Development of gold Immunochromatographic assay strip based on specific polyclonal antibodies against capsid protein for rapid detection of porcine circovirus 2 in Zhejiang province, China.

BACKGROUND: The existing detection methods for porcine circovirus type 2 (PCV2) specific antibodies in serum cannot determine the infection status, thus it is necessary to establish a method for detecting PCV2 antigen. The capsid protein (CAP) of PCV2, as a major structural protein that plays a significant role in viral replication and in inducing host's immune response, is an ideal target antigen to monitor PCV2 infection. Therefore, a gold immunochromatographic assay (GICA) for rapid detection of PCV2 antigen based on the polyclonal antibodies (PAbs) against PCV2-CAP will be developed. RESULTS: The truncated CAP protein (dCAP) was used to immunize rabbits to generate anti-serum. After preliminary purification by caprylic acid/ammonium sulfate precipitation (CAAS), specific PAbs were purified by affinity chromatography column coupled with dCAP and its titer was about two-fold higher than preliminary purified PAbs. Colloidal gold-PAbs conjugate was synthesized under the optimum conditions. The specific anti-dCAP PAbs and goat anti-rabbit antibody (GAR) were then sprayed onto nitrocellulose (NC) membrane as a test line (TL) and a control line (CL), respectively. The visual limit detection (vLOD) of the GICA strips was 5 ng/mL. Specificity assay indicated that the GICA strips had specifically detected PCV2 and was not reactive for porcine epidemic diarrhea virus (PEDV), pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV) or classic swine fever virus (CSFV). A total of 36 porcine serum samples were detected by this GICA and commercial enzyme-linked immunosorbent assay (ELISA) Kit, 9 positive samples were found by the developed strip with the rate of 25.0% comparing with 11 positive samples detected by the commercially ELISA Kit which positive rate was 30.5%, and the receiver operating characteristic (ROC) curve revealed that the relative sensitivity and specificity of this GICA strip were 72.7 and 96.0%, respectively, with an area of 87.2%. CONCLUSIONS: This study established an efficient detection method with high sensitivity and specificity for the clinical diagnosis of PCV2 antigen, that will facilitate a rapid and convenient way to evaluate the infection status of vaccinated pigs.

A live attenuated RHΔompdcΔuprt mutant of Toxoplasma gondii induces strong protective immunity against toxoplasmosis in mice and cats.

BACKGROUND: Toxoplasma gondii is an obligate intracellular apicomplexan parasite and is responsible for zoonotic toxoplasmosis. It is essential to develop an effective anti-T. gondii vaccine for the control of toxoplasmosis, and this study is to explore the immunoprotective effects of a live attenuated vaccine in mice and cats. METHODS: First, the ompdc and uprt genes of T. gondii were deleted through the CRISPR-Cas9 system. Then, the intracellular proliferation and virulence of this mutant strain were evaluated. Subsequently, the immune responses induced by this mutant in mice and cats were detected, including antibody titers, cytokine levels, and subsets of T lymphocytes. Finally, the immunoprotective effects were evaluated by challenge with tachyzoites of different strains in mice or cysts of the ME49 strain in cats. Furthermore, to discover the effective immune element against toxoplasmosis, passive immunizations were carried out. GraphPad Prism software was used to conduct the log-rank (Mantel-Cox) test, Student's t test and one-way ANOVA. RESULTS: The RHΔompdcΔuprt were constructed by the CRISPR-Cas9 system. Compared with the wild-type strain, the mutant notably reduced proliferation (P < 0.05). In addition, the mutant exhibited virulence attenuation in both murine (BALB/c and BALB/c-nu) and cat models. Notably, limited pathological changes were found in tissues from RHΔompdcΔuprt-injected mice. Furthermore, compared with nonimmunized group, high levels of IgG (IgG1 and IgG2a) antibodies and cytokines (IFN-γ, IL-4, IL-10, IL-2 and IL-12) in mice were detected by the mutant (P < 0.05). Remarkably, all RHΔompdcΔuprt-vaccinated mice survived a lethal challenge with RHΔku80 and ME49 and WH6 strains. The immunized sera and splenocytes, especially CD8+ T cells, could significantly extend (P < 0.05) the survival time of mice challenged with the RHΔku80 strain compared with naïve mice. In addition, compared with nonimmunized cats, cats immunized with the mutant produced high levels of antibodies and cytokines (P < 0.05), and notably decreased the shedding numbers of oocysts in feces (95.3%). CONCLUSIONS: The avirulent RHΔompdcΔuprt strain can provide strong anti-T. gondii immune responses, and is a promising candidate for developing a safe and effective live attenuated vaccine.

Anti-Tumor Effect of Parasitic Protozoans.

The immune system may aberrantly silence when against "altered self", which consequently may develop into malignancies. With the development of tumor immunology and molecular biology, the deepened understanding of the relationship between parasites and tumors shifts the attitude towards parasitic pathogens from elimination to utilization. In recent years, the antitumor impact implemented by protozoan parasites and the derived products has been confirmed. The immune system is activated and enhanced by some protozoan parasites, thereby inhibiting tumor growth, angiogenesis, and metastasis in many animal models. In this work, we reviewed the available information on the antitumor effect of parasitic infection or induced by parasitic antigen, as well as the involved immune mechanisms that modulate cancer progression. Despite the fact that clinical trials of the protozoan parasites against tumors are limited and the specific mechanisms of the effect on tumors are not totally clear, the use of genetically modified protozoan parasites and derived molecules combined with chemotherapy could be an important element for promoting antitumor treatment in the future.