Live Tachyzoites Research Articles

Evaluation of the protective immune response of an attenuated strain of Toxoplasma gondii with long-term passages on the Gecko cell line

Toxoplasma gondii is an obligate intracellular parasite that causes the zoonoses disease, named toxoplasmosis, with global prevalence. Until now, no cost-effective treatment method has been found to deal with toxoplasma, and vaccination is the best way to deal with the infection. In the case of pathogenic protozoa, mainly live vaccines have had successful results compared to other vaccine platforms. This study evaluated the efficacy of a live experimental vaccine through long-term passages on the Gecko cell line (Z1) in inducing a protective immune response in BALB/c mice. Thirty mice were divided into three equal groups; G1: the immunized/challenged group (injection of attenuated strain), G2: the immunized/unchallenged group (injection of attenuated strain), and G3: the control group (injection of culture medium).One month after immunization, the studied mice were challenged with 1ₓ103 live tachyzoites of Toxoplasma acute RH strain. We performed Serological investigations, including evaluating antibodies, interferon-gamma (IFN-γ), and interleukins 2, 4, 10, and 12 (IL-2,4,10,12). At the study's end, a molecular test was performed on brain and liver tissues in the immunized groups to check the presence of parasites. The results from the serological tests for the evaluation of antibodies, interferon-gamma (IFN-γ), and interleukins 10 and 12 (IL-10, 12) show a significant difference (p < 0.05) between the vaccinated group and the control group, which are essential indicators of protective immunity against toxoplasma infection. Thus, in the vaccinated group, the survival rate of mice against the challenge was 70%. Also, in group two (G2), the attenuated strain of Toxoplasma gondii had no pathogenicity, and all mice survived until the end of the study period. Molecular results also showed the absence of parasites in the brain and liver tissues in this immunized group and the parasite was found in only one case of liver tissue in G1. Therefore, the attenuated strain has caused significant and protective humoral and cellular immune responses in vaccinated groups. This study showed that with the long-term passage of the acute strain on the Gecko cell line, it is possible to quickly obtain a non-diseased attenuated strain with the ability to induce protective immunity. This successful finding can introduce further research to achieve a promising vaccine in the target animals.

Reactivation and Foetal Infection in Pregnant Heifers Infected with Neospora caninum Live Tachyzoites at Prepubertal Age.

Neospora caninum is recognised for causing cattle abortion, provoking severe economic losses in the livestock industry worldwide. The aim of the present study was to evaluate the reactivation and foetal infection in pregnant heifers inoculated with live N. caninum tachyzoites before puberty. A total of 15 30-month-old pregnant heifers were allocated into four groups: animals inoculated with live tachyzoites of NC-Argentina LP1 isolate before puberty and challenged with live tachyzoites of NC-1 strain at 210 days of gestation (DG) (Group A); animals mock inoculated before puberty and challenged with NC-1 strain at 210 DG (Group B), animals inoculated before puberty but not subsequently challenged (Group C); and noninfected and nonchallenged animals (Group D). The results of this study showed that 100% of animals infected before puberty (Groups A and C) suffered reactivation of the infection at the seventh month of gestation. In addition, in three and two calves from Groups A and C, respectively, congenital infection was confirmed. Interestingly, we provide evidence that the use of live N. caninum tachyzoites in young animals as a strategy to induce protection is neither safe nor effective.

Evaluation of cytotoxic activity of live toxoplasma gondii tachyzoites and toxoplasma antigen on MCF-7 human breast cancer cell line

