Identification of novel proteins in Neospora caninum using an organelle purification and monoclonal antibody approach

Neospora caninum is an important veterinary pathogen that causes abortion in cattle and neuromuscular disease in dogs. Neospora has also generated substantial interest because it is an extremely close relative of the human pathogen Toxoplasma gondii, yet does not appear to infect humans. While for Toxoplasma there are a wide array of molecular tools and reagents available for experimental investigation, relatively few reagents exist for Neospora. To investigate the unique biological features of this parasite and exploit the recent sequencing of its genome, we have used an organelle isolation and monoclonal antibody approach to identify novel organellar proteins and develop a wide array of probes for subcellular localization. We raised a panel of forty-six monoclonal antibodies that detect proteins from the rhoptries, micronemes, dense granules, inner membrane complex, apicoplast, mitochondrion and parasite surface. A subset of the proteins was identified by immunoprecipitation and mass spectrometry and reveal that we have identified and localized many of the key proteins involved in invasion and host interaction in Neospora. In addition, we identified novel secretory proteins not previously studied in any apicomplexan parasite. Thus, this organellar monoclonal antibody approach not only greatly enhances the tools available for Neospora cell biology, but also identifies novel components of the unique biological characteristics of this important veterinary pathogen.     

Identification of three novel Toxoplasma gondii rhoptry proteins

The rhoptries are key secretory organelles from apicomplexan parasites that contain proteins involved in invasion and modulation of the host cell. Some rhoptry proteins are restricted to the posterior bulb (ROPs) and others to the anterior neck (RONs). As many rhoptry proteins have been shown to be key players in Toxoplasma invasion and virulence, it is important to identify, understand and characterise the biological function of the components of the rhoptries. In this report, we identified putative novel rhoptry genes by identifying Toxoplasma genes with similar cyclical expression profiles as known rhoptry protein encoding genes. Using this approach we identified two new rhoptry bulb (ROP47 and ROP48) and one new rhoptry neck protein (RON12). ROP47 is secreted and traffics to the host cell nucleus, RON12 was not detected at the moving junction during invasion. Deletion of ROP47 or ROP48 in a type II strain did not show major influence in in vitro growth or virulence in mice.     

Identification and characterization of a novel Neospora caninum immune mapped protein 1

Immune mapped protein 1 (IMP1) is a newly discovered protein in Eimeria maxima. It is recognized as a potential vaccine candidate against E. maxima and a highly conserved protein in apicomplexan parasites. Although the Neospora caninum IMP1 (NcIMP1) orthologue of E. maxima IMP1 was predicted in the N. caninum genome, it was still not identified and characterized. In this study, cDNA sequence encoding NcIMP1 was cloned by RT-PCR from RNA isolated from Nc1 tachyzoites. NcIMP1 was encoded by an open reading frame of 1182 bp, which encoded a protein of 393 amino acids with a predicted molecular weight of 42.9 kDa. Sequence analysis showed that there was neither a signal peptide nor a transmembrane region present in the NcIMP1 amino acid sequence. However, several kinds of functional protein motifs, including an N-myristoylation site and a palmitoylation site were predicted. Recombinant NcIMP1 (rNcIMP1) was expressed in Escherichia coli and then purified rNcIMP1 was used to prepare specific antisera in mice. Mouse polyclonal antibodies raised against the rNcIMP1 recognized an approximate 43 kDa native IMP1 protein. Immunofluorescence analysis showed that NcIMP1 was localized on the membrane of N. caninum tachyzoites. The N-myristoylation site and the palmitoylation site were found to contribute to the localization of NcIMP1. Furthermore, the rNcIMP1-specific antibodies could inhibit cell invasion by N. caninum tachyzoites in vitro. All the results indicate that NcIMP1 is likely to be a membrane protein of N. caninum and may be involved in parasite invasion.     

