Antibodies Induced by Lipoarabinomannan in Bovines: Characterization and Effects on the Interaction between Mycobacterium Avium Subsp. Paratuberculosis and Macrophages In Vitro

Lipoarabinomannan (LAM) is a major glycolipidic antigen on the mycobacterial envelope. The aim of this study was to characterize the humoral immune response induced by immunization with a LAM extract in bovines and to evaluate the role of the generated antibodies in the in vitro infection of macrophages with Mycobacterium avium subsp. paratuberculosis (MAP). Sera from fourteen calves immunized with LAM extract or PBS emulsified in Freund's Incomplete Adjuvant and from five paratuberculosis-infected bovines were studied. LAM-immunized calves developed specific antibodies with IgG1 as the predominant isotype. Serum immunoglobulins were isolated and their effect was examined in MAP ingestion and viability assays using a bovine macrophage cell line. Our results show that the antibodies generated by LAM immunization significantly increase MAP ingestion and reduce its intracellular viability, suggesting an active role in this model

Detection of bovine IgG isotypes in a PPA-ELISA for Johne's disease diagnosis in infected herds

Johne’s Disease or Paratuberculosis is a chronic granulomatous enteritis disease affecting ruminants. Detection of subclinically infected animals is difficult, hampering the control of this disease. The aim of this work was to evaluate the performance of detection of IgG isotypes in a PPA-ELISA to improve the recognition of cattle naturally infected with Map in different stages. A total of 108 animals from Tuberculosis-free herds were grouped as follows: exposed (n = 30), subclinically infected (n = 26), clinically infected (n = 14), and healthy controls (n = 38). Receiver-operating characteristic (ROC) curves of isotypes/PPA-ELISAs were constructed and areas under the curves were compared to evaluate the performance of each test. Our study demonstrated that the conventional PPA-ELISA (detecting IgG) is the best to identify clinically infected animals with high sensitivity (92.9%) and specificity (100%). Meanwhile, IgG2/PPA-ELISA improved the number of subclinically infected cattle detected as compared with conventional IgG/PPA-ELISA (53.8 versus 23.1%). In addition, it had the maximum sensitivity (65.0%, taking into account all Map-infected cattle). In conclusion, the combination of IgG and IgG2/PPA-ELISAs may improve the identification of Mapinfected cattle in different stages of disease. The usefulness of IgG2 detection in serological tests for Johne’s Disease diagnosis should be further evaluated.Fil: Fernández, Bárbara. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Cátedra de Inmunología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Gilardoni, Liliana Rosa. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Cátedra de Inmunología; ArgentinaFil: Jolly, Ana. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Cátedra de Inmunología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Colavecchia, Silvia Beatriz. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Cátedra de Inmunología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Paolicchi, Fernando Alberto. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce. Agencia de Extensión Rural Balcarce; Argentina. Universidad Nacional de Mar del Plata. Facultad de Cs.agrarias. Departamento de Producción Animal; ArgentinaFil: Mundo, Silvia Leonor. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Cátedra de Inmunología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Immunological findings associated with Argentinean strains of Mycobacterium avium subsp. paratuberculosis in bovine models

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of ruminant paratuberculosis. The aim of this study was to evaluate the biological behavior of different Argentinean strains of MAP in two bovine infection models: macrophage (in vitro) and calf (in vivo) through the evaluation of early immune responses at the peripheral and local levels. Two MAP strains (A and C) were selected taking into account the different patterns of TNF-α and IL-10 secretion displayed by infected bovine macrophages in vitro. Two groups of calves were infected with 250 mg of total wet weight live MAP: strain A infected group (MA, n = 3), strain C infected group (MC, n = 2). Another group of animals was mock-infected (MI, n = 3). Infection was confirmed by MAP culture of feces and microscopic observation of granulomatous lesions in the gut tissue. All infected calves showed positive results in the DTH skin test. A significant increase in peripheral CD4CD25+ cells in MC group on day 150 was detected. The specific cellular immune response developed allowed the identification of the infection as early as 30 days in the MA group. However, the percentage of CD8CD25+ cells was significantly increased on day 120 in MC group. Significant differences between groups in proliferation and cellular responses were also detected in ileocecal lymph node samples. In summary, the strains of MAP employed herein induced differential immune responses in peripheral cells, in the proliferative responses and in cell functionality at the local level. Our findings support the hypotheses that the in vitro behavior displayed by macrophages could be a tool to identify differences among MAP strains infecting bovines and that the host-pathogen interactions occurring upon infection are dependent on the strain of MAP involved.EEA BalcarceFil: Colavecchia, Silvia Beatriz. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Cátedra de Inmunología; ArgentinaFil: Fernández, Bárbara. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Cátedra de Inmunología; ArgentinaFil: Jolly, Ana. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Cátedra de Inmunología; ArgentinaFil: Minatel, Leonardo. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Cátedra de Patología; ArgentinaFil: Hajos, Silvia Elvira. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Cátedra de Inmunología; ArgentinaFil: Paolicchi, Fernando. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Laboratorio de Bacteriología. Universidad Nacional de Mar del Plata. Facultad de Ciencias Agrarias; ArgentinaFil: Mundo, Silvia Leonor. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Cátedra de Inmunología; Argentin

