Parasites

The type II strain CZ was originally isolated from the faeces of a Siberian tiger at the Dvůr Králové Zoo (Czech Republic) in 2005 by Dr. B. Koudela and, subsequently, cloned by limiting dilution to yield clone H3, which has been maintained by passages between HFF, sheep and/or mice, at the University of Zurich ever since. Tachyzoites were grown in confluent HFF monolayers cultivated in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma-Adlrich) with high glucose supplemented with 10% heat inactivated foetal calf serum (FCS), additional 2 mM L-glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin and 250 ng/ml amphotericin B. Tissue cysts for cat inoculations were obtained from brains of sheep or organs and muscles from mice and EES (sampled at day 3, 5 and 7) were harvested as described previously16.

cDNA library construction and RNA-Seq

Samples of tachyzoites and EES of T. gondii for cDNA library preparation and RNA-Seq analysis were prepared as described previously16. RNA quality was assessed using the Agilent RNA 6000 Pico or Nano Kits (Agilent) and a Bioanalyzer 2100 (Agilent). RNA concentration was measured using a Qubit fluorometer (Invitrogen) together with the RNA assay (Invitrogen).

The TruSeq RNA Sample Prep Kit v2 (Illumina Inc.) was used in the succeeding steps. Briefly, total RNA samples (100–1000 ng) were poly-A enriched and then reverse-transcribed into double-stranded cDNA. The cDNA samples were fragmented, end-repaired and polyadenylated before ligation of TruSeq adapters containing the index for multiplexing. Fragments containing TruSeq adapters on both ends were selectively enriched by PCR. The quality and quantity of the enriched libraries were validated using a Qubit (1.0) Fluorometer and the Caliper GX LabChip GX (Caliper Life Sciences, Inc.) and qPCR. The libraries were normalised to 10 nM in Tris-Cl, pH 8.5 with 0.1% Tween-20 based on the qPCR values.

The TruSeq PE Cluster Kit v3-cBot-HS or TruSeq SR Cluster Kit v3-cBot-HS (Illumina, Inc.) was used for cluster generation using 10 pM of pooled normalised libraries on the cBOT. Sequencing were performed on the Illumina HiSeq 2500 paired end using the TruSeq SBS Kit v3-HS (Illumina, Inc.). Tachyzoites were sequenced on quarter lanes at 2 × 100 bp; EES were sequenced on 0.5 or 1 lanes each at 2 × 100 bp or 2 × 125 bp. Where few reads were obtained, the samples were sequenced twice.

Read data were quality controlled with Fastqc and FastqScreen to confirm reads were of high quality and free of contaminants. Read-alignment was done using the STAR-aligner48. As reference, we used the genome of T. gondii strain ME49 (ToxoDB Release 24). We computed gene expression values (normalised counts) with the function featureCounts from the R package Rsubread49. Developmental progress was assessed by hierarchical clustering of the samples and re-assigned to groups EES1–5 (see results, Figure S1). Differential expression was computed using the generalised linear model implemented in the Bioconductor package DESeq 250 yielding the log2 value of the fold changes, p-values and false discovery rates (FDR). We considered a fold change of >4 for differential expression and used a p-value cut-off of 0.05 to determine significance. Genes with more than 10 normalised counts were considered expressed (is present = TRUE). We also calculated an additional value for transcript abundance normalised to transcript length: FPKM (fragments per kilobase of exon model per million mapped reads). The raw data and FPKMs have been deposited in NCBI’s Gene Expression Omnibus51 (Accession number GSE108740). Further annotation was performed by gene ontology mapping using Blast2Go52 v4.1.9 using an upper cut-off e-value of <0.0005.