The aim of this study was to investigate the cytotoxic potency of live Toxoplasma gondii tachyzoites as well as Toxoplasma antigen on MCF-7 human breast cancer cell line. Cancer cell lines are considered an essential preliminary step towards in-vitro investigation of the potential antineoplastic impact of novel chemotherapeutic agents. Pathogens, including viruses, bacteria, and parasites are noticeably under investigation, considering their potential antineoplastic activity. Some have attained a steady position in the clinical field as hepatitis B virus, human papilloma virus and BCG immunization. Toxoplasma gondii is an apicomplexan parasite with promising antineoplastic activity. In this study, live Toxoplasma tachyzoites provoked a direct cytotoxic effect on MCF-7 in a dose dependent manner, while Toxoplasma antigen didn’t induce such impact.&#x0D; Skipping the direct cytotoxic effect of Toxoplasma antigen doesn’t totally divert the possible antineoplastic activity of Toxoplasma antigen. Potential alternative immune mediated mechanisms could be an alternative. Further in-vivo studies in different cancer models are mandatory to investigate the underlying mechanisms of antineoplastic activity of Toxoplasma gondii

Viability of Toxoplasma gondii tachyzoites in different conditions for parasite transportation.

Background and Aim:Toxoplasma gondii tachyzoite is the infective stage that causes acute infection, leading to severe toxoplasmosis. The tachyzoite stage has been extensively used for several inoculation purposes, including antigen production, immunological studies, nutrition mechanisms, and in vitro drug trials. The use of fresh tachyzoites is required for inoculation in either in vitro or in vivo studies. However, there is a lack of information on preserving live tachyzoites during transportation from laboratories to inoculation sites. Therefore, this study aimed to validate suitable preservative conditions for maintaining live parasites by determining the survival and viability of T. gondii tachyzoites on the basis of different media, temperatures, and incubation times.Materials and Methods:The free live T. gondii tachyzoites were evaluated on their viability when maintained in different media without 5% Carbon dioxide (CO2). The purified tachyzoites of the RH and PLK strains were individually suspended in normal saline (NS), phosphate-buffered saline (PBS), minimum essential medium (MEM), and MEM with 10% fetal bovine serum (MEM-FBS) and incubated for 6 h at ice-cold (IC; 3-9°C) and room temperature (RT; 25°C). Parasite survival was measured at the 0, 1st, 2nd, 3rd, 4th, 5th, and 6th h post-incubation using the trypan blue exclusion test.Results:The viability was in the range of 85.0%–91.0% for IC using NS and 81.0%–85.1% (IC) and 75.3%–77.5% (RT) using PBS. The viability was approximately 75.0%–83.0% (IC) and 70.0%–79.0% (RT) using MEM and MEM-FBS. There was a significant difference in the viability between the seven periods on the basis of one-way repeated Analysis of variance and Friedman analyses. Parasite survival slightly reduced (20.0%–30.0%) in NS and MEM-FBS at both temperatures during incubation. Notably, PBS could not support tachyzoite viability after 3 h post-incubation.Conclusion:NS was a suitable preservative for maintaining purified T. gondii tachyzoites during transportation at IC and RT without 5% CO2 supplementation. This could be a valuable medium for parasite transportation, especially when there is a large distance between the laboratory and inoculation site.

Toxoplasma gondii activates NLRP12 inflammasome pathway in the BALB/c murine model

The host resistance against Toxoplasma gondii (T. gondii) infection is related to the initiation of the immune response. The study aimed to investigate the role of the leucine-rich repeat family, pyrin domain -containing protein 12 (NLRP12), and cytoplasmic nucleotide-binding domain in the inflammasome-mediated cell death during murine toxoplasmosis. Groups of BALB/c mice (n = 10) were inoculated intraperitoneally with live tachyzoites, excretory-secretory antigens (ESAs) of T. gondii RH strain, and RPMI. The gene expression levels of NLRP12, caspase-3, caspase-1, IL-1β, IL-18, ASC, and Bcl-2 were measured in the peritoneal cells using quantitative real-time PCR, while the determination of NLRP12 protein level was measured by Western blot. Also, the intracellular reactive oxygen species (ROS) production was investigated. Quantitative and comparative analyses showed that injection of tachyzoites significantly increased NLRP12, caspase-3, caspase-1, IL-1β, IL-18, and ASC genes mRNA expression levels (p<0.01). Contrary to the acute infection, the Bcl-2 gene was significantly expressed in the ESAs group (p<0.0001). The level of NLRP12 protein was significantly higher in the mice that received tachyzoites and ESAs in comparison to the control group (p<0.0001). These findings provide an inside into the host-T. gondii interaction and NLRP12 regulation, which is important for the modulation of the immunological response.

