Identification and Function of Apicoplast Glutaredoxins in Neospora caninum.

Glutaredoxins (GRXs), important components of the intracellular thiol redox system, are involved in multiple cellular processes. In a previous study, we identified five GRXs in the apicomplexan parasite, Neospora caninum. In the present study, we confirmed that the GRXs S14 and C5 are located in the apicoplast, which suggests unique functions for these proteins. Although single-gene deficiency did not affect the growth of parasites, a double knockout (Δgrx S14Δgrx C5) significantly reduced their reproductive capacity. However, there were no significant changes in redox indices (GSH/GSSG ratio, reactive oxygen species and hydroxyl radical levels) in double-knockout parasites, indicating that grx S14 and grx C5 are not essential for maintaining the redox balance in parasite cells. Key amino acid mutations confirmed that the Cys203 of grx S14 and Cys253/256 of grx C5 are important for parasite growth. Based on comparative proteomics, 79 proteins were significantly downregulated in double-knockout parasites, including proteins mainly involved in the electron transport chain, the tricarboxylic acid cycle and protein translation. Collectively, GRX S14 and GRX C5 coordinate the growth of parasites. However, considering their special localization, the unique functions of GRX S14 and GRX C5 need to be further studied.

Glutathione reductase: A cytoplasmic antioxidant enzyme and a potential target for phenothiazinium dyes in Neospora caninum.

Neospora caninum causes heavy losses related to abortions in bovine cattle. This parasite developed a complex defense redox system, composed of enzymes as glutathione reductase (GR). Methylene blue (MB) impairs the activity of recombinant form of Plasmodium GR and inhibits the parasite proliferation in vivo and in vitro. Likewise, MB and its derivatives inhibits Neospora caninum proliferation, however, whether the MB mechanism of action is correlated to GR function remains unclear. Therefore, here, N. caninum GR (NcGR) was characterized and its potential inhibitors were determined. NcGR was found in the tachyzoite cytosol and has a similar structure and sequence compared to its homologs. We verified the in vitro activity of rNcGR (875 nM) following NADPH absorbance at 340 nM (100 mM KH2PO4, pH 7.5, 1 mM EDTA, ionic strength: 600 mM, 25 °C). rNcGR exhibited a Michaelian behavior (Km(GSSG):0.10 ± 0.02 mM; kcat(GSSG):0.076 ± 0.003 s-1; Km(NADPH):0.006 ± 0.001 mM; kcat(NADPH): 0.080 ± 0.003 s-1). The IC50 of MB,1,9-dimethyl methylene blue, new methylene blue, and toluidine blue O on rNcGR activity were 2.1 ± 0.2 µM, 11 ± 2 µM, 0.7 ± 0.1 µM, and 0.9 ± 0.2 µM, respectively. Our results suggest the importance of NcGR in N. caninum biology and antioxidant mechanisms. Moreover, data presented here strongly suggest that NcGR is an important target of phenothiazinium dyes in N. caninum proliferation inhibition.

Comparative assessment of the effects of bumped kinase inhibitors on early zebrafish embryo development and pregnancy in mice.

Bumped kinase inhibitors (BKIs) are effective against a variety of apicomplexan parasites. Fifteen BKIs with promising in vitro efficacy against Neospora caninum tachyzoites, low cytotoxicity in mammalian cells, and no toxic effects in non-pregnant BALB/c mice were assessed in pregnant mice. Drugs were emulsified in corn oil and were applied by gavage for 5 days. Five BKIs did not affect pregnancy, five BKIs exhibited ~15-35% neonatal mortality and five compounds caused strong effects (infertility, abortion, stillbirth and pup mortality). Additionally, the impact of these compounds on zebrafish (Danio rerio) embryo development was assessed by exposing freshly fertilised eggs to 0.2-50 µM of BKIs and microscopic monitoring of embryo development in a blinded manner for 4 days. We propose an algorithm that includes quantification of malformations and embryo deaths, and established a scoring system that allows the calculation of an impact score (Si) indicating at which concentrations BKIs visibly affect zebrafish embryo development. Comparison of the two models showed that for nine compounds no clear correlation between Si and pregnancy outcome was observed. However, the three BKIs affecting zebrafish embryos only at high concentrations (≥40 µM) did not impair mouse pregnancy at all, and the three compounds that inhibited zebrafish embryo development already at 0.2 µM showed detrimental effects in the pregnancy model. Thus, the zebrafish embryo development test has limited predictive value to foresee pregnancy outcome in BKI-treated mice. We conclude that maternal health-related factors such as cardiovascular, pharmacokinetic and/or bioavailability properties also contribute to BKI-pregnancy effects.

