Removal of protozoa, opportunistic pathogens with virulence factors in swine manure using anaerobic digestion: Full-scale investigation and lab-scale optimization

Abstract

Swine manure serves as a significant reservoir of zoonotic protozoa and opportunistic pathogens, posing environmental and public health risks when inadequately treated. In this study, multiple molecular approaches, including quantitative PCR, nested PCR with gp60-based phylogenetic analysis, virulence factor profiling, and metagenome-assembled genome (MAG) reconstruction, were employed to investigate the abundance, diversity, and treatment responses of Cryptosporidium, Giardia, twelve opportunistic pathogens, and associated virulence factors (VFs) in swine manure. Three full-scale anaerobic digestion (AD) systems were investigated, and thermophilic and hyperthermophilic pretreatments were applied to lab-scale AD systems to evaluate the efficiency of biological risk control. Cryptosporidium parvum was identified as the dominant species, with subtype IIaA17G4R1 and related zoonotic subtypes detected in both lab-scale and full-scale samples. Phylogenetic clustering of swine-derived sequences with human and cattle isolates indicates a potential risk of zoonotic transmission through manure-associated environmental contamination. In lab-scale AD, a significant reduction in Cryptosporidium, particularly under hyperthermophilic conditions, was observed, while Giardia was undetectable in both influent and effluent samples. In full-scale systems, persistence of Escherichia coli, Clostridium perfringens, Enterococcus, Salmonella, and multiple VFs was confirmed in the effluents. The hyperthermophilic-mesophilic (70 °C-37 °C) lab-scale treatments achieved a substantial reduction in overall pathogen abundance from 3.40 × 108 to 1.21 × 108 copies/g dry weight and in virulence gene loads from 5.24 to 2.35 copies/cell (P < 0.001), along with the significant removal of pathogenic MAGs such as Enterococcus, Escherichia, Pseudomonas, and Streptococcus. These findings demonstrate the effectiveness of AD for reducing microbial risks and underscore the potential of thermophilic phase digestion as a scalable, biologically effective method for reducing microbial risks associated with livestock manure reuse.