Gliding motility by <i>N. caninum</i> tachyzoites.

<p><b>A</b>. <i>N. caninum</i> deposited surface membrane trails during gliding on a solid substrate. Trails were visualized by staining with anti-SAG1 mAb. Representative micrographs of Nc-1, Nc-Spain 3H and Nc-Liverpool trails are shown. Scale bar: 5 µm. <b>B</b>. <i>N. caninum</i> isolates exhibit significant differences in gliding motility (P<0.0001; one-way ANOVA test), being highest for Nc-Spain 9 (Nc-Spain 9 <i>versus</i> Nc-1, Nc-SweB1, Nc-Spain 3H and Nc-Spain 7; P<0.05-0.0001; Bonferroni's post-test), and lowest for Nc-Spain 3H (Nc-Spain 3H <i>versus</i> all isolates; P<0.05-0.0001; Bonferroni's post-test). The mean length of trails formed by Nc-Liverpool was also superior to that of Nc-1 (P<0.05; Bonferroni's post-test). Trails were measured as relative parasite body lengths. Ten to fifteen trails were measured per strain per experiment. Mean (±SEM) of the determinations from three independent experiments is represented. Asterisks indicate isolates which showed significant differences.</p

<i>In vitro</i> migratory phenotypes of DC infected with <i>N. caninum</i> and <i>T. gondii</i>.

<p><b>A</b>. Human monocyte-derived DC are permissive to <i>N. caninum</i>. Immunofluorescence staining of DC (phalloidin-Alexa Fluor 596) infected with the isolate Nc-1<i>Luc</i> (MOI 3). Parasites were labelled with hyperimmune rabbit antiserum and Alexa Fluor 488 as secondary antibody. Overlay with DAPI (blue). Representative images of 6, 10 and 24 h p.i. are shown. Scale bar: 10 µm. <b>B</b>. Bar diagram shows the transmigration frequencies of human monocytic-derived DC incubated with live tachyzoites (MOI 2) from <i>T. gondii</i> (Tg-RH-LDM and Tg-ME49-PTG strains) and <i>N. caninum</i> (Nc-1, Nc-Liverpool, Nc-SweB1, Nc-Spain 3H, Nc-Spain 4H, Nc-Spain 6, Nc-Spain 7 and Nc-Spain 9 isolates), CM (non-infected cells), LPS (100 ng/ml) and heat-inactivated parasites (heat-Tg-ME49-PTG or heat-Nc-1) as indicated under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032123#s2" target="_blank">Materials and Methods</a>. The black bars indicate <i>T. gondii</i> and the white bars indicate <i>N. caninum</i>. Cell migration (migrated/added) was assessed by optical counting of transmigrated cells across a transwell filter. Means (±SEM) from the replicates from at least three independent experiments are shown. Asterisks indicate treatments which conferred significant differences in transmigration compared with non-infected cells (P<0.001; Dunnett's test). <b>C</b>. Transmigration of BALB/c bone marrow-derived DC infected with <i>T. gondii</i> (Tg-ME49-PTG, MOI 2) or <i>N. caninum</i> (Nc-1, Nc-Spain 4H, Nc-Spain 7 and Nc-Spain 3H, MOI 2) as indicated under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032123#s2" target="_blank">Materials and Methods</a>. Means (±SEM) from the replicates from at least three independent experiments are shown. Asterisks indicate significant differences compared to non-infected cells (P<0.05; Dunnett's test).</p

Characteristics of <i>N. caninum</i> isolates.

