11 research outputs found
Evaluation of a competitive inhibition ELISA based on the recombinant protein tSAG1 to detect anti–Neospora caninum antibodies in cattle
Neospora caninum is a protozoan parasite that causes abortion and important economic losses in cattle worldwide. There are no treatments or vaccines available; disease control is based on diagnosis and herd management strategies. We developed, validated, and evaluated under field conditions a competitive inhibition ELISA based on the truncated SAG1 protein (tSAG1), expressed in Escherichia coli, and the RafNeo5 monoclonal antibody (ciELISAtSAG1). A criterion based on the 3-y sequential serologic analysis of 230 dairy cows by IFAT was used as the gold standard. The assay was validated using 860 serum samples from cows that were consistently positive or negative by IFAT throughout the study period. ciELISAtSAG1 was then used to evaluate the prevalence of neosporosis in 16 beef cow herds (22 samples per herd, 352 total samples). The results were compared with those from IFAT and a commercial cELISA (cELISAVMRD). The ciELISAtSAG1 cutoff was ≥ 29%I, with a diagnostic sensitivity of 98.7% (95% CI = 96.8–99.7%) and a diagnostic specificity of 97.9% (95% CI = 96.4–99.0%). Concordance among IFAT, cELISAVMRD, and ciELISAtSAG1 was 90.3%. The agreement (κ) between ciELISAtSAG1 and the other 2 tests was ≥ 0.81. The overall prevalence of neosporosis in the 16 beef herds was 30% (range: 5–60%). The ciELISAtSAG1 could be useful for large-scale detection of anti–N. caninum antibodies in cattle and seroepidemiologic investigations, given its appropriate sensitivity and specificity, and the simplicity of production.EEA RafaelaFil: Novoa, María Belen. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Valentini, Beatriz. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela; ArgentinaFil: Sarli, Macarena. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Torioni, Susana. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela; ArgentinaFil: Primo, María Evangelina. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Echaide, Ignacio. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela; Argentin
Validation and field evaluation of a competitive enzyme-linked immunosorbent assay for diagnosis of Babesia bovis infections in Argentina
Infections by Babesia bovis limit cattle production and cause important economic losses in tropical and subtropical areas around the world. Monitoring of calf sera can be used to detect unprotected cattle herds and to decide on strategic control measures, as well as for epidemiological studies. Merozoite surface antigen 2c (MSA-2c) is an immunodominant surface protein expressed in B. bovis merozoites and sporozoites and contains B-cell epitopes that are conserved among geographic isolates. A monoclonal antibody against recombinant MSA-2c (rMSA-2c) was previously shown to inhibit the binding of anti-B. bovis antibodies to a parasite B-cell epitope in a competitive enzyme-linked immunosorbent assay (cELISA) format. In the work at hand, the parameters of this cELISA were reevaluated and adjusted when necessary, and a cutoff value was determined by receiver operator characteristic (ROC) curve analysis of a total of 357 bovine sera of known reactivity, as assessed by indirect immunofluorescence assay (IFAT). The established rMSA-2c cELISA demonstrated a specificity of 98% and a sensitivity of 96.2%. An additional set of 303 field bovine sera from regions where ticks are endemic and tick-free regions of Argentina was tested by both rMSA-2c cELISA and IFAT, and the results were shown to be in very good agreement (kappa index, 0.8325). The performance shown by rMSA-2c cELISA in the detection of B. bovis-specific antibodies and its suitability for standardization and large-scale production, as well as the possibility of its application in most veterinary diagnostic laboratories, make the assay a powerful tool for the surveillance of herd immunity as a strategic measure for the control of bovine babesiosis.Fil: Dominguez, Mariana Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Patobiología; ArgentinaFil: Echaide, Ignacio Eduardo. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Santa Fe. Estación Experimental Agropecuaria Rafaela; ArgentinaFil: Torioni de Echaide, Susana Marta. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Santa Fe. Estación Experimental Agropecuaria Rafaela; ArgentinaFil: Wilkowsky, Silvina Elizabeth. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; ArgentinaFil: Zabal, Osvaldo Alfredo. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; ArgentinaFil: Mosqueda, Juan J.. Universidad Autonoma de Queretaro.; MéxicoFil: Schnittger, Leonhard. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Patobiología; ArgentinaFil: Jacobsen, Monica Ofelia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Patobiología; Argentin