Inhibitory action of phenothiazinium dyes against Neospora caninum.

Neospora caninum is an Apicomplexan parasite related to important losses in livestock, causing abortions and decreased fertility in affected cows. Several chemotherapeutic strategies have been developed for disease control; however, no commercial treatment is available. Among the candidate drugs against neosporosis, phenothiazinium dyes, offer a low cost-efficient approach to parasite control. We report the anti-parasitic effects of the phenothiaziums Methylene Blue (MB), New Methylene Blue (NMB), 1,9-Dimethyl Methylene Blue (DMMB) and Toluidine Blue O (TBO) on N. caninum, using in vitro and in vivo models. The dyes inhibited parasite proliferation at nanomolar concentrations (0.019-1.83 µM) and a synergistic effect was achieved when Methylene Blue was combined with New Methylene Blue (Combination Index = 0.84). Moreover, the phenothiazinium dyes improved parasite clearance when combined with Pyrimethamine (Pyr). Combination of Methylene Blue + 1,9-Dimethyl Methylene Blue demonstrated superior efficacy compared to Pyrimethamine based counterparts in an in vivo model of infection. We also observed that Methylene Blue, New Methylene Blue and 1,9-Dimethyl Methylene Blue increased by 5000% the reactive oxygen species (ROS) levels in N. caninum tachyzoites. Phenothiazinium dyes represent an accessible group of candidates with the potential to compound future formulations for neosporosis control.

Immediate Interferon Gamma Induction Determines Murine Host Compatibility Differences between Toxoplasma gondii and Neospora caninum.

Rodents are critical for the transmission of Toxoplasma gondii to the definitive feline host via predation, and this relationship has been extensively studied as a model for immune responses to parasites. Neospora caninum is a closely related coccidian parasite of ruminants and canines but is not naturally transmitted by rodents. We compared mouse innate immune responses to N. caninum and T. gondii and found marked differences in cytokine levels and parasite growth kinetics during the first 24 h postinfection (hpi). N. caninum-infected mice produced significantly higher levels of interleukin-12 (IL-12) and interferon gamma (IFN-γ) by as early as 4 hpi, but the level of IFN-γ was significantly lower or undetectable in T. gondii-infected mice during the first 24 hpi. "Immediate" IFN-γ and IL-12p40 production was not detected in MyD88-/- mice. However, unlike IL-12p40-/- and IFN-γ-/- mice, MyD88-/- mice survived N. caninum infections at the dose used in this study. Serial measures of parasite burden showed that MyD88-/- mice were more susceptible to N. caninum infections than wild-type (WT) mice, and control of parasite burdens correlated with a pulse of serum IFN-γ at 3 to 4 days postinfection in the absence of detectable IL-12. Immediate IFN-γ was partially dependent on the T. gondii mouse profilin receptor Toll-like receptor 11 (TLR11), but the ectopic expression of N. caninum profilin in T. gondii had no impact on early IFN-γ production or parasite proliferation. Our data indicate that T. gondii is capable of evading host detection during the first hours after infection, while N. caninum is not, and this is likely due to the early MyD88-dependent recognition of ligands other than profilin.

Triclosan inhibits the growth of Neospora caninum in vitro and in vivo.

Neospora caninum is an apicomplexan parasite considered one of the main causes of abortion in cattle worldwide; thus, there is an urgent need to develop novel therapeutic agents to control the neosporosis. Enoyl acyl carrier protein reductase (ENR) is a key enzyme of the type II fatty acid synthesis pathway (FAS II), which is essential for apicomplexan parasite survival. The antimicrobial agent triclosan has been shown to be a very potent inhibitor of ENR. In this study, we identified an E. coli ENR-like protein in N. caninum. Multiple sequence alignment showed all the requisite features of ENR existed in this protein, so we named this protein NcENR. Swiss-Model analysis showed NcENR interacts with triclosan. We observed that ENR is localized in the apicoplast, a plastid-like organelle. Similar to the potent inhibition of triclosan on other apicomplexa parasites, this compound markedly inhibits the growth of N. caninum at low concentrations. Further research showed that triclosan attenuated the invasion ability and proliferation ability of N. caninum at low concentrations. The results from in vivo studies in the mouse showed that triclosan attenuated the virulence of N. caninum in mice mildly and reduced the parasite burden in the brain significantly. Taken together, triclosan inhibits the growth of N. caninum both in vitro and in vivo at low concentrations.