<p>NeighborNet phylogenetic network for the <i>N. ca</i>ninum isolates included in this study was based on multilocus genotypes determined by 9 microsatelite markers (MS4, MS5, MS6a, MS6b, MS7, MS8, MS10, MS12 and MS21) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032123#pone.0032123-RegidorCerrillo2" target="_blank">[18]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032123#pone.0032123-Stenlund1" target="_blank">[22]</a>. Phylogenetic network analysis was developed using the shoftware SplitsTree4 (v 4.11.3). <i>N.caninum</i> isolates included the Spanish isolates: Nc-Spain 3H, Nc-Spain 4H, Nc-Spain 6, Nc-Spain 7 and Nc-Spain 9, which were obtained from asymptomatic calves <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032123#pone.0032123-Stenlund1" target="_blank">[22]</a>. Nc-1 was obtained from a clinically affected dog in the United States <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032123#pone.0032123-RegidorCerrillo3" target="_blank">[19]</a>, Nc-Liverpool from a clinically affected dog in the United Kingdom <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032123#pone.0032123-Dubey2" target="_blank">[20]</a> and Nc-SweB1 from a stillborn calf in Sweden <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032123#pone.0032123-Barber1" target="_blank">[21]</a>. The asterisk (*) indicates isolates obtained from asymptomatic animals. The percentages represent neonatal mortality and vertical transmission rates, respectively. The rates were determined in previous studies using a pregnant BALB/c mouse model [16 and unpublished data]. The letter “V” indicates highly virulent isolates according to the significant differences found in neonatal mortality. The rest of the isolates could be considered as lowly/moderately virulent <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032123#pone.0032123-GomezLopez1" target="_blank">[16]</a>.</p

Adoptive transfer of <i>N. caninum</i> tachyzoite-infected DC in mice and parasite transmission to offspring during pregnancy.

<p><b>A</b>. Kinetics of dissemination of <i>N. caninum</i> in BALB/c mice inoculated i.p. with ∼2.5×10⁶ cfu (experiment 1) or ∼1.5×10⁶ cfu (experiment 2) free Nc-1<i>Luc</i> tachyzoites or Nc-1<i>Luc</i>-infected DC. Progression of <i>N. caninum</i> infection and parasite biomass was assessed by bioluminescence imaging (BLI) using the IVIS Spectrum imaging system. Photon emission (photons s⁻¹ cm⁻²) was assessed for 180 s 10–12 min after i.p. injection of d-luciferin. Images are from two experiments with four mice per group on days 1 and 2 p.i. Colour scales indicate photon emission (photons s⁻¹ cm⁻²) during 180 s exposure time. <b>B</b>. Total photon emission analysis of BALB/c mice inoculated i.p. with free Nc-1<i>luc</i> tachyzoites or Nc-1<i>Luc</i>-infected DC on days 1–5 after inoculation. There was a dramatic increase in the parasite burden in mice inoculated with Nc-1<i>Luc</i>-infected DC compared to free Nc-1<i>Luc</i> on days 1 and 2 p.i. (P<0.05; Student's <i>t</i> test). Compiled data are from two independent experiments (day 1 p.i. n = 11; day 2–5 p.i. n = 8). <b>C</b>. Imaging of infected organs <i>ex vivo</i>. Organs were dissected on day 1 p.i from mice infected with free Nc-1<i>Luc</i> tachyzoites or Nc-1<i>Luc</i> -infected DC, respectively. Parasite load was significantly higher in the Nc1<i>Luc</i>-infected DC group. Signals were observed in images of the testis (T), liver (LV), spleen (S), mesenteric lymph nodes (MLN), lung (L), kidney (K) and brain (CNS). No signal was detected in the heart (H). <b>D</b>. Detection of <i>N. caninum</i> DNA in mouse brains on day 1 p.i. by real-time PCR as indicated under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032123#s2" target="_blank">Materials and Methods</a>. Y-axis indicates percentage of mice with positive PCR for mice infected with Nc-1<i>Luc</i>-infected DC or free Nc-1<i>Luc</i> tachyzoites, respectively (n = 11; positive 4/11 <i>versus</i> 0/11; P<0.05, Fisher <i>F</i> test). <b>E</b>. Kaplan–Meier survival curves for neonates born to dams inoculated with ∼2.5×10⁶ cfu of free Nc-Spain 7 tachyzoites or Nc-Spain 7-infected DC, 5×10⁶ non-infected DC or PBS buffer. The curves represent the percentage of animals surviving over a period of 30 days post-partum (pp). Vertical steps downward correspond to days pp when a mouse died or was sacrificed. Symbols indicate censored observations. The number of dead mice was registered daily, and the median survival time of the Nc-Spain 7-infected DC group was significantly shorter than that of the free Nc-Spain 7-infected group (P<0.001, Log-rank test). Compiled data are from two independent experiments (n = 7–14 pregnant mice per group). <b>F</b>. Parasite loads in brain from offspring quantified by real-time PCR and expressed in terms of number of parasites per µg of host DNA. Pups born to mice inoculated with Nc-Spain 7-infected-DC displayed significant higher parasite loads than the free Nc-Spain 7-infected group (P<0.01; Student's <i>t</i> test). The data are represented as individual points and horizontal lines correspond to the mean value. Compiled data are from two independent experiments.</p

