Selection of Neospora caninum antigens stimulating bovine CD4+ve T cell responses through immuno-potency screening and proteomic approaches

Neospora caninum is recognised worldwide as a major cause of bovine infectious abortion. There is a real need to develop effective strategies to control infection during pregnancy which may lead to either abortion or congenital transmission. Due to the intracellular nature of the parasite, cell-mediated immune (CMI) responses involving CD4+ve, CD8+ve, γ/δ TCR+ve T cells and NK cells, as well as production of IFN-γ, are thought to be important for protective immunity. In this study we applied a combination of proteomic and immunological approaches to identify antigens of N. caninum that are recognized by CD4+ve T cell lines derived from infected cattle. Initially, N. caninum tachyzoite Water Soluble Antigens (NcWSA) were fractionated by size-exclusion HPLC and then screened for immune-potency using CD4+ve T cell lines. LC-ESI-MS/MS (liquid chromatography electrospray ionisation tandem mass spectrometry) was employed to catalogue and identify the proteins comprising three immunologically selected fractions and led to the identification of six N. caninum target proteins as well as sixteen functional orthologues of Toxoplasma gondii. This approach allows the screening of biologically reactive antigenic fractions by the immune cells responsible for protection (such as bovine CD4+ve cells) and the subsequent identification of the stimulating components using tandem mass spectrometry

Infected Dendritic Cells Facilitate Systemic Dissemination and Transplacental Passage of the Obligate Intracellular Parasite Neospora caninum in Mice

The obligate intracellular parasite Neospora caninum disseminates across the placenta and the blood-brain barrier, to reach sites where it causes severe pathology or establishes chronic persistent infections. The mechanisms used by N. caninum to breach restrictive biological barriers remain elusive. To examine the cellular basis of these processes, migration of different N. caninum isolates (Nc-1, Nc-Liverpool, Nc-SweB1 and the Spanish isolates: Nc-Spain 3H, Nc-Spain 4H, Nc-Spain 6, Nc-Spain 7 and Nc-Spain 9) was studied in an in vitro model based on a placental trophoblast-derived BeWo cell line. Here, we describe that infection of dendritic cells (DC) by N. caninum tachyzoites potentiated translocation of parasites across polarized cellular monolayers. In addition, powered by the parasite's own gliding motility, extracellular N. caninum tachyzoites were able to transmigrate across cellular monolayers. Altogether, the presented data provides evidence of two putative complementary pathways utilized by N. caninum, in an isolate-specific fashion, for passage of restrictive cellular barriers. Interestingly, adoptive transfer of tachyzoite-infected DC in mice resulted in increased parasitic loads in various organs, e.g. the central nervous system, compared to infections with free parasites. Inoculation of pregnant mice with infected DC resulted in an accentuated vertical transmission to the offspring with increased parasitic loads and neonatal mortality. These findings reveal that N. caninum exploits the natural cell trafficking pathways in the host to cross cellular barriers and disseminate to deep tissues. The findings are indicative of conserved dissemination strategies among coccidian apicomplexan parasites

Prevalence of intestinal parasite infections in stray and farm dogs from Spain.

Dogs play a potential role as reservoirs for zoonotic parasites, being especially problematic uncontrolled dog populations such as stray and farm dogs with access to populated areas. In order to investigate the prevalence of canine intestinal parasites in at-risk dog populations, we tested a total of 233 faecal samples shed by stray and dairy farm dogs from northern Spain. Telemann method was used to detect the presence of eggs and (oo)cysts of common dog intestinal parasites and Cryptosporidium was detected by PCR. One hundred and forty eight out of 233 samples (63.5%) were positive for at least one intestinal parasite, being Ancylostomidae (35.6%; 83/233) and Trichuris (35.2%; 82/233) the parasites most frequently identified. Cryptosporidium DNA was not detected in any of the faecal samples analysed. The overall prevalence was significantly higher in stray dogs than in farm dogs (72.5% vs 58.8%). Specifically, stray dogs had a significantly higher prevalence of Ancylostomatidae, Toxocara, Toxascaris and Taenidae. These dog populations are an important source of environmental contamination with intestinal parasite forms, which could be of significance to animal and human health.This work was supported by project PR1/06-14467-B (Complutense University of Madrid)S

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

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

<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

Use of an immunodominant p17 antigenic fraction of Neospora caninum in detection of antibody response in cattle

A Neospora caninum 17 kDa protein fraction (p17) has been described as an immunodominant antigen (IDA) under reducing and non-reducing conditions. The aim of the present study was to investigate the diagnostic utility of p17 in cattle. In order to achieve this, p17 was purified by electroelution from whole N. caninum tachyzoite soluble extract and a p17-based Western blot (WB-p17) was developed. The p17 recognition was measured by densitometry and expressed as OD values to check the validity of the WB-p17. A total of 131 sera including sequential samples from naturally- and experimentally-infected calves and breeding cattle were analysed by WB-p17 and compared with IFAT using whole formalin-fixed tachyzoites as a reference test. The results obtained highlight the feasibility of using the N. caninum p17 in a diagnostic test in cattle. Firstly, the assay based on the p-17 antigen discriminated between known positive and negative sera from different cattle populations, breeding cattle and calves. Secondly, the p17 antigen detected fluctuations in the antibody levels and seroconversion in naturally- and experimentally-infected cattle. Significant differences in p-17 antigen recognition were observed between naturally infected aborting and non-aborting cattle, as well as significant antibody fluctuations over time in experimentally infected cattle, which varied between groups. Furthermore, the results obtained with WB-p17 are in accordance with the results obtained with the IFAT, as high agreement values were obtained when all bovine subpopulations were included (kappa = 0.86)