Association between Exposure to Leptospira spp. and Abortion in Mares in Croatia.

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.

Mycobacteriosis in a Pet Ferret (Mustela putorius furo) Caused by Mycobacterium xenopi: A Case Report on Neglected Risk of Zoonotic Transmission.

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.

Global distribution of Leptospira serovar isolations and detections from animal host species: A systematic review and online database.

OBJECTIVES: Leptospira, the spirochaete causing leptospirosis, can be classified into >250 antigenically distinct serovars. Although knowledge of the animal host species and geographic distribution of Leptospira serovars is critical to understand the human and animal epidemiology of leptospirosis, current data are fragmented. We aimed to systematically review, the literature on animal host species and geographic distribution of Leptospira serovars to examine associations between serovars with animal host species and regions and to identify geographic regions in need of study. METHODS: Nine library databases were searched from inception through 9 March 2023 using keywords including Leptospira, animal, and a list of serovars. We sought reports of detection of Leptospira, from any animal, characterised by cross agglutinin absorption test, monoclonal antibody typing, serum factor analysis, or pulsed-field gel electrophoresis to identify the serovar. RESULTS: We included 409 reports, published from 1927 through 2022, yielding data on 154 Leptospira serovars. The reports included data from 66 (26.5%) of 249 countries. Detections were from 144 animal host species including 135 (93.8%) from the class Mammalia, 5 (3.5%) from Amphibia, 3 (2.1%) from Reptilia, and 1 (0.7%) from Arachnida. Across the animal host species, Leptospira serovars that were detected in the largest number of animal species included Grippotyphosa (n = 39), Icterohaemorrhagiae (n = 29), Pomona (n = 28), Australis (n = 25), and Ballum (n = 25). Of serovars, 76 were detected in a single animal host species. We created an online database to identify animal host species for each serovar by country. CONCLUSIONS: We found that many countries have few or no Leptospira serovars detected from animal host species and that many serovars were detected from a single animal species. Our study highlights the importance of efforts to identify animal host species of leptospirosis, especially in places with a high incidence of human leptospirosis. We provide an updated resource for leptospirosis researchers.

Global distribution of Leptospira serovar isolations and detections from animal host species: a systematic review and online database.

Objectives: Leptospira, the spirochaete causing leptospirosis, can be classified into >250 antigenically distinct serovars. Although knowledge of the animal host species and geographic distribution of Leptospira serovars is critical to understand the human and animal epidemiology of leptospirosis, currently data are fragmented. We aimed to systematically review the literature on animal host species and geographic distribution of Leptospira serovars to examine associations between serovars with animal host species and regions, and to identify geographic regions in need of study. Methods: Nine library databases were searched from inception through 9 March 2023 using keywords including Leptospira, animal, and a list of serovars. We sought reports of detection of Leptospira, from any animal, characterized by cross agglutinin absorption test, monoclonal antibody typing, serum factor analysis, or pulsed-field gel electrophoresis to identify the serovar. Results: We included 409 reports, published from 1927 through 2022, yielding data on 154 Leptospira serovars. The reports included data from 66 (26.5%) of 249 countries. Detections were from 144 animal host species including 135 (93.8%) from the class Mammalia, 5 (3.5%) from Amphibia, 3 (2.1%) from Reptilia, and 1 (0.7%) from Arachnida. Across the animal host species, Leptospira serovars that were detected in the largest number of animal species included Grippotyphosa (n=39), Icterohaemorrhagiae (n=29), Pomona (n=28), Australis (n=25), and Ballum (n=25). Of serovars, 76 were detected in a single animal host species. We created an online database to identify animal host species for each serovar by country. Conclusions: We found that many countries have few or no Leptospira serovars detected from animal host species and that many serovars were detected from a single animal species. Our study highlights the importance of efforts to identify animal host species of leptospirosis, especially in places with a high incidence of human leptospirosis. We provide an updated resource for leptospirosis researchers.

Serological Surveillance of Equine Leptospirosis in Croatia in the Period From 2012 to 2022: A Key Insight Into the Changing Epizootiology.

