Development And Preliminary Application Of A Rapid Detection Method For Livestock And Poultry Pathogens Based On MinION^TM 16S RDNA Full-Length Amplicon Sequencing

Livestock and poultry bacterial pathogens not only hamper the development of the global farming industry,but also threaten the global food safety and pose a risk of humananimal pathogen transmission.The detection and identification of bacterial pathogens in livestock and poultry samples has been an important aspect in the veterinary field for a long time.Traditional pathogen detection and identification methods exhibit several disadvantages including complicated operation procedures,long detection period,and inability to detect unknown pathogens,which can hardly meet the application scenario of severe epidemiological situation or urgent clinical treatments that highly rely on the rapid and accurate pathogen identification.MinION full-length 16S rDNA amplicon sequencing provides a fast,low-cost,portable solution for complex pathogen detection.In this study,a MinION full-length 16S rDNA amplicon sequencing-based livestock and poultry bacterial pathogen detection procedure was developed,and was applied to the detection of Salmonella in the environmental wipe samples collected from the animal production environments and the detection of pathogens associated with animal gastrointestinal diseases,aiming to evaluate the potential of the efficiency of this developed method in detection and monitoring of livestock and poultry bacterial pathogens.In Chapter 1,we firstly established the optimal DNA extraction protocols for different type of samples,such as fecal samples,pre-enrichment solutions of the fecal samples,and environmental wipe samples.For the fecal samples and their pre-enrichment solutions,the grinding bead method could be used to lyse the bacterial cells,and the generated DNAwas then washed using the over-column method or the magnetic bead method depending on the application scenario and the degree of urgency.The DNA extraction time was mainly deponded the DNA washing method,which took around 40 min for magnetic bead washing method or 1.5 h when the over-column washing method was used.The bioinformatics pipeline of the MinION full-length 16S rDNA amplicon sequencing data analysis was also developed.Firstly,automatic format conversion and data filtering was performed using the Guppy software implemented in the MinION sequencer,and then the generated data was trimmed and quality controlled using Porechop software.Chimeric sequences were filtered using yacrd software.Taxonomic annotation was performed using Minimap 2 software based on the rrnDB database.The pathogenic bacterial species present in the results and their abundance were then determined.The sequencing and associated bioinformatics analysis took around 1.5 h.Then,the from-sample-to-result time of the newly developed MinION full-length 16S rDNA amplicon analysis was determined.It took around 24 plus 6.5 hours for the pre-enrichment-based sample collection and DNA extraction method,including 24 hours for bacteria enrichment from the fecal or wipe samples,1.5 hours for DNA extraction,2 hours for 16S rDNA PCR amplification,1.5 hours for library preparation,and 1.5 hours for sequencing and associated bioinformatics analysis.It took around 6 hours if DNA was directed extracted from the fecal samples using the optimized abrasive beads+magnetic beads method.The sensitivity and specificity of this method were determined based on Salmonella contaminated fecal samples experiments under laboratory conditions.The results showed that no read was identified as for the fecal samples without Salmonella contamination,and the fecal samples contaminated with 100 CFU/g Salmonella were tested as Salmonella positive even with thousands of bacterial species present in the samples,collectively demonstrating the high sensitivity and specificity of the newly developed method.In Chapter 2,this study attempts to apply the technique initially to the detection of Salmonella in environmental wipe samples from pig slaughterhouses and goose farms,and to the detection of gastrointestinal disease samples from animals.The developed sequencing-based method showed a higher sensitivity(positive rate 95.5%)than the cultivation method(45.5%/54.6%)and PCR method(40.9%)for Salmonella detection the environmental wipe samples.The method is capable of detecting Salmonella within 6.5 h from the pre-enrichment solution and of detecting multiple pathogens in the sample simultaneously,which not only avoids the risk of spreading pathogens due to false negatives when using the isolation method,but also shows potential for guiding the isolation of pathogens.This method exhibited advantages to identify the bacterial pathogens in the gastrointestinal tract of diseased animals,and also assist in the diagnosis of inflammatory bowel disease in pets through the microbiota composition analysis,thus showing promising potential in different application scenarios in veterinary clinical applications.Overall,a rapid MinION 16S rDNA full-length amplicon sequencing-based livestock and poultry bacterial pathogen detection method was developed and exhibited promising potential to identify the causal agents of veterinary animal gastrointestinal diseases and the bacterial pathogens in the environmental wipe samples.The technique has high application value in the detection and monitoring of bacterial pathogens in livestock farming and veterinary clinics.