The anthropogenic impact on wildlife is ever increasing. With shrinking habitats, wild populations are being pushed to co-exist in proximity to humans leading to an increased threat of infectious diseases. Therefore, understanding the immune system of a species is key to assess its resilience in a changing environment. The innate immune system (IIS) is the body's first line of defense against pathogens. High variability in IIS genes, like toll-like receptor (TLR) genes, appears to be associated with resistance to infectious diseases. However, few studies have investigated diversity in TLR genes in vulnerable species for conservation. Large predators are threatened globally including leopards and cheetahs, both listed as 'vulnerable' by IUCN. To examine IIS diversity in these sympatric species, we used next-generation-sequencing to compare selected TLR genes in African leopards and cheetahs. Despite differences, both species show some TLR haplotype similarity. Historic cheetahs from all subspecies exhibit greater genetic diversity than modern Southern African cheetahs. The diversity in investigated TLR genes is lower in modern Southern African cheetahs than in African leopards. Compared to historic cheetah data and other subspecies, a more recent population decline might explain the observed genetic impoverishment of TLR genes in modern Southern African cheetahs. However, this may not yet impact the health of this cheetah subspecies.

Central European Institute of Technology University of Veterinary Sciences Brno Brno Czech Republic

Department of Animal Genetics University of Veterinary Sciences Brno Czech Republic

Research Institute of Wildlife Ecology University of Veterinary Medicine Savoyenstraße 1 1160 Vienna Austria

School of Health and Life Science Teesside University Middlesbrough Tees Valley TS1 3BX UK

South African National Biodiversity Institute National Zoological Garden 232 Boom Street Pretoria 0002 South Africa

University of Oulu Pentti Kaiteran Katu 1 90570 Oulu Finland

University of the Free State Bloemfontein Campus Bloemfontein 9300 South Africa

WWF South African Bridge House Boundary Terraces Mariendahl Ave Newlands 7725 Capetown South Africa

Zobrazit více v PubMed

Ausubel FM. Are innate immune signaling pathways in plants and animals conserved? Nat. Immunol. 2005;6:973–979. doi: 10.1038/ni1253. PubMed DOI

Medzhitov R, Janeway CA. Innate immunity: The virtues of a nonclonal system of recognition. Cell. 1997;91:295–298. doi: 10.1016/S0092-8674(00)80412-2. PubMed DOI

Eason DD, et al. Mechanisms of antigen receptor evolution. Semin. Immunol. 2004;16:215–226. doi: 10.1016/j.smim.2004.08.001. PubMed DOI

Lien E, et al. Toll-like receptor 4 imparts ligand-specific recognition of bacterial lipopolysaccharide. J. Clin. Investig. 2000;105:497–504. doi: 10.1172/JCI8541. PubMed DOI PMC

Muzio M, Mantovani A. Toll-like receptors. Microbes Infect. 2000;2:251–255. doi: 10.1016/S1286-4579(00)00303-8. PubMed DOI

Takeda K, Akira S. Toll-like receptors. Curr. Protoc. Immunol. 2015;109:14121–141210. doi: 10.1002/0471142735.im1412s109. PubMed DOI

Buwitt-Beckmann U, et al. TLR1-and TLR6-independent recognition of bacterial lipopeptides. J. Biol. Chem. 2006;281:9049–9057. doi: 10.1074/jbc.M512525200. PubMed DOI

Gazzinelli RT, Denkers EY. Protozoan encounters with Toll-like receptor signalling pathways: Implications for host parasitism. Nat. Rev. Immunol. 2006;6:895–906. doi: 10.1038/nri1978. PubMed DOI

Moresco EMY, LaVine D, Beutler B. Toll-like receptors. Curr. Biol. 2011;21:R488–R493. doi: 10.1016/j.cub.2011.05.039. PubMed DOI

Fitzgerald KA, Kagan JC. Toll-like receptors and the control of immunity. Cell. 2020;180:1044–1066. doi: 10.1016/j.cell.2020.02.041. PubMed DOI PMC

