Histologically, immunohistochemically, ultrastructurally, and molecularly confirmed neosporosis abortion in an aborted equine fetus.

Neosporosis is a common cause of abortion in cattle worldwide but is rare in horses. Here, the first case of histologically, ultrastructurally, immunohistochemically, and molecularly confirmed equine abortion caused by neosporosis is reported. Samples of lung, heart, liver, skeletal muscle, tongue, brain, and the placenta from a female fetus aborted at 280 days of gestation were fixed in formalin and submitted for diagnosis. Histologically, there was disseminated neosporosis with severe lesions in lungs, liver and the heart. Protozoal tachyzoites in all tissues reacted with polyclonal anti-Neospora caninum rabbit antibodies. Transmission electron microscopic observation on lung tissue revealed tachyzoites consistent with Neospora, including many rhoptries. Polymerase-chain reaction (PCR) using primers designed to amplify the rRNA gene internal transcribed spacer 1 (ITS1) of the Sarcocystidae was performed on DNA extracted from fetal tissues. Comparison of the ITS1 amplified from the foal tissue to sequences available in GenBank revealed 100% sequence identity to the ITS1 from three isolates of Neospora hughesi.

In vitro screening of the open source Pathogen Box identifies novel compounds with profound activities against Neospora caninum.

Neospora caninum is a major cause of abortion in cattle and represents an important veterinary health problem of great economic significance. The Medicines for Malaria Venture (MMV) Pathogen Box, an open-source collection of 400 compounds with proven anti-infective properties against a wide range of pathogens, was screened against a N. caninum beta-galactosidase reporter strain grown in human foreskin fibroblasts. A primary screening carried out at 1µM yielded 40 compounds that were effective against N. caninum tachyzoites. However, 30 of these compounds also affected the viability of the host cells. The 10 remaining compounds exhibited IC50 values between 4 and 43nM. Three compounds with IC50 values below 10nM, namely MMV676602, MMV688762 and MMV671636, were further characterized in vitro in more detail with respect to inhibition of invasion versus intracellular proliferation, and only MMV671636 had an impact on intracellular proliferation of tachyzoites. This was confirmed by transmission electron microscopy, showing that the primary target of MMV671636 was the mitochondrion. MMV671636 treatment of experimentally infected mice significantly reduced the number of animals with lung and brain infection, and these mice also exhibited a significantly reduced titer of antibodies directed against N. caninum antigens. Thus, MMV671636 is a promising starting point for the development of a future neosporosis therapy.

Identification and characterization of GRA6/GRA7 of Neospora caninum in MDBK cells.

Neospora caninum, an apicomplexan parasite, is recognized as a major bovine abortifacient. Dense granule antigens (GRAs) play important roles in the formation and modification of parasitophorous vacuoles (PVs) in Toxoplasma gondii. However, a few studies investigating GRAs have been reported in N. caninum. The aim of the present study was to characterize the dense GRA6/GRA7 of N. caninum in PVs using MDBK cells as a host cell model. Neospora caninum was inoculated into MDBK cells, and changes were observed using a transmission electron microscope (TEM). Neospora caninum GRA6/GRA7 were identified and characterized using bioinformatics, cell fractionation, and immunofluorescence. The TEM results revealed that integrated PVs were present in MDBK cells after N. caninum infection. Bioinformatics analysis showed that NcGRA6/NcGRA7 shared 28.76% and 29.66% homology with T. gondii GRA6/GRA7 (TgGRA6/TgGRA7) but had similar signal peptides, transmembrane domains, and motifs. Cell fractionation and subcellular localization analyses both showed that NcGRA6 was distributed in the lumen and intravacuolar network in soluble and transmembrane forms. The transmembrane form of NcGRA7 was observed in the PV membrane. These data lay a foundation for further study on bovine neosporosis and NcGRA6/NcGRA7 function during PV formation.

A new microneme protein of Neospora caninum, NcMIC8 is involved in host cell invasion.

Microneme proteins play an important role in the invasion process of Apicomplexan parasites through adhesion to host cells. We discovered a new N. caninum protein, NcMIC8, which is highly identical to TgMIC8. The NcMIC8 sequence has 2049 bp and no intron in the open reading fragment. It has a molecular weight of 73.8 kDa and contains a signal peptide, a transmembrane region, a low complexity region and 10 epidermal growth factor (EGF) domains. Immuno-fluorescence assay showed that NcMIC8 is located in the microneme. NcMIC8 was secreted to culture medium under stimulation of 1% ethanol, and cleaved to form the mature body of 40 kDa before transporting to microneme or during secretion. Blocking NcMIC8 using anti-NcMIC8 serum effectively inhibited host cell invasion by tachyzoites in vitro. NcMIC8 in the form of mature body interacts with NcMIC3, and the two microneme proteins form a complex probably during transportation. NcMIC8 is a new microneme protein of N. caninum and could be an attractive target for the control of neosporosis.