Identification of Neospora caninum proteins regulated during the differentiation process from tachyzoite to bradyzoite stage by DIGE

Identification of differentially expressed proteins during Neospora caninum tachyzoite–bradyzoite conversion processes may lead to a better knowledge of the pathogenic mechanisms developed by this important parasite of cattle. In the present work, a differential expression proteomic study of tachyzoite and bradyzoite stages was accomplished for the first time by applying DIGE technology coupled with MS analysis. Up to 72 differentially expressed spots were visualized (1.5‐fold in relative abundance, p<0.05, t‐test). A total of 53 spots were more abundant in bradyzoites and 19 spots in tachyzoites. MS analysis identified 26 proteins; 20 of them overexpressed in the bradyzoite stage and 6 in the tachyzoite stage. Among the novel proteins, enolase and glyceraldehyde‐3‐phosphate dehydrogenase (involved in glycolysis), HSP70 and HSP90 (related to stress response) as well as the dense granule protein GRA9, which showed higher abundance in the bradyzoite stage, might be highlighted. On the other hand, isocitrate dehydrogenase 2, involved in the Krebs cycle, was found to be more abundant in tachyzoites extract. Biological functions from most novel proteins were correlated with previously reported processes during the differentiation process in Toxoplasma gondii. Thus, DIGE technology arises as a suitable tool to study mechanisms involved in the N. caninum tachyzoite to bradyzoite conversion.     

Identification of antigenic proteins from Neospora caninum recognized by bovine immunoglobulins M, E, A and G using immunoproteomics

Antigenic proteins of Neospora caninum (N. caninum) against bovine immunoglobulins M, E, A, and G were investigated by using immunoproteomics. Proteins of N. caninum (KBA-2) tachyzoite lysates separated by two-dimensional gel electrophoresis were transferred to polyvinylidene difluoride (PVDF) membranes, probed with different bovine immunoglobulin class and classified. Antigenic spots recognized were also identified by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) analysis. 132, 84, 4, and 40 antigenic protein spots were recognized on N. caninum immunoblot profiles against bovine IgM, IgE, IgA, and IgG, respectively. Of these protein spots, the antigenic proteins recognized by either IgM, IgE, and IgG, or IgM and IgG were HSP70, pyruvate kinase, actin, NCDG-1, tubulin alpha-chain, and putative ribosomal protein S2. On the other hand, IgM, IgE, and IgA reacted with NTPase, HSP60, tubulin beta-chain, putative protein disulfide isomerase, enolase, lactate dehydrogenase, serine-threonine phosphatase, 14-3-3 protein homologue, and GRA2 protein. Most of the antigenic proteins identified were associated with the process of invasion, proliferation, and egression of apicomplexans. In our study, HSP70, actin, NTPase, HSP60, pyruvate kinase, enolase, putative ribosomal protein S2, NCDG-1, and GRA2 proteins were found to be immunodominant proteins, which may contribute to the development of diagnostic markers and vaccine.     

Multilocus microsatellite analysis reveals extensive genetic diversity in Neospora caninum

Neospora caninum is a world-wide parasite that causes neuromuscular disorders in dogs and bovine abortion. Biological diversity among isolates has been proved in both in vivo and in vitro studies. In contrast, little is known about the genetic diversity of this parasite. Microsatellite sequence analysis constitutes a suitable tool that has been used for the genetic analysis of other apicomplexan parasites. In this report, we describe the identification and analysis of 13 microsatellite loci from N. caninum DNA sequences deposited in public databases, which were evaluated with the use of 9 isolates grown in vitro. One microsatellite was monomorphic, and the remaining 12 loci exhibited 3 to 9 separate alleles. Multilocus analysis showed that each of the 9 isolates investigated here displayed a unique profile and revealed no association between the genetic similarity and host or geographic origin. The multilocus analysis approach described here might nevertheless provide the powerful tool needed to study the genetic complexity of N. caninum and the molecular epidemiology of neosporosis.     

