Spatial Distribution and Genetic Diversity of Highly Pathogenic Avian Influenza A (H5N1) in Bulgaria, 2024–2026

Highly pathogenic avian influenza (HPAI) A (H5N1) remains an important threat to poultry production and wildlife health in Europe. This study aimed to characterise the spatial, temporal, and genetic patterns of HPAI A (H5N1) outbreaks in Bulgaria during 2024–2026. Forty-four confirmed outbreaks were analysed using spatial and molecular epidemiological approaches. Spatial aggregation was assessed using the Clark–Evans nearest-neighbour analysis, while kernel density estimation (KDE) was used to identify areas of increased outbreak concentration. DBSCAN clustering was applied to characterise spatially aggregated and geographically isolated outbreaks. Genotype data were available for 35 outbreaks, and associations of genotype with spatial group and recorded host or poultry-production category were assessed using Fisher’s exact tests. Hemagglutinin (HA) sequences were further analysed using phylogenetic reconstruction. Outbreaks demonstrated significant spatial aggregation, with the highest concentration occurring in south-central Bulgaria and a secondary hotspot in south-eastern Bulgaria. DBSCAN identified four spatial clusters and eight geographically isolated outbreaks. EA-2023-DA was the predominant genotype, accounting for 25 of the 35 genotyped outbreaks (71.4%), and showed a broad spatial and temporal distribution. Other genotypes, including EA-2021-AF, EA-2024-DF, EA-2024-DI DI.2, and EA-2024-DI DI.2.1, occurred less frequently and showed more restricted spatial distributions. Genotype composition differed significantly among DBSCAN spatial groups (Fisher’s exact test, p = 0.0152). However, the enrichment of EA-2023-DA in the largest spatial cluster was not statistically significant when analysed as a binary genotype comparison. The overall association between genotype and recorded host or production category was significant (p = 0.0357), but not after excluding unspecified poultry (p = 0.2162) or when restricted to ducks and laying hens (p = 0.7318). Overall, the findings demonstrate pronounced spatial heterogeneity in HPAI A (H5N1) outbreaks in Bulgaria, together with significant differences in genotype composition across spatial groups. The widespread detection of EA-2023-DA, alongside the occurrence of genetically distinct and more geographically restricted genotypes, indicates the concurrent circulation of multiple viral lineages. Integration of spatial, temporal, and molecular surveillance can support improved identification of areas with increased outbreak concentration and strengthen HPAI surveillance and control strategies in Bulgaria.