Microsporidia – Emergent Pathogens in the Global Food Chain

Trends in Parasitology - Tập 32 - Trang 336-348 - 2016
G.D. Stentiford1, -->J.J. Becnel2, L.M. Weiss3, P.J. Keeling4, E.S. Didier5, B-->.A.P. Williams6, S. Bjornson7, M-->.L. Kent8, M.A. Freeman9, M.J.F. Brown10, E-->.R. Troemel11, K. Roesel12, Y. Sokolova13, K.F. Snowden14, L. Solter15
1Pathology and Molecular Systematics Team, Centre for Environment, Fisheries and Aquaculture Science (CEFAS), Barrack Road, Weymouth, Dorset DT4 8UB, UK
2United States Department of Agriculture (USDA) Agricultural Research Center (ARS), Center for Medical, Agricultural, and Veterinary Entomology (CMAVE), 1600 South West 23rd Drive, Gainesville, FL, 32608, USA
3Albert Einstein College of Medicine, 1300 Morris Park Avenue, Forchheimer 504, Bronx, NY 10641, USA
4Canadian Institute for Advanced Research, Botany Department, University of British Columbia, 3529-6270 University Boulevard, Vancouver, BC V6T 1Z4, Canada
5Division of Microbiology, Tulane National Primate Research Center and Department of Tropical Medicine, School of Public Health and Tropical Medicine, Tulane University, 1440 Canal Street, New Orleans, LA 70112, USA
6Biosciences, College of Life and Environmental Sciences, University of Exeter, Geoffrey Pope, Stocker Road, Exeter EX4 4QD, UK
7Department of Biology, Saint Mary's University, 923 Robie Street, Halifax, Nova Scotia, Canada
8Departments of Microbiology and Biomedical Sciences, 220 Nash Hall, Oregon State University, Corvallis, OR 97331, USA
9Ross University School of Veterinary Medicine, St. Kitts, West Indies
10School of Biological Sciences, Royal Holloway, University of London, Egham, Surrey TW20 0EX, UK
11University of California, San Diego, 4202 Bonner Hall, 9500 Gilman Drive #0349, La Jolla, CA 92093-0349, USA
12International Livestock Research Institute, c/o Freie Universität Berlin, Institute of Parasitology and Tropical Veterinary Medicine, Robert-von-Ostertag-Strasse 7-13, Berlin, 14163 Germany
13Department of Comparative Biomedical Sciences, Louisiana State University, School of Veterinary Medicine, 1909 Skip Bertman Drive, Baton Rouge LA 70803, USA
14Texas A&M University, College of Veterinary Medicine and Biomedical Sciences, Department of Veterinary Pathobiology, Mailstop 4467, College Station, TX 77843-4467, USA
15Illinois Natural History Survey, Prairie Research Institute at the University of Illinois at Urbana-Champaign, 1816 South Oak Street, Champaign, IL 61820, USA

Tài liệu tham khảo

Gajadhar, 2006, Overview of food- and water-borne zoonotic parasites at the farm level, Rev. Sci. Tech., 25, 595, 10.20506/rst.25.2.1679 WHO, 2007 Doyle, 2003 Centers For Disease Control and Prevention, 2011 MacKenzie, 1994, A massive outbreak in Milwaukee of Cryptosporidium infection transmitted through the public water supply, N. Engl. J. Med., 331, 161, 10.1056/NEJM199407213310304 Simarro, 2012, Human African trypanosomiasis in non-endemic countries (2000–2010), J. Travel Med., 19, 44, 10.1111/j.1708-8305.2011.00576.x Slifko, 2000, Emerging parasite zoonoses associated with water and food, Int. J. Parasitol., 30, 1379, 10.1016/S0020-7519(00)00128-4 Sak, 2008, First report of Enterocytozoon bieneusi infection on a pig farm in the Czech Republic, Vet. Parasitol., 153, 220, 10.1016/j.vetpar.2008.01.043 Stentiford, 2013, Microsporidia: diverse, dynamic and emergent pathogens in aquatic systems, Trends Parasitol., 29, 567, 10.1016/j.pt.2013.08.005 Vossbrinck, 2005, Molecular phylogeny of the Microsporidia: ecological, ultrastructural