The Expression Pattern of Surface Markers in Canine Adipose-Derived Mesenchymal Stem Cells.

The influence of cultivation on the expression pattern of canine adipose-derived mesenchymal stem cells (cAD-MSCs) surface markers, contributing to, among others, the promotion of growth, proliferation, differentiation and immunomodulatory mechanisms of an excellent therapeutic, is still unknown. To fill the gap, we investigated CD90, CD44, CD73, CD29, CD271, CD105, CD45 and CD14 patterns of expression at the protein level with flow cytometry and mRNA level using a real-time polymerase chain reaction array. Gentle variations of expression occurred during cultivation, along with increased CD90, CD44 and CD29 expression, low and decreasing CD271 and CD73 expression and a decrease of initially high CD105. As expected, CD45 and CD14 were not expressed by cAD-MSCs. Interestingly, we discovered a significant decrease of CD73 expression, compared to early (P1-P3) to late (P4-P6) passages, although the CD73 gene expression was found to be stable. The percentage of positive cells was found to be higher for all positive markers up to P4. As CD73's one important feature is a modulation from a pro-inflammatory environment to an anti-inflammatory milieu, the expression of CD73 in our conditions indicate the need to consider the time cells spend in vitro before being transplanted into patients, since it could impact their favourable therapeutical properties.

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.

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.

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.

In vitro aging alters the gene expression and secretome composition of canine adipose-derived mesenchymal stem cells.

Introduction: Canine adipose-derived mesenchymal stem cells (cAD-MSCs) hold therapeutic promise due to their regenerative potential, particularly within their secretome. However, concerns arise regarding the impact of in vitro cultivation necessitated for storing therapeutic doses, prompting this study to comprehensively explore the impact of in vitro aging on gene expression and secretome composition. Methods: The study involved collecting abdominal adipose tissue samples from nine healthy female dogs, from which cAD-MSCs were extracted and cultured. Stem cells were validated through trilineage differentiation assays and flow cytometry immunophenotyping. Gene expression profiling using RT-qPCR array, and cAD-MSCs secretome LC-MS/MS analysis, were conducted at passages 3 and 6 to reveal gene expression and protein composition alterations during in vitro culture. Results and Discussion: The results demonstrate that the gene expression and secretome composition of cAD-MSCs were impacted by in vitro aging. Among many alterations in gene expression between two passages, two significant downregulations were noted in the MSC-associated PTPRC and IL10 genes. While the majority of proteins and their functional characteristics were shared between passages, the influence of cell aging on secretome composition is highlighted by 10% of proteins being distinctively expressed in each passage, along with 21 significant up- and downregulations. The functional attributes of proteins detected in passage 3 demonstrated a greater inclination towards supporting the regenerative capacity of cAD-MSCs. Moreover, proteins in passage 6 exhibited a noteworthy correlation with the blood coagulation pathway, suggesting an elevated likelihood of coagulation events. To the best of our knowledge, this study presents the first original perspective on the changes in secretome composition that occur when cAD-MSCs age in vitro. Furthermore, it contributes to broadening the currently restricted knowledge base concerning the secretome of cAD-MSCs. In conclusion, our findings show that the regenerative potential of cAD-MSCs, as well as their secretome, may be compromised by in vitro aging. Therefore, our study suggests a preference for earlier passages when considering these cells for therapeutic applications.

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.

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.

An Outstanding Role of Adipose Tissue in Canine Stem Cell Therapy.

Adipose tissue, previously known as connective tissue with a role in energy storage, is currently changing the course of treatments in veterinary medicine. Recent studies have revealed one particularly impressive function among all the newly discovered functions of adipose tissue. The interactive cells hosted by adipose tissue, the stromal vascular fraction (SVF), and their role in treating numerous diseases have provided a prospective course of research with positive outcomes in regenerative veterinary medicine (RVM). This review describes the main features of adipose tissue, emphasizing an eclectic combination of cells within the SVF and its thus far researched therapeutic possibilities in canine RVM. An afterwards focus is on a highly researched component of the SVF, adipose-derived mesenchymal stem cells (ASCs), which were shown to have an extraordinary impact relying on several proposed mechanisms of action on mitigating pathologies in canines. Furthermore, ASC therapy showed the most significant results in the orthopaedics field and in neurology, dermatology, ophthalmology, gastroenterology, and hepatology, which elevates the possibilities of ASC therapy to a whole new level. Therefore, this review article aims to raise awareness of the importance of research on cellular components, within abundant and easily accessible adipose tissue, in the direction of regenerative therapy in canines, considering the positive outcomes so far. Although the focus is on the positive aspects of cellular therapy in canines, the researchers should not forget the importance of identifying the potential negative aspects within published and upcoming research. Safe and standardized treatment represents a fundamental prerequisite for positively impacting the lives of canine patients.

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.

Vaccine failure caused an outbreak of equine influenza in Croatia.

