Molecular Detection of Anaplasma marginale in the Central Region of Venezuela: A PCR-based Assessment of the msp2 and msp5 Genes.

Abstract

Bovine anaplasmosis reduces livestock productivity in cattle, making timely and accurate diagnosis essential for epidemiological surveillance. To explore alternative primers for pathogen detection beyond the reference 19A/19B set targeting the single-copy msp5 gene and to update epidemiologic data we evaluated analytical and field performance of primers designed for the multicopy msp2 gene, using conserved terminal regions (msp2A/B). We also described the prevalence of A. marginale in specific sampled herds from several regions of Venezuela. PCR conditions were standardized, and the analytical sensitivity of msp2A/B and 19A/B was evaluated. Field evaluations involved 208 cattle from small farms in four distinct geographical regions of Venezuela (Aragua, Cojedes, Guárico, and Miranda), divided into two groups with different clinical characteristics. Both primer sets detected the pathogen at concentrations as low as 0.01 ng. In a herd of 24 cattle experiencing an anaplasmosis outbreak, positivity rates were 12.5% and 66.67% using primers msp2A/B and 19A/B, respectively. One of the positive field-sample amplicons was cloned and sequenced to confirm bacterial identity, yielding a 97% BLAST identity with complete coverage to the reference strain Florida. Additionally, 10 and 5 nucleotide substitutions were detected at the 5' and 3' ends, respectively, compared to the reference isolate. This suggests terminal heterogeneity in the gene that may contribute to the reduced diagnostic performance of msp2A/B. Additionally, 184 clinically healthy cattle analyzed solely with 19A/B revealed a subclinical prevalence of 41.85%. In a country with limited recent molecular reports, these findings underscore the epidemiological relevance of persistent infections. This study also constitutes the first report of an msp2 sequence from a Venezuelan A. marginale isolate, providing preliminary insights of the genetic diversity of A. marginale and support using msp5-based PCR for routine field detection and surveillance in Venezuela.