Alen Slavica

Molecular characterization of Leptospira spp. strains isolated from small rodents in Croatia.

We report the isolation and characterization of 16 Leptospira spp. strains isolated from small rodents captured in 11 different regions of inland Croatia. Large NotI and SgrAI restriction fragment allowed us to assign 10 isolates to the serovar istrica, 5 isolates to the serovar tsaratsovo and 1 isolate to the serovar lora. The phylogenetic analysis conducted from the sequences of the first 330 bp from the 16S rDNA gene revealed that the strains belonged to three different species, L. borgpetersenii, L. kirschneri and L. interrogans. Carrier rates in eight rodent species varied from 0 to 71.4%. Mus musculus showed the highest infection level and confirmed its role as a major reservoir of the serogroup Sejroë. For the first time we reported the occurrence of serovars tsaratsovo and lora in Croatia.

Serum biochemical values in fallow deer (Dama dama L.) from different habitats in Croatia

Serum biochemical values were determined in blood samples collected from 48 shot fallow deer from the Brijuni islands and 45 sedated fallow deer (Dama dama L.) from hunting grounds in the continental part of Croatia. The parameters were compared with regard to age, sex and habitat. Statistically significant differences were found for serum total protein concentration, alkaline phosphatase, aspartate aminotransferase and alanine aminotransferase activity between young and adult island deer, as well as for total protein, triacylglyceride, cholesterol concentration and alkaline phosphatase activity between young and adult continental deer. In young animals, island males had higher albumin concentrations, while continental males had higher alkaline phosphatase values than females. In adult animals, island males had a higher blood urea nitrogen concentration, while continental males had higher albumin and cholesterol concentrations, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase and creatine kinase activities. In this group, males exhibited lower bilirubin and triacylglyceride concentrations than females. Our results indicate that besides age, sex and sampling method, nutritional and environmental factors should be considered when evaluating serum biochemical parameters of fallow deer

Detection and genetic characterization of tick-borne encephalitis virus (TBEV) derived from ticks removed from red foxes (Vulpes vulpes) and isolated from spleen samples of red deer (Cervus elaphus) in Croatia.

Tick-borne encephalitis (TBE) is a growing public health concern in central and northern European countries. Even though TBE is a notifiable disease in Croatia, there is a significant lack of information in regard to vector tick identification, distribution as well as TBE virus prevalence in ticks or animals. The aim of our study was to identify and to investigate the viral prevalence of TBE virus in ticks removed from red fox (Vulpes vulpes) carcasses hunted in endemic areas in northern Croatia and to gain a better insight in the role of wild ungulates, especially red deer (Cervus elaphus) in the maintenance of the TBE virus in the natural cycle. We identified 5 tick species (Ixodes ricinus, Ixodes hexagonus, Haemaphysalis punctata, Dermacentor reticulatus, Rhipicephalus sanguineus) removed from 40 red foxes. However, TBE virus was isolated only from adult I. ricinus and I. hexagonus ticks showing a viral prevalence (1.6%) similar to or higher than reported in endemic areas of other European countries. Furthermore, 2 positive spleen samples from 182 red deer (1.1%) were found. Croatian TBE virus isolates were genetically analyzed, and they were shown to be closely related, all belonging to the European TBE virus subgroup. However, on the basis of nucleotide and amino acid sequence analysis, 2 clusters were identified. Our results show that further investigation is needed to understand the clustering of isolates and to identify the most common TBE virus reservoir hosts in Croatia. Sentinel surveys based on wild animal species would give a better insight in defining TBE virus-endemic and possible risk areas in Croatia.

Concentrations of Trace Elements in Tissues of Red Fox (Vulpes vulpes) and Stone Marten (Martes foina) from Suburban and Rural Areas in Croatia

Trace elements concentrations (As, Cd, Cu, Pb and Hg) were determined in the liver, kidney and muscle of 28 red fox (Vulpes vulpes) and 16 stone marten (Martes foina) from suburban and rural habitats from Croatia. Rural and suburban habitats affected Cd and Hg levels in the muscle, liver and kidney of red fox. Significant differences in metal concentrations in the muscle, liver and kidney were detected among species. Suburban stone marten accumulated the highest levels of trace elements (mg/kg w.w.): in muscle 0.019 for Hg; in liver 0.161 for Cd, 36.1 for Cu and 0.349 for Pb; in kidney 1.34 for Cd and 0.318 for Pb. Values observed were higher than those found in suburban red fox and therefore, may represent an important bioindicator for the accumulation of toxic metals in urbanized habitats.