Hawley DM, Sydenstricker KV, Kollias GV, Dhondt AA. Genetic diversity predicts pathogen resistance and cell-mediated immunocompetence in house finches. Biol. Lett. 2005;1:326–329. doi: 10.1098/rsbl.2005.0303. PubMed DOI PMC

Morris KM, Wright B, Grueber CE, Hogg C, Belov K. Lack of genetic diversity across diverse immune genes in an endangered mammal, the Tasmanian devil (S arcophilus harrisii) Mol. Ecol. 2015;24:3860–3872. doi: 10.1111/mec.13291. PubMed DOI

Hughes AL, Hughes MK. Natural selection on the peptide-binding regions of major histocompatibility complex molecules. Immunogenetics. 1995;42:233–243. doi: 10.1007/BF00176440. PubMed DOI

Spielman D, Brook BW, Briscoe DA, Frankham R. Does inbreeding and loss of genetic diversity decrease disease resistance? Conserv. Genet. 2004;5:439–448. doi: 10.1023/B:COGE.0000041030.76598.cd. DOI

Tonteri A, Vasemägi A, Lumme J, Primmer C. Beyond MHC: Signals of elevated selection pressure on Atlantic salmon (Salmo salar) immune-relevant loci. Mol. Ecol. 2010;19:1273–1282. doi: 10.1111/j.1365-294X.2010.04573.x. PubMed DOI

Downing T, Lloyd AT, O’Farrelly C, Bradley DG. The differential evolutionary dynamics of avian cytokine and TLR gene classes. J. Immunol. 2010;184:6993–7000. doi: 10.4049/jimmunol.0903092. PubMed DOI

Villasenor-Cardoso M, Ortega E. Polymorphisms of innate immunity receptors in infection by parasites. Parasite Immunol. 2011;33:643–653. doi: 10.1111/j.1365-3024.2011.01327.x. PubMed DOI

Netea MG, Wijmenga C, O’neill LA. Genetic variation in Toll-like receptors and disease susceptibility. Nat. Immunol. 2012;13:535–542. doi: 10.1038/ni.2284. PubMed DOI

Abrantes J, Areal H, Esteves PJ. Insights into the European rabbit (Oryctolagus cuniculus) innate immune system: Genetic diversity of the toll-like receptor 3 (TLR3) in wild populations and domestic breeds. BMC Genet. 2013;14:73. doi: 10.1186/1471-2156-14-73. PubMed DOI PMC

Dalton DL, Vermaak E, Smit-Robinson HA, Kotze A. Lack of diversity at innate immunity Toll-like receptor genes in the Critically Endangered White-winged Flufftail (Sarothrura ayresi) Sci. Rep. 2016;6:36757. doi: 10.1038/srep36757. PubMed DOI PMC

Grueber CE, Wallis GP, King TM, Jamieson IG. Variation at innate immunity toll-like receptor genes in a bottlenecked population of a New Zealand robin. PLOS ONE. 2012;7:e45011. doi: 10.1371/journal.pone.0045011. PubMed DOI PMC

Liman N, Alan E, Apaydın N. The expression and localization of Toll-like receptors 2, 4, 5 and 9 in the epididymis and vas deferens of a adult tom cats. Theriogenology. 2019;128:62–73. doi: 10.1016/j.theriogenology.2019.02.001. PubMed DOI

Whitney J, Haase B, Beatty J, Barrs VR. Breed-specific variations in the coding region of toll-like receptor 4 in the domestic cat. Vet. Immunol. Immunopathol. 2019;209:61–69. doi: 10.1016/j.vetimm.2019.02.009. PubMed DOI PMC

Robert-Tissot C, et al. The innate antiviral immune system of the cat: Molecular tools for the measurement of its state of activation. Vet. Immunol. Immunopathol. 2011;143:269–281. doi: 10.1016/j.vetimm.2011.06.005. PubMed DOI PMC