In April 2004 an outbreak of equine influenza occurred at the Zagreb hippodrome, Croatia. Clinical respiratory disease of the same intensity was recorded in vaccinated and non-vaccinated horses. The equine influenza vaccine used in Croatia at the time of the outbreak contained the strains A/equine/Miami/63 (H3N8), A/equine/Fontainebleau/79 (H3N8) and A/equine/Prague/56 (H7N7). At the same time, the usual strains in vaccines used in Europe were, in accordance with the recommendation of the World Organisation for Animal Health (OIE) Expert Surveillance Panel on equine influenza, A/equine/Newmarket/1/93 (H3N8) and A/equine/Newmarket/2/93 (H3N8). At the same time, some current vaccines in the USA contained A/equine/Kentucky/97 (H3N8). Genetic characterization of the HA1 portion of the haemagglutinin (HA) gene of virus isolated from the outbreak indicated that the isolate (A/equine/Zagreb/04) was an H3N8 strain closely related to recent representative viruses of the American lineage Florida sub-lineage. In comparison with both H3N8 vaccine strains used in horses at the Zagreb hippodrome, A/equine/Zagreb/04 displayed amino acids changes localised to 4 of the 5 described antigenic sites (A-D) of subunit protein HA1. Comparison of the amino acid sequence of the HA1 subunit protein of the outbreak strain with that of A/equine/Newmarket/1/93 displayed three amino acids changes localised in antigenic sites B and C, while antigenic sites A, D and E were unchanged. The Zagreb 2004 outbreak strain had the same amino acids at antigenic sites of the HA1 subunit protein as the strain A/equine/Kentucky/97. Amino acid changes in antigenic sites between HA1 subunit of the outbreak strain and the strains used in the vaccines likely accounted for the vaccine failure and the same clinical signs in vaccinated and unvaccinated horses. Use of a recent strain in vaccines should limit future outbreaks.

Bovine platelet-activating factor acetylhydrolase (PAF-AH) activity related to fertility.

Plasma platelet-activating factor acetylhydrolase (PAF-AH), the enzyme characterized by the association with plasma lipoproteins, degrades platelet-activating factor (PAF) as well as PAF-like oxidatively fragmented phospholipids produced during oxidative stress. Apart from pro-inflammatory properties, PAF is also related to reproductive processes and successful fertility. In order to get a better insight into the involvement of PAF-AH in the fertility of cows, the aim of the study was to determine the PAF-AH activity as well as the C-reactive protein, cholesterol and high density lipoprotein-cholesterol (HDL-C) in the serum of dairy cows throughout the pregnancy and lactation, as well as in infertile cows. The results showed that serum PAF-AH activity changes throughout pregnancy and lactation with a lower level during periparturient period. It is also found higher PAF-AH activity in lactating cows with reproductive disorders compared to high lactating cows without reproductive disorders. Strong correlation between PAF-AH activity and HDL-C concentration indicates that HDL could have considerable influence on PAF-AH activity in bovine plasma. CRP concentration was also lower during transition period suggesting that lactation might stimulate CRP synthesis in bovine. A higher CRP concentration in cows with reproductive disorders compared to fertile cows at the peak of lactation, demonstrates that milk production is not the only factor influencing CRP in cows. A significant correlation between PAF-AH activity and CRP level shows that both parameters could be influenced by reproductive status of dairy cows.

Genetic analysis of bovine respiratory syncytial virus in Croatia.

Bovine respiratory syncytial virus (BRSV) represents an important causative agent of respiratory tract disease in cattle. This study describes the genetic diversity of BRSV strains detected in beef cattle herds in Croatia during four consecutive years, from the end of 2011 to April 2016. Genetic diversity of circulating Croatian strains is reflected in their clustering within three different genetic subgroups. Analysis of representative BRSV G gene sequences revealed that infections in Croatia were caused by BRSV strains belonging to two new subgroups (VII and VIII identified herein for the first time). In 2014-2016, the subgroup VII strains were replaced with BRSV strains clustered in the previously unidentified subgroup VIII. Furthermore, co-circulation of subgroup II and new subgroup VIII strains in Croatia was recorded in the same time period. Sequences of Croatian BRSV strains within subgroups II and VII revealed unique mutations within an essential immunodominant region, demonstrating continuous evolution of viral mechanisms for immune escape.

Yersiniosis in zoo marmosets (Callitrix jacchuss) caused by Yersinia enterocolitica 4/O:3.

The aim of this research was to describe two fatal cases of Yersinia enterocolitica bioserotype 4/O:3 infection in non-human primates and to characterise the isolates by PCR and PFGE. In July 2004, two marmosets (Callitrix jacchuss) born in captivity in Zagreb Zoo, died following a few days of intermittent diarrhoea in intervals of 2 weeks. The pathomorphological diagnosis of the female (born in 1997) and the male (born in 1995) marmoset, was disseminated miliary necrosis of the liver. Y. enterocolitica 4/O:3 was isolated from both livers showing that monkeys are susceptible to this bioserotype. The ail gene, which is an essential chromosomal virulence factor in pathogenic Y. enterocolitica isolates, was present in the marmoset isolates. Two different PFGE patterns were obtained from the isolates of the male liver with NotI enzyme. One genotype of the male marmoset isolate was indistinguishable from the genotype of the female marmoset isolate when NotI, ApaI and XhoI enzymes were used indicating a common infection source for the marmosets. The genotypes of the marmoset isolates differed only slightly from one human (of seven Croatian isolates) and from one pig isolate (representing a common genotype found among human and porcine isolates in Germany) suggesting that raw pork fed to the marmoset could have been the infection source.

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.

Short report: dual infections with Puumala virus and Leptospira interrogans serovar lora in a bank vole (Clethrionomys glareolus).

Leptospirosis and hemorrhagic fever with renal syndrome are public health problems in Croatia. Diagnosis and epidemiology of these diseases are complicated because these two diseases are sympatric in certain areas. We describe a natural dual infection of Puumala virus and a leptospire in a bank vole (Clethrionomys glareolus).

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.

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.

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.