Preparation of colloidal gold test strips for the detection of antibodies to peste des petits ruminants based on monoclonal antibodies to N protein / 生物工程学报

A simple, fast, and visual method for detecting antibodies against peste des petits ruminants virus (PPRV) using colloidal gold strips was developed. In this study, the pET-32a-N was transformed into Escherichia coli Rosetta (DE3) for expression. Hybridoma cell lines were generated by fusing SP2/0 myeloma cells with splenocytes from immunized mice with the expressed and purified N protein of PPRV. The PPRV N protein was labeled with colloidal gold particles as the gold-labeled antigen. The N protein served as the gold standard antigen and as the test (T) line-coated antigen, while the monoclonal antibody served as the quality control (C) line-coated antibody to assemble the colloidal gold immunochromatographic test strips for detecting antibodies against the N protein of PPRV. Hybridoma cell line designated as 1F1 was able to stably secrete the monoclonal antibody against the N protein of PPRV. The titer of 1F1 monoclonal antibody in ascites was 1:128 000 determined by indirect enzyme-linked immunosorbent assays (ELISA), and the immunoglobulin subtype of the monoclonal antibody was IgG1, with kappa chain. The obtained monoclonal antibody was able to specifically recognize the N protein of PPRV, as shown by Western blotting and indirect immunofluorescent assay (IFA). The developed colloidal gold test strip method was able to detect PPRV antibodies specifically, and there was no difference between different batches of the test strips. Testing of a total of 122 clinical sera showed that the compliance rate of the test strip with ELISA test was 97.6%.The test strip assay developed in this study has good specificity, reproducibility, and sensitivity, and it can be used for the rapid detection of PPRV antibodies.

Cellular responses in the respiratory tract following intranasal administration of peste des petits ruminant vaccine in goats / Respuestas celulares en el tracto respiratorio después de la administración intranasal de la vacuna contra la peste de pequeños rumiantes en cabras

A trial was conducted to compare the cellular responses in the respiratory tract in intranasal vaccination against caprine Peste des petits ruminant lineage 1 variant virus infection with intramuscular and subcutaneous vaccinations in order to elucidate the mechanism of the protection. Twenty four goats were divided into four equal groups. Group 1 was vaccinated intranasaly, group 2 was vaccinated subcutaneously, and group 3 intramuscularly, while Group 4 was the unvaccinated control group. In each group the vaccinations were carried out once. All goats were challenged intratrachealy with PPR virus at a concentration of 106.5 TCID50 two weeks after vaccination and were euthanised 21 days after the challenge. The bronchoalveolar lavage differential count, bronchial associated lymphoid tissue (BALT) responses were measured using standard techniques. Descriptive Statistics and ANOVA was employed and significance was at p < 0.05. The exposure also resulted into significant increase in the number and size of BALT as well as the number of lymphocytes in BALT. This study showed the mechanism of the protective effect of intranasal vaccination of PPR vaccine observed with the strong mucosal and defensive cellular responses in the respiratory tract observed than the subcutaneous and intramuscular routes.

Se realizó un ensayo para comparar las respuestas celulares en las vías respiratorias después de la vacunación intranasal contra la variante caprina de la infección del virus peste de pequeños rumiantes linaje 1 con vacunas intramusculares y subcutáneas con el fin de dilucidar el mecanismo de protección. Veinticuatro cabras fueron divididas en cuatro grupos iguales. El Grupo 1 fue vacunado por vía intranasal, el grupo 2 vía subcutánea, el grupo 3 vía intramuscular y el grupo 4 control no vacunado. En cada grupo se vacunó sólo una vez. Todas las cabras fueron expuestas al virus peste de pequeños rumiantes por vía intratraqueal a una concentración de 106.5 TCID50 2 semanas después de la vacunación, y fueron sometidos a eutanasia 21 días después. Se midieron el recuento diferencial del lavado broncoalveolar y las respuestas de los tejidos linfoides asociados bronquios (BALT) utilizando técnicas estándar. Los resultados se evaluaron por estadística descriptiva y ANOVA, con una significación p<0,05. La exposición también mostró un aumento significativo en el número y tamaño del BALT, así como el número de linfocitos en este. El estudio mostró que el mecanismo del efecto protector de la vacunación intranasal contra el virus peste de pequeños rumiantes posee una respuesta mucosa y celular defensiva en el tracto respiratorio mayor que la observada por vacunación vía subcutánea e intramuscular.