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Development of a diagnostic method for neosporosis in cattle using recombinant Neospora caninum proteins.

ABSTRACT: BACKGROUND: Neosporosis is an infectious disease primarily of cattle and dogs, caused by intracellular parasite, Neospora caninum. Neosporosis appears to be a major cause of abortion in dairy cattle worldwide and causes to huge economic loss to dairy industry. RESULTS: Recombinant surface associated antigen 1 (NcSAG1), NcSAG1 related sequence 2 (NcSRS2) and the dense granule antigen 2 (NcGRA2) of N. caninum were expressed either in silkworm or in Escherichia coli and purified. The purified recombinant proteins bound to the N. caninum-specific antibodies in serum samples from infected cattle as revealed by an enzyme-linked immunosorbent assay (ELISA). By co-immobilizing these recombinant proteins, a novel indirect ELISA was developed for detection of neosporosis. With the use of 32 serum samples, comprising 12 positive serum samples and 20 negative serum samples, the sensitivity and specificity of the assay were found to be 91.7 and 100%, respectively. Seventy-two serum samples from dairy farms were also tested and one was diagnosed with neosporasis with both this method and a commercial assay. CONCLUSIONS: A diagnostic method employing recombinant proteins of N. caninum was developed. The method showed high sensitivity and specificity. Diagnostic test with field serum samples suggested its applicability to the practical diagnosis of neosporosis.

SUBMITTER: Dong J

PROVIDER: S-EPMC3441611 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Development of a diagnostic method for neosporosis in cattle using recombinant Neospora caninum proteins.

Dong Jinhua J Otsuki Takahiro T Kato Tatsuya T Park Enoch Y EY

BMC biotechnology 20120504

<h4>Background</h4>Neosporosis is an infectious disease primarily of cattle and dogs, caused by intracellular parasite, Neospora caninum. Neosporosis appears to be a major cause of abortion in dairy cattle worldwide and causes to huge economic loss to dairy industry.<h4>Results</h4>Recombinant surface associated antigen 1 (NcSAG1), NcSAG1 related sequence 2 (NcSRS2) and the dense granule antigen 2 (NcGRA2) of N. caninum were expressed either in silkworm or in Escherichia coli and purified. The p ...[more]

PMID: 22558916

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Project description:Despite the enormous economic importance of Neospora caninum related veterinary diseases, the number of effective therapeutic agents is relatively small. Development of new therapeutic strategies to combat the economic impact of neosporosis remains an important scientific endeavor. This study demonstrates molecular, structural and phenotypic evidence that N. caninum calcium-dependent protein kinase 1 (NcCDPK1) is a promising molecular target for neosporosis drug development. Recombinant NcCDPK1 was expressed, purified and screened against a select group of bumped kinase inhibitors (BKIs) previously shown to have low IC50s against Toxoplasma gondii CDPK1 and T. gondii tachyzoites. NcCDPK1 was inhibited by low concentrations of BKIs. The three-dimensional structure of NcCDPK1 in complex with BKIs was studied crystallographically. The BKI-NcCDPK1 structures demonstrated the structural basis for potency and selectivity. Calcium-dependent conformational changes in solution as characterized by small-angle X-ray scattering are consistent with previous structures in low Calcium-state but different in the Calcium-bound active state than predicted by X-ray crystallography. BKIs effectively inhibited N. caninum tachyzoite proliferation in vitro. Electron microscopic analysis of N. caninum cells revealed ultra-structural changes in the presence of BKI compound 1294. BKI compound 1294 interfered with an early step in Neospora tachyzoite host cell invasion and egress. Prolonged incubation in the presence of 1294 interfered produced observable interference with viability and replication. Oral dosing of BKI compound 1294 at 50 mg/kg for 5 days in established murine neosporosis resulted in a 10-fold reduced cerebral parasite burden compared to untreated control. Further experiments are needed to determine the PK, optimal dosage, and duration for effective treatment in cattle and dogs, but these data demonstrate proof-of-concept for BKIs, and 1294 specifically, for therapy of bovine and canine neosporosis.

