This study presents the current status of human leptospirosis in Croatia from an epidemiological and clinical viewpoint. Data from annual reports of the Croatian Institute for Public Health as well as archives of the University Hospital for Infectious Diseases 'Dr Fran Mihaljevic' (UHID) in Zagreb were used in this retrospective cohort analysis. The mean yearly incidence of leptospirosis from 1990 to 2007 was 1.83/100 000 inhabitants, with an incidence >2.5/100 000 inhabitants recorded approximately every 3-4 years, making Croatia one of the countries with the highest incidence of human leptospirosis in Europe. In addition to the majority of sporadic cases, two minor outbreaks were recorded. The clinical burden and more detailed epidemiology of 130 patients hospitalised at UHID in the period 1997-2007 were also studied. Clinical presentations were as expected, with an overall case fatality rate (CFR) of 0.77%. The most commonly established infective serovars were Australis followed by Saxkoebing and Grippotyphosa. In comparison with previous periods, the mean yearly number of patients with leptospirosis hospitalised at UHID decreased, but among them a rather higher rate of patients with Weil's disease and a higher CFR was observed.

Altitudinal distributions, population structures and seasonal dynamics of tick fauna at three localities in Continental Croatia (Medvednica and Papuk) and an alpine biogeographic region (Gorski Kotar) were studied. Sampling of questing ticks was performed twice a year (spring and autumn, from 2019 to 2021) at different altitudes (200, 400, 600, 800 and 1000 m above sea level) using the flagging method. In total, 2942 ticks (53.9% larvae, 40.1% nymphs, 6.0% adults) were sampled and 2937 (99.83%) were determined as Ixodes ricinus, 4 (0.14%) as Haemaphysalis concinna and 1 (0.03%) as Ixodes frontalis. Ixodes ricinus was the only species found at all altitudes and sampling sites. The highest tick abundance was recorded at higher altitudes (800–1000 m asl.) on Medvednica and in Gorski Kotar within mixed forests of European beech and European silver fir, while on Papuk most of the ticks were sampled at lower altitudes (200 m asl.) in Sessile oak forest. From 27 pools containing 305 ticks, 1 (3.7%) was positive for Borrelia burgdorferi sensu lato infection. Borrelia burgdorferi s.l. was detected in 20% (1/5) of the pools containing nymphs and adults collected in Gorski Kotar (600 and 800 m asl.). Sequencing of the ospA gene and phylogenetic analysis revealed the presence of the Borrelia burgdorferi sensu stricto genotype.

Leptospirosis is one of the most common zoonotic infections and a major problem in terms of both veterinary medicine and public health. However, the disease is under-recognised and under-diagnosed worldwide, particularly in horses. Clinical leptospirosis in horses is mainly associated with recurrent uveitis (ERU), which has recently been studied more intensively, and reproductive disorders, the epidemiology of which is still relatively poorly understood. To enhance our comprehension of abortions caused by leptospirosis in horses and to identify the causative strains, a serological study was carried out with subsequent molecular characterisation of the isolate obtained. Using the microscopic agglutination test (MAT), serum samples from mares that aborted and foetal fluids (when available) were tested for antibodies against Leptospira spp. Furthermore, bacteria isolation from kidney cultures was conducted. Of 97 mare serum samples, 21 (21.64%) tested positive, with Grippotyphosa and Pomona being the most frequently detected serogroups. A significantly higher seroprevalence was found in aborting mares compared to the healthy horse population from the same geographical area, as well as a pronounced seasonal variation. Leptospiral antibodies were not detected in any of the foetal fluids, but isolation was successful in 1 case out of 39 (2.56%). Genotyping by multilocus sequence typing (MLST) and core genome multilocus sequence typing (cgMLST) identified the obtained isolate as Leptospira kirschneri, serogroup Pomona, serovar Mozdok. Further surveillance and molecular typing of Leptospira strains causing abortion in horses would be invaluable in understanding the prevalence and impact of leptospirosis on equine reproductive health in Europe.

Leptospirosis is a worldwide zoonosis caused by pathogenic Leptospira spp. with small rodents serving as the main reservoir. In Croatia, the serogroup Pomona has been detected most frequently, but its genomic diversity remains insufficiently characterized. This study presents the first whole genome sequencing analysis of 48 Croatian Leptospira spp. isolates collected from small rodents over a 14-year period. Serological typing confirmed that all the isolates belonged to the serogroup Pomona. Genomic analysis assigned them to L. kirschneri based on high genomic similarity using average nucleotide identity (ANI). The isolates were assigned to ST-98 using traditional multilocus sequence typing (MLST), while cgMLST identified seven genotype clusters, many of which showed geographic structuring. Phylogenetic analyses based on single nucleotide polymorphisms (SNPs) supported this structure and revealed a monophyletic clade of Croatian isolates distinct from other global L. kirschneri strains. Serological typing, MLST, and phylogenetic clustering support classification of the isolates as L. kirschneri, serogroup Pomona, most likely serovar Mozdok, although serovar Tsaratsovo cannot be excluded. These results indicate the existence of a geographically restricted and potentially host-adapted lineage of L. kirschneri in Croatia. The integration of ecological, serological, and genomic data in this study emphasizes the value of whole genome sequencing for understanding the population biology of Leptospira spp. serogroup Pomona. Moreover, it supports targeted, country-specific surveillance and control strategies for leptospirosis through the identification of circulating serovars and species in reservoir hosts, in line with a One Health approach.