Leptospirosis is re-emerging zoonotic bacterial disease of global importance that affects domestic and wild animals and humans. Due to the public health importance, control of disease in Croatia is being implemented by monitoring the seroprevalence of equine leptospirosis and it is regulated by the law. In the period from 2012 to 2022, a total of 61,724 serum samples from apparently healthy horses were admitted to the Laboratory for leptospires, Faculty of Veterinary Medicine University of Zagreb. Serum samples were tested for Leptospira spp. antibodies using the microscopic agglutination test (MAT). Samples were considered seropositive with a cut-off titre 1:200 for Bratislava and 1:400 for other Leptospira spp. serovars. Out of 61,724 serum samples tested, 6,665 (10.80%) were found seropositive for at least one Leptospira serovar. In the studied period, seroprevalence varied between 5.00% and 15.94%. The highest seroprevalence was found for serovar Pomona (41.98%) and serovar Grippotyphosa (31.34%), followed by Sejroe (8.03%), Icterohaemorrhagiae (7.05%) and Bratislava (6.47%). Results indicated that horses in Croatia are particularly exposed to Leptospira spp. infections. The most prevalent presumed infective serovar was Pomona increasing each year. Investigated horses were randomly selected and healthy and most seropositive horses have anamnestic titre due to previous infection. This is the first study in Europe reporting such high seropositivity for the serovar Pomona in apparently healthy horses. According to the results of the present study, the question arises of the possible evolutionary adaptation of the pathogenic serovar Pomona as dominant for horses.

Acute lethal leptospirosis in a red fox (Vulpesvulpes).

Leptospirosis, caused by pathogenic spirochaetes belonging to the genus Leptospira, is a globally distributed zoonosis that can affect many species of domestic and wild animals, and humans. The red fox (Vulpes vulpes) is a native species in Croatia and, due to constant food availability and lack of interspecies competition, is becoming more abundant in urban and suburban areas. Although antibodies for Leptospira spp have been detected in red foxes, lethal disease has not been reported. We necropsied a young, male red fox that had jaundice, multifocal haemorrhages in the heart, lungs and urinary bladder, hepatomegaly, non-congestive splenomegaly and slight yellow discolouration of the renal cortex and medulla. Histology revealed multifocal haemorrhages in parenchymal organs, thrombi within lung septal capillaries and other blood vessels, interstitial lymphocytic and plasmacytic nephritis and erosions of the gastric mucosa. A microscopic agglutination test on the post-mortem cardiac blood clot revealed a high titre to Leptospira interrogans serovar Icterohaemorrhagiae, which implies contact with rats, which are natural reservoirs of this serovar in Croatia. The gross and histological findings in this fox were similar to those in dogs with leptospirosis, indicating that fatal leptospirosis can occur in foxes and suggesting that this species can act as a source of infection for other animals and humans.

Pyometra Associated With Staphylococcus Pseudintermedius in Two Bitches.

Staphylococcus pseudintermedius is a commensal bacterium residing on the mucosae and skin surfaces of dogs. Under favorable conditions, the organism may cause serious infections, primarily involving the skin, but it is also isolated in eye and ear infections. This report describes pathologic findings of pyometra associated with S pseudintermedius in two bitches. One bitch was a 6-year-old Pomeranian bitch who died after three days of diarrhea, vomiting, and depression. During necropsy, the uterus of the bitch was diffusely enlarged and filled with 30 mL of thick, yellow, creamy pus. Histology revealed acute to subacute, diffuse, severe, purulent endometritis with intralesional Gram-positive round cocci revealed as S pseudintermedius. The cause of death was pyometra with subsequent septic shock. The other bitch was a 2-year-old West Highland White Terrier who underwent diagnostic laparotomy due to suppurative peritonitis of unknown cause. During the procedure, about 100 mL of yellow, opaque exudate from the abdominal cavity was removed. The distended uterine horns noted during the procedure were removed and submitted for histopathology which revealed necrosis of endometrium with the accumulation of pus and numerous, intralesional S pseudintermedius in the uterine lumen. Focally, uterine wall necrosis was evident, with a spread of bacteria and purulent inflammation into the adjacent mesometrium causing purulent peritonitis. Although S pseudintermedius presents a common cause of skin and mucosal infection, it should be considered a possible etiological agent of canine pyometra.

