The rapid and precise identification of multiple pathogens is critical for ensuring food safety, controlling epidemics, diagnosing diseases, and monitoring the environment. However, traditional detection methods are hindered by complex workflows, the need for skilled operators, and reliance on sophisticated equipment, making them unsuitable for rapid, on-site testing. Optical biosensors, known for their rapid analysis, portability, high sensitivity, and multiplexing capabilities, offer a promising solution for simultaneous multi-pathogenic identification. This paper explores recent advancements in the utilization of optical biosensors for multiple pathogenic detection. First, it provides an overview of key sensing principles, focusing on colorimetric, fluorescence-based, surface-enhanced Raman scattering (SERS), and surface plasmon resonance (SPR) techniques, as well as their applications in pathogenic detection. Then, the research progress and practical applications of optical biosensors for multiplex pathogenic detection are discussed in detail from three perspectives: microfluidic devices, nucleic acid amplification technology (NAAT), and nanomaterials. Finally, the challenges presented by optical biosensing technologies in multi-pathogen detection are discussed, along with future prospects and potential innovations in the field.

Goose astrovirus (GAstV) is an important pathogen causing visceral gout and high mortality in goslings, which has broken out and spread across China. In 2021, a disease characterized by urate deposition on the visceral surface and 30% mortality occurred in commercial adult Landaise geese in Zhejiang Province, China. A systematic study identified an infecting astrovirus, designated ZJCX, that was efficiently isolated from a diseased goose with a chicken hepatocellular carcinoma cell line (LMH). In contrast to other GAstVs originating from goslings, ZJCX caused cytopathogenic effects in LMH cells, and the crystalline arrangement of viral particles was observed through transmission electron microscopy. Indeed, phylogenetic analysis and nucleotide homology comparison revealed that ZJCX isolate belongs to the genotype II cluster of GAstVs and displays 97.8–98.4% identity with other GAstV II strains. However, several specific mutations occurred in the polyprotein and capsid protein regions. Moreover, a pathogenicity assessment of ZJCX with a gosling model was conducted, and typical visceral gout was reproduced and led to 18% mortality. The viral loads of ZJCX in the blood, kidney, and liver were detected with specific primers after inoculation, which demonstrated that the kidney and liver presented viral loads peaking at seven days post-inoculation (dpi). Biochemical parameter examination showed that AST, ALT, γ-GT, UA, and BUN levels were significantly increased by GAstV, whereas body weight was reduced. Overall, this study indicated that the GAstV isolate could infect adult geese, and the results regarding the viral loads and biochemical parameters induced by ZJCX provide insight into GAstV pathogenicity.

Duck plague virus (DPV), a highly contagious α-herpesvirus in the livestock and poultry environment, poses a significant threat to the healthy growth of ducks, potentially causing substantial economic losses. Effective control of DPV requires the development of specific diagnostic tools. A new fluorescent biosensor (R-C-CHA) was developed to detect virulent strains of DPV. It combined recombinase polymerase amplification (RPA), a CRISPR/Cas12a system, and catalytic hairpin assembly (CHA) for signal enhancement. The RPA primers were specifically designed to target the conserved DPV-CHv UL2 gene region, allowing for the rapid, efficient amplification of the target nucleic acids in isothermal conditions. The CRISPR/Cas12a system was used for sequence-specific recognition, activating its lateral cleavage activity. Furthermore, the CHA cascade reaction was utilized for enzyme-free fluorescent signal amplification. The results showed that the R-C-CHA biosensor completed the detection process in 40 min with a detection limit of 0.02 fg/μL, which was an approximate five-fold improvement compared to traditional RPA-CRISPR/Cas12a biosensors. The R-C-CHA biosensor also demonstrated perfect consistency with clinical detection and polymerase chain reaction (PCR) diagnosis, highlighting its strong potential for rapid detection in livestock and poultry farming settings.

Bovine herpesvirus type 1 (BoHV-1) causes severe respiratory and reproductive diseases in cattle, leading to significant economic losses worldwide. Current inactivated vaccines in China fail to induce robust cellular immunity and cannot differentiate infected from vaccinated animals (DIVA). To address these limitations, we constructed two triple-gene-deleted BoHV-1 vaccine candidates, ∆T3 (∆TK-∆gE/gI) and ∆g3 (∆gG-∆gE/gI), using Red/ET two-step recombination technology based on an infectious BAC clone. Their safety and immunogenicity were evaluated in a rabbit model. Both deletion mutants exhibited growth kinetics similar to the wild-type strain but with significantly reduced viral titers and plaque areas. The triple-gene deletion mutants showed superior safety compared with single-gene deletion mutants, with minimal clinical signs and low viral shedding. Immunization with ∆T3 and ∆g3 induced robust gB-specific and neutralizing antibody responses. Following challenge with the virulent BoHV-1 BC01 strain, vaccinated rabbits maintained normal body temperatures, showed significantly reduced viral shedding (approximately 100-fold), and exhibited milder pulmonary lesions. These findings demonstrate that ∆T3 high-dose and ∆g3 low-dose regimens are promising DIVA-compatible vaccine candidates, offering an effective strategy for IBR control and eradication programs.

Streptococcus suis ( S. suis ) is a major bacterial pathogen in swine industry and also an emerging zoonotic agent. S. suis produces an important extracellular component, capsular polysaccharides (CPS). Based on which, dozens of serotypes have been identified. Through virulence genotyping, we uncovered the relatedness between proportions of SS2, SS3 and SS7 strains despite their differences in serotypes. Multi-locus sequence typing (MLST) was used to characterize whole S. suis population, revealing that there is capsule switch between S. suis strains. Importantly, capsule switch occurred in SS2, 3 and 7 strains belonging to CC28 and CC29, which is phylogenetically distinct from the main CC1 SS2 lineage. To further explore capsule switch in S. suis , comparative genomic analyses were performed using available S. suis complete genomes. Phylogenetic analyses suggested that SS2 strains can be divided into two clades (1 and 2), and those classified into clade 2 are colocalized with SS3 and SS7 strains, which is in accordance with above virulence genotyping and MLST analyses. Clade 2 SS2 strains presented high genetic similarity with SS3 and SS7 and shared common competence and defensive elements, but are significantly different from Clade 1 SS2 strains. Notably, although the cps locus shared by Clade 1 and 2 SS2 strains is almost the same, a specific region in cps locus of strain NSUI002 (Clade 2 SS2) can be found in SS3 cps locus, but not in Clade 1 SS2 strain. These data indicated that SS2 strains appeared in CC28 and CC29 might acquire cps locus through capsule switch, which could well explain the distinction of genetic lineages within SS2 population.