Tomi Trilar

Genetic analysis of wild-type Dobrava hantavirus in Slovenia: co-existence of two distinct genetic lineages within the same natural focus

Genetic analysis was performed of wild-type (wt) Dobrava hantavirus (DOB) strains from Slovenia, the country where the virus was first discovered and where it was found to cause haemorrhagic fever with renal syndrome (HFRS), with a fatality rate of 12%. Two hundred and sixty mice of the genus APODEMUS:, trapped in five natural foci of DOB-associated HFRS during 1990-1996, were screened for the presence of anti-hantavirus antibodies and 49 APODEMUS: flavicollis and four APODEMUS: agrarius were found to be positive. RT-PCR was used to recover partial sequences of the wt-DOB medium (M) and small (S) genome segments from nine A. flavicollis and one A. agrarius. Sequence comparison and phylogenetic analysis of the Slovenian wt-DOB strains revealed close relatedness of all A. flavicollis-derived virus sequences (nucleotide diversity up to 6% for the M segment and 5% for the S segment) and the geographical clustering of genetic variants. In contrast, the strain harboured by A. agrarius showed a high level of genetic diversity from other Slovenian DOB strains (14%) and clustered together on phylogenetic trees with other DOB strains harboured by A. agrarius from Russia, Estonia and Slovakia. These findings suggest that the DOB variants carried by the two species of APODEMUS: in Europerepresent two distinct genetic lineages.

Rickettsia hoogstraalii sp. nov., isolated from hard- and soft-bodied ticks

A novel spotted fever group Rickettsia was found in Haemaphysalis sulcata ticks collected from sheep and goats in Croatia in 2006. At the same time, a genetically identical organism was co-isolated with the embryonic cell line CCE3 obtained from the soft tick Carios capensis in Georgia, USA. In this study, further phenotypic and genotypic characteristics of the novel rickettsial strain present in H. sulcata ticks were investigated. Based on the cultivation of bacteria in mosquito and Vero cell cultures, the presence of rickettsiae in tick tissues and cell cultures [confirmed by transmission electron microscopy (TEM)] and the amplification and sequencing of five rickettsial genes, it was demonstrated that the novel Rickettsia strain fulfils the criteria to be classified as a novel species. The name Rickettsia hoogstraalii sp. nov. is proposed for the new strain. Rickettsia hoogstraalii sp. nov., an obligately intracellular bacterium, was grown in Vero cells and arthropod CCE3, ISE6 and C6/36 cell lines. The morphology of the cells of the novel species was typical of SFG rickettsiae. The small coccobacillary appearance of the bacteria was apparent with light microscopy. A Gram-negative bacterial cell wall and a cytoplasmic membrane separated by a narrow periplasmic space were visible by TEM. To date, Rickettsia hoogstraalii sp. nov. has been isolated from two species of ticks, H. sulcata and C. capensis. The novel species appears to be geographically widely distributed, having been detected in Croatia, Spain and Georgia, USA. Although no information is available regarding the possible pathogenicity of the novel species for vertebrate hosts, R. hoogstraalii sp. nov. has a cytopathic effect in Vero, CCE3 and ISE6 cells. Sequence analyses of the 16S rRNA, 17 kDa, gltA, ompA and ompB genes indicated that even though R. hoogstraalii sp. nov. was closely related to Rickettsia felis, it represents a separate species within the spotted fever group. The type strain of R. hoogstraalii sp. nov. is strain Croatica(T) (=DSM 22243(T)=UTMB 00003(T)).

Bioacoustic investigations and taxonomic considerations on the Cicadetta montana species complex (Homoptera: Cicadoidea: Tibicinidae)

Recent bioacoustic investigations have shown that Cicadetta montana Scopoli 1772 is a complex of morphologically similar sister species that are best characterized by their song patterns. At the type locality of C. montana, only mountain cicadas with simple, long lasting song phrases were heard, recorded and collected. Therefore, we have good reasons to suggest that this type of song is characteristic for C. montana s. str. Boulard described a song of C. montana from France with phrases composed of a long and a short echeme; this type of song is characteristic for cicadas morphologically corresponding to C. montana var. brevipennis Fieber 1876; we suggest to raise this taxon to species level. On the basis of specific song, Puissant and Boulard described C. cerdaniensis from Pyrénées. A similar case was the discovery and description of C. montana macedonica Schedl 1999 from Macedonia; since these Macedonian cicadas are sympatric with at least two other cryptic species in the C. montana group and molecular investigations showed substantial genetic differences between C. macedonica and C. montana or C. brevipennis, we conclude that this taxon should also be raised to species level. Songs of closely related C. podolica and Korean mountain cicada are presented as well.

Detection and identification of spotted fever group rickettsiae in ticks collected in southern Croatia

A total of 197 ticks belonging to four species (Haemaphysalis punctata, Hyalomma marginatum, Rhipicephalus bursa and Dermacentor marginatus) collected in October 2000 from domestic animals in southern Croatia were examined for the presence of rickettsiae by molecular techniques. Specific sequences of the rickettsiae were detected in 25 (12.7%) of ticks tested. The prevalence of infection in D. marginatus and H. marginatum ticks was 36.8 and 64.7%, respectively. None of the ticks belonging to the species H. punctata or Rh. bursa were infected. Sequence analysis of amplified products revealed that D. marginatus ticks are infected with Rickettsia slovaca, whereas H. marginatum are infected with R. aeschlimannii. The results of this study extend the knowledge of the geographic distribution of SFG rickettsiae and indicate that at least two of them, with yet uncertain pathogenicity to humans, are present in ticks in southern Croatia.