Characterization of the Neospora caninum NcROP40 and NcROP2Fam-1 rhoptry proteins during the tachyzoite lytic cycle.

Virulence factors from the ROP2-family have been extensively studied in Toxoplasma gondii, but in the closely related Neospora caninum only NcROP2Fam-1 has been partially characterized to date. NcROP40 is a member of this family and was found to be more abundantly expressed in virulent isolates. Both NcROP2Fam-1 and NcROP40 were evaluated as vaccine candidates and exerted a synergistic effect in terms of protection against vertical transmission in mouse models, which suggests that they may be relevant for parasite pathogenicity. NcROP40 is localized in the rhoptry bulbs of tachyzoites and bradyzoites, but in contrast to NcROP2Fam-1, the protein does not associate with the parasitophorous vacuole membrane due to the lack of arginine-rich amphipathic helix in its sequence. Similarly to NcROP2Fam-1, NcROP40 mRNA levels are highly increased during tachyzoite egress and invasion. However, NcROP40 up-regulation does not appear to be linked to the mechanisms triggering egress. In contrast to NcROP2Fam-1, phosphorylation of NcROP40 was not observed during egress. Besides, NcROP40 secretion into the host cell was not successfully detected by immunofluorescence techniques. These findings indicate that NcROP40 and NcROP2Fam-1 carry out different functions, and highlight the need to elucidate the role of NcROP40 within the lytic cycle and to explain its relative abundance in tachyzoites.

In vitro effects of new artemisinin derivatives in Neospora caninum-infected human fibroblasts.

From a panel of 34 artemisinin derivatives tested in vitro, artemisone, GC007 and GC012 were most efficacious at inhibiting Neospora caninum replication (IC50 values of 3-54nM), did not notably impair the invasiveness of tachyzoites and were non-toxic for human foreskin fibroblasts (HFFs). Transmission electron microscopy of drug-treated N. caninum-infected HFFs demonstrated severe alterations in the parasite cytoplasm, changes in the composition of the matrix of the parasitophorous vacuole (PV) and diminished integrity of the PV membrane. To exert parasiticidal activity, parasites had to be cultured continuously in the presence of 5µM artemisone or GC007 for 3 weeks. N. caninum tachyzoites readily adapted to a stepwise increase in concentrations (0.5-10µM) of GC012, but not to artemisone or GC007. Drugs induced the expression of elevated levels of NcBAG1 and NcSAG4 mRNA, but only NcBAG1 could be detected by immunofluorescence. Thus, artemisinin derivatives represent interesting leads that should be investigated further.

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.

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.

Proteins mediating the Neospora caninum-host cell interaction as targets for vaccination.

Neospora caninum is an apicomplexan parasite that is capable of infecting, a wide range of tissues. The fact that Neospora represents an important abortion-causing parasite in cattle has transformed neosporosis research from an earlier, rather esoteric field, to a significant research topic, and considerable investments have been made in the last years to develop an efficacious vaccine or other means of intervention that would prevent infection and abortion due to N. caninum infection in cattle. Antigenic molecules associated with proteins involved in adhesion/invasion or other parasite-host-cell interaction processes can confer protection against Neospora caninum infection, and such proteins represent valuable targets for the development of a vaccine to limit economical losses due to neosporosis. Although not ideal, small laboratory animal models that mimic cerebral infection, acute disease and fetal loss upon infection during pregnancy have been used for the assessment of vaccine candidates, in parallel with studies on experimental infections in cattle. Herein, we review and critically assess these vaccination approaches and discuss potential options for improvements.

Effects of miltefosine treatment in fibroblast cell cultures and in mice experimentally infected with Neospora caninum tachyzoites.