Loots AK, et al. The role of toll-like receptor polymorphisms in susceptibility to canine distemper virus. Mammalian Biol. 2018;88:94–99. doi: 10.1016/j.mambio.2017.11.014. DOI

Odewahn R, Wright BR, Czirják GÁ, Higgins DP. Differences in constitutive innate immunity between divergent Australian marsupials. Dev. Comp. Immunol. 2022;132:104399. doi: 10.1016/j.dci.2022.104399. PubMed DOI

Ripple WJ, et al. Status and ecological effects of the world’s largest carnivores. Science. 2014;343:1241484. doi: 10.1126/science.1241484. PubMed DOI

Tshabalala T, et al. Leopards and mesopredators as indicators of mammalian species richness across diverse landscapes of South Africa. Ecol. Indicators. 2021;121:107201. doi: 10.1016/j.ecolind.2020.107201. DOI

Atkins JL, et al. Cascading impacts of large-carnivore extirpation in an African ecosystem. Science. 2019;364:173–177. doi: 10.1126/science.aau3561. PubMed DOI

Ford AT, et al. Large carnivores make savanna tree communities less thorny. Science. 2014;346:346–349. doi: 10.1126/science.1252753. PubMed DOI

Suraci JP, Clinchy M, Dill LM, Roberts D, Zanette LY. Fear of large carnivores causes a trophic cascade. Nat.Commun. 2016;7:10698. doi: 10.1038/ncomms10698. PubMed DOI PMC

Myers N. Leopard and cheetah in Ethiopia. Oryx. 1973;12:197–205. doi: 10.1017/S0030605300011534. DOI

Verschueren S, et al. Spatiotemporal sharing and partitioning of scent-marking sites by cheetahs and leopards in north-central Namibia. Afr. J. Ecol. 2021;59:605–613. doi: 10.1111/aje.12878. DOI

Hemami M-R, et al. Using ecological models to explore niche partitioning within a guild of desert felids. Hystrix. 2018;29:216.

Vogel JT, Somers MJ, Venter JA. Niche overlap and dietary resource partitioning in an African large carnivore guild. J. Zool. 2019;309:212–223. doi: 10.1111/jzo.12706. DOI

Seoraj-Pillai N, Pillay N. A meta-analysis of human–wildlife conflict: South African and global perspectives. Sustainability. 2016;9:34. doi: 10.3390/su9010034. DOI

Viollaz JS, Thompson ST, Petrossian GA. When human–wildlife conflict turns deadly: Comparing the situational factors that drive retaliatory leopard killings in South Africa. Animals. 2021;11:3281. doi: 10.3390/ani11113281. PubMed DOI PMC

Patterson BD, Kasiki SM, Selempo E, Kays RW. Livestock predation by lions (Panthera leo) and other carnivores on ranches neighboring Tsavo National Parks, Kenya. Biol. Conserv. 2004;119:507–516. doi: 10.1016/j.biocon.2004.01.013. DOI

Wykstra, M. & Fund, C. C. Cheetah Conservation and Human Impact in Kenya. Cheetah Conservation (2005).

Constant N, Bell S, Hill R. The impacts, characterisation and management of human–leopard conflict in a multi-use land system in South Africa. Biodivers. Conserv. 2015;24:2967–2989. doi: 10.1007/s10531-015-0989-2. DOI

Marker LL, Muntifering J, Dickman A, Mills M, Macdonald D. Quantifying prey preferences of free-ranging Namibian cheetahs. South African J. Wildlife Res. 2003;33:43–53.

