Zrinka Štritof

Molecular analysis of Leptospira spp. isolated from humans by restriction fragment length polymorphism, real-time PCR and pulsed-field gel electrophoresis

A total of 17 Leptospira clinical strains isolated from humans in Croatia were serologically and genetically analysed. For serovar identification, the microscopic agglutination test (MAT) and pulsed-field gel electrophoresis (PFGE) were used. To identify isolates on genomic species level, PCR-based restriction fragment length polymorphism (RFLP) and real-time PCR were performed. MAT revealed the following serogroup affinities: Grippotyphosa (seven isolates), Icterohaemorrhagiae (eight isolates) and Javanica (two isolates). RFLP of PCR products from a 331-bp-long fragment of rrs (16S rRNA gene) digested with endonucleases MnlI and DdeI and real-time PCR revealed three Leptospira genomic species. Grippotyphosa isolates belonged to Leptospira kirschneri, Icterohaemorrhagiae isolates to Leptospira interrogans and Javanica isolates to Leptospira borgpetersenii. Genomic DNA from 17 leptospiral isolates was digested with NotI and SgrAI restriction enzymes and analysed by PFGE. Results showed that seven isolates have the same binding pattern to serovar Grippotyphosa, eight isolates to serovar Icterohaemorrhagiae and two isolates to serovar Poi. Results demonstrate the diversity of leptospires circulating in Croatia. We point out the usefulness of a combination of PFGE, RFLP and real-time PCR as appropriate molecular methods in molecular analysis of leptospires.

New trends in human and animal leptospirosis in Croatia, 2009–2014

Leptospirosis is a geographically widespread and globally underestimated zoonosis that affects humans and variety of animals. To identify trends and possible risk factors, joined medical and veterinary teams investigated epidemiology and epizootiology of leptospirosis in Croatia. Retrospective analysis of data obtained from referent diagnostic laboratories included a total of 1917 human and 123964 animal sera tested in the period from 2009 to 2014. We found high human leptospirosis average incidence rate of 1.53/100000 with clear predominance of male patients older than 40 years (sex ratio M/F:3.2; median age 51±15.1years). Statistical analysis revealed seasonal and annual variations of incidence in humans that were primarily associated with favourable weather conditions (temperature 10-19, 9°C and precipitation above 100mm/m2). Majority of infections in humans were caused by serogroups Sejroe, Australis and Icterohaemorrhagiae. Notable variations in seroprevalence and changing trends in prevailing serogroups were recorded in most of the domestic animals and during the entire period of investigation. All of the observed findings underline leptospirosis as a significant human and veterinary public health threat and emphasize the importance of continuous multidisciplinary surveillance. We also argue that only input from both professions improves our overall knowledge on leptospirosis and leads to better and more efficient prevention and control strategies.

The role of fat dormouse ( Glis glis L.) as reservoir host for spirochete Borrelia burgdorferi sensu lato in the region of Gorski Kotar, Croatia

To determine whether some of the B. burgdorferi sensu lato genospecies associate with fat dormouse as a reservoir host, we investigated the prevalence of infection in questing animals. A total of 45 adult fat dormice (30 female and 15 male) were captured by hunters during their hunting season in the region of Gorski Kotar, Croatia. Dead animals were aseptically dissected, and the urinary bladder tissue was used for isolation attempt and for deoxyribonucleic acid (DNA) extraction. Out of 45 DNA samples extracted from urine bladder tissue, we found four (8.88%) to be polymerase chain reaction (PCR) positive. The RFLP analysis of the PCR product after cleavage with DraI and MseI distinguished between the three major genospecies: B. burgdorferi sensu stricto, B. garinii and B. afzelii. All positive samples were typed as B. afzelii with a unique DraI or MseI pattern. The results of the analysis of urinary bladder tissue samples culture for the presence of Borrelia were negative. Results showed that a prevalence of the Borrelia infection among population of fat dormice indicated their epizootiological involvement as a reservoir of Borrelia spirochetes. Furthermore, this work is an initial step in the investigation of the molecular epidemiology/epizootiology of Lyme borreliosis in Croatia.

Phylogenetic characterisation of feline immunodeficiency virus in naturally infected cats in Croatia indicates additional heterogeneity of subtype B in Europe

This study was performed on 29 domestic cats with a variety of clinical signs, possibly related to FIV infection. Blood samples were tested by a rapid immunochromatographic (ICA) procedure for detection of FIV antibodies. Subsequently, polymerase chain reaction (PCR) was performed to amplify a portion of the proviral gag gene. All 11 positive PCR products were sequenced and compared with previously reported FIV sequences. Croatian proviral isolates that could be amplified were clustered within subtype B, and additional heterogeneity was confirmed by the formation of three separate clusters. Phylogenetic analysis of circulating strains in Croatia and in southeast Europe is necessary to improve diagnostic methods and selection of the appropriate vaccinal strains.

Epidemiological and clinical features of leptospirosis in a highly endemic area over three time periods

To present the features of human leptospirosis over three time periods (1970–1975; 2000–2005; 2010–2015), to compare the collected data and to determine whether the incidence, seasonal and spatial distribution, prevalence of presumptive infective serogroups and clinical features have changed over the last 50 years.