Miltefosine was investigated for its activity against Neospora caninum tachyzoites in vitro, and was shown to inhibit the proliferation of N. caninum tachyzoites cultured in human foreskin fibroblasts (HFF) with an IC50 of 5·2 µM. Treatment of infected cells with 25 µM miltefosine for a period of 10 h had only a parasitostatic effect, while after 20 h of treatment parasiticidal effects were observed. This was confirmed by transmission electron microscopy of N. caninum-infected and miltefosine-treated HFF. Administration of miltefosine to N. caninum-infected Balb/c female mice at 40 mg/kg/day for 14 days resulted in 6 out of 10 mice exhibiting weight loss, ruffled coat and apathy between days 7 and 13 post-infection. In the group that received placebo, only 2 out of 8 mice succumbed to infection, but the cerebral burden was significantly higher compared to the miltefosine treatment group. In a second experiment, the time-span of treatment was reduced to 5 days, and mice were maintained without further treatment for 4 weeks. Only 2 out of 9 mice in the miltefosine treatment group exhibited signs of disease, while 8 out of 10 mice succumbed to infection in the placebo group. These results showed that miltefosine hampered the dissemination of parasites into the CNS during experimental N. caninum infection in mice.

Influence of culture medium pH on internalization, growth and phenotypic plasticity of Neospora caninum.

Neospora caninum, a strictly intracellular protozoan, is a major leading cause of parasite-induced abortion in cattle. A widely held view of N. caninum infection is that both cellular proliferation and stage interconversion (tachyzoite-bradyzoite transformation) are triggered, perhaps even modulated by, changes in cultural conditions. This study tested the hypothesis that exposure of N. caninum tachyzoites to different pH culture media affects the parasite's entry, proliferation and cyst formation in cultured cells. The endocytic pathway for N. caninum entry into the K562 cell line was found to be mediated by low pH of culture medium. Internalization of N. caninum by host cells was significantly increased in acidic and alkaline culture medium compared to cells maintained in neutral medium as revealed by transmission electron microscopy. Parasite proliferation within Vero cells was assessed by plaque formation assay and was found to be highest when pH level was optimum, paralleled by a decrease in the number of cysts. In contrast, parasite encystation increased when the pH level was alkaline or acidic, as evaluated by indirect immunofluorescence and immunocytochemical analyses. Acidic pH regardless of state of host cell infection suppressed the rate of host cell division. These findings suggest that culture medium pH has a determinable effect on the host cell-N. caninum interaction and support the hypothesis that pH of culture medium influence the entry, growth, and phenotypic plasticity of N. caninum in mammalian cells.

A new thrombospondin-related anonymous protein homologue in Neospora caninum (NcMIC2-like1).

Neospora caninum is an Apicomplexan protozoan that has the dog as a definitive host and cattle (among other animals) as intermediate hosts. It causes encephalopathy in dogs and abortion in cows, with significant loss in worldwide livestock. As any Apicomplexan, the parasite invades the cells using proteins contained in the phylum-specific organelles, like the micronemes, rhoptries and dense granules. The aim of this study was the characterization of a homologue (denominated NcMIC2-like1) of N. caninum thrombospondin-related anonymous protein (NcMIC2), a micronemal protein previously shown to be involved in the attachment and connection with the intracellular motor responsible for the active process of invasion. A polyclonal antiserum raised against the recombinant NcMIC2-like1 functional core (thrombospondin and integrin domains) recognized the native form of NcMIC2-like1, inhibited the in vitro invasion process and localized NcMIC2-like1 at the apical complex of the parasite by confocal immunofluorescence, indicating its micronemal localization. The new molecule, NcMIC2-like1, has features that differentiates it from NcMIC2 in a substantial way to be considered a homologue.

Novel components of the Apicomplexan moving junction reveal conserved and coccidia-restricted elements.

Apicomplexan parasites generally invade their host cells by anchoring the parasite to the host membrane through a structure called the moving junction (MJ). This MJ is also believed to sieve host proteins from the nascent parasitophorous vacuole membrane, which likely protects the pathogen from lysosomal destruction. Previously identified constituents of the Toxoplasma MJ have orthologues in Plasmodium, indicating a conserved structure throughout the Apicomplexa. We report here two novel MJ proteins, RON5 and RON8. While RON5 is conserved in Plasmodium, RON8 appears restricted to the coccidia. RON8, which is likely essential, co-immunoprecipitates RON5 and known MJ proteins from extracellular parasites, indicating that a preformed complex exists within the parasites. Upon secretion, we show that RON8 within the MJ localizes to the cytoplasmic face of the host plasma membrane. To examine interactions between RON8 and the host cell, we expressed RON8 in mammalian cells and show that it targets to its site of action at the periphery in a manner dependent on the C-terminal portion of the protein. The discovery of RON5 and RON8 provides new insight into conserved and unique elements of the MJ, furthering our understanding of how the MJ contributes to the intricate mechanism of Apicomplexan invasion.

Infection of primary canine duodenal epithelial cell cultures with Neospora caninum.

