Sinah Lückner , Gastón Moré , Iris Marti , Caroline F. Frey , Javier E. Fernandez , Chahrazed Belhout , Walter Basso
{"title":"欧亚狼(Canis lupus lupus)的高流行率:第三代测序解决了混合感染","authors":"Sinah Lückner , Gastón Moré , Iris Marti , Caroline F. Frey , Javier E. Fernandez , Chahrazed Belhout , Walter Basso","doi":"10.1016/j.ijppaw.2025.101140","DOIUrl":null,"url":null,"abstract":"<div><div><em>Sarcocystis</em> spp. (Apicomplexa: Coccidia) are obligate heteroxenous protozoa that infect a wide range of host species. Transmission follows a predator-prey cycle involving an intermediate host (IH) and a definitive host (DH). For many species, only IHs have been identified, while DHs remain unknown. DHs can be infected with multiple <em>Sarcocystis</em> spp. at the same time, which complicates species identification. We aimed to determine the prevalence and species diversity of <em>Sarcocystis</em> infections in free-ranging wolves in Switzerland using both coprological and molecular methods. A further goal was to evaluate the utility of Third-generation sequencing for resolving mixed infections. A total of 87 wolf intestinal content samples were collected between 2017 and 2023 and analyzed coproscopically by a sedimentation-flotation method. <em>Sarcocystis</em> oocysts/sporocysts were detected in 76 % (66/87). DNA was obtained from 57/66 positive samples and 55/57 resulted positive in a <em>Sarcocystis 18S</em> rRNA screening PCR. Additionally, mitochondrial cytochrome <em>c</em> oxidase subunit I (<em>COI</em>) gene PCR and a real-time PCR targeting <em>S. cruzi</em> were performed. PCR products from conventional PCRs were submitted for Sanger sequencing. Monoinfections were identified in 16 % (9/55) and mixed infections in 84 % (46/55) of the samples. A subset of five samples was analyzed by Third-generation sequencing (Pacific Biosciences) of the <em>18S</em> rRNA full-length and <em>COI</em> fragment PCR products. BLAST and phylogenetic analysis were used to validate taxonomic classification. Molecular analysis identified nine known <em>Sarcocystis</em> species: <em>S. tenella, S. arieticanis, S. capreolicanis, S. linearis, S. gracilis, S. cruzi, S. capracanis, S. iberica,</em> and <em>S. venatoria</em>. Newly developed pipelines for the Third-generation sequencing data provided high-resolution species-level identification in samples with mixed infections. These findings confirm the Eurasian wolf as natural DH for multiple <em>Sarcocystis</em> species for the first time, including <em>S. linearis</em>, <em>S. iberica</em>, and <em>S. venatoria</em>. Further complementary studies on prey species are needed to clarify host-parasite dynamics.</div></div>","PeriodicalId":54278,"journal":{"name":"International Journal for Parasitology-Parasites and Wildlife","volume":"28 ","pages":"Article 101140"},"PeriodicalIF":2.2000,"publicationDate":"2025-09-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"High prevalence of Sarcocystis spp. in the Eurasian wolf (Canis lupus lupus): Third-generation sequencing resolves mixed infections\",\"authors\":\"Sinah Lückner , Gastón Moré , Iris Marti , Caroline F. Frey , Javier E. Fernandez , Chahrazed Belhout , Walter Basso\",\"doi\":\"10.1016/j.ijppaw.2025.101140\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><div><em>Sarcocystis</em> spp. (Apicomplexa: Coccidia) are obligate heteroxenous protozoa that infect a wide range of host species. Transmission follows a predator-prey cycle involving an intermediate host (IH) and a definitive host (DH). For many species, only IHs have been identified, while DHs remain unknown. DHs can be infected with multiple <em>Sarcocystis</em> spp. at the same time, which complicates species identification. We aimed to determine the prevalence and species diversity of <em>Sarcocystis</em> infections in free-ranging wolves in Switzerland using both coprological and molecular methods. A further goal was to evaluate the utility of Third-generation sequencing for resolving mixed infections. A total of 87 wolf intestinal content samples were collected between 2017 and 2023 and analyzed coproscopically by a sedimentation-flotation method. <em>Sarcocystis</em> oocysts/sporocysts were detected in 76 % (66/87). DNA was obtained from 57/66 positive samples and 55/57 resulted positive in a <em>Sarcocystis 18S</em> rRNA screening PCR. Additionally, mitochondrial cytochrome <em>c</em> oxidase subunit I (<em>COI</em>) gene PCR and a real-time PCR targeting <em>S. cruzi</em> were performed. PCR products from conventional PCRs were submitted for Sanger sequencing. Monoinfections were identified in 16 % (9/55) and mixed infections in 84 % (46/55) of the samples. A subset of five samples was analyzed by Third-generation sequencing (Pacific Biosciences) of the <em>18S</em> rRNA full-length