An experimental genetically attenuated live vaccine to prevent transmission of  Toxoplasma gondii  by cats

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Abstract

Almost any warm-blooded creature can be an intermediate host for Toxoplasma gondii. However, sexual reproduction of T. gondii occurs only in felids, wherein fertilisation of haploid macrogametes by haploid microgametes, results in diploid zygotes, around which a protective wall develops, forming unsporulated oocysts. Unsporulated oocysts are shed in the faeces of cats and meiosis gives rise to haploid sporozoites within the oocysts. These, now infectious, sporulated oocysts contaminate the environment as a source of infection for people and their livestock. RNA-Seq analysis of cat enteric stages of T. gondii uncovered genes expressed uniquely in microgametes and macrogametes. A CRISPR/Cas9 strategy was used to create a T. gondii strain that exhibits defective fertilisation, decreased fecundity and generates oocysts that fail to produce sporozoites. Inoculation of cats with this engineered parasite strain totally prevented oocyst excretion following infection with wild-type T. gondii, demonstrating that this mutant is an attenuated, live, transmission-blocking vaccine.

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Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2

Michael Love, Wolfgang Huber, Simon Anders (2014)

In comparative high-throughput sequencing assays, a fundamental task is the analysis of count data, such as read counts per gene in RNA-seq, for evidence of systematic changes across experimental conditions. Small replicate numbers, discreteness, large dynamic range and the presence of outliers require a suitable statistical approach. We present DESeq2, a method for differential analysis of count data, using shrinkage estimation for dispersions and fold changes to improve stability and interpretability of estimates. This enables a more quantitative analysis focused on the strength rather than the mere presence of differential expression. The DESeq2 package is available at http://www.bioconductor.org/packages/release/bioc/html/DESeq2.html. Electronic supplementary material The online version of this article (doi:10.1186/s13059-014-0550-8) contains supplementary material, which is available to authorized users.

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Trimmomatic: a flexible trimmer for Illumina sequence data

Anthony M. Bolger, Marc Lohse, Bjoern Usadel (2014)

Motivation: Although many next-generation sequencing (NGS) read preprocessing tools already existed, we could not find any tool or combination of tools that met our requirements in terms of flexibility, correct handling of paired-end data and high performance. We have developed Trimmomatic as a more flexible and efficient preprocessing tool, which could correctly handle paired-end data. Results: The value of NGS read preprocessing is demonstrated for both reference-based and reference-free tasks. Trimmomatic is shown to produce output that is at least competitive with, and in many cases superior to, that produced by other tools, in all scenarios tested. Availability and implementation: Trimmomatic is licensed under GPL V3. It is cross-platform (Java 1.5+ required) and available at http://www.usadellab.org/cms/index.php?page=trimmomatic Contact: usadel@bio1.rwth-aachen.de Supplementary information: Supplementary data are available at Bioinformatics online.

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STAR: ultrafast universal RNA-seq aligner.

Alexander Dobin, Carrie A. Davis, Felix Schlesinger … (2013)

Accurate alignment of high-throughput RNA-seq data is a challenging and yet unsolved problem because of the non-contiguous transcript structure, relatively short read lengths and constantly increasing throughput of the sequencing technologies. Currently available RNA-seq aligners suffer from high mapping error rates, low mapping speed, read length limitation and mapping biases. To align our large (>80 billon reads) ENCODE Transcriptome RNA-seq dataset, we developed the Spliced Transcripts Alignment to a Reference (STAR) software based on a previously undescribed RNA-seq alignment algorithm that uses sequential maximum mappable seed search in uncompressed suffix arrays followed by seed clustering and stitching procedure. STAR outperforms other aligners by a factor of >50 in mapping speed, aligning to the human genome 550 million 2 × 76 bp paired-end reads per hour on a modest 12-core server, while at the same time improving alignment sensitivity and precision. In addition to unbiased de novo detection of canonical junctions, STAR can discover non-canonical splices and chimeric (fusion) transcripts, and is also capable of mapping full-length RNA sequences. Using Roche 454 sequencing of reverse transcription polymerase chain reaction amplicons, we experimentally validated 1960 novel intergenic splice junctions with an 80-90% success rate, corroborating the high precision of the STAR mapping strategy. STAR is implemented as a standalone C++ code. STAR is free open source software distributed under GPLv3 license and can be downloaded from http://code.google.com/p/rna-star/.

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Author and article information

Contributors

Adrian B. Hehl: adrian.hehl@uzh.ch

Peter Deplazes: deplazesp@access.uzh.ch

Nicholas C. Smith:

ORCID: http://orcid.org/0000-0002-7467-1451

nick.smith@parasite.org.au

Journal

Journal ID (nlm-ta): Sci Rep

Journal ID (iso-abbrev): Sci Rep

Title: Scientific Reports

Publisher: Nature Publishing Group UK (London )

ISSN (Electronic): 2045-2322

Publication date (Electronic): 6 February 2019

Publication date PMC-release: 6 February 2019

Publication date Collection: 2019

Volume: 9

Electronic Location Identifier: 1474

Affiliations

[1 ]ISNI 0000 0004 1937 0650, GRID grid.7400.3, Institute of Parasitology, University of Zürich, ; Winterthurerstrasse 266a, 8057 Zürich, Switzerland

[2 ]ISNI 0000 0001 2156 2780, GRID grid.5801.c, Functional Genomics Center Zürich, ; Winterthurerstrasse 190, 8057 Zürich, Switzerland

[3 ]Molecular Parasitology Section, Laboratory of Parasitic Diseases, NIAID, NIH, Bethesda, Maryland USA

[4 ]ISNI 0000 0001 2180 7477, GRID grid.1001.0, Research School of Biology, , Australian National University, ; Canberra, ACT 0200 Australia

[5 ]ISNI 0000 0000 9939 5719, GRID grid.1029.a, School of Science and Health, , Western Sydney University, ; Parramatta South Campus, Sydney, NSW 2116 Australia

Author information

Hubert Rehrauer http://orcid.org/0000-0001-7612-9394

Nicholas C. Smith http://orcid.org/0000-0002-7467-1451

Article

Publisher ID: 37671

DOI: 10.1038/s41598-018-37671-8

PMC ID: 6365665

PubMed ID: 30728393

SO-VID: 34c399c4-850e-407d-84ea-1a71bedac5e2

License:

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

History

Date received : 16 August 2018

Date accepted : 11 December 2018

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An experimental genetically attenuated live vaccine to prevent transmission of Toxoplasma gondii by cats

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Decoding Infection and Transmission

Most cited references 64

Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2

Trimmomatic: a flexible trimmer for Illumina sequence data

STAR: ultrafast universal RNA-seq aligner.

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