Distribution of parasite sexual developmental stages in cat intestinal ileums infected with WT, <i>Δh1</i> or <i>Δh2</i> mutant parasites.

<p>(A) Pooled counts from four (WT, <i>Δh2</i>) or six (<i>Δh1</i>) independent intestinal sections from an infected cat are summarized. (B) The relative proportion of each stage observed across the four or six samples are shown +/- SD. No significant differences in the distribution of parasite stages were seen (<i>P></i> 0.9999, Two-way ANOVA).</p

Development of oocysts following infection of cats.

<p>(A) Yields of oocysts shed from infected cats. The yield of knockout mutants as a whole were significantly reduced relative to the wild-type (Kruskal-Wallis, <i>P</i> ≤0.05), however due to low sample size, pairwise comparisons between each mutant and the WT approached, but did not reach significance (<i>Δh1 P</i> = 0.116, <i>Δh2 P</i> = 0.821, <i>Δh1Δh2 P</i> = 0.116). (B) The sporulation success rate of shed oocysts shows a significant defect in mutant lines (Dunn’s multiple comparisons test, for wild type vs. <i>Δh2 P</i> = 0.0008; and for the wild type vs. <i>Δh1-H1 P</i> = 0.0178 and <i>Δh1Δh2-H1 P</i>< 0.0001). The oocyst yields of <i>Δh1</i> and <i>Δh1Δh2</i> parasites were not sufficient to allow quantification (not done = N.D.). Each result is displayed as the Mean ±SD of three replicate counts of oocysts (n ≥ 50 per count) from one cat.</p

Disruption of the <i>AAH1</i> and <i>AAH2</i> genes.

<p>(A) Schematic of the <i>AAH2</i> knockout strategy in the wild-type ME49 <i>Δhxg</i>::<i>Luc</i> strain (referred to as WT). A CRISPR-Cas9 construct with guide RNAs targeted to the 5’ and 3’ UTRs of <i>AAH2</i> was co-transfected with the <i>pΔaah2</i>::<i>HXG</i> plasmid (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006272#ppat.1006272.s002" target="_blank">S2 Table</a>) and selected for with MPA +Xanthine to delete <i>AAH2</i> to produce the clone <i>Δaah2</i>::<i>HXG</i> (<i>Δh2-HXG</i>). Subsequently, the <i>HXG</i> gene was replaced with either a clean fusion of the <i>AAH2</i> 5’ and 3’ UTRs (<i>pΔaah2</i>) or an <i>AAH2</i> cDNA construct (<i>pAAH2</i>). (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006272#ppat.1006272.s002" target="_blank">S2 Table</a>) using 6-thioxanthine selection against the <i>HXG</i> locus to create the clean knockout clone <i>Δaah2</i> (<i>Δh2</i>) (upper panel) and the complement clone <i>Δaah2</i>::<i>AAH2</i> (<i>Δh2-H2</i>) (lower panel). Yellow Bars: CRISPR targeting sites. Black bars: PCR screening primer target regions (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006272#ppat.1006272.s003" target="_blank">S3 Table</a>). (B) Schematic of the knockout strategy for <i>AAH1</i>. A CRISPR-Cas9 construct with guide RNAs targeted to the 5’ and 3’ UTRs of <i>AAH1</i> was co-transfected with the <i>pΔaah1</i>::<i>DHFR-Ts</i> repair construct (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006272#ppat.1006272.s002" target="_blank">S2 Table</a>) into WT or <i>Δaah2</i> parasites to create the clones <i>Δaah1</i> (<i>Δh1</i>) and <i>Δaah1Δaah2</i> (<i>Δh1Δh2</i>). Transfectants were selected for via pyrimethamine resistance. Subsequently, using <i>pΔuprt</i>::<i>AAH1</i>::<i>HXG</i>, a cDNA copy of <i>AAH1</i> driven by its native 5’ and 3’ UTRs was complemented into the <i>UPRT</i> locus by means of the <i>HXGPRT</i> drug resistance marker selected for with MPA +Xanthine, negative selection against <i>UPRT</i> with FUDR, and a single-cutting CRISPR-Cas9 construct targeted to the <i>UPRT</i> gene (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006272#ppat.1006272.s002" target="_blank">S2 Table</a>), creating the complement clones <i>Δaah1-AAH1</i> (<i>Δh1-H1</i>) and <i>Δaah1Δaah2-AAH1</i> (<i>Δh1Δh2-H1</i>). Brown & Yellow Bars: CRISPR targeting sites. Black bars: PCR screening primer target regions (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006272#ppat.1006272.s003" target="_blank">S3 Table</a>). (C) PCR verification of successful ablation and complementation of knockouts. Expected product sizes: <i>Tubulin</i> (Tub): 0.378kb. <i>AAH1</i> (H1): 0.745kb (Native), 0.278kb (cDNA). <i>AAH2</i> (H2): 0.745kb (Native), 0.278kb (cDNA). (D) Growth assays of parasites seeded into 96-well plates and allowed to proliferate for 24 h, then quantified using a luciferase assay. The WT, <i>Δh1</i>, Δh2, <i>Δh1Δh2</i>, <i>Δh1-H1</i>, <i>Δh2-H2</i>, and <i>Δh1Δh2-H1</i> parasites showed no significant difference in total growth (Kruskal-Wallis test, <i>P</i> = 0.0672, N = 3 per strain).</p

