Darja Duh

Diversity of Babesia Infecting European Sheep Ticks (Ixodes ricinus)

ABSTRACT Questing Ixodes ricinus (Acari: Ixodidae) adult and nymphal ticks collected in various parts of Slovenia were tested for the presence of babesial parasites with a PCR assay based on the nuclear small subunit rRNA gene (nss-ribosomal DNA [rDNA]). Thirteen of 135 ticks were found to contain babesial DNA. Sequence determination and analysis of amplified portions of nss-rDNA revealed their identity with Babesia microti and a high degree of homology withBabesia odocoilei and Babesia divergens. The results of this study represent the first genetic evidence ofB. microti and B. divergens-like parasites in I. ricinus ticks in Europe.

Interacting Roles of Immune Mechanisms and Viral Load in the Pathogenesis of Crimean-Congo Hemorrhagic Fever

ABSTRACT Until now, the pathogenesis of Crimean-Congo hemorrhagic fever (CCHF) has not been well described. However, it has been hypothesized that it could be a result of the direct injury of virus-infected tissues in combination with the indirect effects of host immune responses, including cytokines. To shed more light on the role of viral load and cytokines, differential influences of CCHF virus (CCHFV) RNA load, antibody response, and cytokine production on severity and outcome of the disease were studied in sera of 46 patients with confirmed acute CCHF from Kosovo. In this study, viral load proved to be strongly related to the severity and outcome of the disease, with higher viral loads detected in patients with fatal outcomes than in surviving patients. Also, patients with fatal outcome had on average a weaker antibody response, if one was present at all. High levels of interleukin-10 (IL-10), gamma interferon (IFN-γ), and tumor necrosis factor alpha (TNF-α) were associated with poor outcome, since detected concentrations were highest in patients with fatal outcome and lowest in patients with moderate disease course. Additionally, a positive linear dependence between viral load and these cytokines was observed. Interestingly, reduced levels of IL-12 were detected in all CCHF patients. Our study favors the hypothesis that CCHF could be a result of a delayed and downregulated immune response caused by IL-10, which leads to an increased replication and spread of CCHFV throughout the body. This consequently triggers increased production of IFN-γ and TNF-α, cytokines mediating vascular dysfunction, disseminated intravascular coagulation, organ failure, and shock.

Viral load as predictor of Crimean-Congo hemorrhagic fever outcome.

We used quantitative real-time reverse transcription–PCR to measure viral load in serum from 24 patients in Kosovo who had acute Crimean-Congo hemorrhagic fever. Viral load correlated with clinical disease and antibodies and could be used as a predictor of disease outcome.

Cervids as Babesiae Hosts, Slovenia

We describe cervids as potential reservoir hosts of Babesia EU1 and B. divergens. Both babesial parasites were found in roe deer. Sequence analysis of 18S rRNA showed 99.7% identity of roe deer Babesia EU1 with the human EU1 strain. B. divergens detected in cervids was 99.6% identical to bovine B. divergens.

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)).

Dobrava Virus RNA Load in Patients Who Have Hemorrhagic Fever with Renal Syndrome

To asses the role of virus load in the pathogenesis of hemorrhagic fever with renal syndrome, the serum Dobrava virus RNA load in 46 patients was measured with a novel quantitative real-time reverse-transcriptase polymerase chain reaction assay and compared to the disease severity. The level of viremia, detected in 26 patients, ranged from 10(2)-10(8) copies/mL of serum. The patients with severe disease had, on average, higher viral RNA loads than patients with a milder course of disease (6.15 vs. 4.67 log(10) copies/mL; P = .053). These results suggest that the Dobrava virus load might be associated with the severity of disease.

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.

The complete genome sequence of a Crimean-Congo Hemorrhagic Fever virus isolated from an endemic region in Kosovo

The Balkan region and Kosovo in particular, is a well-known Crimean-Congo hemorrhagic fever (CCHF) endemic region, with frequent epidemic outbreaks and sporadic cases occurring with a hospitalized case fatality of approximately 30%. Recent analysis of complete genome sequences of diverse CCHF virus strains showed that the genome plasticity of the virus is surprisingly high for an arthropod-borne virus. High levels of nucleotide and amino acid differences, frequent RNA segment reassortment and even RNA recombination have been recently described. This diversity illustrates the need to determine the complete genome sequence of CCHF virus representatives of all geographically distinct endemic areas, particularly in light of the high pathogenicity of the virus and its listing as a potential bioterrorism threat. Here we describe the first complete CCHF virus genome sequence of a virus (strain Kosova Hoti) isolated from a hemorrhagic fever case in the Balkans. This virus strain was isolated from a fatal CCHF case, and passaged only twice on Vero E6 cells prior to sequence analysis. The virus total genome was found to be 19.2 kb in length, consisting of a 1672 nucleotide (nt) S segment, a 5364 nt M segment and a 12150 nt L segment. Phylogenetic analysis of CCHF virus complete genomes placed the Kosova Hoti strain in the Europe/Turkey group, with highest similarity seen with Russian isolates. The virus M segments are the most diverse with up to 31 and 27% differences seen at the nt and amino acid levels, and even 1.9% amino acid difference found between the Kosova Hoti and another strain from Kosovo (9553-01). This suggests that distinct virus strains can coexist in highly endemic areas.

Molecular characterization of human pathogen Babesia EU1 in Ixodes ricinus ticks from Slovenia

New cases of human babesiosis were recently reported in Europe. The etiological agent was identified as Babesia EU1, a zoonotic pathogen with previously unreported molecular characteristics. On the basis of a comparison of the complete babesial 18S rRNA gene, we have generated strong molecular evidence that Ixodes ricinus ticks from Slovenia are infected with EU1.

Evidence of an autochthonous Toscana virus strain in Croatia.

BACKGROUND Phleboviruses are large and widespread group of viruses that are transmitted by arthropods and they have been reported to circulate in endemic regions of Mediterranean Basin, including Croatia. OBJECTIVES To investigate the role of Toscana virus, as a cause of the aseptic meningitis, in summer months in Croatia. STUDY DESIGN Samples from 30 patients with aseptic meningitis were retrospectively tested by serology and RT-PCR for TOSV. RESULTS TOSV RNA was detected in 2/30 and TOSV IgM antibodies were found in 4/30 of patients. Phylogenetic analysis of partial L and S segments suggests that TOSV from Croatia represents an autochthonous strain. CONCLUSIONS The study has confirmed the role of TOSV as an agent that causes aseptic meningitis in Croatia, therefore it should be considered by physicians when encountering meningitis or febrile illness among indigenous population or travellers during the summer months.