Fluorescent polarization assay, two versions of indirect and competitive enzyme- linked immunoassay for brucellosis diagnosis in vaccinated buffaloes (bubalus bubalis)
Fil: Martínez, Diana Elina. Universidad Nacional del Nordeste. Facultad de Ciencias Veterinarias; Argentina.Fil: Aguirre, Nerina. Instituto Nacional de Tecnología Agropecuaria. Estación Experimental Agropecuaria Rafaela; Argentina.Fil: Cipolini Galarza, María Fabiana. Universidad Nacional del Nordeste. Facultad de Ciencias Veterinarias; Argentina.Fil: Torioni de Echaide, Susana Marta. Instituto Nacional de Tecnología Agropecuaria. Estación Experimental Agropecuaria Rafaela; Argentina.The fluorescence polarization assay (FPA), two variants (V) of the indirect enzyme-linked immunosorbent assay (I-ELISA) and the competitive enzyme-linked immunosorbent assay (C-ELISA) were evaluated in buffaloes to detect antibodies against Brucella spp. The V1 of I-ELISA identifies them through the monoclonal (M23) anti-bovine IgG (I-ELISAM23) and the V2 through the ProteinA / G (I-ELISA-A/G). Serum samples of 862 buffaloes (Bubalus bubalis) from the Northeast of Argentina (NEA) were analyzed using the complement fixation test (CFT) as the reference. Receiving Operator Characteristic (ROC) analysis defined for the area under the curve (AUC) determined the cutoff points, sensitivity (Se) and specificity (Sp) for each test. CFT identified 107 positive and 755 negative sera. The best AUC (0.986), Concordance with CFT (96.3%) and kappa value (0.843) was obtained by I-ELISA A/G test. This assay showed the highest Se (95.33%) and C-ELISA the highest Sp (97%). FPA failed to measure the antibodies in 23 (2.65%) serum samples due to unsuccessful reading. I-ELISA M23 proved to be ineffective to diagnose brucellosis in bubaline sera. The four serological tests showed cutoff points lower than those standardized for bovines. As conclusion, I-ELISA A/G, C-ELISA and FPA with its limitations would be effective techniques for the diagnosis of brucellosis in buffaloes in the NEA, requiring an appropriate cut-off point to guarantee their maximum performance in this species.O ensaio de polarização de fluorescência (FPA), duas variantes (V) do ensaio imunoenzimático indireto (I-ELISA) e o ensaio imunoenzimático competitivo (C-ELISA), foram avaliados em búfalos para detectar anticorpos contra Brucella spp. O V1 do I-ELISA os identifica através do IgG monoclonal (M23) anti-bovino (I-ELISAM23) e o V2 através da Proteína A / G (I-ELISA-A / G). Amostras de soro de 862 búfalos (Bubalus bubalis) do Nordeste da Argentina (NEA) foram analisadas usando o teste de fixação do complemento (CFT) como referência. A análise Receiving Operator Characteristic (ROC) definida pela área sob a curva (AUC) determinou os pontos de corte, sensibilidade (Se) e especificidade (Sp) de cada teste. A CFT identificou 107 soros positivos e 755 soros negativos. Os melhores valores de AUC (0.986), concordância com CFT (96.3%) e kappa (0.843) foram obtidos pelo teste I-ELISA A / G. Este ensaio mostrou a maior Se (95.33%) e C-ELISA a maior Sp (97%). O FPA falhou em medir os anticorpos em 23 (2,65%) amostras de soro devido à falha na leitura. O I-ELISA M23 provou ser ineficaz para o diagnóstico de brucelose em soros bubalinos. Os quatro testes sorológicos mostraram pontos de corte inferiores aos padronizados para bovinos. Em conclusão, I-ELISA A / G, C-ELISA e FPA com suas limitações seriam técnicas eficazes para o diagnóstico de brucelose em búfalos no NEA, exigindo um ponto de corte adequado para garantir seu desempenho máximo nesta espécie
Efficacy of long-acting oxytetracycline and imidocarb dipropionate for the chemosterilization of Anaplasma marginale in experimentally infected carrier cattle in Argentina
