Detection of antibodies against Anaplasma marginale in milk using a recombinant MSP5 indirect ELISA
Authors: de Echaide, ST; Bono, MF; Lugaresi, C; Aguirre, N; Mangold, A; Moretta, R; Farber, M; Mondillo, C
An indirect enzyme linked immunosorbent assay (iELISA) for diagnosis of anaplasmosis using undiluted individual milk samples from dairy cows was developed. The recombinant 19 kDa major surface protein 5 (rMSP5) of Anaplasma marginale was used as antigen. A monoclonal antibody against bovine IgG1 conjugated with peroxidase and the chromogen 3,5,3',5'-tetra-methylbenzidine were used in the test. Strong and weak, positive and negative milk samples were setup as reference controls. Results were expressed as percentage of positivity (PP) contrasting with the strongest positive control. The test was evaluated in two groups (G1 and G2) of lactating dairy cows from herds located in A. marginale non-endemic areas of Argentina. The infection status of both groups, G1 (n = 128) sampled after anaplasmosis outbreak, and G2 (it = 216) free of anaplasmosis was established by polymerase chain reaction (PCR). Serum samples of cows from G1 and G2 were analyzed by card agglutination test (CAT) and competitive ELISA (cELISA), while the novel iELISA was evaluated in their corresponding milk samples. At a cutoff of 42 PP, the ELISA has 98% sensitivity and 95% specificity. A significant difference (P < 0.0001) was found between the mean PP value of negative samples from G1 (17.4 +/- 14.9), and G2 (8.6 +/- 7.1). The agreement and kappa (kappa) value between iELISA and PCR was 96%, K = 0.919; between iELISA and CAT was 97%, K 0.880; and between iELISA and cELISA was 97%, K = 0.899. These results strongly support the usefulness of iELISA to detect A. marginale antibodies in milk. Additional studies are necessary to define the ability of the milk iELISA to detect not only acutely infected, but also carrier cattle. (c) 2005 Elsevier B.V. All rights reserved.
Plexin-D1 Is a Novel Regulator of Germinal Centers and Humoral Immune Responses
JOURNAL OF IMMUNOLOGY
Authors: Holl, Eda K.; O'Connor, Brian P.; Holl, T. Matt; Roney, Kelly E.; Zimmermann, Albert G.; Jha, Sushmita; Kelsoe, Garnett; Ting, Jenny P. -Y.
Long-lived humoral immune responses depend upon the generation of memory B cells and long-lived plasma cells during the germinal center (GC) reaction. These memory compartments, characterized by class-switched IgG and high-affinity Abs, are the basis for successful vaccination. We report that a new member of the plexin family of molecules, plexin-D1, controls the GC reaction and is required for secondary humoral immune responses. Plexin-D1 was not required for B cell maturation, marginal zone precursor development, dark and light zone formation, Ig lambda(+) and Ig kappa(+) B cell skewing, B1/B2 development, and the initial extrafollicular response. Plexin-D1 expression was increased following B cell activation, and PlxnD1(-/-) mice exhibited defective GC reactions during T-dependent immune activation. PlxnD1(-/-) B cells showed a defect in migration toward the GC chemokines, CXCL12, CXCL13, and CCL19. Accordingly, PlxnD1(-/-) mice exhibited defective production of IgG1 and IgG2b, but not IgG3 serum Ab, accompanied by reductions in long-lived bone marrow plasmacytes and recall humoral memory responses. These data show a new role for immune plexins in the GC reaction and generation of immunologic memory. The Journal of Immunology, 2011, 186: 5603-5611.