Nikola Bradarić

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)).

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.

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.

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.

Demographic and Epidemiologic Features of Mediterranean Spotted Fever Cases in the Region of Split, Croatia

Mediterranean spotted fever (MSF) is an acute, febrile, tick-transmitted rickettsiosis caused by Rickettsia conorii , an obligate intracellular bacterium endemic in Mediterranean countries, Africa, and Asia.The major vector of the disease is the dog brown tick Rhipicephalus sanguineus . 1,2 The first clinical description of spotted fever disease in Croatia was reported in the city of Split (central part of southern Croatia, eastern coast of Adriatic Sea) by Tartaglia in 1935. 3 The first well documented, serologically confirmed cases of MSF in Croatia were reported among inhabitants and tourists in Split and its adjacent suburban-semirural area in 1982. 4 Ever since a prospective study on MSF in the area of Split was carried out at University Hospital Split, the only hospital in the region, where cases of MSF are observed every year. This article presents an overview of data accumulated from 1982 through 1999 and describes demographic and epidemiological features of patients with MSF in the region of Split, Croatia.

A Follow‐up Study on Newly Recognized Spotted Fever Group Rickettsiae in Ticks Collected in Southern Croatia

The aim was to study the impact of environmental conditions on seasonal population dynamics of tick species found in southern Croatia with the consequence on rickettsiae vectored by them. Efficiency of DNA extraction from individual ticks was verified by amplifying the portion of mitochondrial 16S rRNA gene of tick origin. By using PCR assays and subsequent sequence analysis of the portion of four rickettsial genes ; gltA, ompA, ompB and gene for 17-kDa protein, the presence and identity of rickettsiae were determined.A total of 192 ticks belonging to five species (Haemaphysalis punctata, D. marginatus, H. marginatum, Rhipicephalus bursa and Rh. turanicus) were collected in May 2001. Comparing the results from the collection in autumn 2000, a major decline was noticed in the adult H. punctata population with no rickettsial DNA detected in this species. Rh. bursa ticks were not competent vectors for SFG rickettsiae. Three of 33 (9.1 %) Rh. turanicus, a newly recognized species in the region, were infected with R. rhipicephali. As previously determined, D. marginatus and H. marginatum ticks were repeatedly infected with R. slovaca and R. aeschlimannii, respectively. An increase in population of adult D. marginatus and H. marginatum ticks was detected over the follow up. However, the percent of infection in ticks was lower (64.7 % in October 2000 and 26.1 % in May 2001) for R. aeschlimannii and higher (36.8 % in October 2000 and 60.0 % in May 2001) for R. slovaca.In conclusion, importance of the climatic impact on seasonal population dynamics of ticks in southern Coratia with consequence on SFG rickettsiae vectored by them was shown in this study.

Hepatitis B virus infection among pregnant women in Split region

A seroepidemiologic survey of pregnant women in Split region was performed in order to determine a strategy for prenatal screening for HBsAg. Thirty (7.5%) of 400 women had the markers of past or current HBV infection, while 3/400 (0.75%) were carriers. According to demographic data and risk factors they represent low risk population. General prenatal screening for HBsAg is advisable.

Two outbreaks of typhoid fever related to the war in Bosnia and Herzegovina

Two outbreaks of typhoid fever caused by Salmonella typhi of the same phagotype (A, biotype II) and antibiotic susceptibility are reported. Both occurred during the war in Bosnia and Herzegovina. The first outbreak appeared among the refugees from the town of Jajce. The second outbreak appeared among the inhabitants in the village of Vidosi near Livno. This report describes main clinical, epidemioloigcal and laboratory findings for 22 patients treated in Split University Hospital, Croatia, in the period November 1992–January 1993. Possible epidemiological connections between those two outbreaks are discussed.

The impact of IL-6 and IL-28B gene polymorphisms on treatment outcome of chronic hepatitis C infection among intravenous drug users in Croatia

Background Several genes and their single nucleotide polymorphisms (SNPs) are associated with either spontaneous resolution of hepatitis C infection or better treatment-induced viral clearance. We tested a cohort of intravenous drug users (IVDU) diagnosed with chronic hepatitis C virus (HCV) for treatment response and its association with the SNPs in the interleukin-6 (rs1800795-IL6) and the interleukin-28B (rs12979860-IL28B) genes. Methods The study included 110 Croatian IVDU positive for anti-HCV antibody. Genotyping was performed by polymerase chain reaction (PCR) based approach. Patients were treated by standard pegylated-interferon/ribavirin and followed throughout a period of four years, during which sustained virological response (SVR) was determined. All data were analysed with statistical package SPSS 19.0 (IBM Corp, Armonk, NY, USA) and PLINK v1.07 software. Results Patients showed a significantly better response to treatment according to the number of copies of the C allele carried at rs1800795-IL6 (P = 0.034). All but one of the patients with CC genotype achieved SVR (93%), whereas the response rate of patients with GG genotype was 64%. The association of rs1800795-IL6 with SVR status remained significant after further adjustment for patients’ age, fibrosis staging, and viral genotype (OR 2.15, 95% CI 1.16–4.68, P = 0.019). Distributions of allele frequencies at the locus rs12979860-IL28B among the study cohort and the underlying general population were suggestive of a protective effect of CC genotype in acquiring chronic hepatitis C in the Croatian IVDU population. Discussion The rs1800795-IL6 polymorphism is associated with positive response to treatment in IVDU patients positive for HCV infection. A protective role of rs12979860-IL28B CC genotype in acquiring chronic hepatitis C is suggested for Croatian IVDU population.