The expansion of anaplasmosis to non-endemic areas in Argentina has created the need for specific treatments to eliminate Anaplasma marginale from carriers. The most recent studies have failed to chemosterilize A. marginale infections. In this work, we compare the efficacy of long-acting oxytetracycline (OTC) and imidocarb dipropionate (IMD) to chemosterilize the A. marginale infection. For this purpose, twenty steers were randomly clustered into two groups of ten animals each 78 days after A. marginale experimental infection (day 0). Cattle from group 1 (G1) were treated with three doses of 20 mg kg−1 of OTC (Terramycin® LA, 200 mg/ml) 7 days apart by intramuscular injection. Cattle from G2 were treated with two doses of 5 mg kg−1 of IMD (Imizol®, 120 mg/ml) 14 days apart by intramuscular injection. The efficacy of sterilizing treatments was evaluated by detection of DNA by nested PCR, anti-MSP5 antibodies by ELISA and by inoculation of splenectomized calves with blood from the steers 104 days post-treatment (dpt). The results showed 50% efficacy of the OTC treatment to chemosterilize persistent A. marginale infections in cattle and the failure of the IMD treatment under the evaluated conditions. The persistence of specific antibody levels in the sterilized animals (56 dpt) was shorter than the period of DNA detection. The ELISA was the test of choice to confirm the sterilizing outcome after 60 dpt. In spite of its limitations, the sterilization of A. marginale carrier status using OTC, could be useful for high-value bovines in non-endemic areas.Fil: Sarli, Macarena. Instituto Nacional de Tecnologia Agropecuaria. Centro Regional Santa Fe. Estacion Experimental Agropecuaria Rafaela. Agencia de Extension Rural Rafaela; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Novoa, María Belén. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnologia Agropecuaria. Centro Regional Santa Fe. Estacion Experimental Agropecuaria Rafaela. Agencia de Extension Rural Rafaela; ArgentinaFil: Mazzucco, Matilde N.. Instituto Nacional de Tecnologia Agropecuaria. Centro Regional Santa Fe. Estacion Experimental Agropecuaria Rafaela. Agencia de Extension Rural Rafaela; ArgentinaFil: Morel, Nicolas. Instituto Nacional de Tecnologia Agropecuaria. Centro Regional Santa Fe. Estacion Experimental Agropecuaria Rafaela. Agencia de Extension Rural Rafaela; ArgentinaFil: Primo, Maria Evangelina. Instituto Nacional de Tecnologia Agropecuaria. Centro Regional Santa Fe. Estacion Experimental Agropecuaria Rafaela. Agencia de Extension Rural Rafaela; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Investigación de la Cadena Láctea. - Instituto Nacional de Tecnología Agropecuaria. Centro Regional Santa Fe. Estación Experimental Agropecuaria Rafaela. Instituto de Investigación de la Cadena Láctea; ArgentinaFil: Torioni de Echaide, Susana Marta. Instituto Nacional de Tecnologia Agropecuaria. Centro Regional Santa Fe. Estacion Experimental Agropecuaria Rafaela. Agencia de Extension Rural Rafaela; ArgentinaFil: Echaide, Ignacio Eduardo. Instituto Nacional de Tecnologia Agropecuaria. Centro Regional Santa Fe. Estacion Experimental Agropecuaria Rafaela. Agencia de Extension Rural Rafaela; Argentin
Fluorescent polarization assay, two versions of indirect and competitive enzyme- linked immunoassay for brucellosis diagnosis in vaccinated buffaloes (bubalus bubalis)
The fluorescence polarization assay (FPA), two variants (V) of the indirect enzyme-linked immunosorbent assay (I-ELISA) and the competitive enzyme-linked immunosorbent assay (C-ELISA) were evaluated in buffaloes to detect antibodies against Brucella spp. The V1 of I-ELISA identifies them through the monoclonal (M23) anti-bovine IgG (I-ELISAM23) and the V2 through the ProteinA / G (I-ELISA-A/G). Serum samples of 862 buffaloes (Bubalus bubalis) from the Northeast of Argentina (NEA) were analyzed using the complement fixation test (CFT) as the reference. Receiving Operator Characteristic (ROC) analysis defined for the area under the curve (AUC) determined the cutoff points, sensitivity (Se) and specificity (Sp) for each test. CFT identified 107 positive and 755 negative sera. The best AUC (0.986), Concordance with CFT (96.3%) and kappa value (0.843) was obtained by I-ELISA A/G test. This assay showed the highest Se (95.33%) and C-ELISA the highest Sp (97%). FPA failed to measure the antibodies in 23 (2.65%) serum samples due to unsuccessful reading. I-ELISA M23 proved to be ineffective to diagnose brucellosis in bubaline sera. The four serological tests showed cutoff points lower than those standardized for bovines. As conclusion, I-ELISA A/G, C-ELISA and FPA with its limitations would be effective techniques for the diagnosis of brucellosis in buffaloes in the NEA, requiring an appropriate cut-off point to guarantee their maximum performance in this species.O ensaio de polarização