Abstract

Background and Aim: Non-typhoidal Salmonella remains a significant zoonotic pathogen with substantial implications for animal and public health, particularly in regions where domestic animals and wildlife share ecological interfaces. The invasion protein A (invA) gene is widely used as a molecular marker for detecting Salmonella; however, its sequence variability across host species in sub-Saharan Africa remains poorly understood. This study aimed to characterize nucleotide diversity and phylogenetic relationships of the invA gene among Salmonella isolates obtained from domestic animals and free-ranging wildlife in selected regions of Zambia.

Materials and Methods: A total of 12 invA gene amplicons derived from previously confirmed Salmonella isolates from domestic animals (n = 10) and wildlife (n = 2) were selected for sequencing. Polymerase chain reaction products were purified and sequenced using BigDye terminator chemistry on a 3500 Genetic Analyzer. Sequence assembly and editing were performed using GENETYX software. Multiple sequence alignment was performed with ClustalW, and phylogenetic relationships were inferred using maximum-likelihood with the Tamura–Nei model in MEGA7. Comparative analysis included eight reference sequences retrieved from GenBank.

Results: The invA gene sequences demonstrated high nucleotide similarity (98.0%–100.0%) among isolates from domestic animals and wildlife, while showing broader variability (74.0%–100.0%) when compared with global reference strains. Six distinct sequence types were identified, with the majority originating from chicken isolates. Nucleotide substitutions were predominantly observed between positions 40 bp and 250 bp, indicating a potential hotspot for genetic variation. Some substitutions resulted in amino acid changes, suggesting possible structural and functional implications for the invasion protein. Phylogenetic analysis revealed that all Zambian isolates clustered within a single major lineage (Clade A), indicating close genetic relatedness across host species.

Conclusion: This study provides the first evidence of invA gene sequence polymorphism among Salmonella isolates from domestic animals and wildlife in Zambia. The findings highlight the dynamic evolution of Salmonella at the wildlife–livestock interface and underscore the importance of molecular surveillance in understanding zoonotic transmission. The identified sequence variations may have implications for diagnostic accuracy and the development of region-specific detection tools, reinforcing the need for continuous genomic monitoring under a One Health framework.

Keywords: domestic animals, invA gene, molecular diversity, non-typhoidal Salmonella, phylogenetic analysis, wildlife interface, Zambia, zoonotic transmission.