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Brijestova osa listarica je invazivni štetnik, podrijetlom iz Japana i Kine. Unesena je u Europu posredstvom egzotičnih vrsta brijesta. U Bosni i Hercegovini je prvi puta otkrivena ovim nalazom 2017. godine na području općine Vareš na nekoliko lokaliteta. Ni na jednom od lokaliteta štetnik nije uzrokovao značajniju defolijaciju. Budući da je ovo strana i potencijalno invazivna vrsta, može se očekivati njeno širenje po čitavom području Bosne i Hercegovine.

Since the role of wild rodents/small mammals in hepatitis E virus (HEV) epidemiology has been a subject of considerable debate, this study was conducted to investigate the potential presence of HEV RNA in small rodents collected within their natural habitats and to detect if they can be potential reservoirs of the virus. A total of 483 small rodents were captured using snap traps placed at 11 regions in Croatia. Sampling was undertaken in 2008 and repeated from 2010 to 2014. Liver samples were tested for the presence of HEV RNA. HEV RNA was detected in only one liver sample (0.21%) originated from Apodemus flavicollis from the location Medvednica, nearby Zagreb collected in 2014. According to the sequence analysis, the isolate has shown to be a member of Orthohepevirus A species, genotype HEV- 3. The genotyping results confirmed grouping into subtype 3a, general cluster 3abchij. The detected HEV strain showed to be genetically highly related to strains found in humans and/or domestic pigs and wild boars from Croatia. Our finding indicates that wild small mammals could play a role in the epidemiology of HEV-3 infection and therefore should be taken under consideration as potential reservoirs or/and transmitters of the disease. However, further investigation is needed to recognize their potential for maintaining the infection in natural conditions.

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.

U ovome radu opisan je prvi nalaz hrastove mrežaste stjenice (Corythuca arcuata) na području centralne Bosne, na dva lokaliteta. Kako je ovo prvi nalaz ove vrste na području Bosne i Hercegovine, potrebna su dodatna istraživanja biologije i štetnosti ovog insekta. Budući da je ovo strana i potencijalno invazivna vrsta, može se očekivati njeno širenje po teritoriji čitave Bosne i Hercegovine., This research is first record of oak lace bug (Corythuca arcuata) in the municipality of Vareš, Bosnia and Herzegovina on two localities. It was found eggs, nymphs and adult insects of oak lace bug on the leaves of sessile oak. Since this is a foreign and potentially invasive species, its spread throughout the territory of Bosnia and Herzegovina can be expected.

Francisella tularensisis is a causative agent of tularemia a zoonotic disease that infects wide range of hosts including arthropods, mammals and birds. In this study, the prevalence of tularemia among small mammals in Croatia was investigated. The 444 samples of small rodents and insectivores were collected in eight different localities in continental Croatia during the 2-year study. Spleen samples of: 197 Apodemus agrarius (striped field mouse), 78 Apodemus sylvaticus (wood mouse), 92 Apodemus flavicollis (yellow-necked mouse), 17 Myodes glareolus (bank vole), 27 Mycrotus agrestis (field vole), 20 Microtus arvalis (common vole) and 13 Sorex araneus (common shrew) were investigated for the presence of DNA of Francisella spp. using qRT-PCR method. Two striped field mouses and one wood mouse, originated from the same area - locality of Lipovljani, were found to be positive on Francisella spp. revealing the presence of bacteria among small mammals population in Croatia., 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.

The aim of this study was to determine and compare fi eld dressing percentage in free-living wild boars from lowland (Spačva) and hilly (Papuk) habitats in Croatia. The obtained data indicate a better dressing percentage for wild boars from the Papuk region, with mean values of 77.35% for piglets, 79.61% for yearlings, 80.1% for subadults and 81.93% for adults. The dressing percentage for wild boars from the Spačva region was on average 75.10% for piglets, 73.68% for yearlings, 74.85% for subadults and 63.24% for adults. Furthermore, the internal organs were divided into eatable (heart, liver and kidneys) and non-eatable viscera. Of the complete viscera weight, the eatable part represented 17.57% in piglets, 17.97% in yearlings, 13.37% in subadults and 15.69% in adult boars from the Papuk region. The same values for the Spačva region were 19.79% in piglets, 14.06% into yearlings, 12.87% in subadults and 8.67% in adults. The results obtained in this study indicate a better growth rate and dressing percentage for wild boars from the Papuk region. When the similar feeding potential and habitat characteristics of both hunting grounds are taken in consideration, the most possible explanation for the observed differences is crossbreeding of wild boars with domestic pigs. The nomadic keeping of domestic pigs in the area of Spačva remains a huge problem for the preservation of the pure, autochthonous wild boar genome.