de fluorescência (FPA), duas variantes (V) do ensaio imunoenzimático indireto (I-ELISA) e o ensaio imunoenzimático competitivo (C-ELISA), foram avaliados em búfalos para detectar anticorpos contra Brucella spp. O V1 do I-ELISA os identifica através do IgG monoclonal (M23) anti-bovino (I-ELISAM23) e o V2 através da Proteína A / G (I-ELISA-A / G). Amostras de soro de 862 búfalos (Bubalus bubalis) do Nordeste da Argentina (NEA) foram analisadas usando o teste de fixação do complemento (CFT) como referência. A análise Receiving Operator Characteristic (ROC) definida pela área sob a curva (AUC) determinou os pontos de corte, sensibilidade (Se) e especificidade (Sp) de cada teste. A CFT identificou 107 soros positivos e 755 soros negativos. Os melhores valores de AUC (0.986), concordância com CFT (96.3%) e kappa (0.843) foram obtidos pelo teste I-ELISA A / G. Este ensaio mostrou a maior Se (95.33%) e C-ELISA a maior Sp (97%). O FPA falhou em medir os anticorpos em 23 (2,65%) amostras de soro devido à falha na leitura. O I-ELISA M23 provou ser ineficaz para o diagnóstico de brucelose em soros bubalinos. Os quatro testes sorológicos mostraram pontos de corte inferiores aos padronizados para bovinos. Em conclusão, I-ELISA A / G, C-ELISA e FPA com suas limitações seriam técnicas eficazes para o diagnóstico de brucelose em búfalos no NEA, exigindo um ponto de corte adequado para garantir seu desempenho máximo nesta espécie
Development and field evaluation of an ELISA to differentiate Anaplasma marginale–infected from A. centrale–vaccinated cattle
Immunization of calves with Anaplasma centrale is used to prevent acute anaplasmosis caused by A. marginale. Natural and vaccine-acquired immunity is detected through serologic tests based primarily on A. marginale recombinant major surface protein 5 (MSP5m) because it has 91% identity with MSP5 from A. centrale (MSP5c). We developed a displacement, double-antigen, sandwich ELISA (ddasELISA) to detect antibodies against A. marginale or A. centrale. For ddasELISA validation, we analyzed serum samples positive for antibodies against Anaplasma spp. from cattle naturally infected with A. marginale (n = 300) or vaccinated with A. centrale (n = 255). Species-specific nested PCR (nPCR) assays were used to confirm infection. The optical density (OD) values obtained from antibodies directed at unique epitopes of A. marginale (ODAm) or A. centrale (ODAc) were used in the formula ODAm/ODAc. If the derived ratio was > 0.38, the serum sample was considered positive for antibodies against A. marginale, with 98.9% sensitivity and 98.0% specificity. In a field evaluation, we analyzed 702 Anaplasma spp. antibody–positive serum samples from 34 herds by ddasELISA and nPCR; 571 were classified by ddasELISA as A. marginale–infected or A. centrale–vaccinated, with 84% agreement (κ = 0.70) between ddasELISA and nPCR. Our results indicate that ddasELISA could be used as a cost-effective alternative to molecular techniques to confirm infection with A. marginale in countries in which prevention is based on vaccination with A. centrale.Fil: Bellezze, Julio. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Santa Fe. Instituto de Investigacion de la Cadena Lactea. - Instituto Nacional de Tecnologia Agropecuaria. Centro Regional Santa Fe. Estacion Experimental Agropecuaria Rafaela. Instituto de Investigacion de la Cadena Lactea.; ArgentinaFil: Thompson, Carolina Soledad. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Santa Fe. Instituto de Investigacion de la Cadena Lactea. - Instituto Nacional de Tecnologia Agropecuaria. Centro Regional Santa Fe. Estacion Experimental Agropecuaria Rafaela. Instituto de Investigacion de la Cadena Lactea.; ArgentinaFil: Bosio, Anabela Silvina. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Santa Fe. Instituto de Investigacion de la Cadena Lactea. - Instituto Nacional de Tecnologia Agropecuaria. Centro Regional Santa Fe. Estacion Experimental Agropecuaria Rafaela. Instituto de Investigacion de la Cadena Lactea.; ArgentinaFil: Torioni de Echaide, Susana Marta. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Santa Fe. Instituto de Investigacion de la Cadena Lactea. - Instituto Nacional de Tecnologia Agropecuaria. Centro Regional Santa Fe. Estacion Experimental Agropecuaria Rafaela. Instituto de Investigacion de la Cadena Lactea.; ArgentinaFil: Primo, Maria Evangelina. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Santa Fe. Instituto de Investigacion de la Cadena Lactea. - Instituto Nacional de Tecnologia Agropecuaria. Centro Regional Santa Fe. Estacion Experimental Agropecuaria Rafaela. Instituto de Investigacion de la Cadena Lactea.; Argentin
