Josipa Habuš

Leptospirosis is a re-emerging zoonotic disease of increasing importance in both veterinary and public health, with its epidemiology strongly influenced by environmental and climatic factors. The aim of this study was to investigate the temporal and spatial distribution of canine leptospirosis in Croatia over an 11-year period (2013–2023), with particular emphasis on prevalence trends and serovar distribution. A total of 2,444 canine blood samples submitted for diagnostic testing were analysed using the microscopic agglutination test (MAT) for antibodies against 12 Leptospira serovars. Overall, 983 samples (40.22%) were seropositive. Annual prevalence showed marked fluctuations, with the highest numbers of positive samples recorded in 2018, 2020, and 2021. Geographical analysis revealed higher prevalence in central and northwestern Croatia, particularly in the City of Zagreb and surrounding counties. The most frequently detected serovars were Pomona (26.12%), Grippotyphosa (22.20%), Australis (16.96%), and Sejroe (14.95%), while other serovars occurred only sporadically. Temporal changes in serovar distribution were also observed, indicating dynamic epidemiological patterns. The identified fluctuations in prevalence are likely associated with environmental conditions favorable for Leptospira survival and transmission, including periods of increased precipitation and flooding documented in Croatia during the study period. These findings highlight considerable temporal, geographical, and serovar-related variability in canine leptospirosis, and emphasise the importance of continuous surveillance, regionally relevant vaccination strategies, and a One Health approach to disease prevention and control.

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.

Leptospirosis is a re-emerging zoonosis of wide geographic distribution and high prevalence, significant in domestic and wild animals as well as humans. It may have a notable impact on reproductive health in dogs. The clinical spectrum ranges from mild subclinical infections to severe septicemic disease with renal failure and/or pulmonary hemorrhagic syndrome, often with a high mortality rate. Clinical expression depends on the host's susceptibility, immune status, infectious dose, and the virulence of the infecting serovar or strain. Certain serovars, for still unknown reasons, tend to cause more severe disease manifestations. Reproductive disorders-including embryonic resorption, abortion, and stillbirthare rarely described in the literature, although Leptospira spp. is known to cause transplacental infections. Individual cases of abortion associated with serovars Buenos Aires and Bratislava have been reported, but evidence remains limited and primarily anecdotal. Diagnostic approaches include serological testing (MAT), PCR, and histopathology, though interpretation may be hindered by cross-reactivity and post-vaccinal antibodies. Considering increasing indications of the reproductive impact of Leptospira spp., it should be routinely included in the differential diagnosis of abortion in bitches, especially in endemic areas and in late gestation. Prevention relies on vaccination, rodent control, and strict biosecurity measures, with special focus on breeding females.

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.

Ferrets are highly susceptible to a wide range of mycobacteria, mainly M. bovis, M. avium, and M. triplex. Therefore, ferrets pose a risk of transmission of mycobacteriosis, especially zoonotically relevant tuberculosis. The aim of this study was to describe the findings of M. xenopi mycobacteriosis in a pet ferret and emphasize its zoonotic potential. A pet ferret had a history of weight loss, apathy, hyporexia, and hair loss. Abdominal ultrasound revealed splenomegaly with two solid masses and cystic lesions of the liver. Fine-needle aspiration cytology revealed numerous acid-fast bacilli in epithelioid cells, thus leading to the suspicion of mycobacterial infection. Because of its poor general condition, the ferret was euthanized. Necropsy examination revealed generalized granulomatous lymphadenitis, pneumonia, myocarditis, splenitis, and hepatitis. Histologically, in all organs, there were multifocal to coalescing areas of inflammatory infiltration composed of epithelioid macrophages, a low number of lymphocytes, and plasma cells, without necrosis nor multinucleated giant cells. Ziehl–Neelsen staining detected the presence of numerous (multibacillary) acid-fast bacteria, which were PCR-typed as M. xenopi. This is the first study showing the antimicrobial susceptibility testing of M. xenopi in veterinary medicine, describing the resistance to doxycycline. Overall, our results could facilitate further diagnosis and provide guidelines for the treatment protocols for such infections.

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.

