Hyalomma anatolicum and Rhipicephalus microplus are tick species that are important vectors of numerous pathogens affecting both humans and livestock. Endosymbionts, such as Coxiella-like endosymbionts (CLE), Francisella-like endosymbionts (FLE), and Candidatus Midichloria, play a crucial role in the physiology and vector competence of these ticks. In this study, we investigated the microbial composition of H. anatolicum and R. microplus from four geographically distinct regions of Pakistan to assess whether environmental differences influence their microbiomes. We analyzed the ticks’ gut microbiome targeting the V3-V4 hypervariable region of 16S rRNA for Illumina 16S metagenome NGS sequencing and processed overall 144 ticks. Analysis of gut bacterial composition resulted in observation of 1200 R. microplus and 968 H. anatolicum unique amplicon sequencing variants (ASVs). Relative abundance, Alpha diversity (Shannon, Faith’s phylogenetic distance) and beta diversity metrics (Bray–Curtis, Jaccard and UniFrac) were analyzed and revealed that H. anatolicum ticks have significantly unique and diverse microbial communities with Acinetobacter indicus and Francisella-like endosymbionts dominating as opposed to Candidatus Midichloria. Rhipicephalus microplus exhibited results consistent with the previous studies with no major changes in microbiome including Coxiella-like endosymbionts as the major contributor. These findings suggest that geographical and environmental factors play a significant role in shaping the tick microbiome, with potential consequences for disease transmission and tick survivability. Further research is needed to elucidate the functional roles of these microbial shifts and their impact on public health and livestock in affected regions.

Wastewater-based surveillance has emerged as a powerful approach for population-level monitoring of pathogen circulation in a timely and non-invasive manner. In this study, shotgun metagenomic sequencing was applied to wastewater samples collected from a hospital (HP), a university campus (UN), and a wastewater treatment plant (WTP). Viral sequences were taxonomically classified using Kraken2. Specifically, HP samples showed the highest viral richness, followed by WTP and UN samples (HP vs. UN, p = 0.0003; WTP vs. UN, p = 0.0018). Using Jaccard distance, significant differences were observed between WTP and UN (R² = 0.181, p < 0.001), WTP and HP (R² = 0.159, p < 0.001), and UN and HP (R² = 0.223, p < 0.001), and similarly, for Sørensen–Dice dissimilarity: WTP vs. UN (R² = 0.238, p < 0.001), WTP vs. HP (R² = 0.212, p < 0.001), and UN vs. HP (R² = 0.307, p < 0.001). Human-associated viral families were detected across all sources, predominantly Poxviridae, Orthoherpesviridae, Polyomaviridae and Circoviridae. Furthermore, the taxonomic composition of indirectly associated viruses, mainly Anelloviridae and Crassvirales, was examined. Overall, these findings support the potential of wastewater metagenomics as a reliable tool for monitoring viral diversity within environmental and public health contexts, although further research is needed to establish its operational utility for routine surveillance applications within a One Health framework.