Development and evaluation of a double-antigen sandwich ELISA to identify Anaplasma marginale–infected and A. centrale–vaccinated cattle
Bovine anaplasmosis is a worldwide infectious disease caused by the intraerythrocytic bacterium Anaplasma marginale, which is transmitted by ticks and fomites. A. centrale is a less virulent subspecies used as a live vaccine in cohorts of 8- to 10-mo-old calves that did not naturally reach enzootic stability. We developed 3 variants of a double-antigen sandwich ELISA (dasELISA) using a recombinant major surface protein 5 (MSP5) from A. marginale (dasELISAm) or from A. centrale (dasELISAc) or using MSP5 from both organisms (dasELISAmc). Each dasELISA was tested for the detection of antibodies against A. marginale and A. centrale. The tests were validated using serum samples from cattle not infected with Anaplasma spp. (n = 388), infected with A. marginale (n = 436), and vaccinated with A. centrale (n = 358), confirmed by nested PCR. A total of 462 samples were compared with a commercial competitive ELISA (cELISA). For dasELISAm, dasELISAc, and dasELISAmc, specificities were 98.7%, 98.7%, and 97.4%, and overall sensitivities were 92.6%, 85.7%, and 97.4%, respectively. For A. marginale–infected and A. centrale–vaccinated cattle, sensitivities were 97.7% and 86.3% for dasELISAm, and 77.7% and 95.5% for dasELISAc, respectively. Sensitivity of dasELISAmc was similar for both groups (>96%). The agreement rate between dasELISAmc and cELISA was 96.3% (κ = 0.92); the former test allowed earlier detection of seroconversion of vaccinated cattle than did cELISA. Based on these results, the test could be used to 1) determine the enzootic stability or instability of anaplasmosis in calves, 2) conduct epidemiologic studies, and 3) evaluate the immunogenicity of A. centrale live vaccine.Fil: Sarli, Macarena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Santa Fe. Estación Experimental Agropecuaria Rafaela; ArgentinaFil: Thompson, Carolina Soledad. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Santa Fe. Estación Experimental Agropecuaria Rafaela; ArgentinaFil: Novoa, María Belén. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Santa Fe. Estación Experimental Agropecuaria Rafaela; ArgentinaFil: Valentini, Beatriz Susana. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Santa Fe. Estación Experimental Agropecuaria Rafaela; ArgentinaFil: Mastropaolo, Mariano. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias; ArgentinaFil: Echaide, Ignacio Eduardo. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Santa Fe. Estación Experimental Agropecuaria Rafaela; ArgentinaFil: Torioni de Echaide, Susana Marta. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Santa Fe. Estación Experimental Agropecuaria Rafaela; ArgentinaFil: Primo, Maria Evangelina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Santa Fe. Estación Experimental Agropecuaria Rafaela; Argentin
The dynamics of erythrocyte infection in bovine anaplasmosis: a flow cytometry analysis
Anaplasma marginale (A. marginale) is an obligate intracellular bacterium that infects bovine erythrocytes causing extravascular hemolysis and anemia. In the present work, we combine SYTO16 labeling of parasitized cells with the statistical power of flow cytometry to study the evolution of erythrocyte infection during bovine anaplasmosis.Fil: Moretta, Rosalia Ester. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Petrigh, Romina Sandra. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; ArgentinaFil: Ruybal, Paula. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Mesplet, Maria. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Patobiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Meikle Virginia. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; ArgentinaFil: Wilkowsky, Silvina Elizabeth. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; ArgentinaFil: Torioni de Echaide, Susana Marta. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Santa Fe. Estación Experimental Agropecuaria Rafaela; ArgentinaFil: Garbossa, Graciela. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Farber, Marisa Diana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentin
Parasitemia and Associated Immune Response in Pregnant and Non-Pregnant Beef Cows Naturally Infected With Neospora caninum
