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.     

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.     

Identification of a major surface protein on Neospora caninum tachyzoites

Neospora caninum is a recently identified coccidian parasite that is closely related to Toxoplasma gondii. Molecules associated with the surface of N. caninum tachyzoites are likely to be involved in the process of adhesion and invasion of host cells. They probably also participate in the interaction of the parasite with the immune system, and they could play an important role in the pathogenesis of the parasite. To identify such surface molecules, we performed subcellular fractionation studies of isolated N. caninum tachyzoites. Employing the nonionic detergent Triton-X-114, we prepared a membrane fraction. Immunoblot analysis of this fraction using polyclonal antisera directed against tachyzoites of N. caninum and T. gondii resulted in the identification of a protein of approximately 43 kDa (Nc-p43). This molecule was present in two isolates of Neospora (Nc-1 and Liverpool) but was absent in Toxoplasma (RH-strain) tachyzoites. Further immunofluorescence and immunogold transmission electron microscopy (TEM) studies using affinity-purified anti-Nc-p43 antibodies demonstrated the presence of this molecule on the surface of N. caninum tachyzoites.     

Enzyme-linked immunosorbent assays based on Neospora caninum dense granule protein 7 and profilin for estimating the stage of neosporosis

Neospora caninum is an intracellular protozoan parasite that causes bovine and canine neosporosis, characterized by fetal abortion and neonatal mortality and by neuromuscular paralysis, respectively. Although many diagnostic methods to detect parasite-specific antibodies or parasite DNA have been reported, to date no effective serodiagnostic techniques for estimating pathological status have been described. Our study aimed to elucidate the relationship between the parasite-specific antibody response, parasite activation, and neurological symptoms caused by N. caninum infection by using a recombinant antigen-based enzyme-linked immunosorbent assay. Among experimentally infected mice, anti-N. caninum profilin (NcPF) antibody was only detected in neurologically symptomatic animals. Parasite numbers within the brains of the symptomatic mice were significantly higher than those in asymptomatic animals. In addition, anti-NcPF and anti-NcGRA7 antibodies were mainly detected at the acute stage in experimentally infected dogs, while anti-NcSAG1 antibody was produced during both acute and chronic stages. Furthermore, among anti-NcSAG1 antibody-positive clinical dogs, the positive rates of anti-NcGRA7 and anti-NcPF antibodies in the neurologically symptomatic dogs were significantly higher than those in the non-neurologically symptomatic animals. Our results suggested that the levels of anti-NcGRA7 and anti-NcPF antibodies reflect parasite activation and neurological symptoms in dogs. In conclusion, antibodies against NcGRA7 and NcPF may have potential as suitable indicators for estimating the pathological status of neosporosis.     

Development of a polymerase chain reaction assay for the diagnosis of neosporosis using the Neospora caninum 14-3-3 gene

Neospora caninum is a recently described apicomplexan parasite which causes neuromuscular disease in dogs, and abortion and neonatal morbidity in cattle, sheep and horses. Morphological similarites and serological cross-reactivity between N. caninum and the closely related parasite Toxoplasma gondii, have resulted in the frequent misdiagnosis of neosporosis as toxoplasmosis. This report describes the isolation and characterization of an N. caninum cDNA clone encoding a 14-3-3 protein homologue. The 14-3-3 proteins are a class of proteins which show a high degree of amino acid sequence conservation across several eukaryotic taxa. Using less conserved regions of the N. caninum cDNA clone, nested primers were designed for the amplification of a 614-bp N. caninum DNA fragment by the polymerase chain reaction (PCR). The DNA fragment was amplified from N. caninum genomic DNA, but not from T. gondii, Sarcocystis muris, Sarcocystis tenella, or Sarcocystis cruzi genomic DNA. Additionally, the fragment was amplified from DNA prepared from the brains of N. caninum-infected mice, but not from the brain of a mouse infected with T. gondii. These results suggest that this PCR assay may be useful for the diagnosis of neosporosis.     

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.     

