title: The utility of diversity profiling using Illumina 18S rRNA gene amplicon deep sequencing to detect and discriminate Toxoplasma gondii among the cyst-forming coccidia.
abstract: Next-generation sequencing (NGS) has the capacity to screen a single DNA sample and detect pathogen DNA from thousands of host DNA sequence reads, making it a versatile and informative tool for investigation of pathogens in diseased animals. The technique is effective and labor saving in the initial identification of pathogens, and will complement conventional diagnostic tests to associate the candidate pathogen with a disease process. In this report, we investigated the utility of the diversity profiling NGS approach using Illumina small subunit ribosomal RNA (18S rRNA) gene amplicon deep sequencing to detect Toxoplasma gondii in previously confirmed cases of toxoplasmosis. We then tested the diagnostic approach with species-specific PCR genotyping, histopathology and immunohistochemistry of toxoplasmosis in a Risso's dolphin (Grampus griseus) to systematically characterise the disease and associate causality. We show that the Euk7A/Euk570R primer set targeting the V1-V3 hypervariable region of the 18S rRNA gene can be used as a species-specific assay for cyst-forming coccidia and discriminate T. gondii. Overall, the approach is cost-effective and improves diagnostic decision support by narrowing the differential diagnosis list with more certainty than was previously possible. Furthermore, it supplements the limitations of cryptic protozoan morphology and surpasses the need for species-specific PCR primer combinations.
authors.affiliation: Faculty of Veterinary Science, University of Sydney, Sydney, NSW 2006, Australia.
authors.forename: Madalyn K
authors.initials: MK
authors.lastname: Cooper
authors.affiliation: Faculty of Veterinary Science, University of Sydney, Sydney, NSW 2006, Australia.
authors.forename: David N
authors.initials: DN
authors.lastname: Phalen
authors.affiliation: Faculty of Veterinary Science, University of Sydney, Sydney, NSW 2006, Australia.
authors.forename: Shannon L
authors.initials: SL
authors.lastname: Donahoe
authors.affiliation: Australian Registry of Wildlife Health, Taronga Conservation Society Australia, Mosman, NSW 2088, Australia.
authors.forename: Karrie
authors.initials: K
authors.lastname: Rose
authors.affiliation: Faculty of Veterinary Science, University of Sydney, Sydney, NSW 2006, Australia. Electronic address: jan.slapeta@sydney.edu.au.
authors.forename: Jan
authors.initials: J
authors.lastname: Šlapeta
mesh_terms: D046710:Agapornis / Q000469:parasitology; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000469:parasitology / Q000473:pathology; D016054:DNA, Protozoan / Q000737:chemistry / Q000302:isolation & purification; D004275:DNA, Ribosomal / Q000737:chemistry; D004289:Dolphins / Q000469:parasitology*; D046069:Fur Seals / Q000469:parasitology; D014644:Genetic Variation / Q000235:genetics*; D060005:Genotyping Techniques / Q000662:veterinary; D008297:Male; D008394:Marsupialia / Q000469:parasitology; D005810:Multigene Family / Q000235:genetics; D058885:Multilocus Sequence Typing / Q000662:veterinary; D009517:New South Wales; D011110:Polymorphism, Genetic; D012337:RNA, Ribosomal, 18S / Q000737:chemistry / Q000235:genetics*; D016415:Sequence Alignment / Q000662:veterinary; D014122:Toxoplasma / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D014124:Toxoplasmosis, Animal / Q000175:diagnosis / Q000469:parasitology* / Q000473:pathology
chemical_list: D016054:DNA, Protozoan; D004275:DNA, Ribosomal; D012337:RNA, Ribosomal, 18S
keywords: 18S rDNA; Diagnostics; Genotyping; MiSeq; Next-generation sequencing; Pathogen detection; Toxoplasma gondii