Thuo D, et al. An insight into the prey spectra and livestock predation by cheetahs in Kenya using faecal DNA metabarcoding. Zoology. 2020;143:125853. doi: 10.1016/j.zool.2020.125853. PubMed DOI

Cushman SA, Elliot NB, Macdonald DW, Loveridge AJ. A multi-scale assessment of population connectivity in African lions (Panthera leo) in response to landscape change. Landscape Ecol. 2016;31:1337–1353. doi: 10.1007/s10980-015-0292-3. DOI

Durant SM, et al. The global decline of cheetah Acinonyx jubatus and what it means for conservation. Proc. Natl. Acad. Sci. 2017;114:528–533. doi: 10.1073/pnas.1611122114. PubMed DOI PMC

Belbachir F, Pettorelli N, Wacher T, Belbachir-Bazi A, Durant SM. Monitoring rarity: The critically endangered Saharan cheetah as a flagship species for a threatened ecosystem. PLoS One. 2015;10:e0115136. doi: 10.1371/journal.pone.0115136. PubMed DOI PMC

Marker, L. Cheetahs race for survival: Ecology and conservation. in Wildlife population monitoring (IntechOpen, 2019).

Charruau P, et al. Phylogeography, genetic structure and population divergence time of cheetahs in Africa and Asia: Evidence for long-term geographic isolates. Mol. Ecol. 2011;20:706–724. doi: 10.1111/j.1365-294X.2010.04986.x. PubMed DOI PMC

Jacobson AP, et al. Leopard (Panthera pardus) status, distribution, and the research efforts across its range. PeerJ. 2016;4:e1974. doi: 10.7717/peerj.1974. PubMed DOI PMC

Farhadinia MS, et al. The critically endangered Asiatic cheetah Acinonyx jubatus venaticus in Iran: A review of recent distribution, and conservation status. Biodivers. Conserv. 2017;26:1027–1046. doi: 10.1007/s10531-017-1298-8. DOI

Khalatbari, L., Jowkar, H., Yusefi, G. H., Brito, J. C. & Ostrowski, S. The current status of Asiatic cheetah in Iran. (2018).

Gerhold RW, Jessup DA. Zoonotic Diseases associated with free-roaming cats. Zoonoses and Public Health. 2013;60:189–195. doi: 10.1111/j.1863-2378.2012.01522.x. PubMed DOI

Wiethoelter AK, Beltrán-Alcrudo D, Kock R, Mor SM. Global trends in infectious diseases at the wildlife–livestock interface. Proc. Natl. Acad. Sci. 2015;112:9662–9667. doi: 10.1073/pnas.1422741112. PubMed DOI PMC

Pečnerová P, et al. High genetic diversity and low differentiation reflect the ecological versatility of the African leopard. Curr. Biol. 2021;31:1862–1871.e5. doi: 10.1016/j.cub.2021.01.064. PubMed DOI

O’Brien SJ, et al. Genetic basis for species vulnerability in the Cheetah. Science. 1985;227:1428–1434. doi: 10.1126/science.2983425. PubMed DOI

O’Brien SJ, Wildt DE, Bush M. The Cheetah in genetic Peril. Sci. Am. 1986;254:84–95. doi: 10.1038/scientificamerican0586-84. PubMed DOI

Hedrick PW. Bottleneck (s) or metapopulation in cheetahs. Conserv. Biol. 1996;10:897–899. doi: 10.1046/j.1523-1739.1996.10030897.x. DOI

Menotti-Raymond M, O’Brien SJ. Dating the genetic bottleneck of the African cheetah. Proc. Natl. Acad. Sci. 1993;90:3172–3176. doi: 10.1073/pnas.90.8.3172. PubMed DOI PMC

Castro-Prieto A, Wachter B, Sommer S. Cheetah paradigm revisited: MHC diversity in the world’s largest free-ranging population. Mol. Biol. Evol. 2011;28:1455–1468. doi: 10.1093/molbev/msq330. PubMed DOI PMC

Drake G, et al. The use of reference strand-mediated conformational analysis for the study of cheetah (Acinonyx jubatus) feline leucocyte antigen class II DRB polymorphisms. Mol. Ecol. 2004;13:221–229. doi: 10.1046/j.1365-294X.2003.02027.x. PubMed DOI

Meißner, R. et al. The potential and shortcomings of mitochondrial DNA analysis for cheetah conservation management. Conservation Genetics 1–12 (2022). PubMed PMC

