Toward closing a heartwater monitoring gap with a new pan-species ELISA suitable for domestic ruminants, wildlife, and camels.

Heartwater, caused by the tick-borne bacterium Ehrlichia ruminantium, threatens ruminant production in sub-Saharan Africa and the Caribbean. Serological surveillance relies on species-specific ELISAs, so wildlife and camelids, which are potential reservoirs or sentinels, have no validated tools. We developed and evaluated a pan-species indirect ELISA prototype (ID Screen Ehrlichia ruminantium Indirect Multi-species, Innovative Diagnostics) that detects anti-MAP1-B antibodies in all mammals. A positivity threshold of 30% relative positivity percentage (RPP) was empirically established from 707 heartwater-negative cattle, goat and sheep sera from three regions (maximum RPP 22.1%). Against a goat-specific reference ELISA (n = 503), the prototype showed 98.2% sensitivity, 97.2% specificity and 97.6% overall agreement. ROC analysis identified a Youden-optimal cut-off of 29.96%, confirming the empirical threshold. Inter-assay reproducibility was satisfactory (mean CV 7.4%), and qualitative classification remained stable across runs, including for a near-threshold sample. In four vaccinated sheep, the prototype detected seroconversion at the same time points as a sheep-specific ELISA. In non-domestic hosts, the assay gave uniformly low RPP values in 24 zoo animals of ten species from a heartwater-free area. By contrast, 4.5% of 111 dromedary camels from Chad exceeded the threshold. Eight wildlife species from Kruger National Park (n = 405) were tested and five species presented high median RPP values. This single-protocol assay provides a reproducible tool for integrated heartwater surveillance across livestock, camelids and wildlife, in Africa, in the Caribbean and in all countries threatened by the emergence of heartwater.