According to current knowledge, sexual development of the apicomplexan parasite Neospora caninum takes place in the canine intestine. However, to date there is no information on the interaction between the parasite and the canine intestinal epithelium, and, next to the clinical and in vivo research tools, an in vitro model comprised of canine intestinal cells infected with N. caninum would be very helpful for investigations at the cellular level. Following the isolation of cells of neonatal canine duodenum and growth of cell cultures to monolayers for 5-6 days, canine intestinal epithelial cells were exposed to cell culture-derived N. caninum tachyzoites and bradyzoites. The host cells remained viable during in vitro culture for an average of 2 wk. During this time span, N. caninum was found to readily adhere to any surface area of these cells, but infection took mostly place at sites where microvilli-like structures were missing, e.g., at the cell periphery, with tachyzoites exhibiting at least 3-4 times increased invasive capacities compared to bradyzoites. Once intracellular, parasites resided within a parasitophorous vacuole, moved toward the vicinity of the nucleus and the more distal portion of the epithelial cells, and proliferated to form vacuoles of not more than 2-4 parasites, which were surrounded by numerous mitochondria. Immunofluorescence staining and TEM of infected cells showed that the expression of cytokeratins and the structural integrity of desmosomes and tight junctions were not notably altered during infection. Furthermore, no changes could be detected in the alkaline phosphatase activities in cell culture supernatants of infected and noninfected cells. Canine duodenal epithelial cell cultures represent a useful tool for future studies on the characteristics of the intestinal phases of N. caninum infection.

The differential protein expression profiles and immunogenicity of tachyzoites and bradyzoites of in vitro cultured Neospora caninum.

We report a study on the variations in the protein expression profiles of tachyzoites and bradyzoites of Neospora caninum. The in vitro stage conversion of N. caninum-infected Vero cells was induced by continuous treatment of infected cultures with 70 muM sodium nitroprusside (SNP) for up to 9 days. The stage conversion indicated by the expression of the bradyzoite-specific antigen BAG1 was analyzed by immunofluoresence assay. Morphological changes between tachyzoites and bradyzoites and localization of nuclei were demonstrated by transmission electron microscopy. Notably, we showed the differential protein expression profiles of tachyzoites and bradyzoites of N. caninum upon treatment with SNP. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated different protein patterns between tachyzoites and bradyzoites. Furthermore, Western blotting using rabbit polyclonal antibodies directed against tachyzoites revealed several reactive bands, one of which represented a tachyzoite-specific antigen of approximately 40 kDa remarkably expressed in the tachyzoite stage, but was absent from bradyzoites. Moreover, rabbit polyclonal serum raised against bradyzoites recognized a significant increased expression of an antigen with a MW of approximately 25 kDa in bradyzoites by Western blotting, suggesting that this protein is specifically expressed at the bradyzoite stage. Taken together, our data showed that differential protein expression profiling is a useful tool for discriminating between the two stages during tachyzoite-bradyzoite interconversion in N. caninum infections.

Host cells participate in the in vitro effects of novel diamidine analogues against tachyzoites of the intracellular apicomplexan parasites Neospora caninum and Toxoplasma gondii.

The in vitro effects of 19 dicationic diamidine derivatives against the proliferative tachyzoite stages of the apicomplexan parasites Neospora caninum and Toxoplasma gondii were investigated. Four compounds (DB811, DB786, DB750, and DB766) with similar structural properties exhibited profound inhibition of tachyzoite proliferation. The lowest 50% inhibitory concentrations were found for DB786 (0.21 microM against Neospora and 0.22 microM against Toxoplasma) and DB750 (0.23 microM against Neospora and 0.16 microM against Toxoplasma), with complete proliferation inhibition at 1.7 microM for both drugs against both species. DB750 and DB786 were chosen for further studies. Electron microscopy of N. caninum-infected human foreskin fibroblast (HFF) cultures revealed distinct alterations and damage of parasite ultrastructure upon drug treatment, while host cells remained unaffected. For true parasiticidal efficacy against N. caninum, a treatment duration of 3 h at 1.7 microM was sufficient for DB750, while a longer treatment period (24 h) was necessary for DB786. Pretreatment of tachyzoites for 1 h prior to host cell exposure had no effect on infectivity. However, pretreatment of uninfected host cells had a significant adverse effect on N. caninum proliferation: exposure of HFFs to 1.7 microM DB750 for 6, 12, or 24 h, followed by infection with N. caninum tachyzoites and subsequent culture in the absence of DB750, resulted in significantly delayed parasite proliferation. This suggests that either (i) these compounds or their respective active metabolites were still present after the removal of the drugs or (ii) the drug treatments reversibly impaired some functional activities in HFFs that were essential for parasite proliferation and/or survival.

