Significance of molecular and zymography profiling for accurate detection of infected sheep with Anaplasma marginale in South Sinai, Egypt

Abstract

Abstract Anaplasma marginale is a significant tick-borne pathogen affecting ruminants; yet its prevalence and pathological impact in sheep remain underreported. This study aimed to investigate the prevalence of A. marginale in sheep from Southern Sinai, Egypt, along with its molecular characterization and the associated oxidative stress and immune responses. Microscopic examinations and PCR targeting the msp4 and msp5 genes were employed for detection, with sequencing and phylogenetic analysis performed on the msp5 gene. Oxidative stress markers, including malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione (GSH), were quantified using commercial kits. Inflammatory response was assessed via gelatin zymography for matrix metalloproteinase (MMP-2 and MMP-9). Expression levels of candidate genes, DNA methyltransferase 3 A (DNMT3a), IL-1β, and heat shock proteins (HSPs) were measured by qPCR. A high prevalence of A. marginale infection was observed, with 76% positivity by microscopy and 79.85% by PCR. Sequencing of the msp5 gene revealed 95.2–100% identity with global strains, clustering closely with the Florida (USA) strain in the phylogenetic tree. Infected sheep exhibited significantly elevated MDA levels (p < 0.05) and decreased SOD and GSH levels, indicating oxidative stress. MMP-2 and MMP-9 activities were also significantly increased, suggesting inflammatory involvement. Moreover, qPCR revealed significant upregulation of DNMT3a, IL-1β, and HSPs, highlighting transcriptional changes associated with infection. These findings provide valuable insights into molecular epidemiology and host response to A. marginale in sheep. The combination of msp5 sequencing, oxidative stress biomarkers, zymographic profiles, and gene expression analysis of DNMT3a, IL-1β, and HSPs suggests a potential panel of biomarkers for the diagnosis and monitoring of ovine anaplasmosis.