Representative fluorescence microscope images of dityrosine autofluorescence in sporulated and unsporulated oocysts of WT, <i>Δh1</i>, <i>Δh2</i>, and <i>Δh1Δh2</i> oocysts.

<p>All images were taken at 1000-1600ms exposure using a DAPI UV filter, but due to rapid photobleaching and differing levels of background signal in different oocyst fecal suspensions, direct comparison and quantification of fluorescence is not feasible. Scale bar = 10μm.</p

Copy number analysis of <i>AAH2</i> in <i>T</i>. <i>gondii</i> strains.

<p>(A) Copy number variation (CNV) of Tg_ME49_212740 (<i>AAH2</i>) in five representative strains. (B) CNV at each base across the <i>AAH2</i> gene in ME49. Red lines indicate the start and stop codons of <i>AAH2</i>. (C) PCR using primers (Z28+ Z65) (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006272#ppat.1006272.s003" target="_blank">S3 Table</a>) common to both <i>AAH1</i> and <i>AAH2</i> were used against the ME49 genome to amplify both genes for Sanger sequencing and single nucleotide polymorphism (SNP) determination. (D) Sanger sequencing of PCR fragments against the <i>AAH</i> genes of the wild type ME49 <i>Δhxg</i>::<i>Luc</i> strain shows a 2:1 ratio of <i>AAH2</i> to <i>AAH1</i> SNPs, indicating a duplication of the <i>AAH2</i> gene. Each SNP in the chromatograph is marked by a red dot. In the <i>Δaah2</i>::<i>HXG</i> knockout, all <i>AAH2</i> SNPs are no longer visible, indicating loss of both copies of the <i>AAH2</i> gene.</p

Development of bradyzoites <i>in vitro</i> and <i>in vivo</i>.

<p>(A) There was no significant difference in bradyzoite differentiation <i>in vitro</i> across the parasite lines in either tachyzoite conditions (left) (<i>P</i>> 0.99) or bradyzoite conditions (right) (<i>P</i>> 0.99) (Two-way ANOVA).(B) Representative pictures of tachyzoites, partial cysts, and complete cysts produced <i>in vitro</i> as assessed by DBL staining. (C) Brain cyst yields in mice 1–2 months post-infection. All parasite lines produced similar numbers of tissue cysts <i>in vivo</i> (Kruskal-Wallis, ns).</p

Representative images of hematoxylin and eosin-stained tissue sections of cat intestinal ileum infected with WT (A, B), <i>Δh1</i> (C, D), and <i>Δh2</i> (E, F) parasites.

<p>Multiple stages of the parasite’s sexual cycle can be seen. m: merozoite, is: immature schizont, s: schizont, fg: female gamont, mg: male gamont. Scale bar = 5μm.</p

Experimental Toxoplasmosis in Rats Induced Orally with Eleven Strains of <i>Toxoplasma gondii</i> of Seven Genotypes: Tissue Tropism, Tissue Cyst Size, Neural Lesions, Tissue Cyst Rupture without Reactivation, and Ocular Lesions - Fig 7

<p>(A) Meningoencephalitis in Rat D6157 infected with the TgCatCo3 strain. Note meningitis (a), perivascular infiltration of leukocytes (b), and a glial nodule with a degenerative tissue cyst (c). PASH stained. (B) A large inflammatory focus, probably resulting from the host reaction to degenerative tissue cysts in Rat D6162 infected with theGT1 strain and HE stained.</p

Effects of landscape variables on seroprevalence.

<p>A) Observed seroprevalence in mink and otter by study site. B) Observed seroprevalence by degree of presence of domestic cat. C) Observed seroprevalence by habitat. * Number above bars indicate sample size. Y axis show mean seroprevalence.</p

Seroprevalence of <i>Toxoplasma gondii</i>.

<p>Data from 11 study sites with varying degrees of domestic cat presence, (NS = Not samples).</p