Adeel Mumtaz Abbasi

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.

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.

Hyalomma anatolicum is a tick of significant one-health importance due to its role as a vector for various pathogens affecting humans, animals and the environment, such as Theileria annulata, which causes tropical theileriosis in cattle, leading to severe economic losses. When infected with pathogens like T. annulata, the salivary glands of H. anatolicum undergo gene expression changes, secrete modified proteins and activate immune responses, all of which facilitate pathogen survival and transmission by modulating the host immune response and optimizing conditions for pathogen development. Understanding these responses is crucial for developing control strategies for tick-borne diseases. To understand the interaction between H. anatolicum and T. annulata, we performed a differential gene expression analysis of H. anatolicum salivary glands. An average of approximately 25 million raw sequencing reads were generated in each replicate using Illumina Sequencing. The sequenced reads were de novo assembled and the assembled transcriptome yielded approximately 50,231 non-redundant transcripts after clustering with CD-HIT using a sequence identity of 95% and alignment coverage of 90%. The assembly quality was evaluated with BUSCO analysis and found to be 86% complete using the Arachnida dataset and then blasted against non-redundant protein sequence database from NCBI followed by counting of reads and differential expression analysis. Overall, around 2400 and 400 genes were found differentially expressed with logFC > 1 and logFC > 2 respectively at FDR < 0.05. Top up-regulated genes included Calpain, Papilin, Neprilysin, and Ankyrin repeat-containing protein. Top down-regulated genes included Scoloptoxin, and Selenoprotein S and other uncharacterized proteins. Many other up-regulated proteins with high significance were uncharacterized suggesting room for further H. anatolicum functional and structural characterization studies. To our best knowledge, this is the first study of H. anatolicum sialotranscriptome which greatly contributes to sialotranscriptome information not only as sequence database but also indicates the potential targets for development of vaccine against ticks and transmission-blocking vaccines against T. annulata.