Identification of a Neospora caninum microneme protein (NcMIC1) which interacts with sulfated host cell surface glycosaminoglycans

The invasive stages of apicomplexan parasites enter their host cells through mechanisms which are largely conserved throughout the phylum. Host cell invasion is divided into two distinct events, namely, adhesion onto the host cell surface and the actual host cell entry process. The former is mediated largely through microneme proteins which are secreted at the onset of establishing contact with the host cell surface. Many of the microneme proteins identified so far contain adhesive domains. We here present the genomic and corresponding cDNA sequences coding for a 460-amino-acid (aa) microneme protein in Neospora caninum tachyzoites which, due to its homology to MIC1 in Toxoplasma gondii (TgMIC1), was named NcMIC1. The deduced NcMIC1 polypeptide sequence contains an N-terminal signal peptide of 20 aa followed by two tandemly internal repeats of 48 and 44 aa, respectively. Integrated into each repeat is a CXXXCG sequence motif reminiscent of the thrombospondin-related family of adhesive proteins. The positioning of this motif is strictly conserved in TgMIC1 and NcMIC1. The C-terminal part, comprised of 278 aa, was expressed in Escherichia coli, and antibodies affinity purified on recombinant NcMIC1 were used to confirm the localization within the micronemes by immunofluorescence and immunogold transmission electron microscopy of tachyzoites. Immunohistochemistry of mouse brains infected with tissue cysts showed that expression of this protein is reduced in the bradyzoite stage. Upon initiation of secretion by elevating the temperature to 37 degrees C, NcMIC1 is released into the medium supernatant. NcMIC1 binds to trypsinized, rounded Vero cells, as well as to Vero cell monolayers. Removal of glycosaminoglycans from the host cell surface and modulation of host cell surface glycosaminoglycan sulfation significantly reduces the binding of NcMIC1 to the host cell surface. Solid-phase binding assays employing defined glycosaminoglycans confirmed that NcMIC1 binds to sulfated glycosaminoglycans.     

Cell surface antigen CD5 is a marker for activated human B cells

A minor subset of B cells which in vivo express the surface antigen CD5, has attracted much attention because of its involvement in autoimmune responses. On the basis of observations showing self-renewal capacity of such cells in mice and also the absence of a substantial change of CD5 phenotype during B cell activation in vitro, the CD5+ B cells are now generally considered to represent a separate cell lineage. In the present study, CD5- B cells were isolated by cell sorter and then stimulated in vitro with mutagenized EL4 thymoma cells in the presence of T cell supernatant. About 70% of the B cells were CD5+ after 3 days. Thus, the CD5 antigen behaves as a B cell activation marker. In our system we found that the frequency of rheumatoid factor-producing B cells was on average three times higher in CD5+ than in CD5- B cells isolated ex vivo from human peripheral blood. Most likely this reflects frequent activation of such autoreactive B cells in vivo.     

Dense-granule protein NcGRA7, a new marker for the serodiagnosis of Neospora caninum infection in aborting cows

To investigate whether the production of an antigen-specific antibody is associated with Neospora caninum-induced bovine abortion, 62 serum samples were tested with an enzyme-linked immunosorbent assay using the recombinant antigens NcSAG1, NcSRS2, and NcGRA7. Our study suggested that NcGRA7 would be a new marker for the serodiagnosis of N. caninum infection resulting in abortion.     

ALCAM (CD166) is a surface marker for early murine cardiomyocytes

ALCAM (activated leukocyte cell adhesion molecule, CD166) belongs to the immunoglobulin superfamily and is involved in axon guidance, hematopoiesis, immune response and tumor metastasis. During embryogenesis, mRNA encoding ALCAM was expressed in the cardiac crescent and the neural groove at embryonic day (E) 7.75 and predominately in the tubular heart at E8.5. A newly generated monoclonal antibody against the ALCAM molecule (ALC-48) exclusively stained cardiomyocytes at E8.25-10.5. However, ALCAM expression was lost by cardiomyocytes by E12.5 and its expression shifts to a variety of organs during later stages. ALCAM was found to be a prominent surface marker for cardiomyocytes in early embryonic hearts. The transient expression of ALCAM during early developmental stages marks specific developmental stages in cardiomyocyte differentiation.     

