关键词: 微小隐孢子虫, 蓝氏贾第鞭毛虫, 牛艾美球虫, 胶体金免疫层析试纸条, 快速诊断

Abstract:

Objective To establish a rapid, convenient, and reliable immunological test for field screening and early detection of intestinal parasitic diarrhea in livestock, so as to address the practical needs of grassroots disease diagnosis and control. Methods The specific antigen genes of Cryptosporidium parvum cgd2-420, Giardia lamblia α-15 giardin, and Eimeria bovis heat shock protein 90 (hsp90) were amplified and the recombinant plasmids were constructed. The recombinant antigen proteins were expressed in Escherichia coli and subsequently purified. BALB/c mice were immunized with purified proteins to generate monoclonal antibodies, which were subsequently conjugated with colloidal gold nanoparticles. Specific monoclonal antibodies and goat anti-mouse IgG antibody were striped onto nitrocellulose membranes as test lines and control lines to generate a colloidal gold immunochromatographic test strip. The diagnostic performance of the test strip was systematically evaluated following optimization of colloidal gold labeling conditions, antibody labeling amounts, and coating concentrations of test and control lines, including sensitivity, specificity, repeatability, and stability. Results Recombinant C. parvum cgd2-420, G. lamblia α-15 giardin, and E. bovis HSP90 proteins were successfully constructed and expressed, with relative molecular masses of approximately 40 000, 33 000, and 50 000, respectively, which were consistent with their theoretical molecular masses. All these recombinant proteins exhibited good immunogenicity. Following immunization of BALB/c mice, and cell fusion, and screening, four, five, and five specific monoclonal antibodies were obtained targeting the three pathogens, respectively, and monoclonal antibodyclones HP2, CG5, and JD5 with optimal performance were screened for preparation of the test strip. Following optimization of the reaction condition, the best stability of the colloidal gold labeling system was achieved with addition of 5 μL of 0.2 mol/L K₂CO₃, and the optimal antibody labeling amount was 20 μg, while the optimal coating concentrations for the test and control lines were 2 mg/mL and 1.5 mg/mL, respectively. The immunochromatographic test strip exhibited a minimum detectable dilution of 1∶64, and showed no cross-reactivity with non-target pathogens, which produced consistent intra-batch and inter-batch detection results and maintained stable detection performance after storage under dry conditions at 4 ℃ for 60 days. This test strip showed high consistency with PCR assay for detection of 80 clinical samples, with coincidence rates of 93.75% for E. bovis, 96.25% for C. parvum, and 96.25% for G. lamblia, respectivelys. Conclusion A simple, rapid, and stable colloidal gold-based immunochromatographic assay has been successfully established for rapid detection of intestinal protozoa in livestock, which is suitable for field screening and early diagnosis of C. parvum, G. lamblia, and E. bovis. This assay provides a powerful technical support for early identification and control of parasitic diarrhea in livestock.

Key words: Cryptosporidium parvum, Giardia lamblia, Eimeria bovis, Colloidal gold immunochromatographic strip, Rapid diagnosis