secondary antibodies conjugated to Alexa 488 and counter staining with rabbit anti-ROP1 followed by secondary antibodies conjugated to Alexa 594, as described previously [27]. Slides were washed in PBS and mounted in Vectashield with 49,6-diamidino- 2-phenylindole (DAPI) (Vector Laboratories, Inc, Burlingame, CA), examined with a Zeiss Axioscope (Carl Zeiss Inc, Thorn- wood, NY), and images were acquired with an AxioCam CCD (Zeiss) camera using Axiovision software v4.0 (Zeiss) and processed using Photoshop v7.0. Proteins were resolved on 10% polyacrylamide gels using Laemmeli running buffer, transferred onto a nitrocellulose membrane by semi-dry electrotransfer and blocked in PBS containing 5% nonfat dry milk, 5% goat serum and 0.05% Tween 20. Blots were incubated for 1 hr at room temperature with mAb BB2 anti-Ty antibody (1:1000) and M03792 rabbit antibody against actin (1:2000) as a loading control. After washing, the membrane was incubated for 1 hr at room temperature with goat anti-mouse IgG and goat anti-rabbit IgG conjugated to horseradish peroxidase (HRP) (1:10,000) (Jackson ImmunoRe- search Laboratories, Inc., West Grove, PA). The membrane was washed for 30 min and the antibody complexes were revealed by chemiluminescence using enhanced chemiluminescence (ECL SuperSignal; Pierce Biotechnology, Rockford, IL). Mouse Virulence Assays Animals were maintained in an AAALAC-approved facility and experiments were done under approval from the Washington University Animal Care Committee. Acute virulence was monitored by determining cumulative mortality after i.p. injections of 10, 100, or 1000 tachyzoites into groups of 5 outbred CD1 mice/dose, as defined previously [27]. Animals were monitored for 30 days and any surviving animals were serologically tested by Western blot against whole tachyzoite lysate from the RH strain at 1:1000 dilution of serum and 1:10,000 dilution of goat anti-mouse IgG HRP (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). Signals were detected by ECL using SuperSignal (Pierce Biotechnology, Rockford, IL). Samples were compared to uninfected and chronically infected mice. Cumulative mortality was defined as the number of deaths/ number of animals infected (those that died or were seropositive) and plotted as a single line for all doses combined for each transgenic clone. PCR-Based Analysis of the Upstream Region of ROP18 DNA lysates were prepared from freshly egressed parasites, as described previously [7]. To identify the presence of the insert upstream of ROP18 in T. gondii strains, the target DNA sequence was amplified by PCR in a PTC-100 Thermal Cycler, (M.J Research, Reno, NA) using primer pairs specific for detecting the presence (Insert ROP18-F 59 - CACAGCATGAGC TTAA- GAGTTG -39 (Primer C) and Insert ROP18-R 59 - CACCG- CAAGACAGGCTGTCTTC - 39 (Primer D)) or absence (ROP18-F 59 - CTAGCCACGCTATGCACCTCT - 39 (Primer A) and ROP18-R 59 - GCAAGTCACGCATAGTCTCATC -39 (Primer B)) of the insert. Each reaction was carried out in 25 ml of volume containing 106 PCR buffer, 25 mM MgCl2, 2.5 mM dNTPs, 50 mM each of the forward and reverse primers, 5 U/ml of DNA Taq polymerase (Sigma) and 2 ml of DNA lysate of each of the strains. The reaction mixture was first heated to 95uC for 5 min, followed by 35 cycles of 94uC for 30 sec, 56uC for 30 sec and 72uC for 2 min. Upon completion, 5 ml of the PCR products were examined by electrophoresis in 2% agarose gel containing 0.3 mg/ml ethidium bromide and visualized under UV light. Population Strucure and Virulence of T. gondii qPCR Analysis of ROP18 Expression Total RNAs were isolated by TRIzol treatment (Invitrogen, Carlsbad, CA) of freshly harvested parasites and RNA concentra- tions determined by absorbance at 260 nm. RNAs (3 mg/ml) were transcribed into cDNA using 50 mM oligo (dT)20 and 200 units of SuperScipt III reverse transcriptase (RT) (Invitrogen, Carlsbad, CA) in a volume of 20 ml following the manufacturer’s protocol. For negative controls, water was added instead of RT. Real-time quantitative PCR (qPCR) was performed using a SmartCycler (Cepheid, Sunnyvale, CA) in a 25 ml reaction volume containing SYBR green Supermix (Clontech, Mountain View, CA). ROP18 was detected using the primers ROP18-579-F (59- TGA- GAAGGCGGATTCTGGATG - 39) and ROP18-850-R (59 - CCTTAACAGCCAACTCTTCATTCGTC - 39). Primers spe- cific to T. gondii actin (TgACT1) TgACT-F (59 - TCCCGTCTATCGTCGGAAAG - 39) and TgACT-R (59 - CCATTCCGACCATGATACCC - 39) were used as reference sample (internal control). Reaction mixtures containing 2 mL of cDNA and gene-specific primers were subjected to 40 thermal cycles (95uC for 15 sec and 60uC for 60 sec) of PCR amplification with the SmartCycler. Three replicate reactions were performed for each sample and values are reported as means. Threshold cycle (CT) values were calculated for ROP18 using the SmartCycler software (Cepheid). Differences in the levels of gene expression for each of the strains were determined using the following formula. The amount of target gene (ROP18), normalized to the endogenous housekeeping gene (TgACT1), is given by 22DDCT, where DDCT = DCT (sample)2DCT (housekeeping), and DCT is the CT of the target gene subtracted from the CT of the housekeeping gene, as described previously [51]. Network Analysis of ROP18 A phylogenetic network of ROP18 sequences was derived using the median-joining algorithm [52] (with e = 0) as implemented in NETWORK 4.1 (www.flexus_engineeing.com). ROP18 sequences from 37 strains of T. gondii (Table S2 plus additional strains listed in Table S4) were clustered using DNA Alignment v1.1.2.1 (www. flexus_engineeing.com). Aligned sequences were used to construct a network by combining the features of Kruskal’s algorithm for finding minimum spanning trees that favors short connections and Farris’s maximum-parsimony heuristic algorithm. Estimates of Most Recent Common Ancestry (MRCA) The rate of mutation in ROP18 was determined from a collection of 18 clonal isolates (i.e. types I, II and III) (Table S4). We only considered new mutations that arose since the common origin of the clonal types, hence biallelic polymorphisms that define the major allele types were excluded, as described previously [5]. This rate of diversity was compared to the occurrence of new polymorphisms in the antigen encoding genes SAG1, SAG2 and a collection of introns used previously [5,7]. Polymorphism rates were used to independently calculate the MRCA for the clonal lineages based on ROP18, antigens, and introns, respectively. For estimating the common ancestry based on coding sequences, we considered both 2-fold and 4-fold degenerate codons whereas for estimating the ancestry based on intron sequences, all nucleotide changes were calculated using the 4-fold rate (since these regions are noncoding all substitutions are 4-fold regenerate). This model makes the basic assumptions that each lineage is evolving independently and that mutations are accumulated randomly through time and that a Poisson distribution estimates the frequency of such rare events. We estimated the MRCA for different alleles of ROP18 using the synonymous polymorphisms observed between strains of PLoS Genetics | www.plosgenetics.org 12 March 2009 | Volume 5 | Issue 3 | e1000404