IDO, COX and iNOS have an important role in the proliferation of Neospora caninum in neuron/glia co-cultures.

Central nervous system (CNS) is the main site for encystment of Neospora caninum in different animal species. In this tissue, glial cells (astrocytes and microglia) modulate responses to aggression in order to preserve homeostasis and neuronal function. Previous data showed that when primary cultures of glial cells are infected with N. caninum, they develop gliosis and the immune response is characterized by the release of TNF and IL-10, followed by the control of parasite proliferation. In order to elucidate this control, three enzymatic systems involved in parasite-versus-host interactions were observed on a model of neuron/glia co/cultures obtained from rat brains. Indoleamine 2,3-dioxygenase (IDO), induced nitric oxide synthase (iNOS) responsible for the catabolism of tryptophan and arginine, respectively, and cycloxigenase (COX) were studied comparing their modulation by respective inhibitors with the number of tachyzoites or the immune response measured by the release of IL-10 and TNF. Cells were treated with the inhibitors of iNOS (1.5 mM L-NAME), IDO (1 mM 1-methyl tryptophan), COX-1 (1 µM indomethacin) and COX-2 (1 µM nimesulide) before infection with tachyzoites of N. caninum (1:1 cell: parasite). After 72 h of infection, immunocytochemistry showed astrogliosis and a significant increase in the number and length of neurites, compared with uninfected co-cultures, while an increase of IL-10 and TNF was verified. N. caninum did not change iNOS activity, but the inhibition of the basal levels of this enzyme stimulated parasite proliferation. Additionally, a significant increase of about 40% was verified in the IDO activity, whose inhibition caused 1.2-fold increase in parasitic growth. For COX-2 activity, infection of cultures stimulated a significant increase in release of PGE2 and its inhibition by nimesulide allowed the parasitic growth. These data indicate that iNOS, IDO and COX-2 control the proliferation of N. caninum in this in vitro model. On the other hand, the release of IL-10 by glia besides modulating the inflammation also allow the continuity of parasitism.

Effects of Thai piperaceae plant extracts on Neospora caninum infection.

Neosporosis has a worldwide distribution and causes economic losses in farming, particularly by increasing the risk of abortion in cattle. This study investigated the effects of Thai piperaceae (Piper betle, P. nigrum, and P. sarmentosum) extracts on Neospora caninum infections in vitro and in vivo. In an in vitro parasite growth assay based on the green fluorescent protein (GFP) signal, P. betle was the most effective extract at inhibiting parasite growth in human foreskin fibroblast cells (IC50 of GFP-expressing N. caninum parasites, 22.1µg/ml). The P. betle extract, at 25µg per ml, inhibited parasite invasion into host cells. Furthermore, in two independent experiments, treating N. caninum-infected mice with the P. betle extract for 7days post-infection increased their survival. In trial one, the anti-N. caninum effects of the P. betle extract reduced the mouse clinical scores for 30days post-infection (dpi). The survival rate of the mice treated with 400mg/kg was 100% compared with 66.6% for those treated with 100mg/kg and the non-treated controls. In trial two, treating the infected mice with the P. betle extract increased their survival at 50dpi. All mice in the non-treatment group died; however, the survival rates of the 400mg/kg-treated and 100mg/kg-treated mice were 83.3% and 33.3%, respectively. Also, a trend towards a reduced parasite burden was noted in the brains of the P. betle extract-treated mice, compared with the control mice. Therefore P. betle extract has potential as a medicinal plant for treating neosporosis.

Two Novel Calcium-Dependent Protein Kinase 1 Inhibitors Interfere with Vertical Transmission in Mice Infected with Neospora caninum Tachyzoites.

