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

Molecular Identification of Brucella Abortus Bv5 and Strain 19 in Water Buffaloes (Bubalus Bubalis) in Northeast Argentina

Buffalo (Bubalus bubalis) populations are spread across northern Argentina, and they share their habitat with bovines. Both species are susceptible to brucellosis, and they are under a National Plan of Control and Eradication. To characterize the Brucella spp. that infects buffaloes, the blood of 35 animals that tested positive to brucellosis by a complement fixation test was collected. DNA was obtained and analyzed by polymerase chain reaction using different molecular markers. The genera, species, and biovars of Brucella were established by analyzing specific regions of the genes omp31, eri, alkB, and omp2ab. Brucella spp. was identified in 15 of 35 tested buffaloes. The product of the omp31 gene identified the genera. The detection of two fragments of 297 bp and/or 1000 bp from the eri gene confirmed the presence of B. abortus S19 and wild-type B. abortus. The amplification of the alkB gene allowed the identification of B. abortus biovars characterized by fragments of 498 bp (bv1, bv2, or bv4). The simultaneous amplification of 498 bp (alkB) and 1000 bp (eri) products suggested the presence of B. abortus bv1, which is highly prevalent in the cattle of Argentina. Fragments of 827 bp and 857 bp were amplified from the omp2ab gene, and their sequences showed 100% identity with B. melitensis and B. abortus bv5 (GenBank). However, the 721 bp product (alkB) specific for B. melitensis could not be amplified. This is the first report indicating the presence of B. abortus bv5 in Latin America.Fil: Martinez, Diana. Universidad Nacional del Nordeste; ArgentinaFil: Thompson, Carolina. Instituto Nacional de Tecnologia Agropecuaria. Centro Regional Santa Fe. Estacion Experimental Agropecuaria Rafaela; ArgentinaFil: Russo, Ana Maria. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro de Investigaciones y Transferencia de Formosa; ArgentinaFil: Jacobo, Roberto. Universidad Nacional del Nordeste; ArgentinaFil: Torioni de Echaide, Susana. Instituto Nacional de Tecnologia Agropecuaria. Centro Regional Santa Fe. Estacion 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

Genetic diversity of Anaplasma marginale in Argentina

Bovine anaplasmosis caused by Anaplasma marginale is a worldwide major constraint to cattle production. The A. marginale major surface protein 1 alpha (msp1α) gene contains a variable number of tandem repeats in the amino terminal region and has been used for the characterization of pathogen genetic diversity. This study reports the first characterization of A. marginale genetic diversity in Argentina based on msp1α genotypes and its putative relationship with Rhipicephalus (Boophilus) microplus infestations. Herein, we analyzed whole blood bovine samples from anaplasmosis outbreaks in R. microplus infested (9 samples) and eradicated/free (14 samples) regions. Sequence analysis revealed the existence of 15 different msp1α genotypes with 31 different repeat units. Six new repeat sequences were discovered in this study and 13/31 (42%) repeats were unique to Argentinean strains. The analysis of msp1α repeat sequences according to R. microplus infestations resulted in three repeat groups: (i) found in tick-infested regions (20 repeats), (ii) found in tick free regions (6 repeats) and (iii) randomly distributed (5 repeats). Moreover, A. marginale msp1α genetic diversity was higher in tick-infested regions than in tick free areas. These results, together with previous evidence suggesting that A. marginale msp1α repeat units co-evolved with the tick vector, might represent an evidence of the role of tick-mediated transmission for the generation of pathogen genetic diversity.This work was supported by INCO Epigenevac project -UE FP6-, CONICET, ANPCyT and INTA, Argentina, Oklahoma Agricultural Experiment Station Project 1669.Peer reviewe

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

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

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

Detection of Cattle Naturally Infected with Anaplasma marginale in a Region of Endemicity by Nested PCR and a Competitive Enzyme-Linked Immunosorbent Assay Using Recombinant Major Surface Protein 5

A competitive enzyme-linked immunosorbent assay using recombinant major surface protein 5 (rMSP5-cELISA) of Anaplasma marginale was validated in a naturally infected cattle herd in an area of eastern Oregon where A. marginale is endemic. The true positive and negative A. marginale infection status of 235 randomly selected cattle was determined by using a nested PCR (nPCR) coupled with msp5 sequence analysis and hybridization. Judgment of the reliability of the nPCR and hybridization for detection of persistent infections was based on three observations. First, the nPCR was able to detect as few as 30 infected erythrocytes per ml. Second, the nPCR was able to consistently detect low levels of rickettsemia in seven carrier cattle experimentally infected with A. marginale . Third, msp5 sequence analysis showed >95% identity among 30 nPCR amplicons from cattle naturally infected with field strains of A. marginale . The nPCR and hybridization identified 151 infected and 84 uninfected cattle among the 235 animals tested. With a cutoff point of 28%, the rMSP5-cELISA showed a sensitivity of 96% and a specificity of 95%. These results indicate that the rMSP5-cELISA can sensitively and specifically detect cattle with naturally acquired persistent A. marginale infections and suggest that it is an excellent assay for epidemiological studies, eradication programs, and regulation of international cattle movement