The Emergence of SARS-CoV-2 within the Dog Population in Croatia: Host Factors and Clinical Outcome.

Over a year into the COVID-19 pandemic, there is growing evidence that SARS-CoV-2 infections among dogs are more common than previously thought. In this study, the prevalence of SARS-CoV-2 antibodies was investigated in two dog populations. The first group was comprised of 1069 dogs admitted to the Veterinary Teaching Hospital for any given reason. The second group included dogs that shared households with confirmed COVID-19 cases in humans. This study group numbered 78 dogs. In COVID-19 infected households, 43.9% tested ELISA positive, and neutralising antibodies were detected in 25.64% of dogs. Those data are comparable with the secondary attack rate in the human population. With 14.69% of dogs in the general population testing ELISA positive, there was a surge of SARS-CoV-2 infections within the dog population amid the second wave of the pandemic. Noticeably seroprevalence of SARS-CoV-2 in the dog and the human population did not differ at the end of the study period. Male sex, breed and age were identified as significant risk factors. This study gives strong evidence that while acute dog infections are mostly asymptomatic, they can pose a significant risk to dog health. Due to the retrospective nature of this study, samples for viral isolation and PCR were unavailable. Still, seropositive dogs had a 1.97 times greater risk for developing central nervous symptoms.

Seroprevalence of Streptococcus equi subspecies equi in Croatia - Short communication.

Clinical cases resembling strangles are regularly seen in some areas of Croatia. However, there are no data on the prevalence of infection and the clinical forms or geographic distribution of the disease. The aim of this study was to determine the seroprevalence of Streptococcus equi subspecies equi in horses resident in Croatia, in order to estimate the geographic distribution of infection. The study included 291 horse sera from the eight counties where the majority of Croatian horses are kept. Sera were tested by indirect ELISA (iELISA) for the presence of serum antibodies against S. equi protein A (SEQ_2190) and protein C (SeM). Positive horses were detected in all counties. Overall seroprevalence was 16.5 per cent (48/291), ranging from 7.1 to 29.6 per cent. A positive association was observed between the population size of the horses in the counties and the seropositivity rates: the larger the population, the higher the seropositivity. The results of this study suggest that S. equi infection is widespread in Croatia. Further investigation of the clinical manifestations, circulating strains and other characteristics of the disease in Croatia and raising awareness of the disease among horse owners are now required.

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

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-Krizevci 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.

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.

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.

Molecular Survey of Zoonotic Agents in Rodents and Other Small Mammals in Croatia.

Croatia is a focus for many rodent-borne zoonosis. Here, we report a survey of 242 rodents and small mammals, including 43 Myodes glareolus, 131 Apodemus flavicollis, 53 Apodemus agrarius, three Apodemus sylvaticus, six Sorex araneus, four Microtus arvalis, one Microtus agrestis, and one Muscardinus avellanarius, collected at eight sites in Croatia over an 8-year period. Multiplex MassTag polymerase chain reaction (PCR) was used for detection of Borrelia, Rickettsia, Bartonella, Babesia, Ehrlichia, Anaplasma, Francisella tularensis, and Coxiella burnetii. Individual PCR assays were used for detection of Leptospira, lymphocytic choriomeningitis virus, orthopoxviruses, flaviviruses, hantaviruses, and Toxoplasma gondii. Of the rodents, 52 (21.5%) were infected with Leptospira, 9 (3.7%) with Borrelia miyamotoi, 5 (2%) with Borrelia afzelii, 29 (12.0%) with Bartonella, 8 (3.3%) with Babesia microti, 2 (0.8%) with Ehrlichia, 4 (1.7%) with Anaplasma, 2 (0.8%) with F. tularensis, 43 (17.8%) with hantaviruses, and 1 (0.4%) with an orthopoxvirus. Other agents were not detected. Multiple infections were found in 32 rodents (13.2%): dual infections in 26 rodents (10.7%), triple infections in four rodents (2.9%), and quadruple infections in two rodents (0.8%). Our findings indicate that rodents in Croatia harbor a wide range of bacteria and viruses that are pathogenic to humans.