Prevention of vertical transfer of Neospora caninum in BALB/c mice by vaccination

Neosporosis is an important cause of abortion and neonatal morbidity in dairy cattle. The disease is caused by Neospora caninum, an intracellular protozoan parasite. In this report, we describe the use of a mouse model in the preliminary evaluation of vaccination as a means to prevent vertical transfer of N. caninum. Parasites present in the tissues of the offspring were detected using an N. caninum-specific polymerase chain reaction assay. Immunization of dams with a single inoculation of a crude lysate of N. caninum tachyzoites appeared to induce complete protection against infection of the offspring.     

Ingestion of Neospora caninum tissue cysts by Mustela species

Dogs are a definitive host of Neospora caninum, a protozoal parasite that causes abortion in cattle. Mustelids were tested to determine if they could also be definitive hosts. The procedures used were the same as those previously used to test dogs. Ermine (Mustela erminea), weasels (Mustela frenata) and ferrets (Mustela putorius) were fed N. caninum-infected mice. Neospora caninum oocysts were not observed. Mustelid faeces were fed to mice. The mice did not seroconvert and N. caninum was not detected in murine brains using tissue culture and PCR. The hypothesis that Mustela spp. are definitive hosts of N. caninum is not supported.     

Molecular comparison of the dense granule proteins GRA6 and GRA7 of Neospora hughesi and Neospora caninum

Neospora hughesi is a recently described apicomplexan parasite that has been associated with several cases of equine protozoal myeloencephalitis. The biology of this new parasite is just beginning to be defined. Towards this understanding, we report important differences between the nucleotide and deduced amino acid sequences of the dense granule proteins GRA6 and GRA7 of N. hughesi and Neospora caninum. This information can be used to differentiate the two species and contribute to further understanding of the prevalence and biology of N. hughesi. The newly defined proteins of N. hughesi are referred to as NhGRA6 and NhGRA7 in keeping with the protocol for naming homologous proteins of the Apicomplexa. Genes of the two dense granule proteins of N. hughesi (isolate Nh-A1) and four different isolates of N. caninum were isolated via PCR and their DNA sequences were determined. Computer analysis indicated that the two gene sequences were identical among all four N. caninum isolates. However, the gene for NhGRA6 was found to be 96 nucleotides longer at the 3' end than that of NcGRA6, resulting in a protein product that is 32 amino acids larger than NcGRA6. Two tandem repeat sequences were identified at the 3' end of the NhGRA6 gene. These repeat sequences contributed to the lengthening of the carboxy terminus of NhGRA6 in comparison with that of NcGRA6. The larger size of NhGRA6 was further confirmed by Western blot analysis in which NcGRA6 monospecific antibodies recognised a protein of approximately 42 kDa in N. hughesi whole tachyzoite preparation but a protein of 37 kDa in N. caninum whole tachyzoite preparation. Analysis of GRA7 gene sequences indicated a 6 and 14.8% difference at nucleotide and amino acid sequence level, respectively, between NcGRA7 and NhGRA7. Despite the same number of residues in the deduced amino acid sequences of all the GRA7 proteins, Western blot analysis indicated a difference in the migration pattern of NhGRA7 in comparison with NcGRA7. Results of our study indicate that diagnostic tests based on differences in dense granule sequences and antigenicity may have potential to differentiate between N. hughesi and N. caninum. Such diagnostic tests would be valuable tools to aid in our understanding of the epidemiology of these parasites. Additionally, dense granule proteins are immunogenic and they may have potential as use in recombinant vaccines against neosporosis.     