To determine whether some of the B. burgdorferi sensu lato genospecies associate with fat dormouse as a reservoir host, we investigated the prevalence of infection in questing animals. A total of 45 adult fat dormice (30 female and 15 male) were captured by hunters during their hunting season in the region of Gorski Kotar, Croatia. Dead animals were aseptically dissected, and the urinary bladder tissue was used for isolation attempt and for deoxyribonucleic acid (DNA) extraction. Out of 45 DNA samples extracted from urine bladder tissue, we found four (8.88%) to be polymerase chain reaction (PCR) positive. The RFLP analysis of the PCR product after cleavage with DraI and MseI distinguished between the three major genospecies: B. burgdorferi sensu stricto, B. garinii and B. afzelii. All positive samples were typed as B. afzelii with a unique DraI or MseI pattern. The results of the analysis of urinary bladder tissue samples culture for the presence of Borrelia were negative. Results showed that a prevalence of the Borrelia infection among population of fat dormice indicated their epizootiological involvement as a reservoir of Borrelia spirochetes. Furthermore, this work is an initial step in the investigation of the molecular epidemiology/epizootiology of Lyme borreliosis in Croatia.

In total the samples of blood and kidneys of 379 small rodents and 154 wild swine were analysed. The antibodies to different serovars of leptospirosis were determined in 12.7% of small rodents, most often in the species Mus musculus (34.4%), A. agrestis (14.8%), A. flavicolis (10.8%), C. glareolus (9.4%) and A. sylvaticus (6, 5%). Most frequent were the findings of antibodies to sv. pomona (27.1%), sv. sejroe (20.8%) and sv. australis (14.6%)and the antibodies to sv. hardjo, sv. saxkoebing, sv. tarassovi, sv. grippotyphosa, sv. bataviae and sv. icter-haemorrhagiae were also established. Seventeen (4.5%) isolates were identified, belonging to the serogroups sejroe (10 isolates), pomona (4 isolates), and australis (1 isolate) and one isolate was not identified. In wild swine positive reactions were established in 26% of blood sera analysed. Most frequently the antibodies to sv. pomona (47.5%), sv. australis (40%), sv. grippotyphosa (10%) and sv. icterohaemorrhagiae (2.5%) were established. Thirteen (8.4%) isolates belonging to the serogroups pomona (10 isolates), australis (2 isolates) and icterohaemorrhagiae (1 isolate) were identified.

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 I 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 Sejroe. For the first time we reported the occurrence of serovars tsaratsovo and lora in Croatia.

130 kg of acorns of Common Oak (Quercus robur L.) were distributed on an area of 1.44 ha in an oak forest near Zagreb (Croatia), where acorns were lacking. The influence of this surplus of food on the development of mice and voles was observed from September 1999 till February 2000 on the experimental plot and on an untreated control area before and after offering the acorns. The Redbacked Vole Clethrionomys glareolus was the most abundant and dominant species in both observed areas. After distribution of the acorns, all species of mice, e. g. the Yellownecked Fieldmouse Apodemus flavicollis, the Longtailed Fieldmouse A. sylvaticus, and the Striped Field Mouse A. agrarius, increased up to 4 times in number on the area treated with a surplus of acorns. On the same area, however, all species of voles, e. g. the Redbacked Vole C. glareolus, the Field Vole Microtus agrestis, and the Common Vole M. arvalis, decreased. The numbers of captured and recaptured rodents before and after offering acorns on the treated and the untreated control area were compared by statistical analysis (Chi2-test). The results show how food can influence the competitive relations between mice and voles.

SUMMARYAn outbreak of haemorrhagic fever with renal syndrome (HFRS) started on Medvednica mountain near Zagreb in January 2012. In order to detect the aetiological agent of the disease in small rodents and to make the link with the human outbreak, rodents were trapped at four different altitudes. Using nested RT–PCR, Puumala virus (PUUV) RNA was detected in 41/53 (77·4%) bank voles (Myodes glareolus) and Dobrava virus (DOBV) RNA was found in 6/61 (9·8%) yellow-necked mice (Apodemus flavicollis). Sequence analysis of a 341-nucleotide region of the PUUV S segment, obtained from all infected bank voles and five HFRS patients, showed 98·8–100% sequence similarity, indicating that the patients were probably exposed to PUUV on Medvednica mountain. A very large bank-vole population combined with an extremely high infection rate of PUUV was responsible for this unusual winter outbreak of HFRS in Croatia.

The present study reports for the first time on the helminth species occurring in the gastro-intestinal system of fat dormice (Glis glis) in Croatia. Out of 55 dormice, 63.7 % harboured helminths belonging to two species, the nematode Paraheligmonina gracilis (syn. Longistriata elpatievskii) (Heligmonellidae, Trichostrogyloidea) in the prevalence of 52.7 %, and the cestode Hymenolepis sulcata (Hymenolepididae, Cyclophyllidea) in the prevalence of 32.7 %. Concurrent infections of both parasites were found in 12 fat dormice, P. gracilis alone was detected in 17 hosts and H. sulcata alone in 6 samples. No influence of parasitic infestation on animal weight was observed. Glirid helminths do not represent zoonotic pathogens despite the fact that dormice occasionally inhabit cottages and village houses, and are used in human nutrition.