and <em>COI</em> fragment PCR products. BLAST and phylogenetic analysis were used to validate taxonomic classification. Molecular analysis identified nine known <em>Sarcocystis</em> species: <em>S. tenella, S. arieticanis, S. capreolicanis, S. linearis, S. gracilis, S. cruzi, S. capracanis, S. iberica,</em> and <em>S. venatoria</em>. Newly developed pipelines for the Third-generation sequencing data provided high-resolution species-level identification in samples with mixed infections. These findings confirm the Eurasian wolf as natural DH for multiple <em>Sarcocystis</em> species for the first time, including <em>S. linearis</em>, <em>S. iberica</em>, and <em>S. venatoria</em>. Further complementary studies on prey species are needed to clarify host-parasite dynamics.</div></div>\",\"PeriodicalId\":54278,\"journal\":{\"name\":\"International Journal for Parasitology-Parasites and Wildlife\",\"volume\":\"28 \",\"pages\":\"Article 101140\"},\"PeriodicalIF\":2.2000,\"publicationDate\":\"2025-09-22\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"International Journal for Parasitology-Parasites and Wildlife\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/S2213224425001051\",\"RegionNum\":3,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q3\",\"JCRName\":\"ECOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"International Journal for Parasitology-Parasites and Wildlife","FirstCategoryId":"3","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S2213224425001051","RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"ECOLOGY","Score":null,"Total":0}
High prevalence of Sarcocystis spp. in the Eurasian wolf (Canis lupus lupus): Third-generation sequencing resolves mixed infections
Sarcocystis spp. (Apicomplexa: Coccidia) are obligate heteroxenous protozoa that infect a wide range of host species. Transmission follows a predator-prey cycle involving an intermediate host (IH) and a definitive host (DH). For many species, only IHs have been identified, while DHs remain unknown. DHs can be infected with multiple Sarcocystis spp. at the same time, which complicates species identification. We aimed to determine the prevalence and species diversity of Sarcocystis infections in free-ranging wolves in Switzerland using both coprological and molecular methods. A further goal was to evaluate the utility of Third-generation sequencing for resolving mixed infections. A total of 87 wolf intestinal content samples were collected between 2017 and 2023 and analyzed coproscopically by a sedimentation-flotation method. Sarcocystis oocysts/sporocysts were detected in 76 % (66/87). DNA was obtained from 57/66 positive samples and 55/57 resulted positive in a Sarcocystis 18S rRNA screening PCR. Additionally, mitochondrial cytochrome c oxidase subunit I (COI) gene PCR and a real-time PCR targeting S. cruzi were performed. PCR products from conventional PCRs were submitted for Sanger sequencing. Monoinfections were identified in 16 % (9/55) and mixed infections in 84 % (46/55) of the samples. A subset of five samples was analyzed by Third-generation sequencing (Pacific Biosciences) of the 18S rRNA full-length and COI fragment PCR products. BLAST and phylogenetic analysis were used to validate taxonomic classification. Molecular analysis identified nine known Sarcocystis species: S. tenella, S. arieticanis, S. capreolicanis, S. linearis, S. gracilis, S. cruzi, S. capracanis, S. iberica, and S. venatoria. Newly developed pipelines for the Third-generation sequencing data provided high-resolution species-level identification in samples with mixed infections. These findings confirm the Eurasian wolf as natural DH for multiple Sarcocystis species for the first time, including S. linearis, S. iberica, and S. venatoria. Further complementary studies on prey species are needed to clarify host-parasite dynamics.
期刊介绍:
The International Journal for Parasitology: Parasites and Wildlife (IJP-PAW) publishes the results of original research on parasites of all wildlife, invertebrate and vertebrate. This includes free-ranging, wild populations, as well as captive wildlife, semi-domesticated species (e.g. reindeer) and farmed populations of recently domesticated or wild-captured species (e.g. cultured fishes). Articles on all aspects of wildlife parasitology are welcomed including taxonomy, biodiversity and distribution, ecology and epidemiology, population biology and host-parasite relationships. The impact of parasites on the health and conservation of wildlife is seen as an important area covered by the journal especially the potential role of environmental factors, for example climate. Also important to the journal is ''one health'' and the nature of interactions between wildlife, people and domestic animals, including disease emergence and zoonoses.