We present the effects of two novel bumped kinase inhibitors, BKI-1517 and BKI-1553, against Neospora caninum tachyzoites in vitro and in experimentally infected pregnant mice. These compounds inhibited tachyzoite proliferation of a transgenic beta-galactosidase reporter strain cultured in human foreskin fibroblasts with 50% inhibitory concentrations (IC50s) of 0.05 ± 0.03 and 0.18 ± 0.03 µM, respectively. As assessed by an alamarBlue assay, fibroblast IC50s were above 20 µM; however, morphological changes occurred in cultures treated with >5 µM BKI-1517 after prolonged exposure (>6 days). Treatment of intracellular tachyzoites with 5 µM BKI-1553 for 6 days inhibited endodyogeny by interfering with the separation of newly formed zoites from a larger multinucleated parasite mass. In contrast, parasites treated with 5 µM BKI-1517 did not form large complexes and showed much more evidence of cell death. However, after a treatment duration of 10 days in vitro, both compounds failed to completely prevent the regrowth of parasites from culture. BALB/c mice experimentally infected with N. caninum Spain7 (Nc-Spain7) and then treated during 6 days with BKI-1517 or BKI-1553 at different dosages showed a significant reduction of the cerebral parasite load. However, fertility was impaired by BKI-1517 when applied at 50 mg/kg of body weight/day. At 20 mg/kg/day, BKI-1517 significantly inhibited the vertical transmission of N. caninum to pups and increased the rate of survival of offspring. BKI-1553 was less detrimental to fertility and also provided significant but clearly less pronounced protection of dams and offspring. These results demonstrate that, when judiciously applied, this compound class protects offspring from vertical transmission and disease.

In contrast to Toxoplasma gondii, Neospora caninum tachyzoites did not sustain multiplication in vitro at increased incubation temperatures.

Neospora caninum and Toxoplasma gondii are coccidian parasites that infect a wide range of mammalian and avian species. While viable T. gondii has been in vitro isolated in natural infections from wild and domestic birds, attempts to isolate N. caninum from naturally-infected birds were unsuccessful. We speculate that body temperatures of birds, which are usually higher than those of mammals, may impair the multiplication of N. caninum. In contrast to N. caninum, T. gondii can grow in vitro at temperatures higher than 37°C. To test the hypothesis that N. caninum tachyzoites are impaired to grow in vitro at high temperatures, three strains of N. caninum (NC-1, NC-Liverpool, and NC-Bahia) and three of T. gondii (RH, ME-49 and NED) were cultivated at gradually increasing temperatures starting at 37°C up to 41.5°C. A permanent chicken cell line was chosen for the study. Parasites were observed microscopically and their presence in culture was evaluated by species-specific conventional PCRs. In a second experiment, growth rates of T. gondii (RH strain) and N. caninum (NC-1 strain) were evaluated after direct passage of tachyzoites from 37°C to 41.5°C, and quantified by real-time PCR. In addition to comparisons between N. caninum and T. gondii, growth rates of three T. gondii strains were compared at high temperatures. Neospora caninum tachyzoites could not sustain multiplication at temperatures between 39°C and 41.5°C. Toxoplasma gondii tachyzoites continued to multiply at the same experimental conditions. Direct passage of N. caninum tachyzoites from 37°C to 41.5°C caused a significant decrease in the number of parasites during 96h of observation, while T. gondii had a significant increase in the number of stages after the same period of time. T. gondii RH strain (clonal type I) presented a different growth rate at 41.5°C when compared with type II and type III strains. In conclusion, multiplication of N. caninum tachyzoites in vitro was inhibited at temperatures similar to those of chickens, what may be one of the reasons that isolation of the parasite is difficult in avian species. In contrast to N. caninum, T. gondii continued to grow at 41.5°C.

Activity of several kinds of drugs against Neospora caninum.

Neosporosis caused by Neospora caninum is a serious disease in cattle and dogs worldwide. It is the major cause of abortion and neonatal mortality in cattle. In this study, we evaluated the anti-N. caninum activity of Chinese medicine extracts (curcumin, artemether), herbicides (atrazine, glyphosate), anticoccidiosis drugs (toltrazuril and ponazuril), cyclophosphamide, diminazene aceturate and praziquantel in vitro using parasite growth, replication and host cell invasion assays in human foreskin fibroblast cultures. Curcumin, artemether, atrazine, toltrazuril and ponazuril exhibited inhibitory activity with 50% growth inhibitory concentration (IC50) of 1.1±0.4, 1.0±0.05, 11.2±2.7, 30.3±2.0 and 33.3±4.1µg/ml, respectively, in the growth inhibition assay. They were also active against protozoa replication, but only curcumin was effective against host cell invasion. Glyphosate, cyclophosphamide, diminazene aceturate and praziquantel were ineffective. In an in vivo infection model, curcumin showed no activity against N. caninum infection. We showed that curcumin, artemether, atrazine, toltrazuril, and ponazuril exhibited anti-N. caninum activity in vitro, providing important information for further studies on anti-N. caninum drugs.

