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Background: The dynamic nature of zoonotic emergence, spillover and spread necessitates multisectoral coordination beyond national borders to encompass cross-boundary and regional cooperation. Designated points of entry (POEs), specifically ground crossings, serve as critical locales for establishing and maintaining robust prevention, detection, notification, coordination, and response mechanisms to transboundary emerging and re-emerging disease threats. In order to better assess One Health capacities for transboundary zoonotic diseases (TZD) prevention, detection and response we adapted an existing tool, One Health Systems Assessment for Priority Zoonoses (OHSAPZ), for a cross-border, POE setting in North Africa. Methods: The One Health Transboundary Assessment for Priority Zoonoses (OHTAPZ) tool was used to support prioritization of transboundary zoonoses and analyze operational capacities between national and subnational-level human and animal health stakeholders from Libya and Tunisia. Country partners jointly identified and prioritized five TZDs of concern. Case study scenarios for each priority pathogen were used to elicit current disease operations, as well as multisectoral and bilateral engagement networks. Finally, a gap analysis was performed to determine bilateral strengths and weaknesses to TZDs. Results: The five priority TZDs jointly confirmed to undergo One Health assessment were avian influenza (low and high pathogenic strains); brucellosis; Rift Valley fever; Crimean-Congo hemorrhagic fever; and rabies. Using the qualitative information collected, a transboundary systems map schematic was developed outlining the movement of human patients, animals, diagnostic samples, and routes of communication and coordination both within and between countries for zoonotic diseases. Conclusions: Analysis of current operations (prevention, detection, surveillance, laboratory capacity, quarantine/isolation, and response) and the resulting transboundary systems map schematic helped identify existing capacity strengths for certain priority pathogens, as well as challenges to timely information-sharing and coordination. We developed targeted recommendations to address these limitations for joint action planning between Libya and Tunisia. [ABSTRACT FROM AUTHOR]