Development and evaluation of recombinant GRA8 protein for the serodiagnosis of Toxoplasma gondii infection in goats

BackgroundThe development of sensitive and specific methods for detecting Toxoplasma gondii infection is critical for preventing and controlling toxoplasmosis in humans and other animals. Recently, various recombinant proteins have been used in serological tests for diagnosing toxoplasmosis. The production of these antigens is associated with live tachyzoites obtained from cell cultures or laboratory animals for genomic extraction to amplify target genes. Synthetic genes have gained a key role in recombinant protein production. For the first time, we demonstrated the production of the recombinant protein of the T. gondii dense granular antigen 8 (TgGRA8) gene based on commercial gene synthesis. Recombinant TgGRA8 plasmids were successfully expressed in an Escherichia coli system. The recombinant protein was affinity-purified and characterized via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Furthermore, the diagnostic potential of the recombinant protein was assessed using 306 field serum samples from goats via indirect enzyme-linked immunosorbent assay (iELISA) and the latex agglutination test (LAT).ResultsWestern blotting using known positive serum samples from goats identified a single antigen at the expected molecular weight of TgGRA8 (27 kDa). iELISA illustrated that 15.40% of goat samples were positive for T. gondii-specific IgG antibodies. In addition, TgGRA8 provided high sensitivity and specificity, with significant concordance (91.83) and kappa values (0.69) compared with the results obtained using LAT.ConclusionOur findings highlight the production of a recombinant protein from a synthetic TgGRA8 gene and the ability to detect T. gondii infection in field samples. The sensitivity and specificity of TgGRA8 demonstrated that this protein could be a good serological marker for detecting specific IgG in goat sera.

Delayed-type hypersensitivity skin test against Neospora caninum in heifers with undetectable specific antibodies

Delayed-type hypersensitivity (DTH) has been used in human and veterinary medicine as a skin testing for evaluating in vivo cell-mediated immune responses (CMIR). Whereas CMIR is a key process to control intracellular pathogens, its value at identifying cattle exposed to the abortigenic intracellular coccidian parasite Neospora caninum is unknown. In this work, we have evaluated a DTH skin testing in cattle exposed to N. caninum and still seronegative. Female calves were experimentally sensitized by subcutaneous (SC) inoculation with live tachyzoites of N. caninum (NC-Argentina LP1) in sterile phosphate-buffered saline (PBS) (group A; n: 8) whereas other calveswere mock-sensitized with PBS (group B; n: 6). Two DTH skin tests were performed by intradermal inoculation with a soluble lysate of N. caninum tachyzoites (NC-Argentina LP1) in the neck region at 60d and 960 d after sensitization. Skinfold thickness at the intradermal inoculation site was measured at 0, 24, 48 h post each DTH skin test and skin biopsies taken for microscopic evaluation. Specific N. caninum antibodies kinetics was evaluated all throughthe experiment. We found that whereas N. caninum specific antibodies remained below the ELISA cut-off, a distinctive skinfold thickness increase was detected in sensitized animals (group A) at the DTH skin test site, showing induration, swelling and inflammatory infiltration. Mock sensitized animals (group B) showed no skinfold thickness growth and lacked specific antibody response. Thus, N. caninum DTH skin testing could be a useful diagnostic tool for the detection of CMIR during N. caninum infection in non-humoral responders.