Rabbit anti-deer polyclonal antibody applied to the diagnosis of Mycobacterium avium subsp. paratuberculosis in red deer (Cervus elaphus)

The diagnosis of Johne's Disease by isolating Mycobacterium avium subsp. paratuberculosis from feces requires 6 months and high-cost reagents, and therefore an indirect technique like ELISA, is often being used to avoid these obstacles. The aim of this work was the production of a rabbit anti-deer polyclonal antibody (anti-deer pAb) for use in an in-house ELISA, applying protoplasmic antigen paratuberculosis (PPA) as antigen. Two rabbits were immunized subcutaneously with three doses of deer semipurified gamma globulin emulsified with incomplete Freund's adjuvant. Rabbit serum was purified with protein A. Titers of anti-deer pAb against deer serum and cross-reaction against sera from other species were studied by ELISA and immunoblot. Samples of feces and sera from 16 deer were evaluated by fecal culture and the in-house PPA-ELISA. The results were analyzed statistically using ROC curves and the Kappa index to estimate sensitivity (Se), specificity (Sp) and the agreement level between both techniques. Subsequently, sera from three to five years old stags (n = 155) from a herd with reports of Johne's Disease were evaluated using the in-house PPA-ELISA. Anti-deer pAb showed a specific titer of 2.56 × 105. Cross-reaction against cattle, sheep, llama and goat was detected by ELISA and immunoblot. The in-house PPA-ELISA showed a Se = 85.71 % and Sp = 88.89 %. The agreement level with fecal culture was substantial (κ = 0.62 ± 0.19) and the assay was able to identify 76.77 % of the deer in the suspected herd as positive. The anti-deer pAb obtained was not only useful in the development of the in-house PPA-ELISA, which allows us to reduce the costs of Johne's Disease diagnosis, but it could also be used in other diagnosis tests in deer as well as other ruminant species.Fil: Hermida, Hernán Santiago. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Cátedra de Inmunología; ArgentinaFil: Colavecchia, Silvia Beatriz. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Cátedra de Inmunología; ArgentinaFil: Fernández, Bárbara. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Cátedra de Inmunología; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Instituto de Investigacion y Tecnología en Reproducción Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; ArgentinaFil: Suhevic, Jorge Federico. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Instituto de Investigacion y Tecnología en Reproducción Animal; ArgentinaFil: Martinez Vivot, Marcela. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; ArgentinaFil: Mereb, Guillermo. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias; ArgentinaFil: Mundo, Silvia Leonor. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario; Argentin

Humoral immune response of pigs infected with Toxocara cati

Fil: Sierra, Maria Florencia. Universidad de Buenos Aires, Facultad de Ciencias Veterinarias, Cátedra de Salud Pública; Argentina.Fil: Ricoy, Gerardo. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Parasitología. Servicio inmunología parasitaria; Argentina.Fil: Sosa, Sonia. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Parasitología. Servicio inmunología parasitaria; Argentina.Fil: Colavecchia, Silvia Beatriz. Universidad de Buenos Aires, Facultad de Ciencias Veterinarias, Cátedra de Inmunología; Argentina.Fil: Santillan, Graciela. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Parasitología. Servicio inmunología parasitaria; Argentina.Fil: López, Clara Maria. Universidad de Buenos Aires, Facultad de Ciencias Veterinarias, Cátedra de Salud Pública; Argentina.Fil: Mundo, Silvia Leonor. Universidad de Buenos Aires, Facultad de Ciencias Veterinarias, Cátedra de Inmunología; Argentina.Fil: Sommerfelt, I E. Universidad de Buenos Aires, Facultad de Ciencias Veterinarias, Cátedra de Salud Pública; Argentina.Toxocara cati is one of the causative agents of human toxocariasis. Serological methods are used for diagnosis in paratenic hosts like humans but the humoral immune response triggered by this parasite is unknown. We characterized the humoral immune response to T. cati excretory-secretory antigens (TES) in pigs as animal model during the acute and chronic stages of infection. ELISA and Western Blot techniques were used to determine antibody response. Pigs were experimentally inoculated with 100,000 infective Toxocara cati eggs. Blood was collected at 7, 14, 21 and 28 days post-inoculation (d.p.i.) to assess the acute stage of infection and 90, 120 and 180 d. p.i. for chronic stage analysis. ELISA showed values higher than the cut-off of specific IgM and IgG at 7 d. p.i. with significant differences at 0 and 7 d. p.i. for IgM and at 14, 21 and 28 d. p.i. for IgG in the acute stage. Higher and stable levels were detected in the chronic stage. Western Blot showed bands from 102 to 38 kDa detected by specific IgM and IgG. More immunogenic bands were identified by specific IgG. In the chronic stage of infection a band near 31 kDa was the only band detected by IgM until 150 d. p.i. Specific IgG recognized bands between 102 and 31 kDa. This study demonstrates how the humoral immune response evolves in the acute and chronic stages of infection and provides evidence on the role of the pig as a paratenic host of T. cati