and taxonomic considerations, Folia Parasitol., 52, 131, 10.14411/fp.2005.017 Stentiford, 2013, Plastic parasites: extreme dimorphism creates a taxonomic conundrum in the phylum Microsporidia, Int. J. Parasitol., 43, 339, 10.1016/j.ijpara.2012.11.010 Cavalier-Smith, 1983, A 6-kingdom classification and a unified phylogeny, 1027 Keeling, 2009, Five things to know about Microsporidia, PLoS Pathog., 5, e1000489, 10.1371/journal.ppat.1000489 Katinka, 2001, Genome sequence and gene compaction of the eukaryote parasite Encephalitozoon cuniculi, Nature, 414, 450, 10.1038/35106579 Thomarat, 2004, Phylogenetic analysis of the complete genome sequence of Encephalitozoon cuniculi supports the fungal origin of microsporidia and reveals a high frequency of fast-evolving genes, J. Mol. Evol., 59, 780, 10.1007/s00239-004-2673-0 Williams, 2002, A mitochondrial remnant in the microsporidian Trachipleistophora hominis, Nature, 418, 865, 10.1038/nature00949 Gill, 2006, Assessing the microsporidia-fungi relationship: Combined phylogenetic analysis of eight genes, Gene, 375, 103, 10.1016/j.gene.2006.02.023 Tanabe, 2002, Are Microsporidia really related to Fungi? A reappraisal based on additional gene sequences from basal fungi, Mycol. Res., 106, 1380, 10.1017/S095375620200686X Capella-Gutierrez, 2012, Phylogenomics supports microsporidia as the earliest diverging clade of sequenced fungi, BMC Biol., 10, 47, 10.1186/1741-7007-10-47 Jones, 2011, Discovery of novel intermediate forms redefines the fungal tree of life, Nature, 474, 200, 10.1038/nature09984 Jones, 2011, Validation and justification of the phylum name Cryptomycota phyl. nov, IMA Fungus, 2, 173, 10.5598/imafungus.2011.02.02.08 Karpov, 2014, Morphology, phylogeny, and ecology of the aphelids (Aphelidea, Opisthokonta) and proposal for the new superphylum Opisthosporidia, Front. Microbiol., 5, 112, 10.3389/fmicb.2014.00112 Karpov, 2013, Obligately phagotrophic aphelids turned out to branch with the earliest-diverging Fungi, Protist, 164, 195, 10.1016/j.protis.2012.08.001 Haag, 2014, Evolution of a morphological novelty occurred before genome compaction in a lineage of extreme parasites, Proc. Natl. Acad. Sci. U.S.A., 111, 15480, 10.1073/pnas.1410442111 Keeling, 2014, Phylogenetic place of Microsporidia in the tree of eukaryotes, 195 Becnel, 2014, Checklist of available generic names for microsporidia with type species and type hosts, 671 Vossbrinck, 2014, Phylogeny of the Microsporidia, 203 Vavra, 2013, Microsporidia and ‘the art of living together’, Adv. Parasitol., 82, 254 Didier, 2014, The immunology of microsporidiosis in mammals, 307 Bednarska, 2014, Occurrence of intestinal microsporidia in immunodeficient patients in Poland, Ann. Agric. Environ. Med., 21, 244, 10.5604/1232-1966.1108584 Sprague, 1974, Nosema connori n. sp., a microsporidian parasite of man, Trans. Am. Microscop. Soc., 93, 400, 10.2307/3225442 Desportes, 1985, Occurrence of a new microsporidan: Enterocytozoon bieneusi n.g., n. sp., in the enterocytes of a human patient with AIDS, J. Protozool., 32, 250, 10.1111/j.1550-7408.1985.tb03046.x Orenstein, 1997, Disseminated microsporidiosis in AIDS: are any organs spared?, AIDS, 11, 385 Fayer, 2014, Epidemiology of microsporidia in human infections, 135 Sak, 2011, Latent microsporidial infection in immunocompetent individuals – a longitudinal study, PLoS Negl. Trop. Dis., 5, e1162, 10.1371/journal.pntd.0001162 Lobo, 2012, Microsporidia as emerging pathogens and the implication for public health: a 10-year study on HIV-positive and -negative patients, Int. J. Parasitol., 42, 197, 10.1016/j.ijpara.2011.12.002 Matos, 2012, Epidemiology of Enterocytozoon bieneusi infection in humans, J. Parasitol. Res., 2012, 981424, 10.1155/2012/981424 Lores, 2002, Intestinal microsporidiosis due to Enterocytozoon bieneusi in elderly human immunodeficiency virus-negative patients from Vigo, Spain, Clin. Infect. Dis., 34, 918, 10.1086/339205 National Institute of Aging WHO, 2011 Didier, 2011, Microsporidiosis: not just in AIDS patients, Curr. Opin. Infect. Dis., 24, 490, 10.1097/QCO.0b013e32834aa152 Li, 2003, Infectivity of microsporidia spores stored in water at environmental temperatures, J. Parasitol., 89, 185, 10.1645/0022-3395(2003)089[0185:IOMSSI]2.0.CO;2 Graczyk, 2004, Human waterborne parasites in zebra mussels (Dreissena polymorpha) from the Shannon River drainage area, Ireland, Parasitol. Res., 93, 385, 10.1007/s00436-004-1142-4 Lucy, 2008, Biomonitoring of surface and coastal water for Cryptosporidium, Giardia and human virulent microsporidia using molluscan shellfish, Parasitol. Res., 103, 1369, 10.1007/s00436-008-1143-9 Santin, 2009, Enterocytozoon bieneusi genotype nomenclature based on the internal transcribed spacer sequence: a consensus, J. Eukaryot. Microbiol., 56, 34, 10.1111/j.1550-7408.2008.00380.x Stentiford, 2011, Hepatospora eriocheir (Wang & Chen, 2007) gen. et comb. nov. from European Chinese mitten crabs (Eriocheir sinensis), J. Invertebr. Pathol., 108, 156, 10.1016/j.jip.2011.07.008 Jedrzejewski, 2007, Quantitative assessment of contamination of fresh food produce of various retail types by human-virulent microsporidian spores, Appl. Environ. Microbiol., 73, 4071, 10.1128/AEM.00477-07 Lee, 2008, Molecular detection of Enterocytozoon bieneusi and identification of a potentially human-pathogenic genotype in milk, Appl. Environ. Microbiol., 74, 1664, 10.1128/AEM.02110-07 Decreane, 2012, First reported foodborne outbreak associated with microsporidia, Sweden, October 2009, Epidemiol. Infect., 140, 519, 10.1017/S095026881100077X Coyle, 2004, Fatal myositis due to the microsporidian Brachiola algerae, a mosquito pathogen, N. Engl. J. Med., 351, 42, 10.1056/NEJMoa032655 Choudary, 2011, Tubulinosema sp. microsporidian myositis in immunosuppressed patient, Emerg. Infect. Dis., 17, 1727, 10.3201/eid1709.101926 Vávra, 1998, Microsporidia of the genus Trachipleistophora – causative agents of human microsporidiosis: description of Trachipleistophora anthropophthera n. sp. (Protozoa: Microsporidia), J. Eukaryot. Microbiol., 45, 273, 10.1111/j.1550-7408.1998.tb04536.x Cali, 2003, Ultrastructure and development of Pleistophora ronneafiei n. sp., a microsporidium (Protista) in the skeletal muscle of an immuno-compromised individual, J. Eukaryot. Microbiol., 50, 77, 10.1111/j.1550-7408.2003.tb00237.x Chupp, 1993, Myositis due to Pleistophora (Microsporidia) in a patient with AIDS, Clin. Infect. Dis., 16, 15, 10.1093/clinids/16.1.15 Reetz, 2002, First detection of the microsporidium Enterocytozoon bieneusi in non-mammalian hosts (chickens), Int. J. Parasitol., 32, 785, 10.1016/S0020-7519(02)00045-0 Fayer, 2003, Detection of Encephalitozoon hellem in feces of experimentally infected chickens, J. Eukaryot. Microbiol., 50, 574, 10.1111/j.1550-7408.2003.tb00634.x Zhang, 2011, Identification and genotyping of Enterocytozoon bieneusi in China, J. Clin. Microbiol., 49, 2006, 10.1128/JCM.00372-11 Cama, 2007, Transmission of Enterocytozoon bieneusi between a child and guinea pigs, J. Clin. Microbiol., 45, 2708, 10.1128/JCM.00725-07 