Identification of novel lysosomal matrix proteins by proteome analysis

The lysosomal matrix is estimated to contain about 50 different proteins. Most of the matrix proteins are acid hydrolases that depend on mannose 6-phosphate receptors (MPR) for targeting to lysosomes. Here, we describe a comprehensive proteome analysis of MPR-binding proteins from mouse. Mouse embryonic fibroblasts defective in both MPR (MPR 46-/- and MPR 300-/-) are known to secrete the lysosomal matrix proteins. Secretions of these cells were affinity purified using an affinity matrix derivatized with MPR46 and MPR300. In the protein fraction bound to the affinity matrix and eluted with mannose 6-phosphate, 34 known lysosomal matrix proteins, 4 candidate proteins of the lysosomal matrix and 4 non-lysosomal contaminants were identified by mass spectrometry after separation by two-dimensional gel electrophoresis or by multidimensional protein identification technology. For 3 of the candidate proteins, mammalian ependymin-related protein-2 (MERP-2), retinoid-inducible serine carboxypeptidase (RISC) and the hypothetical 66.3-kDa protein we could verify that C-terminally tagged forms bound in an M6P-dependent manner to an MPR-affinity matrix and were internalized via MPR-mediated endocytosis. Hence these 3 proteins are likely to represent hitherto unrecognized lysosomal matrix proteins.     

Analysis of green pit viper (Trimeresurus alborabris) venom protein by LC/MS-MS

Green pit viper venom has major effect on the hematological system having a thrombin-like effect. Thus, this study is designed to analyze the composition of Trimeresurus albolabris venom by performing gel filtration and LC/MS-MS. The purified protein was then digested by trypsin, and the tryptic fragments were analyzed by iontrap spectrophotometry. This study found four types of proteins, namely jerdonitin, stejaggregin-A beta chain-1, stejnobin, and stejnihagin-A, as the components of T. albolabris venom. All of these toxins played a greater or lesser role in clot formation or otherwise contributed to cross-reactions in antivenom production.     

Review of Neospora caninum and neosporosis in animals

Neospora caninum is a coccidian parasite of animals. It is a major pathogen for cattle and dogs and it occasionally causes clinical infections in horses, goats, sheep, and deer. Domestic dogs are the only known definitive hosts for N. caninum. It is one of the most efficiently transmitted parasite of cattle and up to 90% of cattle in some herds are infected. Transplacental transmission is considered the major route of transmission of N. caninum in cattle. Neospora caninum is a major cause of abortion in cattle in many countries. To elicit protective immunity against abortion in cows that already harbor a latent infection is a major problem. This paper reviews information on biology, diagnosis, epidemiology and control of neosporosis in animals.     

The in vitro development of Neospora caninum bradyzoites

Neospora caninum is a recently identified apicomplexan protozoan parasite that is closely related to Toxoplasma gondii. Neospora caninum is of significant economic importance as it causes neurological disease and abortion in numerous animals. Antibodies to BAG1/hsp30 (also known as BAG5), a T. gondii bradyzoite-specific protein, have been demonstrated to react with N. caninum tissue cysts in vivo. Bradyzoite differentiation of N. caninum in vitro was investigated using culture conditions previously utilised for T. gondii in vitro bradyzoite development. Utilising the NC-Liverpool isolate of N. caninum, cyst-like structures developed within 3-4 days of culture of this parasite in human fibroblasts. In addition, an antigen reacting with mAb 74.1.8 (anti-BAG1) and rabbit anti-recombinant BAGI was demonstrable by immunofluorescence, fluorescence-activated cell sorter, and immunoblot analyses. Expression of this antigen was increased by stress conditions, similar to that which has been described for T. gondii bradyzoite induction. Cyst-wall formation in vitro, as assayed by lectin binding, did not occur as readily for N. caninum as it does for T. gondii.     

