Detection and msp5-based diversity of Anaplasma marginale in cattle-associated arthropods from Rio de Janeiro, Brazil

Bovine anaplasmosis is caused by Anaplasma marginale, whose primary biological vector is Rhipicephalus microplus. Nevertheless, hematophagous arthropods may contribute to the circulation of pathogens in livestock production systems. The present study investigated the molecular occurrence, msp5-based genetic variation, and phylogenetic relationships of A. marginale in R. microplus, Stomoxys calcitrans, and Haematobia irritans collected from dairy cattle in the municipalities of Seropédica and Paracambi, Rio de Janeiro State, Brazil. DNA extracted from tick salivary glands and fly proboscises was subjected to nested PCR (nPCR) targeting the msp5 gene. Of the 164 arthropods analyzed, 71 (43.3%) tested positive for A. marginale, including 54/96 (56.3%) R. microplus, 10/39 (25.6%) S. calcitrans, and 7/29 (24.1%) H. irritans. All successfully sequenced amplicons were confirmed as A. marginale, showing nucleotide identities ranging from 98.1% to 100% compared to sequences available in GenBank. Phylogenetic analyses clustered all sequences within the A. marginale clade, revealing two well-supported subclades and no phylogenetic structure according to arthropod species. Analysis of the final 28-sequence alignment identified 11 haplotypes. Moderate haplotype diversity (Hd = 0.706) and low nucleotide diversity (π = 0.01423) were observed, indicating the circulation of closely related genetic variants. Identical or closely related haplotypes were shared between ticks and flies. These findings support the circulation of genetically related A. marginale variants among different hematophagous arthropods associated with dairy cattle and highlight the importance of integrated approaches for improving our understanding of the eco-epidemiology of bovine anaplasmosis.