Prost S, et al. Genomic analyses show extremely perilous conservation status of African and Asiatic cheetahs (Acinonyx jubatus) Mol. Ecol. 2022;31:4208–4223. doi: 10.1111/mec.16577. PubMed DOI PMC

Mirdita M, et al. ColabFold: Making protein folding accessible to all. Nat. Methods. 2022;19:679–682. doi: 10.1038/s41592-022-01488-1. PubMed DOI PMC

Sugimoto T, et al. Noninvasive genetic analyses for estimating population size and genetic diversity of the remaining Far Eastern leopard (Panthera pardus orientalis) population. Conserv. Genet. 2014;15:521–532. doi: 10.1007/s10592-013-0558-8. DOI

Wetzler LM. The role of Toll-like receptor 2 in microbial disease and immunity. Vaccine. 2003;21:S55–S60. doi: 10.1016/S0264-410X(03)00201-9. PubMed DOI

Heine H, et al. Cutting edge: Cells that carry a null allele for toll-like receptor 2 are capable of responding to endotoxin1. J. Immunol. 1999;162:6971–6975. doi: 10.4049/jimmunol.162.12.6971. PubMed DOI

Farhat K, et al. Heterodimerization of TLR2 with TLR1 or TLR6 expands the ligand spectrum but does not lead to differential signaling. J. Leukocyte Biol. 2008;83:692–701. doi: 10.1189/jlb.0807586. PubMed DOI

Ma Y, Haynes RL, Sidman RL, Vartanian T. TLR8: An innate immune receptor in brain, neurons and axons. Cell Cycle. 2007;6:2859–2868. doi: 10.4161/cc.6.23.5018. PubMed DOI PMC

Smirnova I, Poltorak A, Chan EK, McBride C, Beutler B. Phylogenetic variation and polymorphism at the Toll-like receptor 4 locus (TLR4) Genome Biol. 2000;1:research002.1. doi: 10.1186/gb-2000-1-1-research002. PubMed DOI PMC

Creel S, et al. Changes in African large carnivore diets over the past half-century reveal the loss of large prey. J. Appl. Ecol. 2018;55:2908–2916. doi: 10.1111/1365-2664.13227. DOI

Woodroffe R. Predators and people: Using human densities to interpret declines of large carnivores. Anim. Conserv. 2000;3:165–173. doi: 10.1111/j.1469-1795.2000.tb00241.x. DOI

Searle CE, et al. Leopard population density varies across habitats and management strategies in a mixed-use Tanzanian landscape. Biol. Conserv. 2021;257:109120. doi: 10.1016/j.biocon.2021.109120. DOI

Morris, D. R. et al. Gene flow connects key leopard (Panthera pardus) populations despite habitat fragmentation and persecution. Biodiversity and Conservation 1–19 (2022).

Tricorache P, Yashphe S, Marker L. Global dataset for seized and non-intercepted illegal cheetah trade (Acinonyx jubatus) 2010–2019. Data in Brief. 2021;35:106848. doi: 10.1016/j.dib.2021.106848. PubMed DOI PMC

Tong M, et al. Transcript profiling of toll-like receptor mRNAs in selected tissues of mink (Neovison vison) J. Microbiol. Biotechnol. 2016;26:2214–2223. doi: 10.4014/jmb.1604.04063. PubMed DOI

Wlasiuk G, Nachman MW. Adaptation and constraint at toll-like receptors in primates. Mol. Biol. Evol. 2010;27:2172–2186. doi: 10.1093/molbev/msq104. PubMed DOI PMC

Bagheri M, Zahmatkesh A. Evolution and species-specific conservation of toll-like receptors in terrestrial vertebrates. Int. Rev. Immunol. 2018;37:217–228. doi: 10.1080/08830185.2018.1506780. PubMed DOI

Vasseur E, et al. The selective footprints of viral pressures at the human RIG-I-like receptor family. Hum. Mol. Genet. 2011;20:4462–4474. doi: 10.1093/hmg/ddr377. PubMed DOI