The aim of this longitudinal study was to characterize the parasitemia of Neospora caninum and the associated immunological parameters in naturally infected beef cows for 10 months. The following groups were established: Neospora caninum seropositive pregnant cows (+Preg, n = 7), seropositive non-pregnant cows (+Npreg, n = 7), seronegative pregnant cows (−Preg, n = 4), and seronegative non-pregnant cows (−Npreg, n = 4). Several samples were obtained for absolute and relative leukocyte counting, cytokines IL-10, IL-12, α-TNF, and γ-IFN quantification, specific IgG, IgG1, and IgG2 and avidity and N. caninum DNA molecular detection and quantification. The +Preg group had a higher frequency and concentration of N. caninum DNA in PBMC in the last third of pregnancy compared to +Npreg (p <0.05), with 22 and 8% of detection, respectively. Parasitemia correlated positively with IgG titers and negatively with IgG1/IgG2 ratio (p <0.05). On day 222 of the assay, the +Preg group had the lowest total leukocyte counting (p <0.05). The +Preg group had a higher concentration of IgG and higher avidity in the last third of gestation compared to +Npreg (p <0.05). Avidity correlated with total IgG and IgG2 (p <0.05). All +Preg cows gave birth to clinically healthy but seropositive calves before colostrum intake, therefore, the congenital transmission was 100% efficient. Only a complete N. caninum genotype from a placenta and a partial genotype from cow #3 of the group +Preg were achieved by multilocus microsatellite analysis. Overall, N. caninum parasitemia is frequent in seropositive beef cows during the last third of gestation. This correlates with higher antibody levels and a decrease in total leukocyte counting. The precise timing of the parasitemia may be used for diagnosis purposes and/or for design strategies to avoid vertical transmission. Further studies are needed to identify the immune molecular mechanisms that favor parasitemia during gestation in chronically infected cattle.Fil: Gual, Ignacio. Universidad Nacional de Mar del Plata. Facultad de Ciencias Agrarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata; ArgentinaFil: Campero, Lucía María. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Innovación para la Producción Agropecuaria y el Desarrollo Sostenible - Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce. Instituto de Innovación para la Producción Agropecuaria y el Desarrollo Sostenible; ArgentinaFil: Hecker, Yanina Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Innovación para la Producción Agropecuaria y el Desarrollo Sostenible - Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce. Instituto de Innovación para la Producción Agropecuaria y el Desarrollo Sostenible; ArgentinaFil: Regidor Cerrillo, Javier. Universidad Complutense de Madrid; EspañaFil: Leunda, Maria Rosa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Innovación para la Producción Agropecuaria y el Desarrollo Sostenible - Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce. Instituto de Innovación para la Producción Agropecuaria y el Desarrollo Sostenible; ArgentinaFil: Odeón, Anselmo Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Innovación para la Producción Agropecuaria y el Desarrollo Sostenible - Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce. Instituto de Innovación para la Producción Agropecuaria y el Desarrollo Sostenible; Argentina. Universidad Nacional de Mar del Plata. Facultad de Ciencias Agrarias; ArgentinaFil: Campero, Carlos Manuel. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce; ArgentinaFil: Torioni de Echaide, Susana Marta. Instituto Nacional de Tecnologia Agropecuaria. Centro Regional Santa Fe. Estacion Experimental Agropecuaria Rafaela. Agencia de Extension Rural Rafaela; ArgentinaFil: Echaide, Ignacio Eduardo. Instituto Nacional de Tecnologia Agropecuaria. Centro Regional Santa Fe. Estacion Experimental Agropecuaria Rafaela. Agencia de Extension Rural Rafaela; ArgentinaFil: Estein, Silvia Marcela. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Ciencias Veterinarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil. Centro de Investigación Veterinaria de Tandil. Universidad Nacional del Centro de la Provincia de Buenos Aires. Centro de Investigación Veterinaria de Tandil. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Científicas. Centro de Investigación Veterinaria de Tandil; ArgentinaFil: Ortega Mora, Luis Miguel. Universidad Complutense de Madrid; EspañaFil: Moore, Dadin Prando. Universidad Nacional de Mar del Plata. Facultad de Ciencias Agrarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Innovación para la Producción Agropecuaria y el Desarrollo Sostenible - Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce. Instituto de Innovación para la Producción Agropecuaria y el Desarrollo Sostenible; Argentin
