Over the last decade, microbiology has developed in many areas at an extraordinary rate, and the Veterinary Faculty, together with the National Veterinary Institute, has followed this development within its financial capabilities. The development of molecular methods and the introduction of new technologies, such as next-generation sequencing (NGS) and MALDI-TOF, make it possible to carry out investigations that our predecessors could only dream of. Close connection and cooperation with other research institutions around Europe and the world, as well as the continuous improvement of our experts, guarantees that we will continue to meet the challenges that will be posed to our profession and country. However, the profession is developing and rising to a higher level, especially with the enthusiasm of senior and young researchers, so we must continue to enable them in the future to get their opportunities in solving modern problems.
D.04 Initiative to set up a new research area in Slovenia
COBISS.SI-ID: 4921722At the Veterinary Faculty, the Ion Torrent NGS technology was implemented on the Ion PGM platform. Different methods of sample preparation, nucleic acid extraction, and library preparation was tested. We analyzed the obtained readings according to their quality and removed readings or parts of readings with poor quality by software Torrent Suite (Ion Torrent). We used the Newbler and SPAdes program to recompile readings into neighborhoods. Then we used the Newbler and Geneious program to fit the readings to the reference genome. Using the BLASTN and BLASTX algorithms, nucleotide sequences of readings or neighborhoods were compared with sequences in NCBI GenBank databases. Clinical samples of different animal species in samples of viral isolates propagated on cell cultures and embryonic hen eggs were tested with NGS. We have successfully determined the entire genome of different viruses by RNA in the DNA genome. The whole genome of rotaviruses was determined in two samples of deer faeces. The entire enteric coronavirus genome was determined in the rectal swab of a ferret with diarrhea, while the entire genome of the coronavirus was determined in a ferret with clinical signs of systemic disease. The entire genome of the new coronavirus was also determined in a quail cloacal swab sample.
F.18 Transfer of new know-how to direct users (seminars, fora, conferences)
COBISS.SI-ID: 4387194Next-generation sequencing (NGS) is based on parallel sequencing of large number of short sequences that are later assembled on the basis of overlapping reads. Within the project ''Identification and characterization of pathogens of infectious diseases with the method of next generation sequencing'' several strategies are using: the identification of individual pathogens or their mixed populations in the selected samples and the metagenomic approach. For some diseases of viral etiology, the complete viral genome sequences of the economically important viruses such as porcine epidemic diarrhea, bovine viral diarrhea, rabies, classical swine fever, lumpy skin disease, porcine reproductive and respiratory disease, sapeloviruses, enteroviruses, bovine respiratory syncytial virus, avian influenza and bee viruses have already been determined from isolates or directly from samples with clinical manifestation of the disease. For bacteria, virulence and resistance determinants in animal samples and in addition the possible role of animal or environmental isolates in human infections are investigated. The bacterial whole genome sequences of methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus pseudintermedius (MRSP) and Clostridium difficile transmissions are determined and studied. Samples from animals with clinical manifestation of intestinal and respiratory diseases or unknown etiology are collected for testing and determining of the usefulness of this new technology. The main results of running project J4-8224 have been presented.
F.02 Acquisition of new scientific knowledge
COBISS.SI-ID: 4818554Traditional microbiological methods, which are routinely used in clinical laboratories for identification and characterization of pathogens, have several important limitations as they are based on the cultivability of microbes. They are often time-consuming and restricted to a limited number of selected target species. Molecular methods brought significant improvements to the diagnostics of infectious diseases, but they can only be used for determining the known pathogens. Next-generation sequencing (NGS) is based on parallel sequencing of large number of short sequences that are later assembled based on overlapping reads. Within the project ''Identification and characterization of pathogens of infectious diseases with the method of next generation sequencing'' several strategies were used: the identification of individual pathogens or their mixed populations in the selected samples and the metagenomic approach. For some diseases of viral etiology, the complete viral genome sequences of the economically important viruses such as porcine epidemic diarrhea, bovine viral diarrhea, rabies, classical swine fever, lumpy skin disease, porcine reproductive and respiratory disease, sapeloviruses, enteroviruses, bovine respiratory syncytial virus, avian influenza and bee viruses have already been determined from isolates or directly from samples with clinical manifestation of the disease. For bacteria, virulence and resistance determinants in animal samples and in addition the possible role of animal or environmental isolates in human infections were investigated. The bacterial whole genome sequences of methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus pseudintermedius (MRSP) and Clostridium difficile transmissions were determined and studied. Samples from animals with clinical manifestation of intestinal and respiratory diseases or unknown etiology were collected for testing and determining of the usefulness of this new technology. The main results of the project J4-8224 were presented.
B.03 Paper at an international scientific conference
COBISS.SI-ID: 34150659Surveillance of animal and human viruses to determine the composition and proportion of circulating viruses in specific mosquitoes in a selected region is a necessary challenge for formulating strategies on prediction, preparedness, prevention, and eventual control of emerging viral diseases. Ion Torrent high-throughput next-generation sequencing (NGS) technology was used to determine virome in one selected pool mosquitoes (Culex pipiens) sample. Our results showed that mosquito sample contains 15,526 reads (1.9 % of total reads) belonging to viruses. Virus sequences from six different virus families and two unclassified viruses were identified. The NGS technology was able to detect Usutu virus (USUV) reads, with 99.26 to 100 % nucleotide identity with USUV strains detected in Austria (MF063042), Hungary (MF063043) and Italy (KU573074), while West Nile virus (WNV) was not detected in the tested sample of mosquitos. This is the first attempt to determine the virome in mosquito sample by NGS in our territory.
B.03 Paper at an international scientific conference
COBISS.SI-ID: 4831610