotype diversity than the ones found in African leopards. The confirmation of the screening results for blood parasites in Namibian cheetah and leopard samples with Babesia-specific primersrevealed an additional infection with Hepatozoon felis. The project continued with the analysis of whole genome data and immune response genes in jaguars. Our genome-wide analyses revealed three distinct populations corresponding to Central America, South American lowland, and South American highland regions. While the highland population displayed lower overall immunome-wide variability, specific innate (Natural killer cell complex, TLRs and adaptive (Major histocompatibility complex-class-II) immune genes crucial for adaptive responses showed promising diversity. In cooperation with the Senkenberg Research Institute, Germany, the analysis of diversity in innate and adaptive IR genes in European Wildcat showed lower diversity in wildcats than in domestic cats. At the partner University of Veterinary Science in Brno, the comparative genomic analysis of the Natural Killer Complex in carnivores as well as the characterization of the Leukocyte Receptor complex (LRC) and cytotoxic T lymphocytes has been completed.