Projects / Programmes
Identification and characterisation of pathogens of infectious diseases with the method of next generation sequencing
| Code |
Science |
Field |
Subfield |
| 4.04.00 |
Biotechnical sciences |
Veterinarian medicine |
|
| Code |
Science |
Field |
| B230 |
Biomedical sciences |
Microbiology, bacteriology, virology, mycology |
| Code |
Science |
Field |
| 4.03 |
Agricultural and Veterinary Sciences |
Veterinary science |
next generation sequencing, metagenome, complete genome, samples handling, viruses, bacteria
Researchers (30)
Organisations (3)
Abstract
Traditional microbiological methods, which are routinely used in clinical laboratories for identification and characterisation of pathogens, suffer from several important limitations as they are based on the culturability of microbes. They are often time-consuming and restricted to a limited number of selected target species. Therefore, these methods are neglecting all pathogens additionally present in samples. Molecular methods brought significant improvements to the diagnostics of infectious diseases and do not require prior isolation of pathogens. However, in most cases they can only be used for determining the known pathogens. Fast and reliable methods for the detection of various pathogens are important for the disease surveillance and prevention of their dissemination, which is essential in preventing large economic losses and assuring animal or human health.
An important improvement in the diagnostics of infectious diseases was introduced in 2005 by the next-generation sequencing (NGS). Currently, the leading technologies are Illumina and Ion Torrent. Unlike Sanger sequencing, NGS is based on parallel sequencing of large number of short sequences that are later assembled on the basis of overlapping. The first set of NGS applications in microbiology includes the direct and random sequencing of the entire DNA or cDNA in the selected samples (metagenomski approach). In this way, new pathogens and mixed infections can be discovered. The second set of NGS applications covers the typing of microbial isolates and determination of the various genetic determinants that enable the investigation of evolution and dissemination routes of pathogens.
With the metagenomic approach, the presence of individual pathogens or their mixed populations in the samples will be investigated. For diseases of viral etiology, the viral whole genome sequences will be determined to search for genetic characteristics that affect the clinical manifestation of disease. For bacteria, virulence and resistance determinants in addition to the possible role of animal or environmental isolates in human infections will be investigated. The bacterial whole genome sequences will be employed for phylogenetic analyses and typing. Project will include samples from animals with clinical manifestation of intestinal and respiratory diseases as being the most common cause of infectious diseases in humans and animals worldwide. Samples from deceased or diseased animals with unknown etiology and severe clinical picture will also be included. Among bacteria, the methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus pseudintermedius (MRSP) will be studied as becoming an increasing problem in human and veterinary medicine. Since Clostridium difficile isolates previously associated with animal hosts are increasingly occurring in humans, these will also be included. For viral infections, the field isolates of different species of viruses, obtained from clinically affected animals, will be investigated: the economically important viruses such as the porcine reproductive and respiratory syndrome (PRRS) virus and the bovine viral diarrhea (BVD) virus to research on their epidemiology and disease control, bluetongue virus (BTV) and rabies viruses, and the newly emerging enteric viruses.
The main purpose of the project is to update and expand the existing methods with the new NGS technology, aiming to develop, optimize and implement NGS for rapid detection of pathogens in clinical samples with the metagenomic approach. The whole genome sequences of viruses and bacteria will also be determined and the importance of genetic characteristics for the onset of clinical signs of the disease and the epidemiological connection between individual strains of pathogens will be investigated. Therefore, in the scope of the project the main challenge beside successful preparation of the samples for NGS sequencing will be the bioinformatic analysis of the obtained data.
Significance for science
Research partners (academic institutions) need to proliferate the area of ??applied research, provide long-term co-operation with industry and be competitive in obtaining European and other international projects. The original scientific contributions gained through the project will be presented to both national and international professional and scientific community by publications and presentations. At least three scientific papers should be published in journals with high impact factors, as well as all the microbial genomes sequenced as a part of project work will be published in the Gen Bank and thus accessible to researchers worldwide. The program of the project is entirely compliant with the priorities of the National research programme of the Republic of Slovenia, which describes dissemination of knowledge, scientific prominence and economic efficiency as favorable areas for future development of Slovenia. The study is also in line with the priorities of the third pillar of the Slovenian technology platform - Food for life. In addition, the program H2020 contains also the area of ??public health that is related to safe food. With development of a new methodology as a part of proposed project, we should be able to participate in the project consortium, that would focus on the research of safe food and public health.
The planed research of the proposed project should also lead to new and increased knowledge, particularly about the complete genetic characterization of pathogens permanently or temporarily present in the region. The obtained data will also be compared with that in the international arena. The knowledge about the relationship between infectious agents and their hosts should further help in creating future guidelines how to protect the health of both animals and humans. The results obtained through the project should also allow a critical evaluation of the already established genotyping methods. The development of the new methods for detection and characterization of pathogens through the project should improve the diagnostic procedures that are used for the research and diagnostic internationally.
The results of the project should also contribute to the international exchange of knowledge and to the promotion of Slovenian science. The new insights will be transmitted and included in industry, research and also in the study programs of veterinary medicine, medicine, microbiology and other related disciplines.
Significance for the country
Fast and reliable methods for the detection of various pathogens, for epidemiological studies and for disease monitoring are essential to ensure animal and human health. New diagnostic tools will enable more efficient and faster diagnostics of transmissible diseases with potential for very serious and rapid spread and other important diseases in animals and humans. They will also be the basis for disease control measures, which will lead to the reduction of animal loss and reduce the human infection risk.
New insights on mutual intertwining relationships of various pathogenic and potentially pathogenic microorganisms in animals and the discovery of new pathogenic agents will form the basis for the design of more effective measures for the prevention of respiratory and enteric diseases, in particular, in intensive animal farming, where these diseases can lead to major economic losses. New knowledge will help improve animal husbandry, limit health problems, improve the correct choice of therapy, and contribute to cleaner environment and to the production of cheaper and safer food. Additionally, a positive impact of the project on the international competitiveness of our livestock production can be expected. The new technology enables fast characterization of the whole pathogen genome, which is important in the case of introduction and spread of a particularly dangerous viral infection. Fast characterization of the whole genomes is also becoming a requirement of the reference laboratories. Limiting the outbreaks and spread of the animal infectious diseases will also affect the economics of food production and enhance the international competitiveness of Slovenian livestock production being under a lot of pressure in recent years.
The important point of view for animal health and even more important for human health, is the emergence of especially resistant bacterial strains such as MRSA and C. difficile. The characterization and genotyping of their strains will be the basis to plan and implement effective epidemiological disease control measures, which will lead to the reduction of animal losses and limit the risk of human infection. Human infections with MRSA and C. difficile present a relatively heavy burden for the Slovenian health care system. Research on the mechanisms of resistance to antimicrobial drugs and determinants of pathogenicity will be of interest to a wider veterinary and medical society, especially for the Administration for Food Safety, Veterinary and Plant Protection and the inter-ministerial Commission for the prudent use of antimicrobials in the frame of the Ministry of Health. They can serve as a basis for guidelines for the rational use of antimicrobial agents in the intensive animal farming.
Most important scientific results
Interim report,
final report
Most important socioeconomically and culturally relevant results
Interim report,
final report