Characterization of GASA-1, a new vaccine candidate antigen of Babesia bovis
Surface proteins bound to the cell membrane by glycosylphosphatidylinositol (GPI) anchors are considered essential for the survival of pathogenic protozoans. In the case of the tick-transmitted hemoparasite Babesia bovis, the most virulent causative agent of bovine babesiosis, the GPI-anchored proteome was recently unraveled by an in silico approach. In this work, one of the identified proteins, GASA-1 (GPI-Anchored Surface Antigen-1), was thoroughly characterized. GASA-1 is 179 aa long and has the characteristic features of a GPI-anchored protein, including a signal peptide, a hydrophilic core and a hydrophobic tail that harbors a GPI anchor signal. Transcriptomic analysis shows that it is expressed in pathogenic and attenuated B. bovis strains. Notably, the gasa-1 gene has syntenic counterparts in B. bigemina and B. ovata, which also encode GPI-anchored proteins. This is highly unusual since all piroplasmid GPI-anchored proteins described so far have been found to be species-specific. Sequencing of gasa-1 alleles from B. bovis geographical isolates originating from Argentina, USA, Brazil, Mexico and Australia showed over 98 % identity in both nucleotide and amino acid sequences. A recombinant form of GASA-1 (rGASA-1) was generated in E. coli and anti-rGASA-1 antibodies were raised in mice. Fixed and live immunofluorescence assays showed that GASA-1 is expressed in in vitro cultured B. bovis merozoites and surface-exposed. Moreover, incubation of B. bovis in vitro cultures with anti-GASA-1 antibodies partially, but significantly, reduced erythrocyte invasion, indicating that this protein bears neutralization-sensitive antibody epitopes. Splenocytes of rGASA-1-inoculated mice showed a specific proliferative response when exposed to the recombinant protein, indicating that GASA-1 bears T-cell epitopes. Finally, sera from a group of B. bovis-infected cattle reacted with the recombinant protein, demonstrating that GASA-1 is expressed during natural infection of bovines with B. bovis, and suggesting that it is immunodominant. The high degree of conservation among B. bovis isolates and the presence of syntenic genes in other Babesia species suggest a relevant role of GASA-1 and GASA-1-like proteins for parasite survival, especially considering that, due to their surface location, they are exposed to the selection pressure of the host immune system. The highlighted features of GASA-1 make it an interesting candidate for the development of vaccines against bovine babesiosis.Instituto de PatobiologíaFil: Flores, Daniela. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología; ArgentinaFil: Rodriguez, Anabel Elisa. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología; ArgentinaFil: Tomazic, Mariela Luján. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología; ArgentinaFil: Torioni, Susana Marta. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela; ArgentinaFil: Echaide, Ignacio Eduardo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela; ArgentinaFil: Zamorano, Patricia Ines. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología; Argentina. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología; ArgentinaFil: Langellotti, Cecilia Ana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología; Argentina. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología; ArgentinaFil: Araujo, Flabio Ribeiro de. Empresa Brasileira de Pesquisa Agropecuária (Embrapa). Gado de Corte; BrasilFil: Rolls, Peter. Tick Fever Centre. Department of Agriculture & Fisheries; AustraliaFil: Schnittger, Leonhard. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología; ArgentinaFil: Florin-Christensen, Monica. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patobiología; ArgentinaFil: Flores, Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET); ArgentinaFil: Tomazic, Mariela Luján. Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET); ArgentinaFil: Schnittger, Leonhard. Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET); ArgentinaFil: Florin-Christensen, Monica. Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET); Argentin