Serological response over time to recombinant Neospora caninum antigens in cattle after a neosporosis-induced abortion.

Recombinant Neospora caninum tachyzoite antigens were evaluated in an enzyme-linked immunosorbent assay (ELISA) for recognition by serum antibodies (Ab) from Neospora-infected cattle. Serum samples were obtained every 2 to 3 weeks for 8 to 15 months from 10 cows with histories of Neospora-associated abortion. Serum samples were also obtained from offspring of these animals and from a large number of cows that had aborted a fetus, due to infection by Neospora or other organisms, at various times during gestation. All 10 cows had positive ELISA Ab titers to both recombinant N. caninum tachyzoite antigens after abortion, during subsequent gestation, and after parturition. In three cows, there was a noticeable peak in Ab titers early in gestation. Calves born to Neospora-infected cows also had positive titers of Ab to the recombinant tachyzoite antigens, and these titers remained elevated for at least 4 months after birth. A portion of the serum immunoglobulin in calves may have been derived from colostrum of infected cows. A calf born from a seronegative mother had a positive ELISA titer only after being fed colostrum from a seropositive cow. However, precolostral titers in calves born from Neospora-infected cows were high at birth, suggesting that the parasite was transmitted to the fetus via the placenta and induced a humoral immune response therein. The recombinant tachyzoite antigens were also useful for corroborating clinical diagnoses of Neospora-induced abortion. A significant difference (P < 0.05) between anti-recombinant antigen Ab titers in cows that aborted due to Neospora and those in cows that aborted from other causes was found.     

Analysis of Entamoeba histolytica excretory-secretory antigen and identification of a new potential diagnostic marker

Serodiagnosis of amoebiasis remains the preferred method for diagnosis of amoebic liver abscess (ALA). However, the commercially available kits are problematic in areas of endemicity due to the persistently high background antibody titers. Human serum samples (n = 38) from patients with ALA who live in areas of endemicity were collected from Hospital Universiti Sains Malaysia during the period of 2008 to 2010. Western blots using excretory-secretory antigen (ESA) collected from axenically grown Entamoeba histolytica were probed with the above serum samples. Seven antigenic proteins of ESA with various reactivities were identified, i.e., 152 kDa, 131 kDa, 123 kDa, 110 kDa, 100 kDa, 82 kDa, and 76 kDa. However, only the 152-kDa and 110-kDa proteins showed sensitivities above 80% in the Western blot analysis. All the antigenic proteins showed undetectable cross-reactivity when probed with healthy human serum samples (n = 30) and serum samples from other infections (n = 33). From the matrix-assisted laser desorption ionization-two-stage time of flight (MALDI-TOF/TOF) analysis, the proteins were identified as heavy subunits of E. histolytica lectin and E. histolytica pyruvate phosphate dikinase, respectively. Use of the E. histolytica lectin for diagnosis of ALA has been well reported by researchers and is being used in commercialized kits. However, this is the first report on the potential use of pyruvate phosphate dikinase for diagnosis of ALA; thus, this molecule merits further evaluation on its diagnostic value using a larger panel of serum samples.     

Epidemiology of neosporosis in dairy cattle in Galicia (NW Spain)