Construction of HAP2 knockout and sequencing

We employed a CRISPR/Cas9 strategy to insert a frameshift within the first 20 nt of the first exon of hap2 in the CZ clone H3 T. gondii, with consequential disruption of the final translated HAP2 protein. First, the tubulin A1 promoter from the CZ clone H3 genome was amplified and inserted into KpnI, NsiI, digested plasmid to exchange the SAG1 promoter in the vector described previously53. Inverse PCR was used to exchange the sgRNA of UPRT with the sgRNA for HAP2 with Ph-sgRNA_TgHAP2mutF (5′-TGGATCCACCACTGCCGCGAGTTTTAGAGCTAGAAATAGC-3′) and Ph-genCas9mutR (5′-AACTTGACATCCCCATTTAC-3′) to yield plasmid pTub1::CAS9-U6::sgHAP2 (Fig. 4b). Transfection of CZ clone H3 was carried out as described53. Twenty-four hours post-transfection, transiently transfected GFP + parasites were purified by flow cytometry as described previously54 (Figure S4) and the HAP2 KO clone E6 clone was further purified using two rounds of limiting dilution cloning. Sanger sequencing of PCR products using HAP2ko-dia-F (5′-GAAACAGCACTACAGCTCTTCGC-3′) and HAP2ko-dia-R (5′ ATGCATGAACAAGGTATGGTTCTGC-3′) was used to confirm the knockout. Sanger sequencing of PCR products using HAP2ko-dia-F (5′-GAAACAGCACTACAGCTCTTCGC-3′) and HAP2ko-dia-R (5′ ATGCATGAACAAGGTATGGTTCTGC-3′) was used to confirm disruption of the hap2 ORF.

For whole genome sequencing, genomic DNA from in vitro cultured parasites from HAP2 KO and CZ clone H3 was extracted with the QIAamp DNA Mini Kit (QIAGEN) using the manufacturer’s protocol. DNA was sheared using Covaris Adaptive Focused AcousticsTM (AFA) technology using settings specific to the fragment size of 550 bp. The fragmented DNA was size selected using AMpure beads (Beckman-Coulter), end-repaired and polyadenylated. Using the TruSeq HT DNA NanoSample Prep Kit v2 (Illumina Inc.), TruSeq adapters containing the index for multiplexing were ligated to the fragmented DNA which was then selectively enriched by PCR. The quality and quantity of the enriched libraries were validated using Qubit® (1.0) Fluorometer and the Tapestation (Agilent). The libraries were normalised to 10 nM in Tris-Cl 10 mM, pH 8.5 with 0.1% Tween-20. The Hiseq. 4000 PE Cluster Kit (Illumina) was used for cluster generation using 8 pM of pooled normalised libraries on the cBOT V2. Paired end sequencing with 2 × 150 bp was performed on the Illumina HiSeq. 4000 using the HiSeq. 3000/4000 SBS Kit (Illumina).

Using Trimmomatic55, raw reads were trimmed of the sequencing adapters and further trimmed 50 bases from the 3′ end to remove low-quality ends. A minimum average Phred quality of 25 and a minimum length of 25 bases were adopted. The sequences passing these filters were aligned to the genome of T. gondii ME49 (Release 33) using Bowtie256 with default settings. Putative variants were then identified by following GATK best practices for DNA-sequencing57,58 and variants with an alternative allele frequency below 10% in the CZ clone H3 and above 90% in the HAP2 KO were marked as HAP2 KO-specific variants.

Sporulation assay

Sporulation of oocysts of T. gondii was assessed by seeding small Erlenmeyer flasks, containing 5 ml of 2% sulphuric acid, with oocysts of the CZ clone H3 or the HAP2 KO parasites. The flasks were left at room temperature and stirred twice per day. For the CZ clone H3 oocysts, four flasks were set up and around 100 oocysts were assessed for sporulation under a light microscope from each flask on day 2, 4 and 8 after seeding. In the case of the HAP2 KO oocysts, it was not possible to set up a series of Erlenmeyer flasks because of the low number of oocysts recovered; hence, in this case, sporulation was undertaken in a Petri dish and 1000 oocysts were assessed for sporulation.