Incidence of leptospiral antibodies in different game species over a 10-year period (1996– 2005) in Croatia

During the 10-year survey (1996–2005), a total of 868 blood samples from different game species in Croatia were analyzed for the presence of leptospiral antibodies. The specific antibodies (AB) were detected in 242 samples (27.88%). According to the species in red deer (Cervus elaphus), the antibodies against six different leptospiral serovars were found in 43 of 226 analyzed sera (19.02%). The most frequent antigen serovars in the deer population were Pomona and Ballum (with the same frequency of 23.6%), whereas the highest titer was recorded for serovar Sejroe (1:800). In the analyzed roe deer (Capreolus capreolus) serum samples, a low level of leptospiral antibodies (6.07%) was determined, with just two AB for antigen serovars—Australis and Sejroe. In wild boar (Sus scrofa), leptospiral antibodies were detected in 151 of 431 samples analyzed (35.03%), with AB for nine antigen serovars. The serovars most frequently found were Australis (48.70%) and Pomona (22.70%), and these serovars also recorded the highest titer (1:3,200). Among brown bear (Ursus arctos) samples, leptospiral antibodies were detected in 25.00% of the samples, with four AB for antigen serovars, of which the most frequent was Icterohaemorrhagiae (>40%). This serovar had the highest recorded titer (1:400). From 112 analyzed red fox (Vulpes vulpes) samples, leptospiral antibodies were found in 35 samples (31.25%). The determined antibodies were specific for four antigen serovars, of which the most frequent (46.2%) and with highest titer (1:1600) was serovar Australis. No antibodies (28/0) were recorded in mouflon (Ovis musimon). The most important game species from an epizootiological point of view in the studied area were certainly wild boar and red foxes. With strong serological reactions, these two species could be emphasized as important hosts for Leptospira interrogans sv. Australis in Croatia, but for their declaration as ‘maintaining hosts,’ isolation of sv. Australis is needed. According to aerial distribution, the highest number of positive samples from different game species was recorded in the central and eastern parts of Croatia, known as the ‘historical natural foci’ of leptospirosis—the regions of Posavina, Podravina, Slavonija, and Baranja. In contrast, the areas of Kordun and Gorski Kotar are declared as leptospira low-risk regions for the game species studied.

Tusk abnormalities in wild boar (Sus scrofa L.)

This paper describes pathological alterations in the permanent canines (tusks) of four male wild boars. The mandibular tusks of all individuals, and also some of the maxillary tusks, exhibited an extended enamel hypoplasia in their apical portion, denoting an impairment of secretory ameloblast function. Moreover, the pulp cavities of the mandibular tusks were exposed through cleft-like openings in the wear (whetting) surfaces of the teeth. Presence of a plug of reparative dentine within the pulp cavity was observed in a split mandibular tusk of one individual. In a second boar, the presence of a plug of reparative dentine within the pulp cavity of the mandibular tusks was indicated radiographically. These findings suggested a reparation process attempting to demarcate a vital, apical pulp portion from a necrotic, incisal portion. The enamel hypoplasias observed in the teeth are regarded to be sequelae of the pulp inflammation caused by bacterial invasion in the mandibular tusks. Most likely, bacterial invasion of the dental pulp occurred through the cleft-like openings in the tusks’ whetting surfaces, the openings resulting from insufficient formation of secondary dentine. It is, however, also conceivable that pulp inflammation and partial necrosis occurred as a consequence of bacterial invasion of patent dentinal tubules, and that the openings in the whetting surface developed secondarily as a consequence of the pulp changes. One mandibular tusk showed marked signs of resorption apically, suggesting a spread of the inflammation from the pulp into the periodontium.