Survey for hantaviruses, tick-borne encephalitis virus, and Rickettsia spp. in small rodents in Croatia.

In Croatia, several rodent- and vector-borne agents are endemic and of medical importance. In this study, we investigated hantaviruses and, for the first time, tick-borne encephalitis virus (TBEV) and Rickettsia spp. in small wild rodents from two different sites (mountainous and lowland region) in Croatia. In total, 194 transudate and tissue samples from 170 rodents (A. flavicollis, n=115; A. agrarius, n=2; Myodes glareolus, n=53) were tested for antibodies by indirect immunofluorescence assays (IIFT) and for nucleic acids by conventional (hantaviruses) and real-time RT-/PCRs (TBEV and Rickettsia spp.). A total of 25.5% (24/94) of the rodents from the mountainous area revealed specific antibodies against hantaviruses. In all, 21.3% (20/94) of the samples from the mountainous area and 29.0% (9/31) from the lowland area yielded positive results for either Puumala virus (PUUV) or Dobrava-Belgrade virus (DOBV) using a conventional RT-PCR. All processed samples (n=194) were negative for TBEV by IIFT or real-time RT-PCR. Serological evidence of rickettsial infection was detected in 4.3% (4/94) rodents from the mountainous region. Another 3.2% (3/94) rodents were positive for Rickettsia spp. by real-time PCR. None of the rodents (n=76) from the lowland area were positive for Rickettsia spp. by real-time PCR. Dual infection of PUUV and Rickettsia spp. was found in one M. glareolus from the mountainous area by RT-PCR and real-time PCR, respectively. To our knowledge, this is the first detection of Rickettsia spp. in small rodents from Croatia. Phylogenetic analyses of S- and M-segment sequences obtained from the two study sites revealed well-supported subgroups in Croatian PUUV and DOBV. Although somewhat limited, our data showed occurrence and prevalence of PUUV, DOBV, and rickettsiae in Croatia. Further studies are warranted to confirm these data and to determine the Rickettsia species present in rodents in these areas.

Epizootiological survey of small mammals as Leptospira spp. reservoirs in Eastern Croatia.

In this survey we investigated a population of small mammals in Eastern Croatia in order to determine Leptospira carriage rates and identify circulating serovars. Out of 67 trapped animals, 20 (29.9%) isolates were obtained. Identification of isolates using microscopic agglutination test, pulsed field gel electrophoresis and multi locus sequence typing revealed that 10 (50.0%) isolates belong to serogroup Pomona, serovar Mozdok, 6 (30.0%) isolates to serogroup Australis, serovar Jalna, 2 (10.0%) isolates to serogroup Sejroe, serovar Saxkoebing, and 1 (5.0%) isolate to serogroup Grippotyphosa, serovar Grippotyphosa. One isolate from serogroup Bataviae was unable to be identified to the serovar level. Amplification of a 331-bp region of the locus LA0322 using real-time polymerase chain reaction determined that 12 (60.0%) isolates belong to L. kirschneri, 6 (30.0%) isolates to L. interrogans, and 2 (10.0%) isolates to L. borgpetersenii. Leptospira carriage rate was high (29.9%), which corresponds to a high incidence of human and domestic animal leptospirosis in Eastern Croatia. Furthermore, 90.0% of the isolates belong to serogroups Pomona, Australis and Sejroe which are also the most prevalent serogroups in humans in this area. These findings suggest that small mammals might be an important source of Leptospira spp. infection in Eastern Croatia.

Multiple co-infections of rodents with hantaviruses, Leptospira, and Babesia in Croatia.

