Subcellular localization and functional characterization of Nc-p43, a major Neospora caninum tachyzoite surface protein.

Neospora caninum is a recently identified coccidian parasite which shares many features with, but is clearly distinct from, Toxoplasma gondii. N. caninum tachyzoites infect a wide range of mammalian cells both in vivo and in vitro. The mechanisms by which infection is achieved are largely unknown. Recent evidence has suggested that a receptor-ligand system in which one or several host cell receptors bind to one or several parasite ligands is involved. Parasite cell surface-associated molecules such as the recently identified Nc-p43 antigen are prime suspects for being implicated in this physical interaction. In this study it is shown that invasion of Vero cell monolayers by N. caninum tachyzoites in vitro is impaired on incubation of parasites with subagglutinating amounts of affinity-purified antibodies directed against Nc-p43. Postembedding immunogold labeling with anti-Nc-p43 antibodies demonstrated that Nc-p43 is localized not only on the parasite cell surface but also within dense granules and rhoptries. The fate of Nc-p43 during intracellular proliferation of N. caninum tachyzoites and subsequent maturation of the parasitophorous vacuole was also studied.     

Serological diagnosis of bovine neosporosis by Neospora caninum monoclonal antibody-based competitive inhibition enzyme-linked immunosorbent assay.

Neospora caninum, a protozoan parasite closely related to Toxoplasma gondii, causes abortion and congenital infection in cattle. To investigate specific methods of antemortem diagnosis, the antibody responses of infected cows were evaluated by immunoblot assay and competitive inhibition enzyme-linked immunosorbent assay (CI-ELISA) by using a monoclonal antibody (MAb), MAb 4A4-2, against N. caninum tachyzoites. MAb 4A4-2 bound diffusely to the exterior surface of N. caninum tachyzoites and recognized a single 65-kDa band in immunoblots. MAb 4A4-2 was unreactive to antigens of two closely related apicomplexan protozoa, Toxoplasma gondii and Sarcocystis cruzi. Binding of MAb 4A4-2 was inhibited by mild periodate treatment of N. caninum antigen, demonstrating the carbohydrate nature of the epitope. Immunoblot analysis of N. caninum tachyzoite antigens with sera from cows with confirmed Neospora-induced abortion revealed at minimum 14 major antigens ranging from 11 to 175 kDa. Although the recognized antigens varied from cow to cow, antigens of 116, 65, and 25 kDa were detected in all cows with abortion confirmed to be caused by N. caninum. The binding of MAb 4A4-2 to N. caninum tachyzoite antigen was consistently inhibited by sera from Neospora-infected cows in a CI-ELISA format and was not inhibited by sera from Neospora antibody-negative cows. Furthermore, sera from cattle experimentally infected with T. gondii, S. cruzi, Sarcocystis hominis, or Sarcocystis hirsuta, which had cross-reactive antibodies recognizing multiple N. caninum antigens by immunoblot assay, did not inhibit binding of MAb 4A4-2 in the CI-ELISA. Thus, MAb 4A4-2 binds a carbohydrate epitope on a single N. caninum tachyzoite surface antigen that is recognized consistently and specifically by Neospora-infected cattle.     

Identification of ribosomal phosphoprotein P0 of Neospora caninum as a potential common vaccine candidate for the control of both neosporosis and toxoplasmosis

The characterization of the cross-reactive antigens of two closely related apicomplexan parasites, Neospora caninum and Toxoplasma gondii, is important to elucidate the common mechanisms of parasite-host interactions. In this context, a gene encoding N. caninum ribosomal phosphoprotein P0 (NcP0) was identified by immunoscreening of a N. caninum tachyzoite cDNA expression library with antisera from mice immunized with T. gondii tachyzoites. The NcP0 was encoded by a gene with open reading frame of 936 bp, which encoded a protein of 311 amino acids. The NcP0 gene existed as a single copy in the genome and was interrupted by a 432 bp intron. The NcP0 showed 94.5% amino acid identity to T. gondii P0 (TgP0). Anti-recombinant NcP0 (rNcP0) sera recognized a native parasite protein with a molecular mass of 34 kDa in Western blot analysis. Immunofluorescence analysis showed that the NcP0 was localized to the surface of N. caninum tachyzoites. A purified anti-rNcP0 IgG antibody inhibited the growth of N. caninum and T. gondii in vitro in a concentration-dependent manner. These results indicate that P0 is a cross-reactive antigen between N. caninum and T. gondii and a potential common vaccine candidate to control both parasites.     