The evaluation of attenuated Neospora caninum by long-term passages on murine macrophage cell line in prevention of vertical transmission in mice

To date, there is no effective vaccine to prevent abortion or vertical transmission associated with neosporosis in cattle. In the present study, the efficacy of a live experimental vaccine of Neospora caninum attenuated (NCa) by long-term serial passages on a murine macrophage cell line was evaluated in the prevention of vertical transmission and abortion in the mouse model. Forty non-pregnant mice were randomly divided into four equal groups including non-immunized/challenged (injected with PBS); positive control (inoculated with un-attenuated NC-1 tachyzoites); immunized/challenged (inoculated with NCa attenuated strain) and immunized/non-challenged or vaccinated (inoculated with NCa) groups. Following pregnancy synchronization, both the immunized and control mice were challenged with virulent live NC-1 tachyzoites (2.5 × 106) in the mid-pregnancy stage. The number of abortions and post-natal pup mortalities was recorded. Serological, molecular, and histopathologic examinations were employed to evaluate the efficacy of the vaccine and the vertical transmission rates. Results indicated that the live attenuated N. caninum strain (NCa) could significantly reduce the risk of abnormal parturitions and fetal mortality in the vaccinated group (20 %) compared to the non-immunized/challenged group (80 %). Also, the NCa strain reduced the lesion score in the brain of the offspring (0.3 vs 1.9) compared to the non-immunized/challenged group (P < 0.05). The molecular assay showed a decrease in the parasite DNA detection rates from 83 % and 77 % in the non-immunized/challenged group to 27 % and 0 % in the vaccine group in the brain and liver tissues, respectively. While in the immunized/non-challenged group no parasite DNA was detected in the brain tissue samples of the pups. Serological analyses showed that NCa strain was able to stimulate the humoral immunity and create effective protection against neosporosis with a moderate systemic IFN-γ response. In conclusion, the NCa strain could significantly (P < 0.05) reduce the risk of vertical transmission and proved to be a safe vaccine while conferring significant levels of protection in the laboratory mice.

Cell mediated and innate immune responses in pigs following vaccination and challenge with Toxoplasma parasites

Toxoplasma gondii has a worldwide distribution and can infect almost all warm blooded animals including pigs and humans. This study aims to examine the immune responses induced in pigs following vaccination (live S48 tachyzoites) and/or challenge with T. gondii oocysts, through the examination of changes in levels of transcription in CD4, CD8α, IFN-γ, IL-12p35, CXCR3, MyD88. The experiment involved four groups of animals; pigs in group 1 (Challenged) (Chal) were challenged orally with (1 × 103 oocysts) on day 28 of the experiment. Pigs in group 2 (Vaccinated /Challenged) (Vac/Chal) were vaccinated (S48 isolate tachyzoites) on day 0, then challenged on day 28. The group 3 (Vaccinated) (Vac) animals were vaccinated (S48 isolate tachyzoites) on day 0 of the experiment. Finally the group 4 (control) pigs remained non-vaccinated and non-challenged. All animals were culled 6 weeks post challenge. At post mortem samples of retropharyngeal lymph node (RLN), mesenteric LN (MLN) and spleen were collected, RNA was extracted and cDNA synthesised. The results showed significant increases in IFN-γ expression in samples from groups 1 (Chal) and 2 (Vac/Chal) (RLN) and groups 1, 2 and 3 (Vac) (spleen) and in MyD88 expression (RLN) in samples from groups 1, 2 and 3 compared to the group 4 (control) animals. Significant increases were also observed in CD8α expression in group 1 (Chal) (RLN) and groups 1 and 2 (Vac/Chal) (RLN and MLN) compared against group 4 (control) and group 3 (Vac) respectively. Conversely, significant down regulation of CD4 and/or IL-12p35 transcription was found in at least one sample from groups 1 (Chal), 2 (Vac/Chal) and 3 (Vac) compared to group 4 (control) pigs. This study demonstrates that cell mediated and innate immune responses are generated in pigs following exposure to T. gondii parasites (oocysts or tachyzoites), key amongst them appear to be IFN-γ, MyD88 and CD8α.