Flavonoids modulate the proliferation of Neospora caninum in glial cell primary cultures.

Neospora caninum (Apicomplexa; Sarcocystidae) is a protozoan that causes abortion in cattle, horses, sheep, and dogs as well as neurological and dermatological diseases in dogs. In the central nervous system of dogs infected with N. caninum, cysts were detected that exhibited gliosis and meningitis. Flavonoids are polyphenolic compounds that exhibit antibacterial, antiparasitic, antifungal, and antiviral properties. In this study, we investigated the effects of flavonoids in a well-established in vitro model of N. caninum infection in glial cell cultures. Glial cells were treated individually with 10 different flavonoids, and a subset of cultures was also infected with the NC-1 strain of N. caninum. All of the flavonoids tested induced an increase in the metabolism of glial cells and many of them increased nitrite levels in cultures infected with NC-1 compared to controls and uninfected cultures. Among the flavonoids tested, 3',4'-dihydroxyflavone, 3',4',5,7-tetrahydroxyflavone (luteolin), and 3,3',4',5,6-pentahydroxyflavone (quercetin), also inhibited parasitophorous vacuole formation. Taken together, our findings show that flavonoids modulate glial cell responses, increase NO secretion, and interfere with N. caninum infection and proliferation.

The chloramphenicol acetyltransferase vector as a tool for stable tagging of Neospora caninum.

Neospora caninum is an obligate intracellular Apicomplexa, a phylum where one of the current methods for functional studies relies on molecular genetic tools. For Toxoplasma gondii, the first method described, in 1993, was based on resistance against chloramphenicol. As in T. gondii, we developed a vector constituted of the chloramphenicol acetyltransferase gene (CAT) flanked by the N. caninum dihydrofolate reductase-thymidylate synthase (DHFR-TS) 5' coding sequence flanking region. Five weeks after transfection and under the selection of chloramphenicol the expression of CAT increased compared to the wild type and the resistance was retained for more than one year. Between the stop codon of CAT and the 3' UTR of DHFR, a Lac-Z gene controlled by the N. caninum tubulin 5' coding sequence flanking region was ligated, resulting in a vector with a reporter gene (Ncdhfr-CAT/NcTub-tetO/Lac-Z). The stability was maintained through an episomal pattern for 14 months when the tachyzoites succumbed, which was an unexpected phenomenon compared to T. gondii. Stable parasites expressing the Lac-Z gene allowed the detection of tachyzoites after invasion by enzymatic reaction (CPRG) and were visualised macro- and microscopically by X-Gal precipitation and fluorescence. This work developed the first vector for stable expression of proteins based on chloramphenicol resistance and controlled exclusively by N. caninum promoters.

Comparison of host cell invasion and proliferation among Neospora caninum isolates obtained from oocysts and from clinical cases of naturally infected dogs.

In a previous study we have shown that the in vitro invasion rate (IR) and tachyzoite yield (TY) are associated with the virulence phenotypes of Neospora caninum isolates of bovine origin. In addition, we recently observed marked differences in virulence when canine isolates were compared in a pregnant BALB/c mouse model. In this study, we investigated whether invasion and proliferation capacities could be used as virulence-related N. caninum phenotypic traits. Of the isolates compared in mice, four canine isolates obtained from oocysts (Nc-Ger2, Nc-Ger3, Nc-Ger-6, Nc-6 Arg) had shown a low-moderate virulence, and two further isolates obtained from dogs with neurological signs (Nc-Bahia, Nc-Liv) were highly virulent. The IR for each isolate was determined by a plaque assay and the counting of immunofluorescence-labeled parasitophorous vacuoles at 3 days post-inoculation (p.i.). The TY was determined by the quantification of tachyzoites at 56 h p.i. by real-time PCR. Most of the canine isolates showed similar IR values under controlled invasion conditions for 4h and 72 h p.i., indicating a limited time period for invasion similar to that observed for bovine isolates. The Nc-Ger3, Nc-Bahia, and Nc-Liv isolates showed a significantly higher IR and TY than the Nc-Ger2 and Nc-Ger6 isolates (P<0.0001). A correlation was found between the IRs and TY (ρ>0.885, P<0.033), as well as between the TY and both dam morbidity (ρ=0.8452, P<0.033) and pup mortality (ρ>0.8117, P<0.058) in mice. These results demonstrate the importance both the invasive and proliferative capacities have on the virulence of canine N. caninum isolates.