Apical membrane antigen 1 is a cross-reactive antigen between Neospora caninum and Toxoplasma gondii, and the anti-NcAMA1 antibody inhibits host cell invasion by both parasites

The cross-reactive antigens of Neospora caninum and Toxoplasma gondii are important in the exploration to determine the common mechanisms of parasite-host interaction. In this study, a gene encoding N. caninum apical membrane antigen 1 (NcAMA1) was identified by immunoscreening of a N. caninum tachyzoite cDNA expression library with antisera from mice immunized with recombinant T. gondii apical membrane antigen 1 (TgAMA1). NcAMA1 was encoded by an open reading frame of 1695 bp, which encoded a protein of 564 amino acids. The single-copy NcAMA1 gene was interrupted by seven introns. NcAMA1 showed 73.6% amino acid identity to TgAMA1. Mouse polyclonal antibodies raised against the recombinant NcAMA1 (rNcAMA1) recognized a 69-kDa native parasite protein by Western blotting. Immunofluorescence analysis showed that NcAMA1 was localized to the apical end of tachyzoites. Two-dimensional electrophoresis and Western blotting indicated that an approximately 57-kDa cleavage product was released into the excretory/secretory products of N. caninum. Preincubation of free tachyzoites with anti-rNcAMA1 IgG antibodies inhibited the invasion into host cells by N. caninum and T. gondii. These results indicated that AMA1 is a cross-reactive antigen between N. caninum and T. gondii and a potential common vaccine candidate to control two parasites.     

Identification and characterization of Neospora caninum tachyzoite antigens useful for diagnosis of neosporosis.

The purpose of the present study was to identify antigens of the protozoan Neospora caninum that could be useful for the diagnosis of neosporosis in domestic animals. As revealed by immunoblotting, immune sera from a wide range of animal species exhibited a similar recognition pattern of four major and several minor N. caninum antigens. In contrast to preinoculation sera, all tested immune sera recognized nonreduced immunodominant 17-, 29-, 30-, and 27-kDa antigens. A 46-kDa protein which showed faint recognition by preimmune sera also exhibited a strong response by immune sera. Immunolocalization of the four immunodominant N. caninum antigens was investigated by immunogold electron microscopy using monospecific polyclonal antisera. The 17-kDa antigen appears to be associated with the body part of the rhoptries, while the 29- and 30-kDa antigens were associated with the dense granules, network, and limiting membrane of the parasitophorous vacuole. Studies were also conducted to compare antibody responses to N. caninum and the related protozoan Toxoplasma gondii. Although N. caninum and T. gondii (RH strain) tachyzoites shared a few cross-reacting antigens, the immunodominant antigens of both parasites were not recognized by heterologous sera. Also, immunogold staining with rabbit anti-Neospora hyperimmune serum exhibited almost no labeling of external membranes of Neospora tachyzoites compared with the very marked labeling seen when Toxoplasma tachyzoites (RH strain) were incubated with rabbit anti-Toxoplasma hyperimmune serum. These unique antigenic differences should be useful in developing a diagnostic assay for N. caninum.     

Sensitive and specific identification of Neospora caninum infection of cattle based on detection of serum antibodies to recombinant Ncp29

Neosporosis is an economically important disease of dairy cattle caused by the protozoan Neospora caninum. Diagnostic tests for neosporosis are complicated by the potential for cross-reaction of antibodies to antigens that are similar between N. caninum and closely related parasites Toxoplasma gondii and Sarcocystis cruzi. To provide a sensitive and specific assay for detecting antibodies to N. caninum in the serum of infected animals, we have investigated a recombinant form of the antigen known as Ncp29 (rNcp29), which is a major surface protein of the parasite. Ncp29 is encoded by a gene that is homologous to the SAG1 gene previously characterized from T. gondii. An enzyme-linked immunosorbent assay (ELISA) was used to screen animals for the presence of serum antibodies specific to rNcp29. The rNcp29 ELISA readily distinguished between cattle known to be infected with N. caninum (optical density [OD] > 1.2 at 1:500 or greater dilution) and negative controls (OD < 0.5 at 1:500). Additionally, sera from animals that were infected with T. gondii or S. cruzi were negative. The rNcp29 ELISA developed here provides a specific and sensitive assay for detecting neosporosis in cattle.     