Quantitative real-time PCR

To extract genomic DNA, oocysts were suspended in ice cold water and neat Triton X-100 was added to the suspension until foam appeared. The detergent was washed away and the oocysts were treated with 14% bleach on ice for 10 min. The bleach was then diluted with H 2 O, and oocysts were washed with ice cold H 2 O before DNA extraction or freeze drying. Genomic DNA was extracted from either freshly treated or freeze-dried oocysts using the Faecal DNA Miniprep kit from Zymo following the manufacturer’s protocol. Real-time PCR was performed using the QuantiFast SYBR Green PCR Kit (QIAGEN) on the QuantStudio 7 Flex Real-Time PCR System (Applied Biosystems) and the primers Act1-F (5′-GGCGAACCGTGAGAGAATGA-3′) with Act1-R (5′-ACAGAGAAAGAACGGCCTGG-3′) or clp-F (5′-ATTATGCGGTCCAAGCGGAA-3′) with clp-R (5′-CTACATATCCTGGAGGCGCTC-3′), resulting in amplicons of 89 bp and 158 bp, respectively. The cycling conditions used included denaturation at 95 °C for 5 min followed by 40 cycles of 10 s at 95 °C and 30 s at 60 °C. Melt curve determination was performed for 15 s at 95 °C, 1 min at 60 °C and 15 s at 95 °C. All reactions were done in triplicate. Data were analysed using the comparative Ct method31.

Inoculation and challenge trial design

Fourteen 9-week old European specific pathogen free (SPF) cats were purchased from Isoquinem S.L. (Barcelona, Spain) and allowed 3 weeks to acclimatise to their new surroundings at the University of Zurich before the challenge trial. The cats were tested by ELISA (see below) for the presence of antibodies to T. gondii and for oocysts in their faeces prior to the start of the inoculation and challenge trial; all cats were seronegative and all faeces examined were oocyst-free. Each cat was vaccinated twice with Feligen CRP ad us. vet. (Virbac AG, Switzerland) prior to commencement of the challenge trial. The cats were free to move about, in three groups of four, five and five cats, in three rooms of approximately 10 m2, 11 m2 and 11 m2, respectively. Figure 5a outlines the design of the inoculation and challenge trial: one group of five cats was inoculated at 12 and 17 weeks of age with tissue cysts of T. gondii CZ clone H3 and challenged at 22 weeks of age with tissue cysts of T. gondii CZ clone H3; the group of four cats was inoculated at 12 and 17 weeks of age with tissue cysts of T. gondii HAP2 KO and challenged at 22 weeks of age with tissue cysts of T. gondii CZ clone H3; and the final group of five cats was left uninoculated (i.e., naïve) until challenged at 22 weeks of age with tissue cysts of T. gondii CZ clone H3. Blood samples were taken from each cat immediately prior to inoculation and challenge at 12, 17 and 22 weeks of age as well as when the cats were 26 and 30 weeks old; these samples were analysed for antibodies by ELISA (see below). Faeces were collected at 2 day intervals after inoculation and challenge and oocysts counted (see below).