Metabolic footprinting of extracellular metabolites of brain endothelium infected with Neospora caninum in vitro.

BACKGROUND: The survival of the intracellular protozoan parasite Neospora caninum depends on its ability to adapt to changing metabolic conditions of the host cell. Thus, defining cellular and metabolic changes in affected target tissues may aid in delineating pathogenetic mechanism. We undertook this study to assess the metabolic response of human brain microvascular endothelial cells (HBMECs) to N. caninum infection in vitro. METHODS: HBMECs were exposed to N. caninum infection and the cytotoxic effects of infection were analyzed by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazoliumbromidin (MTT) assay and lactate dehydrogenase (LDH) release assay. Metabolic footprinting of the extracellular metabolites of parasite-infected and non-infected culture supernatant was determined by using targeted (Randox RX Imola clinical chemistry analyser) and unbiased RS (Raman microspectroscopy) approaches. RESULTS: The MTT assay did not reveal any cytotoxic effect of N. caninum challenge on host cell viability. Measurement of LDH activity showed that N. caninum significantly induced loss of cell membrane integrity in a time-dependent and dose-dependent manner compared to control cells. Targeted biochemical analysis revealed that beta hydroxybutyrate, pyruvate, ATP, total protein, non-esterified fatty acids, and triglycerides are significantly different in infected cells compared to controls. RS-based footprinting with principal component analysis (PCA) were able to correctly distinguish extracellular metabolites obtained from infected and control cultures, and revealed infection-related spectral signatures at 865 cm-1, 984 cm-1, 1046 cm-1, and 1420 cm-1, which are attributed to variations in the content of lipids and nucleic acids in infected cultures. CONCLUSIONS: The changing pattern of extracellular metabolites suggests that HBMECs are target of metabolic alterations in N. caninum infection, which seem to reflect the changing metabolic state of infected cells and constitute a level of information exchange that host and parasite use to coordinate activities.

Comparison of an ELISA assay for the detection of adhesive/invasive Neospora caninum tachyzoites.

Neospora caninum belongs to the phylum Apicomplexa, the causative agent of neosporosis, which leads to economic impacts on cattle production. A common feature among apicomplexan parasites is the invasive process driven mostly by the parasite. As a first evaluation of candidate molecules that play a possible role by interfering in this invasive process, the in vitro invasion assay is a fast and direct way to screen future agonists or antagonists. This work involved the development of a new cell culture ELISA and transient ß-galactosidase activity applied to the semi-quantitative detection of N. caninum in Vero cell culture. Cell culture ELISA is based on histochemistry and immunology, resulting in a colorimetric reaction. The ß-galactosidase activity was obtained by the transient transfection of the lacZ gene under control of RPS13 promoter of N. caninum. These methods were used to evaluate the effects of temperature (37°C and 85°C) on the invasion and adhesion of tachyzoites. The three tested methods (real time PCR, ß-galactosidase activity and ELISA) showed a similar pattern, indicating that different methods may be complementary.

Comparison of an ELISA assay for the detection of adhesive/invasive Neospora caninum tachyzoites / Comparação de um ensaio de ELISA para a detecção de taquizoítas adesivos/invasivos de Neospora caninum

Neospora caninum belongs to the phylum Apicomplexa, the causative agent of neosporosis, which leads to economic impacts on cattle production. A common feature among apicomplexan parasites is the invasive process driven mostly by the parasite. As a first evaluation of candidate molecules that play a possible role by interfering in this invasive process, the in vitro invasion assay is a fast and direct way to screen future agonists or antagonists. This work involved the development of a new cell culture ELISA and transient β-galactosidase activity applied to the semi-quantitative detection of N. caninum in Vero cell culture. Cell culture ELISA is based on histochemistry and immunology, resulting in a colorimetric reaction. The β-galactosidase activity was obtained by the transient transfection of the lacZ gene under control of RPS13 promoter of N. caninum. These methods were used to evaluate the effects of temperature (37°C and 85°C) on the invasion and adhesion of tachyzoites. The three tested methods (real time PCR, β-galactosidase activity and ELISA) showed a similar pattern, indicating that different methods may be complementary.