Reduction in transplacental transmission of Neospora caninum in outbred mice by vaccination

Infection with the protozoan parasite Neospora caninum is an important cause of abortion in cattle. A major source of infection is transplacental transfer of the parasite from mother to offspring during pregnancy. This study describes investigations on the immunisation of outbred Qs mice before pregnancy with live or a crude lysate of N. caninum (NC-Nowra isolate) to prevent transplacental transfer of a challenge infection administered during pregnancy. Parasites present in the brains of pups from mice challenged with N. caninum (NC-Liverpool) were detected by PCR. Injection of live NC-Nowra tachyzoites before pregnancy dramatically reduced transplacental transfer from 75 to 0.8% in one experiment and from 76 to 8% in a second experiment. Injection of a crude lysate of NC-Nowra tachyzoites reduced transplacental transfer from 67 to 53% in one experiment and from 76 to 63% in a second experiment. Analysis of N. caninum-specific IgG1 and IgG2a antibody levels prior to pregnancy and challenge showed that NC-Nowra lysate induced a response skewed towards IgG1 whereas live parasites induced both IgG1 and IgG2a antibodies. After pregnancy and a challenge infection, a similar IgG1/IgG2a response was seen in all challenged groups. These results provide further positive support for the hypothesis that transplacental transmission of this parasite is preventable by vaccination.     

Identification of the antigenic region of Neospora caninum dense granule protein 7 using ELISA

Dense granule protein 7 (NcGRA7) is a potent diagnostic antigen of Neospora caninum. Some studies have reported on the difficult expression, low yield, and variable degree of solubility of recombinant NcGRA7. We aimed to unravel the possible causes for these issues and tested NcGRA7 antigenicity in enzyme linked immunosorbent assays (ELISAs). The NcGRA7 coding sequence (217 amino acids) was split into five amino acid regions: NcGRA7m (27–217), NcGRA7m3 (27–160), NcGRA7m4 (27–135), NcGRA7m5 (161–190), and NcGRA7m6 (188–217). Three fragments, NcGRA7m, NcGRA7m3 and NcGRA7m4, exhibited high antigenic properties when tested against experimentally-infected mice and dog sera by ELISA. High levels of IgG2 antibodies against NcGRA7m3 were observed in field dog sera. In experimentally and naturally-infected cattle, the N. caninum-specific sera only reacted with NcGRA7m, indicating that this antigenic region differs among the three animal species. This study presents valuable information about the antigenic properties and topology of NcGRA7, and highlights its suitability for diagnostic purposes.     

Seroprevalence of Neospora caninum antibodies in dogs in India

Neospora caninum is one of most important causes of abortion in cattle worldwide, and dogs are an important risk factor for N. caninum infection in cattle. Antibodies to N. caninum were determined in 184 (126 rural, 58 urban) dogs from the Punjab State, India, using commercial monoclonal antibody-based competitive ELISA and found in 16.8% of the animals. The prevalence of N. caninum antibodies was significantly higher in rural dogs (21.4%, 27 of 126) than city dogs (6.9%, 4 of 58). To our knowledge this is the first report of N. caninum infection in canines from India.     

Neospora caninum and Toxoplasma gondii: a novel adhesion/invasion assay reveals distinct differences in tachyzoite-host cell interactions

This paper describes an adhesion/invasion assay, based on combined pyrrolidine dithiocarbamate (PDTC) and antibody treatment of parasites followed by quantitative real-time PCR. This PDTC-PCR assay can be used to comparatively assess the participation of host cell- and parasite-associated components during host cell adhesion and entry by Neospora caninum and Toxoplasma gondii tachyzoites, respectively, and is potentially applicable to any other apicomplexan parasite. The assay allows to determine the parasite invasion rate in relation to the overall number of parasites which interact with host cells in any given experiment, and thus represents a significant improvement to conventional microscopic assays in terms of accuracy and reproducibility. Using this assay it was possible to show that adhesion and invasion of N. caninum tachyzoites are two distinct and separated events, in that N. caninum tachyzoites preferentially utilise host cell surface chondroitin sulphates for adhesion, but not for the host cell invasion process. Application of the PDTC-PCR assay also demonstrated that N. caninum and T. gondii tachyzoites differ largely with regard to the functional involvement of proteases in adhesion and invasion of host cells. Thus, although phylogenetically closely related, N. caninum and T. gondii are biologically quite different and exhibit distinct dissimilarities with regard to host cell interactions.