Francisella tularensisis uzročnik je zoonoze tularemije, koja ima široki raspon domaćina, uključujući člankonošce, sisavce i ptice. Dvije podvrste F. tularensis, subsp. tularensis (Tip A) i subsp. holartica (Tip B), opisane su kao najčešći uzročnici bolesti u ljudi (Kingry and Petersen, 2014; Kuehn et al., 2013). Tip A i Tip B razlikuju se po svojim biokemijskim značajkama, patogenosti, ekologiji, epidemiologiji te geografskoj rasprostranjenosti. Rasprostranjenost Tipa A uglavnom je ograničena na sjevernoamerički kontinent, za razliku od Tipa B, koji se javlja duž cijele sjeverne polutke, a ujedno je i najčešći uzročnik tularemije u Europi(Keim et al., 2007; Kuehn et al., 2013). No ekologija i kruženje ove bakterije u prirodi do danas još nisu u potpunosti razjašnjeni. Različiti glodavci (voluharice, miševi, tekunice, bizantski štakori, dabrovi) te dvojezupci (kunić i zec) opisani su kao mogući rezervoari F.tularensis. Zadnja epidemiološka i epizootiološka istraživanja tularemije u Hrvatskoj datiraju iz šezdesetih i sedamdesetih godina prošloga stoljeća(Borcić et al., 1975). Stoga je cilj ovoga rada bio prikupiti nove podatke o prevalenciji tularemije u populaciji sitnih sisavaca u Hrvatskoj. Ukupno 444 uzoraka sitnih glodavaca i kukcojeda sakupljeno je u razdoblju od dvije godine na osam različitih lokaliteta u šumama kontinentalne Hrvatske: Lipovljani, Nova Subocka, Velika Gorica, Stara Gradiška, Županja, Sunja, Koprivnica i Čakovec (Slika 1). Pretraženi su uzorci slezene od: 197 Apodemus agrarius (poljski miš), 78 Apodemus sylvaticus (šumski miš), 92 Apodemus flavicollis (žutogrli miš), 17 Myodes glareolus (šumska voluharica), 27 Mycrotus agrestis (livadna voluharica), 20 Microtus arvalis (poljska voluharica) te 13 Sorex araneus (rovka). Uzorcisu pretraženi na prisutnost DNA Francisella spp. koristeći qRT-PCR metodu. Dobiveni rezultati pokazuju da su od ukupno 444 prikupljene životinje tri uzorka (0.67%), od kojih dva poljska miša te jedan šumski miš, bili pozitivni su na francizelu (Tablica 1). Sva tri pozitivna uzorka prikupljena su na istom području – lokalitetu Lipovljani. Tularemija je bolest koja se primarno povezuje s „nizinskim“ i „vodenim“ ekosustavima (Borcić, 1973). Područje duž toka rijeke Save (Posavina) opisuje se kao prirodno žarište tularemije u Hrvatskoj (Borcic et al., 1976), akao glavni rezervoar tularemije na tom području navodi se poljska voluharica (Borcic et al., 1976). Trimiša pozitivna na F. tularensis upućuju da je ova bakterija i dalje prisutna u populaciji sitnih glodavaca u Hrvatskoj. Nadalje, svi pozitivni uzorci prikupljeni su na lokalitetu Lipovljani, smještenom u srednjem dijelu toka rijeke Save, stoga ova regija zadržava obilježje prirodnog žarišta tularemije u Hrvatskoj.

Objectives: 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.Methods: Epidemiological and clinical data obtained from patients hospitalised and treated in a well‐known endemic focus of leptospirosis, Koprivnica‐Križevci County in Croatia, were analysed.Results: We observed a steady decline in the overall incidence of leptospirosis and a change in the patient age distribution, with the age ratio changing in favour of middle‐aged and older patients. Although leptospirosis was most frequently diagnosed in August in all time periods, the number of cases increased in autumn. The most prevalent serogroup during the first and the second time period was Icterohaemorrhagiae, while in the third time period, the serogroup Australis prevailed. We also noted an increase in the number of severe clinical manifestations.Conclusions: This retrospective research demonstrates a continuous decline in the incidence of human leptospirosis in Croatia. The pattern of disease has changed from predominantly mild clinical forms observed in children to more severe clinical forms observed in middle‐aged to older patients, especially those working in agriculture. Additional epidemiological changes included an increase in the number of cases during the autumn months and changes in prevailing serogroups. Statistical analysis revealed a significant relationship between the severity of the clinical picture, patient age and presumed sources of infection.

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.