Wild rats, including the brown rat (Rattus norvegicus) and black rat (Rattus rattus), are known reservoirs for various zoonotic pathogens. Traditional detection methods typically rely on molecular techniques targeting a limited number of specific pathogens. This narrow focus can allow emerging zoonotic threats to go unnoticed. To address this gap, broader microbiome profiling techniques offer an innovative solution by enabling comprehensive analysis of a sample’s bacterial or viral composition. This study explores the use of 16S rRNA gene amplicon sequencing as a tool for detecting zoonotic bacteria in wild rats and employs virome-enriched sequencing to identify zoonotic viruses.
Methodology Overview
We collected DNA from kidney samples of 147 wild brown rats and 42 black rats. To enhance the specificity of our bacterial detection, blocking primers were developed to minimize the amplification of rat host DNA. The bacterial composition of the kidney samples was assessed using alpha and beta diversity metrics, analyzed statistically through PERMANOVA and SIMPER analyses. Our sequencing efforts led to the identification of 14 bacterial genera with zoonotic potential, confirming the presence of zoonotic Leptospira spp. and Bartonella tribocorum through qPCR and Sanger sequencing. Notably, over 65% of the samples were predominantly composed of three bacterial taxa: Streptococcus, Mycoplasma, and Leptospira.
Zoonotic Pathogen Landscape
Wild rats can harbor a variety of pathogens, such as Seoul orthohantavirus and Leptospira spp., posing significant public health risks. The synanthropic lifestyle of these rodents facilitates pathogen transmission to humans, making surveillance essential. Current diagnostic methods, like qPCR, while sensitive, are limited in their capacity to detect multiple pathogens simultaneously. This limitation can result in overlooked zoonotic threats. As such, the development of advanced techniques like metagenomic deep sequencing is vital, as it allows for broader screening of bacterial and viral compositions without prior knowledge of specific pathogens.
Pilot Study and Sample Collection
Between 2013 and 2018, pest control agencies captured rats across various locations in the Netherlands using live traps and snap traps. A total of 189 rats were included in this study based on species and trapping location type, which varied between urban, rural, agricultural, and industrial environments. Each rat was anesthetized and subsequently euthanized, with detailed data collected on species, sex, body weight, and organ samples stored for analysis.
DNA Extraction and Sequencing
The procedure for DNA extraction involved cutting a small cross-section of each kidney, followed by the use of a lysis buffer tailored to the sample weight. The extraction process applied stringent controls to minimize contamination risk. A specialized blocking primer was developed to inhibit rat mitochondrial DNA amplification, significantly enhancing the yield of bacterial DNA from the samples. The 16S rRNA gene was amplified and sequenced using the Illumina MiSeq platform, generating extensive data that underwent rigorous quality control and analysis.
Identification of Potentially Zoonotic Bacteria
Analysis of the sequencing data revealed 14 bacterial genera with zoonotic potential. Among these, Streptococcus, Mycoplasma, and Leptospira were the most prevalent. We further validated the presence of zoonotic species through qPCR, which aligned closely with findings from the 16S rRNA sequencing. For instance, the prevalence of Leptospira spp. was found to be 55% via sequencing and 46% via qPCR, indicating a strong correlation between the two methodologies.
Understanding Factors Influencing Pathogen Presence
The study also examined internal and external factors that might correlate with zoonotic pathogen carriage. Notably, body weight showed a positive correlation with Leptospira carriage, while species differences indicated that brown rats had a higher prevalence of Bartonella compared to black rats. Furthermore, beta diversity analyses revealed significant differences in kidney bacterial composition based on species and location type, with distinct patterns emerging across urban and rural environments.
Conclusion
This study underscores the potential of microbiome profiling as a valuable tool for detecting zoonotic pathogens in wildlife. By leveraging advanced sequencing techniques, researchers can gain a more comprehensive understanding of pathogen diversity and distribution among wild rat populations. As zoonotic threats continue to evolve, enhancing surveillance methods is crucial for safeguarding public health.
- Key Takeaways:
- Traditional pathogen detection methods may overlook emerging threats.
- Microbiome profiling can provide a more comprehensive view of zoonotic pathogens.
- Specific factors like species and body weight influence pathogen prevalence in wild rats.
- Advanced sequencing methods enhance the ability to detect a wide range of pathogens simultaneously.
Read more → pmc.ncbi.nlm.nih.gov