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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.

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.

Abstract. 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.

During the 10-year survey (1996)(1997)(1998)(1999)(2000)(2001)(2002)(2003)(2004)(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.