Patterns of Tick-Borne Encephalitis Virus Infection in Rodents in Slovenia

Tick-borne encephalitis virus (TBEV) is the most important causative agent of arboviral infection in Europe, causing neurologic symptoms. The incidence of the disease has greatly increased over the past decades, and in the meantime, some changes in spatial distribution of TBE cases have been observed. Therefore, it is important to recognize the distribution of endemic areas, to use preventive measures successfully. In this study, rodents from all over Slovenia were evaluated as suitable sentinels for TBEV distribution. Rodents from four species (Myodes glareolus, Apodemus flavicollis, Apodemus sylvaticus, and Apodemus agrarius) were screened for the presence of TBEV antibodies with immunofluorescence assay; the antibodies were detected in 5.9% of sera. The prevalence of infection varied according to the rodent species and according to the region of trapping. Select rodents were also screened for the presence of TBEV RNA in several organs. Both analyses showed higher rate of infection in bank voles, which also produced higher titers of anti-TBEV antibodies and a higher TBEV RNA viral load compared with mice. The regional prevalence of infection in rodents can be correlated with the incidence of disease. Molecular results indicate that the virus can be detected in the organs of the rodents for longer periods, indicating prolonged infections of the rodent hosts by the virus. Rodents can therefore be used as a useful indicator of the circulation of TBEV in an area.

The molecular evidence of Babesia microti infection in small mammals collected in Slovenia

In Europe, the zoonotic cycle of Babesia microti has not been determined so far. Recently, B. microti was detected in Ixodes ricinus ticks in Slovenia by using molecular methods. In order to investigate the mammalian hosts of B. microti in Slovenia we collected 261 small mammals representing 11 species. They were tested for the presence of babesial parasites with a PCR assay based on the nuclear small subunit rRNA gene (nss-rDNA). The bank vole (Clethrionomys glareolus) and yellow-necked mouse (Apodemus flavicollis) were infected with B. microti. The prevalence rate was 15.9% for C. glareolus and 11.8% for A. flavicollis. Nucleotide sequences of amplified portions of B. microti nss-rDNA from C. glareolus and A. flavicollis were indistinguishable from each other and identical with those previously described in I. ricinus ticks collected in Slovenia. The results of this study represent molecular evidence of B. microti in small mammals in Europe.

Prevalence and molecular characterization of tick-borne encephalitis virus in Ixodes ricinus ticks collected in Slovenia.

The hard tick Ixodes ricinus is the principal vector of tick-borne encephalitis virus (TBEV) in Slovenia; but until now, there was no information about the prevalence of TBEV infection in Slovenian ticks. We conducted a 2-year survey in 2005 and 2006, during which we were collecting I. ricinus ticks monthly in eight different locations of Slovenia. A total of 4777 I. ricinus ticks were collected: 1515 in year 2005 and 3262 in year 2006. The collected ticks were pooled into groups from which total RNA was extracted. Viral RNA was detected using real-time RT-polymerase chain reaction (PCR). Ticks infected with TBEV were found in six of eight locations. Viral RNA was detected in 8 of the 230 pools of ticks collected in 2005 and in 14 of the 442 pools collected in 2006. Prevalence of TBEV infection in Slovenian ticks was determined as 0.47%: 0.54% in 2005 and 0.43% in 2006. The detected infection rate in ticks significantly correlates with the TBEV incidence rates in selected areas. Using the method of sequencing, we have confirmed that the TBEV in ticks is genetically related to the TBEV in Slovenian patients.

The hantaviral load in tissues of naturally infected rodents.

Hantaviruses cause a lifelong and asymptomatic infection in naturally infected hosts as well as in experimentally infected rodents. Understanding the ecology and pathogenesis of hantaviruses requires an interdisciplinary research approach, which links laboratory experiments with results gained from field studies. Although several studies report hantavirus persistence and tissue infection patterns for experimentally infected rodents, field data is very limited. For this reason, the aim of our study was to investigate Puumala, Dobrava and Saaremaa virus RNA loads and tissue infection patterns in their natural reservoirs. Hantavirus RNA was demonstrated in all tested internal organs and blood samples of 14 naturally infected rodent hosts. However, the concentration of a specific virus differs depending on the virus, the host and the organ tested. Above all, the Dobrava virus showed a considerably higher viral load in all internal organs and blood samples of infected Apodemus flavicollis hosts. Results obtained in the study support the thesis that virus RNA load reaches its peak in the first month after infection, presumably after the virus has spread throughout all internal organs. This also implies that recently infected rodents are more important for transmission of the virus in the community.

Molecular Identification of Rickettsia felis‐like Bacteria in Haemaphysalis sulcata Ticks Collected from Domestic Animals in Southern Croatia

Abstract:  Haemaphysalis sulcata ticks collected from sheep and goats in southern Croatia were found infected with rickettsiae. Molecular analysis of the complete gltA gene and portion of 17 kDa and ompB genes revealed the presence of Rickettsia felis‐like bacteria in up to 26% of tested ticks.

First molecular evidence of Tula hantavirus in Microtus voles in Slovenia.

Different Microtus species, present in a worldwide range habitat populating North America, Europe, Asia, and few other species have been recognized previously as a hantavirus reservoir. Tula hantavirus was first reported in Microtus arvalis and Microtus rossiaemeridionalis from Central Russia and later discovered in several European countries. Using molecular techniques we have demonstrated the presence of Tula hantavirus in three different Microtus species in Slovenia. Phylogenetic analyses of partial S segment placed Slovenian strains in the same genetic lineage as Austrian and Croatian strains.