Phenotypic characterization of immune cells in fetal tissues of cattle immunized and challenged with Neospora caninum

The purpose of this work was to characterize the cellular phenotype in inflammatory infiltrates of fetal tissues from pregnant heifers immunized and experimentally challenged with Neospora caninum. Fetuses from 20 heifers separated into 5 groups were obtained. The experiment was designed as follow: Group A, heifers inoculated intravenously with live tachyzoites of Argentine strain NC-6 (n = 4); Group B heifers inoculated subcutaneously with soluble native antigen from the same strain formulated with immune stimulant complexes (ISCOMs) (n = 4); Group C heifers inoculated with recombinant proteins, rNcSAG1, rNcHSP20, rNcGRA7 formulated with ISCOMs (n = 4), Group D heifers inoculated subcutaneously with sterile phosphate buffered solution (n = 4) and Group E heifers inoculated subcutaneously with antigen-free ISCOMs (n = 4). Experimental challenge was performed at 70 days of gestation and all heifers were euthanized 34 days later. Fetal tissues were taken for histological studies. Inflammatory lesions were observed in brain and lung, and immunhistochemistry was used to identify CD3+, CD20+ and MHC II+ cells. The majority of the cells that infiltrate and circumscribe the lesions in the brain and lung tissue expressed MHC II antigen; varying between 70–90% of the total cellular infiltrate. CD3+ cells were also present within the lesions, contributing to up to 30% of the inflammatory cells. CD20+ cells appeared as a marginal group, in some cases, with a range between 10 and 25%. As expected, the immunolabeling of MHC II + and CD3 + cells in fetal tissues was associated with fetal infection with N. caninum. There were statistically significant differences in the distribution and population of the inflammatory infiltrate in relation to the immunogenic treatment and the type of tissue, with inflammatory cells being markedly less extensive fetuses from group A (dams previously exposed to N. caninum) and in brain tissue. This work showed that Neospora-infection induced MHC II+ and CD3+ cells in bovine fetuses from dams receiving experimental vaccines.

Immune response to Neospora caninum live tachyzoites in prepubertal female calves.

The aim of the present study was to characterize the specific immune response in prepubertal female calves inoculated with Neospora caninum. Forty-eight N. caninum-seronegative 6-month-old Angus female calves were randomly allocated into two groups: group A calves were inoculated subcutaneously (sc) with 1 × 106 tachyzoites of the low virulence NC-Argentina LP1 isolate in sterile phosphate-buffered saline (PBS); group B calves were mock inoculated sc with sterile PBS. Calves from group A developed a specific immune response characterized by the production of IgG antibodies and the expression of IFN-γ and TNF-α cytokines. Animals did not present any febrile reaction or reactions at the site of inoculation. Although chronic N. caninum infection was developed in 50% of calves of group A after inoculation, according to the presence of antibodies against rNc-SAG4, antigen characteristic of bradyzoites, N. caninum antibodies dropped below the cut-off of ELISA from day 210 post-inoculation onwards. Future trials using the same group of inoculated animals will allow the characterization of the evolution of the immune response during pregnancy and to determine whether the immunization with the local isolate is able to prevent congenital transmission and to protect against heterologous challenges.

Propranolol efficacy as a novel adjuvant for immunization against Toxoplasma gondii tachyzoites

Severe or lethal damages, caused by Toxoplasma gondii infection in congenital cases and immunocompromised patients implies the necessity for development of a vaccine and an appropriate adjuvant would be needed to elicit a protective Th1 biased-immune response. The adjuvant activity of propranolol was surveyed and compared with alum by immunization of BALB/c mice with protein components of T. gondii tachyzoites. Five groups of BALB/c mice were immunized with phosphate buffered saline (negative control), Toxoplasma lysate antigen (TLA), alum plus TLA, Propranolol plus TLA, and alum, propranolol and TLA. Immunization efficacy was evaluated by lymphocyte proliferation and DTH tests, challenge with live tachyzoites, IFN-γ production by spleen cells, serum TNF-α concentration and anti- Toxoplasma total IgG, IgG1 and IgG2a measurements. Mice of the PRP-TLA group induced significantly more IFN-γ and TNF-α production and lymphocyte proliferation than other groups. This group of mice also showed more anti-T. gondii IgG2a and DTH responses and showed a significantly increased survival time after challenge. These findings indicate that propranolol as an adjuvant in combination with TLA, may enhance cellular immunity against T. gondii.