This comprehensive study of neosporosis in dairy cattle in Galicia (NW Spain) included: (1) a comparative study of three serological techniques for detection of Neospora caninum antibodies (direct agglutination, enzyme-linked immunosorbent assay and indirect immunofluorescence); (2) a cross-sectional serological survey in which 276 herds and 5,196 animals were tested; (3) a study of N. caninum antibody dynamics; (4) the isolation of viable tachyzoites of N. caninum. Data were analysed to determine the risk factors associated with the infection. A total of 219 herds (79.3%) and 816 heads of cattle (15.7%) were found to be seropositive. Seropositivity was higher on farms with dogs than on farms without dogs, and there was a negative correlation between the size of the herds and seroprevalence. Co-infection with Toxoplasma gondii increased the risk of seropositivity. Cows infected with N. caninum were 5.3 times more likely to abort than non-infected cows. The dynamics study showed an increase in anti-N. caninum antibody titres during the third trimester of pregnancy. Viable tachyzoites were isolated from brain samples. These results indicate that the economic impact of N. caninum is high in Galicia, and therefore, the inclusion of control measures for neosporosis in the official control health programmes is strongly recommended.     

Sputum microbiota as a potential diagnostic marker for multidrug-resistant tuberculosis

The prevalence of drug-resistant Mycobacterium tuberculosis (Mtb) strains makes disease control more complicated, which is the main cause of death in tuberculosis (TB) patients. Early detection and timely standard treatment are the key to current prevention and control of drug-resistant TB. In recent years, despite the continuous advancement in drug-resistant TB diagnostic technology, the needs for clinical rapid and accurate diagnosis are still not fully met. With the development of sequencing technology, the research of human microecology has been intensified. This study aims to use 16 rRNA sequencing technology to detect and analyze upper respiratory flora of TB patients with anti-TB drug sensitivity (DS, n = 55), monoresistance isoniazide (MR-INH, n = 33), monoresistance rifampin (MR-RFP, n = 12), multidrug resistance (MDR, n = 26) and polyresistance (PR, n = 39) in southern China. Potential microbial diagnostic markers for different types of TB drug resistance are searched by screening differential flora, which provides certain guiding significance for drug resistance diagnosis and clinical drug use of TB. The results showed that the pulmonary microenvironment of TB patients was more susceptible to infection by external pathogens, and the infection of different drug-resistant Mtb leads to changes in different flora. Importantly, seven novel microorganisms (Leptotrichia, Granulicatella, Campylobacter, Delfitia, Kingella, Chlamydophila, Bordetella) were identified by 16S rRNA sequencing as diagnostic markers for different drug resistance types of TB. Leptotrichia, Granulicatella, Campylobacter were potential diagnostic marker for TB patients with INH single-resistance. Delftia was a potential diagnostic marker for TB patients with RFP single drug-resistance. Kingella and Chlamydophila can be used as diagnostic markers for TB patients with PR. Bordetella can be used as a potential diagnostic marker for identification of TB patients with MDR.     

Podoplanin (D2-40) is a novel marker for follicular dendritic cell tumors

Podoplanin, recognized by monoclonal antibody D2-40, may be a useful marker for follicular dendritic cell (FDC) tumors. Paraffin sections of 125 dendritic cell, histiocytic, and spindle cell lesions were studied, including 11 FDC tumors, 5 interdigitating dendritic cell tumors, 10 histiocytic sarcomas, 5 Langerhans cell histiocytosis, 5 sinus histiocytosis with massive lymphadenopathy, 5 inflammatory pseudotumors of lymph node or spleen, 9 nodal Kaposi sarcomas, 6 inflammatory myofibroblastic tumors (IMTs), 29 gastrointestinal stromal tumors (GISTs), and 10 cases each of malignant peripheral nerve sheath tumor, leiomyosarcoma, monophasic synovial sarcoma (SS), and solitary fibrous tumor. All FDC tumors and Kaposi sarcomas showed strong immunoreactivity for podoplanin (predominantly membranous). Podoplanin expression was only occasionally observed in the other tumor types, including 7 GISTs (24%), 2 IMTs (33%), and 3 SS (30%), and was generally weak and cytoplasmic. Reactivity for podoplanin was more common in spindle cell GISTs (5/13 [38%]) than in epithelioid or mixed-type GISTs (2/16 [13%]). Podoplanin is a highly sensitive marker for FDC tumors and may be useful to help confirm the diagnosis in conjunction with conventional FDC markers, particularly in the differential diagnosis of dendritic cell and histiocytic lesions.     

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.