In vitro isolation and characterisation of the first canine Neospora caninum isolate in Australia.

Neospora caninum was isolated and established in vitro from the skin lesion of a naturally infected dog. The identity of the parasite was evaluated by immunofluorescent antibody test (IFAT), microscopy, Western blotting and polymerase chain reaction (PCR). N. caninum DNA was detected in the whole blood, serum, skin lesion, rectal scrapings and faeces of the infected dog utilising a nested PCR targeting the Nc-5 gene of N. caninum. Antigenic and genetic characterisation of the isolate, designated WA-K9, at a number of loci including the Nc-5 gene, heat shock protein 70 (HSP-70) gene, alpha-tubulin and beta-tubulin genes revealed no variation between this isolate and two N. caninum isolates from different geographic areas. Clinical aspects of this case, which included cutaneous and neurological disease, are also discussed.

Monoclonal antibody directed against Neospora caninum tachyzoite carbohydrate epitope reacts specifically with apical complex-associated sialylated beta tubulin.

Monoclonal antibodies (mabs) were generated against whole sonicated Neospora caninum tachyzoites as immunogen. Initial ELISA screening of the reactivity of hybridoma culture supernatants using the same antigen and antigen treated with sodium periodate prior to antibody binding resulted in the identification of 8 supernatants with reactivity against putative carbohydrate epitopes. Following immunoblotting, mab6D12 (IgG1), binding a 52/48-kDa doublet, and mab6C6 (IgM), binding a 190/180-kDa doublet, were selected for further studies. Immunofluorescence of tachyzoite-infected cultures localized the corresponding epitopes not to the surface, but to interior epitopes at the apical part of N. caninum tachyzoites. During in vitro tachyzoite to bradyzoite stage conversion, mab6C6 labeling translocated toward the cyst periphery, while for mab6D12 no changes in localization were noted. Upon extraction of tachyzoites with the nonionic detergent Triton-X-100, the 52-kDa band recognized by mab6D12 was present exclusively in the insoluble, cytoskeletal fraction of both N. caninum and Toxoplasma gondii tachyzoites. Tandem mass spectrometry analysis identified this protein as N. caninum beta tubulin. The 48-kDa band labeled by mab6D12 was a Vero cell protein contamination. The protein(s) reacting with mab6C6 could not be conclusively identified by mass spectrometry. Immunofluorescence consistently failed to label T. gondii tachyzoites, indicating that beta tubulin in T. gondii and N. caninum could be differentially modified or that the reactive epitope in T. gondii is masked. Immunogold TEM of isolated apical cytoskeletal preparations and dual immunofluorescence with antibody to tubulin confirmed that mab6D12 binds to the anterior part of apical complex-associated microtubules. The sodium periodate sensitivity of the beta tubulin associated epitope was confirmed by immunoblotting and ELISA, and treatment of N. caninum cytoskeletal proteins with sialidase prior to mab6D12 labeling resulted in a profound loss of antibody binding, suggesting that mab6D12 reacts with sialylated beta tubulin.

Subcellular fractionation and molecular characterization of the pellicle and plasmalemma of Neospora caninum.

A characteristic structural feature of Toxoplasma gondii and Neospora caninum is the presence of a triple-membrane pellicle, on the zoite stages of their complex life-cycle. Here we report the results of electron microscopic studies which show that the pellicle is made of a typical plasmalemma covered on its cytoplasmic side by a system of flattened vesicles named the inner membrane complex. Using methods described previously for the purification of pellicle and plasmalemma fractions from T. gondii, we have evaluated the same methodology for the preparation of pellicles and plasmalemma from N. caninum. The approach used involved subcellular fractionation and sucrose gradient centrifugation to prepare fractions containing pellicles. Plasmalemma was prepared by extraction of this fraction with a high salt glycerol treatment. Fractions containing membrane structures were identified by electron microscopy, and the proteins and antigens present in them were subsequently studied by SDS-PAGE and Western blotting. Electron microscopy of the pellicle fractions of N. caninum demonstrated preservation of the triple-membrane structure which is identical to that found in T. gondii. SDS-PAGE of the pellicle fractions revealed it contained several major proteins. Analyses revealed that the plasmalemma of N. caninum contained 2 abundant proteins in addition to other much lower abundance antigens detectable by monoclonal antibodies. These studies therefore report, for the first time, a detailed molecular characterization of the pellicle and plasmalemma of N. caninum.