Velásquez, 2012, First case report of infection caused by Encephalitozoon intestinalis in a domestic cat and a patient with AIDS, Vet. Parasitol., 190, 583, 10.1016/j.vetpar.2012.06.034 Bjørnson, 2014, Microsporidia biological control agents and pathogens of beneficial insects, 635 Geden, 1992, Rapid deterioration of searching behavior, host destruction, and fecundity of the parasitoid Muscidifurax raptor (Hymenoptera: Pteromalidae) in culture, Ann. Entomol. Soc. Am., 85, 179, 10.1093/aesa/85.2.179 Geden, 1995, Nosema disease of the parasitoid Muscidifurax raptor (Hymenoptera: Pteromalidae): prevalence, patterns of transmission, management, and impact, Biol. Control, 5, 607, 10.1006/bcon.1995.1072 van Huis, 2013 Potts, 2010, Global pollinator declines: impacts, trends and drivers, Trends Ecol. Evol., 25, 345, 10.1016/j.tree.2010.01.007 Cornman, 2012, Pathogen webs in collapsing honeybee colonies, PLoS ONE, 7, e43562, 10.1371/journal.pone.0043562 Fries, 1993, Nosema apis – a parasite in the honeybee colony, Bee World, 74, 5, 10.1080/0005772X.1993.11099149 Fries, 1996, Nosema ceranae n. sp. (Microspora, Nosematidae), morphological and molecular characterization of a microsporidian parasite of the Asian honeybee Apis cerana (Hymenoptera, Apidae), Eur. J. Protistol., 32, 356, 10.1016/S0932-4739(96)80059-9 Van Engelsdorp, 2009, Colony collapse disorder: a descriptive study, PLoS ONE, 4, e6481, 10.1371/journal.pone.0006481 Dainat, 2012, Colony collapse disorder in Europe, Environ. Microbiol. Rep., 4, 123, 10.1111/j.1758-2229.2011.00312.x Cameron, 2011, Patterns of widespread decline in North American bumble bees, Proc. Natl. Acad. Sci. U.S.A., 108, 662, 10.1073/pnas.1014743108 Daszak, 2000, Emerging infectious diseases of wildlife – threats to biodiversity and human health, Science, 287, 443, 10.1126/science.287.5452.443 Ratnieks, 2010, Clarity on honey bee collapse?, Science, 327, 152, 10.1126/science.1185563 Fürst, 2014, Disease associations between honeybees and bumblebees as a threat to wild pollinators, Nature, 506, 364, 10.1038/nature12977 Brown, 2011, The trouble with bumblebees, Nature, 469, 169, 10.1038/469169a Murray, 2013, Pathogen prevalence in commercially reared bumble bees and evidence of spillover in conspecific populations, Biol. Conserv., 159, 269, 10.1016/j.biocon.2012.10.021 Kent, 2014, Microsporidia in fish, 493 Palenzuela, 2014, A new intranuclear microsporidium, Enterospora nucleophila n. sp., causing an emaciative syndrome in a piscine host (Sparus aurata), prompts the redescription of the family Enterocytozoonidae, Int. J. Parasitol., 44, 189, 10.1016/j.ijpara.2013.10.005 Ramsay, 2009, Pseudoloma neurophilia infections in zebrafish Danio rerio: effects of stress on survival, growth, and reproduction, Dis. Aquat. Org., 88, 69, 10.3354/dao02145 Wongtavatchai, 1995, Effects of the microsporidian Enterocytozoon salmonis on the immune response of chinook salmon, Vet. Immunol. Immunopathol., 48, 367, 10.1016/0165-2427(95)05435-9 Stentiford, 2012, Disease will limit future food supply from global crustacean fishery and aquaculture sectors, J. Invertebr. Pathol., 110, 141, 10.1016/j.jip.2012.03.013 Stentiford, 2010, Myospora metanephrops (n. g., n. sp.) from marine lobsters and a proposal for erection of a new order and family (Crustaceacida; Myosporidae) in the class Marinosporidia (Phylum Microsporidia), Int. J. Parasitol., 40, 1433, 10.1016/j.ijpara.2010.04.017 Stentiford, 2014, Areospora rohanae n.gn. n.sp. (Microsporidia; Areosporiidae n. fam.) elicits multi-nucleate giant-cell