Prevalence and burden of <i>O. ochengi</i> in vaccinated and control animals at 22 months post-exposure.

a<p>Bold type indicates statistical significance at the <i>P</i><.05 level.</p>b<p>Fisher's exact test.</p>c<p>Mann-Whitney <i>U</i> test.</p>d<p>One animal was negative at this time-point but had been positive on prior occasions.</p

Total area-under-curve (OD_{405 nm}) for IgG1 and IgG2 responses to eight <i>Onchocerca ochengi</i> recombinant antigens.

<p>‘Vaccinated, patent’ (<i>n</i> = 7) or ‘vaccinated, non-patent’ (<i>n</i> = 5) refers to presence or absence of dermal microfilariae at 22 months post-exposure, respectively; all adjuvant-control animals had patent infections (<i>n</i> = 13). Area-under-curve was calculated from data obtained at 0, 4 and 21 months post-exposure. Box-and-whisker plots display the median line, 25^th–75^th IQR (box), highest and lowest values within 1.5× IQR (whiskers), outliers (○; >1.5–3.0×IQR) and extreme values (▹; >3.0×IQR).</p

Schedule of injections.

<p>Notes FCA, Freund's complete adjuvant; FIA, Freund's incomplete adjuvant; PBS, phosphate-buffered saline.</p>a<p>Preceding natural exposure to infection.</p>b<p>Adjuvant controls received an equivalent volume of PBS instead of antigen, in combination with vehicle, following an identical schedule to vaccinated animals.</p>c<p>Final injection in the Freund's series.</p

Protein-coding gene content and metabolic activity are largely conserved between the two species.

<p>(A) Most protein-coding genes in <i>N. caninum</i> have a one-to-one orthologous relationship (yellow) with a gene of <i>T. gondii</i>. A larger proportion of the <i>T. gondii</i> genome consists of genes with no <i>N. caninum</i> homologue than vice versa (organism-specific genes in red). The increase in shared multi-gene families (blue) in <i>N. caninum</i> reflects the expansion of SRS genes in this organism. The increase in organism-specific multigene families (red) in <i>T. gondii</i> reflects, for instance, the TSF gene family identified by us in this work. (B) Of the one-to-one orthologues shared by <i>T. gondii</i> and <i>N. caninum</i> we identified those which have orthologues in three or more non-apicomplexan eukaryotes (yellow), are not present in three or more apicomplexans but in all apicomplexan groups sequenced to date (grey), are in at least one other apicomplexan group (blue) or are specific to <i>T. gondii</i> and <i>N. caninum</i> (red). (C) Pooled day three and four RNAseq experiments were used to determine orthologous genes differentially expressed between <i>T. gondii</i> and <i>N. caninum</i>. Differentially expressed genes were examined for enrichment with enzymes from different KEGG pathways as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002567#s4" target="_blank">methods</a>. No pathways were identified with a p-value less than 0.01, although putative differences were identified at a p-value cutoff of 0.05. The width of bars in the chart relates to the number of genes in the pathway which were differentially expressed, e.g. <i>Porphyrin metabolism</i> had three enzymes differentially expressed, while <i>Nitrogen metabolism</i> had two and <i>Tyrosine metabolism</i> one. Only pathways with a p-value (adjusted for multiple hypothesis testing) below 0.1 are shown. (D) Gene Ontology terms over represented amongst genes upregulated in <i>N. caninum</i> and <i>T. gondii</i>. All terms shown are significantly upregulated (P<0.05). The terms ‘membrane’, ‘regulation of transcription, DNA-dependent’ and ‘ATP binding’ are found more often than expected in genes upregulated in <i>T. gondii</i>. SRS surface antigens, rhoptry kinases and AP2 transcription factors respectively are associated with these terms, suggesting that SRSs, ROPs and AP2s are amongst the most highly upregulated groups of genes in <i>T. gondii</i> relative to <i>N. caninum</i>. The term ‘protein amino acid phosphorylation’ is overrepresented amongst genes upregulated in <i>N. caninum</i> relative to <i>T. gondii</i>. Many of the genes in this group are rhoptry kinases suggesting that while some are upregulated in <i>T. gondii</i>, others are upregulated in <i>N. caninum</i>. These findings are explored in more detail in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002567#ppat-1002567-g003" target="_blank">Figure 3</a>.</p

Repertoires and differential expression of known and predicted host-interaction genes and AP2 transcription factors.

<p>It was only possible to reliably identify orthologues for 22 SRS genes due to the way they have expanded, often being in large tandem arrays subject to gene conversion. While the AP2 transcription factors are not directly involved in host-parasite interaction they may be important in regulating expression of invasion genes. Each report card details the comparative repertoires of a particular group of genes in these species, the names of the genes specific to each organism and those which are differentially expressed between organisms. Further details of these relationships, including reference numbers, are included in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002567#ppat.1002567.s013" target="_blank">Table S4</a>. Arrows show the fold change in expression (RPKM; Reads Per Kilobase per Million mapped reads) between the two species on a log₂ scale. The ticks are 2, 6 and 8 on this scale. Green arrows highlight increased expression in <i>N. caninum</i> tachyzoites. Red arrows highlight genes with increased expression in <i>T. gondii</i> tachyzoites vs. <i>N. caninum</i> tachyzoites. A fold change is infinite where the gene is not expressed at all in one organism.</p

Chromosomal alignment of <i>N. caninum</i> Nc-Liv and <i>T. gondii</i> Me49 highlighting surface antigen gene families.

<p>(A) Aligned chromosomes of <i>N. caninum</i> (above) and <i>T. gondii</i> (below) showing conservation of synteny and distribution of SRS and SUSA surface antigen gene families. Tandemly repeated genes are shown clustered together. Uncoloured genes had less than 20% unique sequence and expression levels could not be accurately determined. 49 additional NcSRSs were found in UnAssigned Contigs (UACs), while three further TgSRSs were not assigned to chromosomes. (B) Shows putative rearrangements between <i>N. caninum</i> and <i>T. gondii</i> chromosomes. Large (>30 kb) insertions in one genome relative to the other are numbered on the chromosomes of <i>N. caninum</i> (orange) and <i>T. gondii</i> (blue). Red ribbons show regions of protein sequence similarity between these regions. The plot shows that most insertions have a pairwise relationship, e.g. region 13 from <i>T. gondii</i> chromosome VIIa is putatively orthologous to region 24 in <i>N. caninum</i> chromosome IX. Thus these regions are shared and not specific to one organism. The arrow symbol refers to sequence similarity with parts of the comparator genome not currently assigned to chromosomes (UACs). A capital ‘T’ identifies a region with no similarity in the comparator genome. These regions include genes belonging to novel families (TSF and KRUF).</p