Characteristic pro-inflammatory cytokines and host defence cathelicidin peptide produced by human monocyte-derived macrophages infected with Neospora caninum.

Neospora caninum is a coccidian intracellular protozoan capable of infecting a wide range of mammals, although severe disease is mostly reported in dogs and cattle. Innate defences triggered by monocytes/macrophages are key in the pathogenesis of neosporosis, as these cells are first-line defenders against intracellular infections. The aim of this study was to characterize infection and innate responses in macrophages infected with N. caninum using a well-known cell model to study macrophage functions (human monocyte THP-1 cells). Intracellular invasion of live tachyzoites occurred as fast as 4 h (confirmed with immunofluorescence microscopy using N. caninum-specific antibodies). Macrophages infected by N. caninum had increased expression of pro-inflammatory cytokines (TNFα, IL-1β, IL-8, IFNγ). Interestingly, N. caninum induced expression of host-defence peptides (cathelicidins), a mechanism of defence never reported for N. caninum infection in macrophages. The expression of cytokines and cathelicidins in macrophages invaded by N. caninum was mediated by mitogen-activated protein kinase (MEK 1/2). Secretion of such innate factors from N. caninum-infected macrophages reduced parasite internalization and promoted the secretion of pro-inflammatory cytokines in naïve macrophages. We concluded that rapid invasion of macrophages by N. caninum triggered protective innate defence mechanisms against intracellular pathogens.

Toll-like receptors 3, 7 and 8 are upregulated in the placental caruncle and fetal spleen of Neospora caninum experimentally infected cattle

Innate immune responses at the maternal-fetal interface are key in the pathogenesis of Neospora caninum, an obligate parasite that causes abortion in cattle. Herein, we determined the gene expression of endosomal Toll-like receptors (TLRs) in the placenta and fetuses from both non-infected pregnant heifers and pregnant heifers intravenously challenged with live tachyzoites of N. caninum on day 70 of gestation. On day 104 of pregnancy, mRNA expression of TLRs 3 and 8, as well as that of TLRs 7 and 9, was high in the spleen of fetuses from N. caninum-infected heifers. Gene expression levels of endosomal TLRs were also detectable in the placenta and the maternal caruncle from infected heifers, being TLRs 3, 7 and 8 particularly upregulated, mostly in the caruncle. Basal TLR levels were higher in fetal spleen than in placental tissues. This study provides novel information on how innate TLR responses are induced at the maternal-fetal interface of cattle in response to intracellular N. caninum.

Protective immunity against toxoplasmosis in mice induced by single-dose immunization with rSAG1/2 protein released from poly(lactide-co-glycolide) microparticles.

Triphasic sustained release of tachyzoite chimeric protein, rSAG1/2, from poly(lactide-co-glycolide) (PLG)-encapsulated rSAG1/2 (PLG-rSAG1/2) microparticles (MPs) is a promising characteristic for developing a single-dose vaccine against Toxoplasma gondii in domestic animals. In the present study, we aimed to evaluate whether single immunization with PLG-rSAG1/2 MPs in BALB/c mice would achieve effective immunity and protection against T. gondii. Peritoneal immunization of mice with a single dose of PLG-rSAG1/2 MPs enhanced serum IgG titers and lymphocyte proliferation in a triphasic model over a long 12-week period. In addition, 12 weeks after immunization, significant production of IFN-γ was also monitored in mice vaccinated with one dose of PLG-rSAG1/2 MPs. More importantly, the immunity induced by one dose of PLG-rSAG1/2 MPs protected 70% of mice (14/20) against a lethal subcutaneous challenge of 1 × 104 live tachyzoites of T. gondii (RH strain). In conclusion, a single dose of PLG-rSAG1/2 MPs capable of sustaining triphasic release of rSAG1/2 protein induces long-lasting triphasic immunity against T. gondii in mice. Our data indicate the feasibility of PLG-rSAG1/2 MPs to be developed as a single-dose vaccine against T. gondii for potential use in domestic animals.