Preparation of infectious inocula

Eight to ten-week old CBA/Rj mice (Janvier, France) and Fischer 344/IcoCrl rats (Charles River, Italy) were infected by intraperitoneal injection of 200 μl PBS containing 1,000 tachyzoites of either T. gondii CZ clone H3 (mice and rats) or the HAP2 KO isolate (mice only). The use of CBA/Rj mice and Fischer 344/IcoCrl rats as hosts for T. gondii has been described previously59,60. At 48 or 49 days post-injection, blood samples from five randomly-chosen mice and rats were taken and tested for antibody production by ELISA (see below); all were antibody-positive. At 50 days post-injection, rodents were killed and brains, eyes, hearts and muscles collected and diced finely until homogenised. The presence of T. gondii in the inocula was confirmed by PCR, as described previously61. For the first inoculation of cats, brains, eyes and hearts plus 320 g of skeletal muscle from 32 T. gondii CZ clone H3 strain infected mice and brains, eyes and hearts plus 200 g of skeletal muscle from 20 T. gondii CZ HAP2 KO strain infected mice were used. For the second inoculation of cats, brains, eyes and hearts plus 300 g of skeletal muscle from 30 mice infected with T. gondii CZ strain, and brains and hearts plus 300 g muscles of 30 T. gondii CZ HAP2 KO strain infected mice were used. For the challenge infections of cats, brains, eyes and hearts plus 560 g skeletal muscle from 39 rats infected with T. gondii CZ strain were used. For each inoculation, the preparations were homogenised and divided into equal portions, which were fed to cats individually. It should be noted that, on two out of three occasions, a significant percentage (13% and 91%) of CBA/Rj mice infected with the CZ clone H3, and on one of two occasions, a significant percentage (48%) of CBA/Rj mice infected with the HAP2 KO strain of T. gondii either died unexpectedly overnight or had to be euthanised under our animal care and ethics protocols due to the exhibition of symptoms of acute toxoplasmosis (including ruffled fur, hunched posture and > 20% weight loss), even after administration of sulfadiazine62 and despite the fact that brain cysts could not be observed by light microscopy in infected mice. In contrast, large brain cysts were detected readily in infected Fischer 344/IcoCrl rats and only a single rat, of sixty infected with T. gondii CZ clone H3, developed mild ocular symptoms. Thus, we used tissue cysts from rats for the final challenge inocula in our trial and now prefer this host for the production of tissue cysts of T. gondii.

Specific antibody measurement

ELISA plates (NUNC Maxisorp, Thermo Scientific, Roskilde DK) were coated with 100 µl per well using 10 µl of T. gondii tachyzoite antigen in 1 ml coating buffer (0.1 M carbonate/bicarbonate, pH 9.6) overnight at 4 °C. Production of this antigen was as described previously63. The plates were washed and subsequently blocked with 300 μl buffer II (PBS (pH 7.2) with 0.02% (w/v) NaN 3 , 0.05% (w/v) bovine haemoglobin (Fluka/Sigma Aldrich, Switzerland) and 0.3% (w/v) Tween-20) per well for 30 min. All further incubation steps took place in a 37 °C humid chamber for 1 h, followed by four washes with ELISA wash buffer (physiological saline containing 0.3% Tween-20). Sera were then used in a 1:200 dilution in buffer II. Alkaline phosphatase labeled goat anti-feline IgG H + L (Southern Biotech, USA) diluted 1:4000, goat anti-mouse IgG A3562 (Sigma-Aldrich, Switzerland) diluted 1:10,000 and goat anti-rat igG A8438 (Sigma-Aldrich, Switzerland) diluted 1:4000 in buffer II were used as conjugate. Antibody reactions were visualised by adding 100 µl of substrate (1 mg/ml of 4-nitrophenylphosphate (Sigma-Aldrich, Switzerland) in 0.05 M carbonate/bicarbonate buffer (pH 9.8) containing 1 mM MgCl 2 ) and the absorbance was read at 405 nm in an ELISA reader (Multiscan RC, BioConcept, Allschwil, Switzerland) after 10 min of incubation at 37 °C.

Oocyst counting

Since we could not house cats in individual cages under our animal experimentation protocols, we had to devise novel solutions to allow us to determine individual oocyst excretion rates in our infected cats. Hence, individual cats infected with T. gondii were fed different coloured plastic beads in every meal. These coloured beads proved readily detectable and allowed us to separate faeces from each cat. Faeces were collected from 2 days post-inoculation until no oocysts were observed for at least 4 days. Faeces from individual cats were pooled every 2 days, i.e., day 1 + 2 post-inoculation, day 3 + 4 post-inoculation, day 5 + 6 post-inoculation, etc. Faeces were soaked in 0.1% Tween-80 (Sigma-Aldrich) overnight at room temperature.