Identification of the cross-reactive and species-specific antigens between Neospora caninum and Toxoplasma gondii tachyzoites by a proteomics approach

The characterization of the cross-reactive and species-specific antigens of Neospora caninum and Toxoplasma gondii is important in the exploration to determine the common mechanisms of parasite-host interaction and to improve the serological diagnosis; it is also useful for the selection of the cross-reactive antigens that could be used in the development of vaccines or drugs for controlling the diseases caused by these two parasites. In this study, cross-reactive and species-specific antigens between N. caninum and T. gondii tachyzoites were comprehensively investigated using a proteomics approach with the application of two-dimensional gel electrophoresis, immunoblot analysis, matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS), and MALDI-TOF/TOF-MS analysis. Immunoblotting and mass spectrometry analysis revealed that at least 42 individual protein spots of N. caninum were reacted with the anti-N. caninum serum, among which at least 18 protein spots were cross-reacted with the anti-T. gondii serum. Moreover, at least 31 protein spots of T. gondii were reacted with the anti-T. gondii serum, among which at least 19 protein spots were cross-reacted with the anti-N. caninum serum. Furthermore, some new specific proteins were also identified in the N. caninum protein profile by searching Toxoplasma sequences or sequences from other organisms. This study substantiates the usefulness of proteomics in the immunoscreening of the cross-reactive or species-specific antigens of both parasites. In addition, the present study showed that there was significant homology in the antigenic proteome profiles between the two parasites. These observations have implications for the design of multicomponent common vaccines against both parasite infections.     

Neospora caninum-infected cattle develop parasite-specific CD4+ cytotoxic T lymphocytes

Cattle infected with Neospora caninum readily experience transplacental parasite transmission, presumably after maternal parasitemia, leading to abortion or birth of congenitally infected calves. Cytotoxic T lymphocytes (CTL) are important mediators of protective immunity against Toxoplasma gondii, an intracellular apicomplexan protozoan closely related to N. caninum. In this study, N. caninum-specific CTL expanded from peripheral blood mononuclear cells of two major histocompatibility complex-mismatched, experimentally infected cattle were identified by using a (51)Cr release cytotoxicity assay. Enrichment and blocking of CD4(+)- and CD8(+)-T-lymphocyte effector subsets indicated that CD4(+) CTL killed N. caninum-infected, autologous target cells and that killing was mediated through a perforin/granzyme pathway. Detection and characterization of CTL responses to N. caninum in the natural, outbred, bovine host will facilitate identification of immunogens and design of immunization strategies to induce parasite-specific CTL against transplacental N. caninum transmission in cattle.     

Comparison of the internal transcribed spacer, ITS 1, from Toxoplasma gondii isolates and Neospora caninum

The internal transcribed spacer (ITS1) region and the 5′ part of the 5.8S ribosomal RNA gene of the ribosomal DNA repeat from 20 Toxoplasma gondii isolates was sequenced and found to be identical in all isolates, independent of host origin or virulence to mice. The ITS1 region from the closely related coccidian parasite Neospora caninum differed in 22% of its nucleotides. Hence, the ITS1 region provides a good marker for the distinction of T. gondii and N. caninum but is not useful for epidemiology studies of T. gondii.     

Identification and characterization of Neospora caninum cyclophilin that elicits gamma interferon production

Gamma interferon (IFN-gamma) response is essential to the development of a host protective immunity in response to infections by intracellular parasites. Neosporosis, an infection caused by the intracellular protozoan parasite Neospora caninum, is fatal when there is a complete lack of IFN-gamma in the infected host. However, the mechanism by which IFN-gamma is elicited by the invading parasite is unclear. This study has identified a microbial protein in the N. caninum tachyzoite N. caninum cyclophilin (NcCyP) as a major component of the parasite responsible for the induction of IFN-gamma production by bovine peripheral blood mononuclear cells (PBMC) and antigen-specific CD4(+) T cells. NcCyP has high sequence homology (86%) with Toxoplasma gondii 18-kDa CyP with a calculated molecular mass of 19.4 kDa. NcCyP is a secretory protein with a predicted signal peptide of 17 amino acids. Abundant NcCyP was detected in whole-cell N. caninum tachyzoite lysate antigen (NcAg) and N. caninum tachyzoite culture supernatant. In N. caninum tachyzoite culture supernatant, three NcCyP bands of 19, 22, and 24 kDa were identified. NcAg stimulated high levels of IFN-gamma production by PBMC and CD4(+) T cells. The IFN-gamma-inducing effect of NcAg was blocked by cyclosporine, a specific ligand for CyP, in a dose-dependent manner. Furthermore, cyclosporine abolished IFN-gamma production by PBMC from na?ve cows as well as PBMC and CD4(+) T cells from infected/immunized cows. These results indicate that the N. caninum tachyzoite naturally produces a potent IFN-gamma-inducing protein, NcCyP, which may be important for parasite survival as well as host protection.     