Neospora caninum Activates p38 MAPK as an Evasion Mechanism against Innate Immunity.

Due to the high prevalence and economic impact of neosporosis, the development of safe and effective vaccines and therapies against this parasite has been a priority in the field and is crucial to limit horizontal and vertical transmission in natural hosts. Limited data is available regarding factors that regulate the immune response against this parasite and such knowledge is essential in order to understand Neospora caninum induced pathogenesis. Mitogen-activated protein kinases (MAPKs) govern diverse cellular processes, including growth, differentiation, apoptosis, and immune-mediated responses. In that sense, our goal was to understand the role of MAPKs during the infection by N. caninum. We found that p38 phosphorylation was quickly triggered in macrophages stimulated by live tachyzoites and antigen extracts, while its chemical inhibition resulted in upregulation of IL-12p40 production and augmented B7/MHC expression. In vivo blockade of p38 resulted in an amplified production of cytokines, which preceded a reduction in latent parasite burden and enhanced survival against the infection. Additionally, the experiments indicate that the p38 activation is induced by a mechanism that depends on GPCR, PI3K and AKT signaling pathways, and that the phenomena here observed is distinct that those induced by Toxoplasma gondii’s GRA24 protein. Altogether, these results showed that N. caninum manipulates p38 phosphorylation in its favor, in order to downregulate the host’s innate immune responses. Additionally, those results infer that active interference in this signaling pathway may be useful for the development of a new therapeutic strategy against neosporosis.

Tracing amino acid exchange during host-pathogen interaction by combined stable-isotope time-resolved Raman spectral imaging.

This study investigates the temporal and spatial interchange of the aromatic amino acid phenylalanine (Phe) between human retinal pigment epithelial cell line (ARPE-19) and tachyzoites of the apicomplexan protozoan parasite Toxoplasma gondii (T. gondii). Stable isotope labelling by amino acids in cell culture (SILAC) is combined with Raman micro-spectroscopy to selectively monitor the incorporation of deuterium-labelled Phe into proteins in individual live tachyzoites. Our results show a very rapid uptake of l-Phe(D8) by the intracellular growing parasite. T. gondii tachyzoites are capable of extracting l-Phe(D8) from host cells as soon as it invades the cell. l-Phe(D8) from the host cell completely replaces the l-Phe within T. gondii tachyzoites 7–9 hours after infection. A quantitative model based on Raman spectra allowed an estimation of the exchange rate of Phe as 0.5–1.6 × 104 molecules/s. On the other hand, extracellular tachyzoites were not able to consume l-Phe(D8) after 24 hours of infection. These findings further our understanding of the amino acid trafficking between host cells and this strictly intracellular parasite. In particular, this study highlights new aspects of the metabolism of amino acid Phe operative during the interaction between T. gondii and its host cell.

Neospora caninum: Chronic and congenital infection in consecutive pregnancies of mice

Neospora caninum, the causative agent of bovine neosporosis is the major cause of abortion in cattle worldwide. The principal route of transmission is via in utero infection of the offspring. Congenitally-infected dams remain persistently infected for life and might undergo abortions in consecutive pregnancies. In the present study, the effect of N. caninum in chronic and congenital infection was examined. CD1 mice were infected intra-peritoneally with live tachyzoites of the NcIs491 isolate, while non-infected mice served as a control. There were no clinical signs of infection observed following inoculation, but high titers of specific anti- N. caninum antibodies were detected. A month after infection, when chronic-infection was established, mice were mated. Fertility, litter size and mortality rate were monitored within two generations of four consecutive pregnancies. During a nine months period of the study all females maintained high level of antibodies, while the non- infected control mice remained seronegative. There was no difference in the fertility rate of the dams, or in the litter size of infected and control mice. Mortality of offspring of the first and second generations of the infected dams was observed within the two first weeks of life. The vertical transmission was analyzed by PCR assay of offspring brains. PCR positive results were found in all 13 litters of the first generation tested during four consecutive pregnancies. The rate of vertical transmission slightly decreased in successive pregnancies being 74.2%, 59.5%, 48.1% and 40% for the first to fourth pregnancies respectively. In the second generation 21 out of 28 litters were found positive and the overall rate of vertical transmission was 28.5%. In chronically and congenitally infected dams N. caninum infection was maintained during all successive pregnancies for about 9 months. The results show that CD-1 outbred mice are a suitable model for studying chronic and congenital neosporosis.

