Galleria mellonella as a novel invertebrate model for studying Ehrlichia ruminantium pathogenesis and host-pathogen interactions.

Ehrlichia ruminantium, the causative agent of heartwater disease, is an obligate intracellular bacterium that poses significant economic threats to livestock production in endemic regions. Current research models present substantial ethical, logistical, and economic constraints, particularly for studying host-pathogen interactions within arthropod vectors. Here, we establish Galleria mellonella larvae as a tractable invertebrate infection model for Ehrlichia ruminantium, enabling experimental investigation of pathogen persistence and host-pathogen interactions in an arthropod system. Following infection, G. mellonella proved susceptible to E. ruminantium with moderate mortality and remarkable bacterial persistence. Using rhodamine-labeled bacteria and fluorescence microscopy, we tracked bacterial dissemination from injection sites to systemic distribution in characteristic segmental patterns throughout the larval body. Rhodamine-labeled bacteria were consistently associated with hemocytes, the primary immune cells of G. mellonella, although widefield imaging cannot establish intracellular localization. Quantitative PCR revealed stable bacterial loads over the study period, and bacteria recovered from infected larvae remained viable and infectious for endothelial cells. These findings show that E. ruminantium persists in G. mellonella for at least two weeks without inducing a detectable melanization response. The fluorescence adopted a reproducible segmental distribution whose biological basis remains to be determined. This model offers significant advantages: ethical acceptability, cost-effectiveness, experimental tractability, and compatibility with high-throughput screening approaches. The G. mellonella system represents a valuable complement to existing mammalian models and provides a unique platform for investigating arthropod-specific aspects of E. ruminantium biology, screening antimicrobial compounds, and testing hypotheses of immune evasion that may inform strategies for heartwater disease control.