Infection of mice with Neospora caninum (Protozoa: Apicomplexa) does not protect against challenge with Toxoplasma gondii.

Neospora caninum and Toxoplasma gondii are structurally related protozoal parasites of mammals that may cause abortion and neonatal morbidity and mortality. Groups of mice were subcutaneously inoculated with 10(5) live zoites of the NC-1 or NC-3 isolates of N. caninum and reinoculated with an identical number of live zoites 2 weeks later. Groups of mice which were injected subcutaneously with Hanks balanced salt solution served as controls. Three weeks after the final N. caninum inoculation, one-half of the mice were inoculated subcutaneously with 2.5 x 10(4) zoites of the RH isolate of T. gondii and the other half were inoculated subcutaneously with 2.5 x 10(4) zoites of the GT-1 isolate of T. gondii. Serum samples taken from mice on the day of T. gondii inoculation were negative for specific antibodies to T. gondii, but mice inoculated with N. caninum had reciprocal titers of greater than or equal to 800 to this protozoan. All of the mice died after challenge with T. gondii, and no significant differences (P greater than 0.05) between the survival times of mice inoculated with either isolate of N. caninum and those of control mice were seen. This study indicates that N. caninum and T. gondii are distinct biologic entities and not closely related isolates.     

Evaluation of two Neospora caninum recombinant antigens for use in an enzyme-linked immunosorbent assay for the diagnosis of bovine neosporosis.

Neospora caninum is a recently described apicomplexan parasite which causes paralysis and death in dogs. Neospora parasites also cause abortion and neonatal morbidity in cattle, sheep, goats, and horses, and neosporosis is emerging as an important cause of bovine abortion worldwide. The purpose of this study was to identify N. caninum cDNA clones encoding antigens that would be useful for the immunodiagnosis of bovine neosporosis. Two N. caninum tachyzoite cDNA clones expressing antigens that were recognized by serum from naturally and experimentally infected cattle were identified. The DNA sequences of these clones were determined, and the inserts were subcloned into the plasmid expression vector pTrcHisB. Both recombinant antigens, expressed as fusion proteins with a His6 tag, were purified on a nickel-chelating affinity column and evaluated in separate enzyme-linked immunosorbent assays (ELISAs). Both recombinant antigen ELISAs were capable of distinguishing between sera from Neospora-infected cows and sera from uninfected control cows. Furthermore, both assays were able to detect an antibody response in animals that were experimentally inoculated with N. caninum. Neither antigen showed evidence of cross-reactivity with serum from animals inoculated with the closely related parasites Toxoplasma gondii, Sarcocystis cruzi, Sarcocystis hominis, and Sarcocystis hirsuta.     

Application of Real-Time Fluorescent PCR for Quantitative Assessment of Neospora caninum Infections in Organotypic Slice Cultures of Rat Central Nervous System Tissue

The previously described Nc5-specific PCR test for the diagnosis of Neospora caninum infections was used to develop a quantitative PCR assay which allows the determination of infection intensities within different experimental and diagnostic sample groups. The quantitative PCR was performed by using a dual fluorescent hybridization probe system and the LightCycler Instrument for online detection of amplified DNA. This assay was successfully applied for demonstrating the parasite proliferation kinetics in organotypic slice cultures of rat brain which were infected in vitro with N. caninum tachyzoites. This PCR-based method of parasite quantitation with organotypic brain tissue samples can be regarded as a novel ex vivo approach for exploring different aspects of cerebral N. caninum infection.     