We adapted elements of three published protocols for enumerating oocysts in cat faeces32,64,65. In brief, faecal samples were first homogenised using a wooden palette stick and one-tenth of each was passed through a metal strainer, washed with tap water and re-suspended to a final volume of 200 ml. A 50 ml sub-sample of this suspension was centrifuged at 1000 x g for 10 min at room temperature (Allegra X-15R centrifuge, Beckman Coulter). The supernatants of each sample were discarded and the pellets re-suspended in sucrose solution (specific density = 1.2) to a final volume of 50 ml, followed by centrifugation at 1600 x g for 10 min at room temperature, without brakes, to guarantee optimal floatation. The top 10 ml of each supernatant was collected and mixed, and 1 ml was transferred into a mini-FLOTAC chamber. The mini-FLOTAC device was left undisturbed for 10 min to allow oocysts to float to the top of the counting chamber. Two different quarters were counted under a light microscope and the mean value of these two counts was multiplied by 1,600 to obtain the total number of oocysts in the original faecal sample. In cases where oocyst numbers were particularly high, the final subsample in sucrose was diluted 1:10 or 1:100 with additional sucrose solution for counting. Every sample was counted twice, by two different assessors to ensure precision. Additionally, internal protocol tests were conducted regularly, by repeated counts of samples seeded with four different known concentrations of oocysts. In cases where oocysts could not be seen in the mini-FLOTAC chambers, a 10 μl sample was drawn from the very top of the final 10 ml flotation sample, after this sample had rested undisturbed for at least 10 mins to ensure floatation of all oocysts; this sample was placed on a glass microscope slide, with coverslip, and screened under a light microscope for the presence of oocysts. This adapted protocol for oocyst enumeration proved to be efficient and reliable, with appropriate sensitivity of detection for vaccination trials.

Tissue cyst purification and genotyping

Freshly harvested cat brains were washed several times in ice cold PBS. Approximately, 1/6th of the volume was cut into small pieces and suspended in 12–15 ml ice cold PBS and syringe homogenised using 16 G, 18 G, 20 G and 22 G needles. Ice cold PBS (to a total volume of 18–23 ml) and 0.33% Tween-80 were then added to the homogenate. Each sample was divided into two and tissue cysts were fractionated on a 90%/30% Percoll gradient as described previously40. The interface containing the tissue cysts was harvested and washed twice with ice cold PBS at 2000 rpm for 10 min at 4 °C. The washed fraction was syringe homogenised using 18 G, 22 G, 24 G and 26 G needles and used to infect HFFs; the resulting tachyzoites were propagated in vitro. Genomic DNA was extracted from tachyzoites using the QIAamp DNA mini kit (QIAGEN) as per the manufacturer’s protocol. PCRs were then performed using HAP2ko-dia-F (5′-GAAACAGCACTACAGCTCTTCGC-3′) and HAP2ko-dia-R (5′- ATGCATGAACAAGGTATGGTTCTGC-3′). Control PCRs were performed with HAP2 KO and CZ clone H3 genomic DNA from tachyzoites. Diagnostic digestions of the PCR products were performed with XcmI (NEB Biolabs) and PCR products were analysed for nucleotide sequence by Sanger sequencing (Microsynth, Balgach, Switzerland). Image acquisition was performed with an imager from the Alpha Innotech Corporation using the AlphaEase FC software (version 6.0.0).

Ethics statement

Animal experiments were performed under the direct supervision of a veterinary specialist, and according to Swiss law and guidelines on Animal Welfare and the specific regulations of the Canton of Zurich under permit numbers 130/2012 and 019/2016, as approved by the Veterinary Office and the Ethics Committee of the Canton of Zurich (Kantonales Veterinäramt Zürich, Zollstrasse 20, 8090 Zürich, Switzerland).