Hantaviruses, Leptospira spp., and Babesia spp. are rodent-borne pathogens present worldwide. We studied multiple co-infections of small rodents in Croatia with all three pathogens. Twenty-eight Apodemus flavicollis and 16 Myodes glareolus were tested for the presence of hantavirus RNA by real-time RT-PCR, Leptospira strains by renoculture method and Babesia DNA by PCR. Anti-hantavirus antibodies and anti-Leptospira antibodies were detected by serological methods. Very high infection rates with each pathogen were found in A. flavicollis: 20 of 28 rodents (71%) were infected with Dobrava virus, 13 rodents (46%) were infected with Leptospira, and 5 rodents (18%) were infected with Babesia. Multiple co-infections with all three pathogens were found in 3 of 28 (11%) A. flavicollis animals, suggesting that the same rodent host can be infected with several pathogens at the same time. Dual infections with both hantaviruses and Leptospira were found in 7 of 44 rodents (16%), with hantaviruses and Babesia in 2 rodents (5%), and double infection with both Leptospira and Babesia were found in 1 rodent (2%). Since hantaviruses, Leptospira, and Babesia have similar geographical distributions, it is to be expected that in other parts of the world multiple co-infections, representing a serious threat to public health, can be found.

Electrocardiographic changes in hospitalized patients with leptospirosis over a 10-year period.

BACKGROUND: The aim of this study was to investigate the incidence and type of ECG changes in patients with leptospirosis regardless of clinical evidence of cardiac involvement. MATERIAL/METHODS: A total of 97 patients with serologically confirmed leptospirosis treated at the University Hospital for Infectious Diseases "Dr. Fran Mihaljevic" in Zagreb, Croatia, were included in this retrospective study. A 12-lead resting ECG was routinely performed in the first 2 days after hospital admission. Thorough past and current medical history was obtained, and careful physical examination and laboratory tests were performed. RESULTS: Abnormal ECG findings were found in 56 of 97 (58%) patients. Patients with abnormal ECG had significantly elevated values of bilirubin and alanine aminotransferase, lower values of potassium and lower number of platelets, as well as more frequently recorded abnormal chest x-ray. Non-specific ventricular repolarization disturbances were the most common abnormal ECG finding. Other recorded ECG abnormalities were sinus tachycardia, right branch conduction disturbances, low voltage of the QRS complex in standard limb leads, supraventricular and ventricular extrasystoles, intraventricular conduction disturbances, atrioventricular block first-degree and atrial fibrillation. Myopericarditis was identified in 4 patients. Regardless of ECG changes, the most commonly detected infection was with Leptospira interrogans serovar Australis, Leptospira interrogans serovar Saxkoebing and Leptospira kirschneri serovar Grippotyphosa. CONCLUSIONS: The ECG abnormalities are common at the beginning of disease and are possibly caused by the direct effect of leptospires or are the non-specific result of a febrile infection and metabolic and electrolyte abnormalities. New studies are required for better understanding of the mechanism of ECG alterations in leptospirosis.

Serum platelet-activating factor acetylhydrolase and paraoxonase-1 activity in horses infected with Leptospira spp.

The aim of the study was to investigate the effect of infection with Leptospira spp. in horses on activities of platelet-activating factor acetylhydrolase (PAF-AH) and paraoxonase-1 (PON1) considering the anti-inflammatory/anti-oxidative properties of both enzymes. A total of 63 sport and leisure horses' sera were chosen among those routinely serologically examined on leptospirosis during the year 2009 in Croatia. Sera were divided into three groups according to the estimated level of antibody titre against Leptospira spp; group 1: sera serologically negative to leptospirosis (antibody titre<50) (n=21); group 2: sera with residual or anamnestic antibody titre (50-200) (n=23); group 3: sera with high antibody titre (≥1600) indicating recent Leptospira infection (n=19). Serum PAF-AH and PON1 activity was not significantly different between investigated groups (p>0.05). There were no statistical differences in lipid status parameters (p>0.05) among study groups as well. However, significant positive correlations (p<0.05) of PAF-AH and PON1 with total cholesterol and HDL-C were found. Results may indicate low levels of systemic inflammatory response and oxidative stress in horses with subclinical leptospirosis. Further studies with clinically manifested disease are needed to elucidate the potential role of PAF-AH and PON1 as prognostic markers of the leptospirosis outcome.

Human leptospirosis in Croatia: current status of epidemiology and clinical characteristics.

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.

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.