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).

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.

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.

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.

International audience; In Europe, Dobrava-Belgrade (DOBV), Saaremaa (SAAV) and Puumala (PUUV) viruses are known to cause hemorrhagic fever with renal syndrome (HFRS). All three hantaviruses are now found in Croatia. Lung tissue samples of 315 Apodemus mice trapped in 2003-2004 were screened for the presence of hantaviral N-Ag and 20 mice (6.3%) were found either strongly positive or weak/suspected-positive. Partial sequences of hantavirus M and S segments were recovered by RT-PCR from six mice and subjected to (phylo)genetic analysis that revealed the presence of four novel strains of DOBV and one of SAAV. Curiously, one of the newly described DOBV strains was found in A. agrarius mouse, i.e. not in the traditional host, A. flavicollis mice, suggesting a spillover event. S segment sequences recovered previously from HFRS cases (Markotic et al., 2002) were confirmed as DOBV sequences; one of which appeared particularly close to the prototype Slovenian DOBV isolate. Taken together with earlier data on PUUV in Croatia, these results show a co-circulation of three European hantavirus pathogens in this country. So far, not a single SAAV sequence has been recovered from HFRS patients either in Croatia or neighbouring Slovenia and Hungary nor in Slovakia suggesting a somewhat lower fequency of acute SAAV infection in humans in this part of Europe than for example in the Baltics.

Babesia divergens and B. divergens-like organisms are the main causative agents of human babesiosis in Europe. Recently, the first case of human infection with Babesia microti was confirmed in Germany, implicating the presence of zoonotic isolates. To estimate the presence of zoonotic B. microti in Croatia we analyzed 120 small wild mammals that serve as its reservoir by polymerase chain reaction. Yellow-necked mice (Apodemus flavicollis) and bank voles (Myodes glareolus) were both found to be infected with prevalence of 16.2%. Sequence analysis of the portion of 18S rDNA gene demonstrated that six polymerase chain reaction-positive samples, detected in both rodent species, were identical to that of the human Jena/Germany strain (EF413181). The other two isolates were identical to the nonzoonotic Munich strain (AB071177). The results of this study indicate the presence of zoonotic B. microti in A. flavicollis and M. glareolus in Croatia and a potential risk for human health.

HFRS is an endemic disease throughout Croatia. The incidence of HFRS varies in a cyclic fashion, with peaks occurring every couple of years, coinciding with peaks in vole populations. PUUV was shown to be dominant pathogen during the last HFRS outbreak in Croatia in 2002. We focused our research on two newly discovered localities (Okučani and Nova Gradiška) with a high number of reported HFRS cases and a significant increase in rodent population. PUUV infection was verified in 84.2% of patients at this region during the 2002 outbreak. Genetic analysis of wild-type (wt) PUUV strains was performed. Fifty seven bank voles Clethrionomys glareolus originating from PUUV-associated HFRS areas were screened for the presence of PUUV N antigen and 15 (26 %) were found positive. Total RNA isolated from rodent lung tissues was reverse transcribed followed by PCR amplification with primers specific for PUUV medium (M) or small (S) genome segments. Partial PUUV M segment sequences (approximately 450 bp long) were recovered from five bank voles and partial S segment sequences (app. 250 nt long) - from two bank voles. Genetic analysis of Croatian wt-PUUV strains revealed their close relatedness suggesting that the two localities belong to the same natural focus of infection. On phylogenetic trees Croatian PUUV strains clustered together with the strains from Slovenia and Austria forming distinct Alpe-Adrian genetic lineage.

The fat dormouse (Glis glis) is a traditional game species in the Republic of Croatia. Although today the fat dormouse is not frequently caught as game, it is still a source of animal protein in human nutrition in certain rural areas of Croatia. In this paper the chemical analysis of fat dormouse meat is presented. The average values for the quantity of water, fat, protein and ash in dormouse meat are comparable with the chemical composition of the meat of rabbits and brown hares, except for the important fact that rabbit and hare meat contains a greater quantity of fat on average. According to our results, the meat of fat dormice can be categorised as dietary food, characterised by a small percentage of fat (mean 2.83%) and a high amount of protein (mean 21.01%).

Fourteen new polymorphic di- and di-tetra complex microsatellite loci were isolated and characterized from the edible dormouse (Glis glis). The markers were tested on 427 individuals representing populations from Croatia and Poland. All loci were polymorphic, with the number of alleles per locus ranged from 4 to 31, with a mean of 10. Observed and expected heterozygosities ranged from 0.208–0.957 to 0.228–0.907, respectively. No evidence of linkage disequilibrium between loci has been found. These highly polymorphic markers could provide a powerful tool for detailed genetic studies of this endangered species.