Liver enzymes and blood metabolites in a population of free-ranging red deer (Cervus elaphus) naturally infected with Fascioloides magna

We investigated the effects of Fascioloides magna infection on the serum biochemistry values of the naturally infected red deer population in eastern Croatia. The investigation was performed on 47 red deer with F. magna infection confirmed patho-anatomically in 27 animals (57.4%). Fibrous capsules and migratory lesions were found in 14 deer while only fibrous capsules without migratory lesions were found in 13 deer. In 13 deer both immature and mature flukes were found, in 5 deer only immature flukes were found and in 9 deer only mature flukes were found. Fascioloides magna infected deer with fibrous capsules and migratory lesions had significantly higher values for lactate dehydrogenase (LDH), glutamate dehydrogenase (GLDH) and globulin, and lower values for albumin/globulin ratio and glucose compared to uninfected deer. Fascioloides magna infected deer with fibrous capsules without the presence of migratory lesions had higher values for alanine aminotransferase (ALT) and globulin, and lower values for albumin/globulin ratio and glucose, than the uninfected deer. The number of immature flukes was positively correlated with values of aspartate aminotransferase (AST), γ-glutamyltransferase (GGT), LDH, GLDH, urea and triglycerides. The number of migratory lesions was positively correlated with GGT, GLDH, globulin and urea values. The creatinine value was positively correlated with the number of mature flukes. The trial showed that F. magna infection causes significant changes in serum biochemistry. Moreover, these changes do not completely resemble changes following F. hepatica infection. Further investigation of changes in liver enzymes and other serum metabolites in controlled, experimentally induced fascioloidosis in red deer is needed to better understand the pathogenesis of F. magna.

Serologic evidence of Leptospira spp. serovars in brown bears (Ursus arctos) from Croatia.

Serum samples from 52 free-ranging brown bears (Ursus arctos) collected in Croatia over a period of 10 yr (1998–2007) were tested by microscopic agglutination test for specific antibodies (Ab) to 12 Leptospira spp. pathogenic serovars. At titers ranging from 1:100 to 1:2,000, 19 samples (36.5%) were Ab-positive to at least one serovar. Antibodies for 10 Leptospira spp. serovars were detected: Icterohaemorrhagiae, Australis, Sejroe, Canicola, Poi, Hardjo, Ballum, Saxkoebing, Pomona, and Grippotyphosa. In comparison to previous reports, the prevalence of Ab to serovar Icterohaemorrhagiae (52.6%) was significantly higher. Other common serovars were Australis (47.4%) and Sejroe (42.1%). High Ab titers for serovars Canicola (1:500) and Grippotyphosa (1:1,000) were detected for the first time in free-ranging bears from Croatia. A significant correlation between the age of the bears and detection of Ab to Leptospira spp. serovars suggested the presence of pathogenic agents in the natural habitats, whereas increasing trends of Ab prevalence for specific serovars (Icterohaemorrhagiae, Australis, and Sejroe) confirmed cohabitation of bears with rats and other small terrestrial mammals on garbage dumps and at bear feeding stations. To prevent cohabitation of bears and rodents, improvements in Croatian waste treatment, big game management, and rodent control programs are strongly recommended, especially in Lika and Gorski Kotar, regions that have high-quality natural habitats for brown bears in Croatia.

Hematology and serum biochemistry of European mouflon (Ovis orientalis musimon) in Croatia.

The aim of the present study was to determine the reference intervals for the most commonly used hematological and biochemical parameters of European mouflon from a closed hunting ground in the eastern part of the Republic of Croatia. Blood samples were collected from 39 live, physically restrained, clinically normal European mouflon, as well as from 50 domestic sheep. The distribution of values within each parameter was determined and statistical differences in values between sexes were also determined. For each sample, 14 hematological and 18 biochemical parameters were analyzed. Hematology and biochemistry values of the European mouflon were also compared with the values of domestic sheep. In further studies, the established values might be useful for the health assessment of mouflon.

Repetitive sequences in Eurasian lynx (Lynx lynx L.) mitochondrial DNA control region

Mitochondrial DNA (mtDNA) control region (CR) of numerous species is known to include up to five different repetitive sequences (RS1–RS5) that are found at various locations, involving motifs of different length and extensive length heteroplasmy. Two repetitive sequences (RS2 and RS3) on opposite sides of mtDNA central conserved region have been described in domestic cat (Felis catus) and some other felid species. However, the presence of repetitive sequence RS3 has not been detected in Eurasian lynx (Lynx lynx) yet. We analyzed mtDNA CR of 35 Eurasian lynx (L. lynx L.) samples to characterize repetitive sequences and to compare them with those found in other felid species. We confirmed the presence of 80 base pairs (bp) repetitive sequence (RS2) at the 5′ end of the Eurasian lynx mtDNA CR L strand and for the first time we described RS3 repetitive sequence at its 3′ end, consisting of an array of tandem repeats five to ten bp long. We found that felid species share similar RS3 repetitive pattern and fundamental repeat motif TACAC.