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Identification of a highly antigenic region of subtilisin-like serine protease 1 for serodiagnosis of Neospora caninum infection.

ABSTRACT: Neospora caninum is an apicomplexan parasite that causes abortion in cattle; hence, accurate diagnosis of this pathogen is important to the cattle farming industry. Our previous proteomics and immunoscreening analyses revealed that the N. caninum subtilisin-like serine protease 1 (NcSUB1) has potential as a serodiagnostic tool for Neospora. Consequently, we expressed two fragments containing five NcSUB1 tandem repeat copies covering amino acids (aa) 524 to 843 (NcSUB1t) and 555 to 679 (NcSUB1tr) to identify the antigenic regions. The serodiagnostic performances of NcSUB1t and NcSUB1tr were compared with that of N54, which contains a single copy of the repeats (aa 649 to 784), and with the truncated NcSAG1 (NcSAG1t), which lacks a signal peptide and C-terminal hydrophobic regions, as a positive reference. Serum samples from N. caninum experimentally infected cattle and mice and cattle from a farm with confirmed cases of Neospora abortion were tested by enzyme-linked immunosorbent assay (ELISA) with the four antigens. In the N. caninum experimentally infected cattle, the highest IgG1 antibody titers were detected against NcSUB1t, while specific IgG1 antibodies were detectable from 16 days postinfection (dpi), with levels peaking at 36 dpi for all of the antigens. On the other hand, the levels of anti-NcSUB1 IgG2 antibodies were lower than those of anti-SAG1t IgG2 antibodies. The ELISA with NcSUB1t and NcSUB1tr had good sensitivity (94.59 to 95.95%) and specificity (80 to 100%) with bovine serum field samples compared to NcSAG1t and showed no cross-reactions with sera from Toxoplasma gondii experimentally infected mice. Moreover, IgG antibodies against NcSUB1t were detected during parturition in the NcSAG1t antibody-positive cattle, and NcSUB1t-specific antibody transfer was observed from a mother to her calf. Our results show that the NcSUB1 tandem repeat is potentially useful for serodiagnosis of N. caninum.

SUBMITTER: Ybanez RH

PROVIDER: S-EPMC3807189 | biostudies-literature | 2013 Oct

REPOSITORIES: biostudies-literature

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Identification of a highly antigenic region of subtilisin-like serine protease 1 for serodiagnosis of Neospora caninum infection.

Ybañez Rochelle Haidee D RH Terkawi Mohamad Alaa MA Kameyama Kyohko K Xuan Xuenan X Nishikawa Yoshifumi Y

Clinical and vaccine immunology : CVI 20130821 10

Neospora caninum is an apicomplexan parasite that causes abortion in cattle; hence, accurate diagnosis of this pathogen is important to the cattle farming industry. Our previous proteomics and immunoscreening analyses revealed that the N. caninum subtilisin-like serine protease 1 (NcSUB1) has potential as a serodiagnostic tool for Neospora. Consequently, we expressed two fragments containing five NcSUB1 tandem repeat copies covering amino acids (aa) 524 to 843 (NcSUB1t) and 555 to 679 (NcSUB1tr) ...[more]

PMID: 23966554

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Project description:BackgroundNeospora caninum is an intracellular parasite that causes significant economic losses in cattle industry. Understanding the host resistance mechanisms in the innate immune response to neosporosis could facilitate the exploration of approaches for controlling N. caninum infection. The NLR inflammasome is a multiprotein platform in the cell cytoplasm and plays critical roles in the host response against microbes.MethodsNeospora caninum-infected wild-type (WT) macrophages and Nlrp3 -/- macrophages, and inhibitory approaches were used to investigate inflammasome activation and its role in N. caninum infection. Inflammasome RT Profiler PCR Arrays were used to identify the primary genes involved in N. caninum infection. The expression of the sensor protein NLRP3, processing of caspase-1, secretion of IL-1β and cell death were detected. Neospora caninum replication in macrophages was also assessed.ResultsMany NLR molecules participated in the recognition of N. caninum, especially the sensor protein NLRP3, and further study revealed that the NLRP3 distribution became punctate in the cell cytoplasm, which facilitated inflammasome activation. Inflammasome activation-mediated caspase-1 processing and IL-1β cleavage in response to N. caninum infection were observed and were correlated with the time of infection and number of infecting parasites. LDH-related cell death was also observed, and this death was regarded as beneficial for the clearance of N. caninum. Treatment of N. caninum-infected macrophages with caspase-1, pan-caspase and NLRP3 inhibitors led to the impaired release of active IL-1β and a failure to restrict parasite replication. And Neospora caninum infected peritoneal macrophages from Nlrp3-deficient mice displayed greatly decreased release of active IL-1β and the failure of caspase-1 cleavage.ConclusionsThe NLRP3 inflammasome can be activated in N. caninum-infected macrophages, and plays a protective role in the host response to control N. caninum.

Dataset Information

Identification of a highly antigenic region of subtilisin-like serine protease 1 for serodiagnosis of Neospora caninum infection.

Publications

Identification of a highly antigenic region of subtilisin-like serine protease 1 for serodiagnosis of Neospora caninum infection.

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