Heinrich SK, et al. Cheetahs have a stronger constitutive innate immunity than leopards. Sci. Rep. 2017;7:44837. doi: 10.1038/srep44837. PubMed DOI PMC

Schwensow N, Castro-Prieto A, Wachter B, Sommer S. Immunological MHC supertypes and allelic expression: How low is the functional MHC diversity in free-ranging Namibian cheetahs? Conserv. Genet. 2019;20:65–80. doi: 10.1007/s10592-019-01143-x. DOI

McDade TW, Georgiev AV, Kuzawa CW. Trade-offs between acquired and innate immune defenses in humans. Evol. Med. Public Health. 2016;2016:1–16. doi: 10.1093/emph/eov033. PubMed DOI PMC

De Volo SB, Reynolds RT, Douglas MR, Antolin MF. An improved extraction method to increase DNA yield from molted feathers. The Condor. 2008;110:762–766. doi: 10.1525/cond.2008.8586. DOI

Dobrynin P, et al. Genomic legacy of the African cheetah, Acinonyx jubatus. Genome Biol. 2015;16:1–20. doi: 10.1186/s13059-015-0837-4. PubMed DOI PMC

Kõressaar T, et al. Primer3_masker: Integrating masking of template sequence with primer design software. Bioinformatics. 2018;34:1937–1938. doi: 10.1093/bioinformatics/bty036. PubMed DOI

Chen S, Zhou Y, Chen Y, Gu J. fastp: An ultra-fast all-in-one FASTQ preprocessor. Bioinformatics. 2018;34:i884–i890. doi: 10.1093/bioinformatics/bty560. PubMed DOI PMC

Li H, et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics. 2009;25:2078–2079. doi: 10.1093/bioinformatics/btp352. PubMed DOI PMC

Institute, B. Picard toolkit. Broad Institute, GitHub repository (2019).

Li H. A statistical framework for SNP calling, mutation discovery, association mapping and population genetical parameter estimation from sequencing data. Bioinformatics. 2011;27:2987–2993. doi: 10.1093/bioinformatics/btr509. PubMed DOI PMC

Danecek P, et al. Twelve years of SAMtools and BCFtools. Gigascience. 2021;10:giab008. doi: 10.1093/gigascience/giab008. PubMed DOI PMC

Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nat. Methods. 2012;9:357–359. doi: 10.1038/nmeth.1923. PubMed DOI PMC

Milne I, et al. Using Tablet for visual exploration of second-generation sequencing data. Brief. Bioinform. 2013;14:193–202. doi: 10.1093/bib/bbs012. PubMed DOI

Librado P, Rozas J. DnaSP v5: A software for comprehensive analysis of DNA polymorphism data. Bioinformatics. 2009;25:1451–1452. doi: 10.1093/bioinformatics/btp187. PubMed DOI

Larsson A. AliView: A fast and lightweight alignment viewer and editor for large datasets. Bioinformatics. 2014;30:3276–3278. doi: 10.1093/bioinformatics/btu531. PubMed DOI PMC

Nguyen L-T, Schmidt HA, von Haeseler A, Minh BQ. IQ-TREE: A fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol. Biol. Evol. 2015;32:268–274. doi: 10.1093/molbev/msu300. PubMed DOI PMC

Kosakovsky Pond SL, Posada D, Gravenor MB, Woelk CH, Frost SDW. Automated phylogenetic detection of recombination using a genetic algorithm. Mol. Biol. Evol. 2006;23:1891–1901. doi: 10.1093/molbev/msl051. PubMed DOI

Murrell B, et al. Detecting individual sites subject to episodic diversifying selection. PLOS Genet. 2012;8:e1002764. doi: 10.1371/journal.pgen.1002764. PubMed DOI PMC

Weaver S, et al. Datamonkey 2.0: A modern web application for characterizing selective and other evolutionary processes. Mol. Biol. Evol. 2018;35:773–777. doi: 10.1093/molbev/msx335. PubMed DOI PMC

Comparative genomics of the Natural Killer Complex in carnivores