Development and evaluation of a double-antigen sandwich ELISA to identify Anaplasma marginale–infected and A. centrale–vaccinated cattle

Abstract

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.

A vaccine using Anaplasma marginale subdominant type IV secretion system recombinant proteins was not protective against a virulent challenge

“…Three proteins, major surface protein 5 (MSP5) of A. marginale, surface antigen 1 (SAG1) of Neospora caninum, and merozoite surface antigen 2c (MSA2c) of Babesia bovis were expressed in E. coli and purified by pseudo-affinity on a Ni +2 -NTA agarose column following the protocol descripted for MSP5 protein [30]. These proteins share the sequence (FKIEGRH HHHHH) in the C-terminal extreme with four of the proteins used as immunogens, and were used for the anti-His-tag adsorption step or for the determination of the presence of these antibodies at the post-absorption step.…”

Descripción de dos casos de babesiosis cerebral en terneros de hasta 15 días de edad

“…Adicionalmente, se extrajeron muestras de sangre para serología de 20 vacas en cada establecimiento (E1 y E2), entre ellas la madre del ternero sometido a necropsia en E1. La presencia de anticuerpos específicos contra B. bovis y B. bigemina en estas muestras se determinó por la técnica de ELISA indirecto descripta por Echaide et al (2004), mientras que los anticuerpos contra A. marginale se determinaron por la técnica de ELISA de doble paratope (Sarli et al, 2019). Por último, se obtuvieron también extendidos de sangre periférica en nueve vacas del E1 y seis vacas del E2, los cuales se procesaron de igual forma que para los terneros (Benavides Ortiz et al, 2012).…”

Immunoinformatic Analysis to Identify Proteins to Be Used as Potential Targets to Control Bovine Anaplasmosis

“…With regards to tick vaccination, recent studies have shown that antitick recombinant vaccines such as aquaporin, subolesin, and tick gut glycoprotein Bm86 have a synergistic effect in reducing the engorgement of tick larvae in vitro [ 6 , 7 , 8 ]. So, an integral view of the tick-pathogen relationship should be considered to propose not only effective control measures but also successful diagnostic and vaccination methods as those recently reported [ 8 , 9 , 10 ].…”