Metagenomic Surveillance of Blood-Fed Mosquitoes for Assessing Zoonotic Risk in Managed Animal Settings

Vector-borne zoonotic diseases remain a major global public health concern, particularly at interfaces where humans, domestic animals, and wildlife interact closely. Conventional surveillance approaches often fail to detect early zoonotic spillover events, especially in farm and zoological settings. Blood-fed mosquitoes, which feed on diverse vertebrate hosts, offer a unique opportunity for non-invasive environmental surveillance through xenosurveillance. This study evaluates the feasibility of using blood-fed mosquitoes as biological samplers to assess zoonotic risk in managed animal settings in eastern India.

A total of 185 blood-fed female mosquitoes were collected from livestock farms and zoological enclosures in West Bengal, India, and grouped based on host association (cattle, buffalo, goat, poultry, zebra, and deer). Mosquito species were identified using mitochondrial cytochrome c oxidase I (COI) gene sequencing. Host-group-wise pooled DNA from mosquito heads and abdomens was subjected to shotgun metagenomic sequencing using Oxford Nanopore MinION technology. Taxonomic classification was performed using Kraken 2, and microbial diversity was analyzed through alpha and beta diversity metrics using phyloseq.

Six mosquito species were identified, including Culex tritaeniorhynchus, Culex vishnui, and Mansonia uniformis, known vectors of zoonotic pathogens. Metagenomic analysis revealed diverse microbial communities dominated by Actinobacteria, Proteobacteria, and Firmicutes, with significant host-associated variation in microbial composition. Buffalo- and zebra-associated mosquitoes exhibited the highest microbial richness, while cattle-associated mosquitoes showed comparatively lower diversity. Genomic fragments corresponding to potential zoonotic and veterinary pathogens-including Plasmodium relictum, Babesia bigemina, and Clostridium botulinum-were detected across multiple host groups. Beta diversity analysis demonstrated clear host-driven clustering of mosquito-associated microbiomes.

This pilot study demonstrates that blood-fed mosquitoes can serve as effective non-invasive biological samplers for detecting environmental DNA signatures of potential zoonotic pathogens in managed animal settings. While the detection of pathogen-associated genomic fragments does not confirm active infection or transmission, the findings highlight the utility of mosquito-based metagenomic surveillance as an early warning and risk-detection tool within a One Health framework. Integrating such approaches with targeted diagnostics and epidemiological surveillance may strengthen preparedness for emerging vector-borne zoonotic threats.