Cell mediated immune responses in the placenta following challenge of vaccinated pregnant heifers with Neospora caninum

The aim of the present study was to investigate and correlate the cell-mediated immune response and pathological changes at the maternal-fetal interface of Neospora-challenged pregnant cattle previously immunized with live and inactivated experimental vaccines. Pregnant heifers naïve to Neospora caninum were divided in 5 groups of 4 animals, each one immunized before mating: Group A heifers were intravenously (iv) immunized with 6.25×107 live tachyzoites of the NC-6 strain; group B heifers were immunized twice subcutaneously (sc) 3 weeks apart with native antigen extract of the NC-6 strain formulated with ISCOMs; group C heifers were sc immunized twice 3 weeks apart with three recombinant proteins (rNcSAG1, rNcHSP20, rNcGRA7) of the NC-1 strain formulated with ISCOMs; group D heifers were sc injected with sterile phosphate-buffered saline (PBS) and group E heifers received sc ISCOM-matrix (ISCOMs without antigen). All groups were iv-challenged with 4.7×107 NC-1 tachyzoites at 70 days of gestation. Heifers were culled at day 104 of gestation and placentomes were examined to evaluate lesions and local cellular immune responses using histopathology, immunohistochemistry and real time-PCR. Immunohistochemistry was performed using bovine leucocyte specific antibodies. Cytokine expression and levels (IFN-γ, IL-4, IL-10, IL-12 and TNF-α) were measured using real-time reverse transcription-PCR and ELISA, respectively. Minimal inflammation was observed in group A placentomes; while placentomes from group B, C, D and E had moderate to severe infiltration with CD3+, CD4+, γδ-T cells, CD8+ cells and macrophages being more numerous in groups B and E placentomes, when compared with groups C and D (P<0.001). Cytokine levels were significantly increased in the caruncles of animals of groups B and C in comparison with the other animal groups (P<0.001). The results from this study showed that the strongest cellular immune responses were observed in the placentomes of animals that were immunized with inactivated vaccines (groups B and C) and in the placentomes of animals that were sc-sham-inoculated (groups D and E). On the other hand, animals that were immunized with live tachyzoites showed a milder immune cell infiltration to the placenta possibly due to the existence of a protective systemic maternal immune response that helped to minimize N. caninum infection at the maternal-fetal interface.

The effect of a live Neospora caninum tachyzoite vaccine in naturally infected pregnant dairy cows

Neosporosis, caused by the intracellular protozoan Neospora caninum, is a major cause of abortion and reproductive failure in cattle worldwide. The principal route of transmission of neosporosis is via in utero infection of the offspring. There is no effective prophylactic treatment or vaccine available against bovine neosporosis. A N. caninum NcIs491 isolate was examined for its ability to immunize and reduce abortions in naturally infected dairy cows under field conditions. N. caninum-seropositive pregnant dams were inoculated with 108 live tachyzoites during mid-term pregnancy. A total of 520 N. caninum seropositive dams were included in this study, of these, 146 were immunized and 374 cows served as a non-vaccinated control group. A significantly lower incidence of abortion was observed in vaccinated compared to non-vaccinated cows, 16 and 26% respectively (P=0.01), with a vaccine efficacy of 39%. However, the number of seropositive offspring remained similar in both groups. Overall, this field trial suggests that vaccination with live N. caninum tachyzoites should be considered as an effective measure to reduce abortions caused by neosporosis in naturally infected cows.