新孢子虫核酸检测方法的建立及应用评价

以新孢子虫Nc-5基因为目的基因,建立检测新孢子虫的PCR方法并初步应用。根据Gen Bank发布的新孢子虫Nc-5特异性基因序列设计引物,以新孢子虫基因组DNA为模板,利用梯度PCR法优化反应条件,建立PCR检测方法。以弓形虫、疟原虫、牛环形泰勒虫、包拉米虫和马尔太虫为模板进行扩增以验证建立方法的特异性。采用紫外分光光度计测定新孢子虫基因组DNA浓度和纯度,倍比稀释后的DNA进行PCR扩增,产物电泳分析确定建立方法的敏感性;选择高浓度和低浓度的新孢子虫基因组DNA重复检测3次,分析建立方法的重复性和稳定性。利用该方法对实验室建立的小鼠感染模型进行初步应用,评价建立方法的检测效果。结果成功建立新孢子虫的核酸扩增检测方法,扩增序列与Gen Bank(LN714476.1)中Nc-5基因序列一致性为100%,建立的新孢子虫核酸扩增检测方法与弓形虫、疟原虫、牛环形泰勒虫、包拉米虫和马尔太虫均无交叉反应,最低能检测新孢子虫DNA浓度为0.5788 pg/μL,应用该方法检测感染新孢子虫的BALB/c小鼠模型,可在肺和脑组织中检测到新孢子虫的感染。结果表明,建立的新孢子虫核酸扩增检测方法有效,并为后续研究奠定基础。     

Detection of immunoglobulin G antibodies to Neospora caninum in humans: high seropositivity rates in patients who are infected by human immunodeficiency virus or have neurological disorders

Considering that little is known about the epidemiology of Neospora caninum infection in humans, particularly in populations with high Toxoplasma gondii infection rates, the present study aimed to investigate the presence of antibodies to N. caninum in T. gondii-seropositive and -seronegative individuals. A total of 256 serum samples divided into four groups (61 samples from human immunodeficiency virus [HIV]-positive patients, 50 samples from patients with neurological disorders, 91 samples from newborns, and 54 samples from healthy subjects) were assessed for N. caninum and T. gondii serologies by indirect fluorescent-antibody test, enzyme-linked immunosorbent assay, and immunoblotting (IB). Immunoglobulin G antibodies to N. caninum were predominantly detected in HIV-infected patients (38%) and patients with neurological disorders (18%), while newborns and healthy subjects showed lower seropositivity rates (5% and 6%, respectively). Seropositivity to N. caninum was significantly associated with seropositivity to T. gondii in both HIV-infected patients and patients with neurological disorders. Seroreactivity to N. caninum was confirmed by IB, with positive sera predominantly recognizing the 29-kDa antigen of N. caninum. The results of this study indicate the presence of N. caninum infection or exposure in humans, particularly in HIV-infected patients or patients with neurological disorders, who could have opportunistic and concurrent infections with T. gondii. These findings may bring a new concern for the unstable clinical health of HIV-infected patients and the actual role of N. caninum infection in immunocompromised patients.     

Molecular analyses on Neospora caninum-triggered NETosis in the caprine system

Neospora caninum is an obligate intracellular protozoan parasite causing serious reproductive disorders in large and small ruminants worldwide. Polymorphonuclear neutrophils (PMN) react against multiple invading pathogens through different mechanisms including the release of neutrophil extracellular traps (NETs). Here, in vitro interactions of caprine PMN and N. caninum tachyzoites were studied. Scanning electron microscopic- and immunofluorescence-analyses demonstrated that caprine PMN undergo NETosis upon contact with tachyzoites of N. caninum, extruding filaments that entrap parasites. Detailed co-localization studies of N. caninum tachyzoite-induced NETs revealed the presence of PMN-derived DNA being decorated with histones (H1, H2A/H2B, H3,H4) and neutrophil elastase (NE) corroborating the molecular characteristics of classical mammalian NETs. As a new result for parasite-induced NETosis, we identified pentraxin and cathepsin B in N. caninum-triggered NETs. Nonetheless, functional inhibition assays revealed that during caprine NET formation triggered by N. caninum different molecular signaling pathways are induced, when compared to other apicomplexan parasites or host species. As such, N. caninum-induced NETosis appears to be influenced by MPO but independent of NADPH oxidase, SOCE, ERK1/2 and p38 MAPK activities. Furthermore, the inhibition of PMN autophagy via blockage of the PI3K-mediated signaling pathway failed to influence tachyzoite-induced NETosis. Since N. caninum-tachyzoites induced caprine NETosis, this effector mechanism should be considered as an early host immune response during acute caprine neosporosis.