MOESM1 of Experimental caprine neosporosis: the influence of gestational stage on the outcome of infection

Additional file 1. Summary of individual results. Individual clinical signs, parasite DNA detection and serological results in dams and foetuses/kids

MOESM4 of Experimental ovine toxoplasmosis: influence of the gestational stage on the clinical course, lesion development and parasite distribution

Additional file 4 Individual frequency of parasite DNA detection in infected animals. Table showing the individual frequency of parasite DNA detection in infected animals

MOESM3 of Experimental ovine toxoplasmosis: influence of the gestational stage on the clinical course, lesion development and parasite distribution

Additional file 3 Box-plot graph T. gondii burdens measured at the same period post infection and comparing between the three groups at the placenta and foetal viscera. Box-plot graphs represent the median burden, the lower and upper quartiles (boxes) and minimum and maximum values (whiskers). (*) indicates P < 0.05 significant differences between groups in each period post infection

Relationships among the <i>N</i><i>. caninum</i> MLGs (n = 82) estimated by eBURST analysis.

<p>Each complete MLG is represented by a dot and the genotype number assigned in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072678#pone.0072678.s003" target="_blank">Table S1</a> (Numbers 1-11 represent worldwide MLGs; 12-64 Spanish MLGs; 65-80 Argentinean MLGs; 81-88 Scottish MLGs; and 89-95 German MLGs). MLG dots were also coloured according to their geographical origin (see legend). The dot diameter is proportional to the number of samples with identical MLG (see legend). Single locus variants (SLV) are connected by black lines and double loci variants (DLV) by blue lines. MLGs clusters (n=9) are represented. Main MLGs clusters (n=3) are showed by clear circles. Number and name in the squares identifies the herd origin and worldwide isolates, respectively (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072678#pone.0072678.s003" target="_blank">Table S1</a>). Note that singletons were excluded from the snapshot representation (worldwide n=7; Spanish n=7; Argentinean n=2; Scottish n=3; and German n=2).</p

MOESM3 of Systemic and local immune responses in sheep after Neospora caninum experimental infection at early, mid and late gestation

Additional file 3. Summary of lesion and PCR detection and quantification of N. caninum in placenta and foetal liver and brain. dpi: days post infection when abortion occurred; ASF: Average size of focus; %LES: Percentage of section affected by lesions. * All lambs from this group gave birth to viable lambs between days 142 and 155 of gestation. Three of nine lambs were born prematurely prior to day 145, and exhibited weakness, recumbency and unresponsiveness to external stimuli. 1 Percentage of the section affected by lesions. 2 Fractions represent the number of positive animals/total number of animals checked by nested-ITS1 PCR, and figures within brackets represent the median values of parasite burden (tachyzoites/mg tissue). a, b Values followed by unlike superscripts differ significantly by Dunn’s test for pairwise comparisons. c, d Fractions determined for positive animals followed by unlike superscripts differ significantly by Fisher’s exact test

Not Available

Not AvailableFive classical clustering method s: four hierarchical-single linkage, average-between linkage, average-within linkage, Wards-and one non-hi era rchical-k-means-using five different distance measures: squared Euclidean, city block, Chebychev's, Pearson correlation and Minkowski have been compared on the basis of simu lated multivariate data on paddy crop genotypes. The performance of different clustering methods was compared based on the average percentage probability of misclassification an its standard error. The performance of different hierarchical clustering methods varied with distance measures used and it was found that squared Euclidean performed best among the five distances followed by city block distance in majority of cases. Among the five methods, the Ward's method performed best with least average percentage probability of misdassification followed by non-hierarchical k-means method irrespective of the sample size. Among the different distance measures used under hierarchical clustering methods, the squared Euclidean distance showed least average percentage probability of misclassification followed by city block distance.Not Availabl