Project description:Neospora caninum is a protozoan parasite closely related to Toxoplasma gondii Neosporosis caused by N. caninum is considered one of the main causes of abortion in cattle and nervous-system dysfunction in dogs, and identification of the virulence factors of this parasite is important for the development of control measures. Here, we used a luciferase reporter assay to screen the dense granule proteins genes of N. caninum, and we found that NcGRA6, NcGRA7, and NcGRA14 are involved in the activation of the NF-κB, calcium/calcineurin, and cAMP/PKA signals. To analyze the functions of these proteins and Neospora cyclophilin, we successfully knocked out their genes in the Nc1 strain using plasmids containing the CRISPR/Cas9 components. Among the deficient lines, the NcGRA7-deficient parasites showed reduced virulence in mice. An RNA sequencing analysis of infected macrophage cultures showed that NcGRA7 mainly regulates the host cytokine and chemokine production. The levels of gamma interferon in the ascites fluid, CXCL10 expression in the peritoneal cells, and CCL2 expression in the spleen were lower 5 days after infection with the NcGRA7-deficient parasite than after infection with the parental strain. The parasite burden and the degree of necrosis in the brains of mice infected with the NcGRA7-deficient parasite were also lower than in those of the parental strain. Collectively, our data suggest that both the NcGRA7-dependent activation of the inflammatory response and the parasite burden are important in Neospora virulence.IMPORTANCENeospora caninum invades and replicates in a broad range of host species and cells within those hosts. The effector proteins exported by Neospora induce its pathogenesis by modulating the host immunity. We show that most of the transcriptomic effects in N. caninum-infected cells depend upon the activity of NcGRA7. A deficiency in NcGRA7 reduced the virulence of the parasite in mice. This study demonstrates the importance of NcGRA7 in the pathogenesis of neosporosis.

Project description:Prevalence of disease in wildlife populations, which is necessary for developing disease models and conducting epidemiologic analyses, is often understudied. Laboratory tests used to screen for diseases in wildlife populations often are validated only for domestic animals. Consequently, the use of these tests for wildlife populations may lead to inaccurate estimates of disease prevalence. We demonstrate the use of Bayesian latent class analysis (LCA) in determining the specificity and sensitivity of a competitive enzyme-linked immunosorbent assay (cELISA; VMRD (®), Inc.) serologic test used to identify exposure to Neospora caninum (hereafter N. caninum) in three wildlife populations in southeastern Ohio, USA. True prevalence of N. caninum exposure in these populations was estimated to range from 0.1% to 3.1% in American bison (Bison bison), 51.0% to 53.8% in Père David's deer (Elaphurus davidianus), and 40.0% to 45.9% in white-tailed deer (Odocoileus virginianus). The accuracy of the cELISA in American bison and Père David's deer was estimated to be close to the 96% sensitivity and 99% specificity reported by the manufacturer. Sensitivity in white-tailed deer, however, ranged from 78.9% to 99.9%. Apparent prevalence of N. caninum from the test results is not equal to the true prevalence in white-tailed deer and Père David's deer populations. Even when these species inhabit the same community, the true prevalence in the two deer populations differed from the true prevalence in the American bison population. Variances in prevalence for some species suggest differences in the epidemiology of N. caninum for these colocated populations. Bayesian LCA methods could be used as in this example to overcome some of the constraints on validating tests in wildlife species. The ability to accurately evaluate disease status and prevalence in a population improves our understanding of the epidemiology of multihost pathogen systems at the community level.

Dataset Information

Development of a diagnostic method for neosporosis in cattle using recombinant Neospora caninum proteins.

Publications

Development of a diagnostic method for neosporosis in cattle using recombinant Neospora caninum proteins.

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