Validation and field evaluation of a double-antigen sandwich ELISA for the detection of antibodies against Anaplasma spp. in individual and bulk tank milk samples in dairy cattle

• Autores: Bellezze, Julio; Martínez Pería, Marcelo; Thompson, Carolina Soledad; Torioni, Susana Marta; Vanzini, Victor Rene; Echaide, Ignacio Eduardo; Primo, María Evangelina
• Año de publicación: 2026
• Idioma: inglés
• Tipo de recurso: artículo
• Fuente: Veterinary Parasitology: Regional Studies and Reports 68 : 101420. (February 2026)
• Materia: Ganado de Leche; Enfermedades de los Animales; Anaplasmosis Bovina; Anticuerpos; Leche; Antígenos; Dairy Cattle; Animal Diseases; Anaplasma; Bovine Anaplasmosis; ELISA; Antibodies; Milk; Antigens
• Institución: Instituto Nacional de Tecnología Agropecuaria

Bovine anaplasmosis is a worldwide infectious disease caused by the intraerythrocytic bacterium Anaplasma marginale, which is transmitted by ticks and fomites. The aim of this work was to validate a double-antigen sandwich ELISA (dasELISA) for the identification of Anaplasma spp. antibodies on individual and bulk tank milk (BTM) samples to use as a screening method. A dasELISA for the detection of antibodies against Anaplasma spp. in milk was validated using individual samples (milk, whole blood, and serum) from A. marginale-infected (n = 29) and uninfected (n = 100) cows, using nested PCR as a reference standard technique. Analogous samples from 76 A. centrale-vaccinated cows were used to evaluate cross-reactivity. The concordance between serum and milk results was evaluated by analyzing paired samples from 293 dairy cows. dasELISA performance on BTM was evaluated using samples from 114 dairy herds from the anaplasmosis low endemicity region. The Anaplasma spp. infection status of the herd was established by testing 29–30 cows per herd by serological and molecular techniques. Finally, 3,049 BTM samples were analyzed by dasELISA to assess the proportion of dairy herds infected (PDHI) with Anaplasma spp. in the main dairy area of Argentina, which is located between 28 and 34°S latitude. The dasELISA cut-off in individual milk samples was ≥3 % of positivity (%P), with a diagnostic sensitivity (DSe) of 96.5 % (95 % CI, 82.2–99.9) and a diagnostic specificity (DSp) of 86.0 % (95 % CI, 77.6–92.1). The concordance between dasELISA on milk and serum results was 85.7 %, with a κ value of 0.71 (95 % CI = 0.63–0.78). The DSe of the dasELISA in BTM samples from herds of known status, using a cut-off ≥3 %P, was 89.5 %, and the DSp was 95.6 %. dasELISA detected 100 % of herds in which A. marginale had been confirmed by nPCR. When the field study was performed, 20 % of the BTM samples were positive by dasELISA. The PDHI decreased as the latitude increased, which is in agreement with the heterogeneity of the area evaluated regarding the endemicity of A. marginale and the presence/absence of its main vector, Rhipicephalus microplus. This PDHI would be overestimated by infection with A. centrale in herds where vaccination is usually carried out. dasELISA could be used as a screening assay for anti-Anaplasma spp. antibodies detection in individual milk samples and for epidemiologic surveillance in BTM samples.
EEA Rafaela
Fil: Bellezze, Julio. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela. Instituto de Investigación de la Cadena Láctea (IDICAL); Argentina
Fil: Bellezze, Julio. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigación de la Cadena Láctea (IDICAL); Argentina
Fil: Martínez Pería, Marcelo. Nuevo Laboratorio LAVER (Villa María, Córdoba); Argentina
Fil: Thompson, Carolina. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela. Instituto de Investigación de la Cadena Láctea (IDICAL); Argentina
Fil: Thompson, Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigación de la Cadena Láctea (IDICAL); Argentina
Fil: Torioni, Susana. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela. Instituto de Investigación de la Cadena Láctea (IDICAL); Argentina
Fil: Torioni, Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigación de la Cadena Láctea (IDICAL); Argentina
Fil: Vanzini, Víctor René. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela. Instituto de Investigación de la Cadena Láctea (IDICAL); Argentina
Fil: Vanzini, Víctor René. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigación de la Cadena Láctea (IDICAL); Argentina
Fil: Echaide, Ignacio Eduardo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela. Instituto de Investigación de la Cadena Láctea (IDICAL); Argentina
Fil: Echaide, Ignacio Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigación de la Cadena Láctea (IDICAL); Argentina
Fil: Primo, María Evangelina. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela. Instituto de Investigación de la Cadena Láctea (IDICAL); Argentina
Fil: Primo, María Evangelina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigación de la Cadena Láctea (IDICAL); Argentina