Neospora caninum, parasita do filo Apicomplexa, é causador da neosporose, doença responsável por perdas econômicas importantes na pecuária. Um fator comum entre os apicomplexas é o processo de invasão majoritariamente dirigido pelo parasita. Dentre as primeiras avaliações de moléculas candidatas, que possivelmente interferem no processo de invasão, o ensaio de invasão in vitro é um meio rápido e direto de selecionar futuros agonistas ou antagonistas. Este trabalho desenvolveu um novo ELISA baseado em cultura (Cell-culture ELISA) e um ensaio que mede a atividade transiente de β-galactosidase, aplicados para a detecção semiquantitativa de N. caninum em células Vero. Cell-culture ELISA é baseado em histoquímica e imunologia, resultando em uma reação colorimétrica. A atividade da β-galactosidase foi obtida pela transfecção transiente do gene LacZ sob controle do promotor RPS13 de N. caninum. Esses métodos avaliaram os efeitos da temperatura (37°C e 85°C) sobre a invasão e adesão. Os três métodos testados (real time PCR, atividade de β-galactosidase e ELISA) mostraram um padrão similar, indicando que diferentes métodos podem ser complementares. Adicionalmente, esse ELISA é adequado para aplicação em laboratórios carentes de uma complexa estrutura para métodos de detecção moleculares.

Mucosal and systemic T cell response in mice intragastrically infected with Neospora caninum tachyzoites.

The murine model has been widely used to study the host immune response to Neospora caninum. However, in most studies, the intraperitoneal route was preferentially used to establish infection. Here, C57BL/6 mice were infected with N. caninum tachyzoites by the intragastric route, as it more closely resembles the natural route of infection through the gastrointestinal tract. The elicited T-cell mediated immune response was evaluated in the intestinal epithelium and mesenteric lymph nodes (MLN). Early upon the parasitic challenge, IL-12 production by conventional and plasmacytoid dendritic cells was increased in MLN. Accordingly, increased proportions and numbers of TCRαß+CD8+IFN-γ+ lymphocytes were detected, not only in the intestinal epithelium and MLN, but also in the spleen of the infected mice. In this organ, IFN-γ-producing TCRαß+CD4+ T cells were also found to increase in the infected mice, however later than CD8+ T cells. Interestingly, splenic and MLN CD4+CD25+ T cells sorted from infected mice presented a suppressive activity on in vitro T cell proliferation and cytokine production above that of control counterparts. These results altogether indicate that, by producing IFN-γ, TCRαß+CD8+ cells contribute for local and systemic host protection in the earliest days upon infection established through the gastrointestinal tract. Nevertheless, they also provide substantial evidence for a parasite-driven reinforcement of T regulatory cell function which may contribute for parasite persistence in the host and might represent an additional barrier to overcome towards effective vaccination.

In vitro effects of novel ruthenium complexes in Neospora caninum and Toxoplasma gondii tachyzoites.

Upon the screening of 16 antiproliferative compounds against Toxoplasma gondii and Neospora caninum, two hydrolytically stable ruthenium complexes (compounds 16 and 18) exhibited 50% inhibitory concentrations of 18.7 and 41.1 nM (T. gondii) and 6.7 and 11.3 nM (N. caninum). To achieve parasiticidal activity with compound 16, long-term treatment (22 to 27 days at 80 to 160 nM) was required. Transmission electron microscopy demonstrated the rapid impact on and ultrastructural alterations in both parasites. These preliminary findings suggest that the potential of ruthenium-based compounds should thus be further exploited.

Suspension culture of Neospora caninum by Theileria annulata-infected cell line.

There are some limiting aspects of scaling up the Neospora caninum tachyzoites in continuous cell lines, particularly as related to the absence of surface attachment. In this study, suspension cell culture of Theileria annulata-infected lymphoblastoid (TIL) was used as a host cell for the continous production of N. caninum tachyzoites. The numbers of free tachyzoites in the medium supernatant were showed regularly increased up to the day 6 post-cultivation. Transmission electron microscopy demonstrated that N. caninum tachyzoites invaded the TIL cells and multiplied intracellularly. This showed that the tachyzoites were successfully proliferated in TIL cells and were released in complete Dulbecco's modified Eagle's medium. This is a successful report of in vitro cultivation of N. caninum tachyzoites achieved by using suspension host cell culture.