formation in crabs, J. Invert. Pathol., 118, 1, 10.1016/j.jip.2014.02.004 Tourtip, 2009, Enterocytozoon hepatopenaei sp. nov. (Microspora: Enterocytozoonidae), a parasite of the black tiger shrimp Penaeus monodon (Decapoda: Penaeidae): fine structure and phylogenetic relationships, J. Invert. Pathol., 102, 21, 10.1016/j.jip.2009.06.004 Tangprasittipap, 2013, The microsporidian Enterocytozoon hepatopenaei is not the cause of white feces syndrome in whiteleg shrimp Penaeus (Litopenaeus) vannamei, BMC Vet. Res., 9, 139, 10.1186/1746-6148-9-139 Stentiford, 2007, Enterospora canceri n.gen., n.sp., an intranuclear microsporidian infecting European edible crab (Cancer pagurus), Dis. Aquat. Org., 75, 61, 10.3354/dao075061 Doyle, 2014, Inbreeding and disease in tropical shrimp aquaculture: a reappraisal and caution, Aquacult. Res., 2014, 1 Santin, 2009, A longitudinal study of Enterocytozoon bieneusi in dairy cattle, Parasitol. Res., 105, 141, 10.1007/s00436-009-1374-4 Jeong, 2007, Occurrence and genotypic characteristics of Enterocytozoon bieneusi in pigs with diarrhea, Parasitol. Res., 102, 123, 10.1007/s00436-007-0740-3 Snowden, 2014, Microsporidia in higher vertebrates, 469 Cislakova, 2001, Prevalence of antibodies to Encephalitozoon cuniculi (Microsporidia) in angora goats – a potential risk of infection for breeders, Ann. Agric. Environ. Med., 8, 289 Goodwin, 2006, Prevalence of antibodies to Encephalitozoon cuniculi in horses from Brazil, Vet. Parasitol., 142, 380, 10.1016/j.vetpar.2006.07.006 Santin, 2010, A zoonotic genotype of Enterocytozoon bieneusi in horses, J. Parasitol., 96, 157, 10.1645/GE-2184.1 Ozcan, 2011, Encephalitozoonosis in New Zealand rabbits and potential transmission risk, Vet. Parasitol., 179, 234, 10.1016/j.vetpar.2011.02.007 Bass, 2015, Diverse applications of environmental DNA methods in parasitology, Trends Parasitol., 31, 499, 10.1016/j.pt.2015.06.013 Vávra, 2014, Structure of microsporidia, 1 Larsson, 2014, The primitive microsporidia, 605 Cali, 2014, Developmental morphology and life cycles of the microsporidia, 71 Cali, 2010, Human vocal cord infection with the microsporidium Anncaliia algerae, J. Eukaryot. Microbiol., 57, 562, 10.1111/j.1550-7408.2010.00510.x Watts, 2014, Anncaliia algerae microsporidial myositis, Emerg. Infect. Dis., 20, 185, 10.3201/eid2002.131126 Franzen, 2006, Transfer of the members of the genus Brachiola (microsporidia) to the genus Anncaliia based on ultrastructural and molecular data, J. Eukaryot. Microbiol., 53, 26, 10.1111/j.1550-7408.2005.00066.x Margileth, 1973, Disseminated nosematosis in an immunologically compromised infant, Arch. Pathol., 95, 145 Cali, 1998, Brachiola vesicularum, n. g., n. sp., a new microsporidium associated with AIDS and myositis, J. Eukaryot. Microbiol., 45, 240, 10.1111/j.1550-7408.1998.tb04532.x Hocevar, 2014, Microsporidiosis acquired through solid organ transplantation: a public health investigation, Ann. Intern. Med., 160, 213, 10.7326/M13-2226 Norhayati, 2008, A preliminary study on the prevalence of intestinal microsporidiosis in patients with and without gastrointestinal symptoms in Malaysia, Trans. R. Soc. Trop. Med. Hyg., 102, 1274, 10.1016/j.trstmh.2008.05.019 Orenstein, 2005, Fatal pulmonary microsporidiosis due to Encephalitozoon cuniculi following allogeneic bone marrow transplantation for acute myelogenous leukemia, Ultrastruct. Pathol., 29, 269, 10.1080/01913120590951257 Sharma, 2011, Microsporidial keratitis: need for increased awareness, Surv. Ophthalmol., 56, 1, 10.1016/j.survophthal.2010.03.006 Chabchoub, 2009, Genetic identification of intestinal microsporidia species in immunocompromised patients in Tunisia, Am. J. Trop. Med. Hyg., 80, 24, 10.4269/ajtmh.2009.80.24 Didier, 1991, Isolation and characterization of a new human microsporidian, Encephalitozoon hellem (n. sp.), from three AIDS patients with keratoconjunctivitis, J. Infect. Dis., 163, 617, 10.1093/infdis/163.3.617 Sharma, 2014, Ocular microsporidiosis, 403 Cali, 1993, Septata intestinalis n. g., n. sp., an intestinal microsporidian associated with chronic diarrhea and dissemination in AIDS patients, J. Eukaryot. Microbiol., 40, 101, 10.1111/j.1550-7408.1993.tb04889.x Hamamci, 2015, Prevalence of Encephalitozoon intestinalis and Enterocytozoon bieneusi in cancer patients under chemotherapy, Mikrobiyol. Bul., 49, 105, 10.5578/mb.8787 Jimenez-Gonzalez, 2012, Microsporidia in pediatric patients with leukemia or limphoma, Rev. Invest. Clin., 64, 25 Orenstein, 1992, Systemic dissemination by a newly recognized intestinal microsporidia species in AIDS, AIDS, 6, 1143, 10.1097/00002030-199210000-00013 Sivgin, 2013, Encephalitozoon intestinalis: a rare cause of diarrhea in an allogeneic hematopoietic stem cell transplantation (HSCT) recipient complicated by albendazole-related hepatotoxicity, Turk. J. Hematol., 30, 204, 10.4274/Tjh.90692 Teachey, 2004, Pulmonary infection with microsporidia after allogeneic bone marrow transplantation, Bone Marrow Transpl., 33, 299, 10.1038/sj.bmt.1704327 Carlson, 2004, Disseminated microsporidiosis in a pancreas/kidney transplant recipient, Arch. Pathol. Lab. Med., 128, e41, 10.5858/2004-128-e41-DMIAKT Galvan, 2011, First cases of microsporidiosis in transplant recipients in Spain and review of the literature, J. Clin. Microbiol., 49, 1301, 10.1128/JCM.01833-10 Rabodonirina, 1996, Enterocytozoon bieneusi as a cause of chronic diarrhea in a heart-lung transplant recipient who was seronegative for human immunodeficiency virus, Clin. Infect. Dis., 23, 114, 10.1093/clinids/23.1.114 Cali, 1991, Corneal microsporidioses: characterization and identification, J. Protozool., 38, 215S Pariyakanok, 2005, Keratitis caused by Trachipleistophora anthropopthera, J. Infect., 51, 325, 10.1016/j.jinf.2004.08.030 Choudhary, 2011, Tubulinosema sp. microsporidian myositis in immunosuppressed patient, Emerg. Infect. Dis., 17, 1727, 10.3201/eid1709.101926 Shadduck, 1990, Isolation of a microsporidian from a human patient, J. Infect. Dis., 162, 773, 10.1093/infdis/162.3.773 Fernandes, 2013, Polymicrobial and microsporidial keratitis in a pathien using Boston scleral contact lens for Sjögren's syndrome and ocular pemphigoid, Cont. Lens Anterior Eye, 36, 95, 10.1016/j.clae.2012.10.082 Hartskeerl, 1993, Genetic evidence for the occurrence of extra-intestinal Enterocytozoon bieneusi infections, Nucleic Acids Res., 21, 4150, 10.1093/nar/21.17.4150 Del Aguila, 1997, Identification of Enterocytozoon bieneusi spores in respiratory samples from an AIDS patient with a 2-year history of intestinal microsporidiosis, J. Clin. Microbiol., 35, 1862, 10.1128/JCM.35.7.1862-1866.1997 Rutrecht, 2009, Differential virulence in a multiple-host parasite of bumble bees: resolving the paradox of parasite survival?, Oikos, 118, 941, 10.1111/j.1600-0706.2009.17392.x Mayack, 2009, Energetic stress in the honeybee Apis mellifera from Nosema ceranae infection, J. Invert. Pathol., 100, 185, 10.1016/j.jip.2008.12.001 Huang, 2013, Nosema ceranae escapes fumagillin control in honey bees, PLOS Pathog., 9, e1003185, 10.1371/journal.ppat.1003185