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    Map in Croatia indicating the regions where the rodents were trapped.

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    Maximum likelihood phylogenetic tree based on a 388-nucleotide fragment of Bartonella gltA gene. All sequences obtained in this study are indicated by * and the six species they cluster into are shown in gray. Only select relevant sequences from GenBank are shown in the tree.

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

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  • University Hospital for Infectious Diseases “Dr. Fran Mihaljević,” Zagreb, Croatia; Center for Infection and Immunity, Mailman School of Public Health, Columbia University, New York, New York; Faculty of Forestry, University of Zagreb, Zagreb, Croatia; Faculty of Veterinary Medicine, University of Zagreb, Zagreb, 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.

Introduction

Rodents are hosts to a wide range of zoonotic pathogens. Rodent-borne agents can be transmitted to humans directly through contact with rodents and their excretions or through rodent bites and indirectly by arthropod vectors including ticks, fleas, and mites. Croatia is a natural focus for many rodent-borne zoonoses because of its diverse forest ecology and wide range of rodent species. Different rodent species in Croatia have been implicated as reservoirs to several clinically important bacteria, protozoa, and viruses. The main rodent-associated bacteria include species of Anaplasma, Borrelia, Bartonella, Coxiella, Ehrlichia, Francisella, Leptospira, and Rickettsia. Infections with Leptospira and Borrelia are the most frequently reported from among this group. With a mean yearly infection incidence of 1.7 per 100,000 inhabitants, Croatia has one of the highest recorded incidences of human leptospirosis in the world.1 Borrelia infections result in two distinct tick-transmitted diseases, Lyme borreliosis and relapsing fever (RF). Both are endemic in Croatia with majority of cases of borreliosis reported in the northwest of the country.2 Q fever, caused by Coxiella burnetii is endemic in parts of southern Croatia.3,4 Limited data exist on tick-transmitted Rickettsia, Anaplasma, and Ehrlichia.5 Human cases of anaplasmosis and rickettsiosis have been reported in Croatia, but little data are available on ehrlichiosis.6,7 Infections with Francisella tularensis, the agent of tularemia, are occasionally reported in Croatia, particularly in Sava Valley in the central part of the country.8 Bartonella spp. are hemotropic bacteria detected in many mammals, predominantly rodents.9 Many species and subspecies of Bartonella circulate in European rodents, although the pathogenicity of many of these species is as yet undetermined. In addition, the diversity of Bartonella spp. present in rodents in Croatia has not been examined.

Hantaviruses are the viruses most frequently associated with rodents in Croatia. These include Dobrava (DOBV) and Puumala (PUUV) viruses, implicated in hemorrhagic fever with renal syndrome, and the nonpathogenic Tula (TULV) and Saaremaa viruses.1014 Rodents can also be infected with flaviviruses, orthopoxviruses, and lymphocytic choriomeningitis virus (LCMV); however, to our knowledge, there are no published reports of investigation for the presence of these viruses in Croatia.1517 Rodents in Croatia are also reservoirs of protozoan Apicomplexa parasites, including Toxoplasma and Babesia that cause toxoplasmosis and babesiosis, respectively, mainly in immunocompromised individuals.

Here, we report results of analysis of rodents and small mammals trapped in eight localities in Croatia for the presence of a wide range of known zoonotic human pathogens.

Materials and Methods

Sample collection.

This survey was an extension of a project designed to assess the distribution of small rodents in diverse forest ecosystems in Croatia. Between 2003 and 2011, rodents were trapped in eight different localities in Croatia: Ivanić-Grad, Mikanovci, Ilok, Cerna, Papuk, Žutica, Draganić, and Sušica (Figure 1). In each of three selected geographic transects at each location, 100 traps were placed. The distance between the traps was approximately 7 m and the distance between the transects was approximately 150 m. Each transect was at least 50 m away from the edge of the forest stands. Each of these localities includes forest ecosystems without proximity to urban areas. All rodents were trapped using Sherman-type live traps and were morphologically characterized to species.18 Species identification of Apodemus flavicollis and Apodemus sylvaticus was confirmed by PCR (polymerase chain reaction) targeting the mitochondrial cytochrome b gene and sequencing of PCR products.19,20 A total of 242 animals were collected, including 131 yellow-necked mouse (A. flavicollis), 53 striped field mouse (Apodemus agrarius), three wood mouse (A. sylvaticus), 43 bank voles (Myodes glareolus), six common shrews (Sorex araneus), four common voles (Microtus arvalis), one field vole (Microtus agrestis), and one common dormouse (Muscardinus avellanarius). Animal experimentation guidelines of the American Society of Mammologists was followed during all animal work.21 After euthanasia, kidney and lung samples were collected from individual specimens and stored at −80°C.

Figure 1.
Figure 1.

Map in Croatia indicating the regions where the rodents were trapped.

Citation: The American Society of Tropical Medicine and Hygiene 94, 2; 10.4269/ajtmh.15-0517

Nucleic acid extraction and complementary DNA synthesis.

A total of 323 organ specimens were available for PCR analysis. These consisted of lung samples acquired from 237 animals and kidney samples from 86 animals. At least one organ (lung or kidney) was available for PCR analysis from all 242 animals. In cases where both lung and kidney samples were available from an individual animal, both were tested by PCR. Tissues were homogenized using TissueLyser and 5-mm beads for homogenization (Qiagen, Hilden, Germany). DNA and RNA were extracted from each sample using the AllPrep DNA/RNA Mini Kit (Qiagen). DNA from each sample was eluted in 100 μL EB Buffer and RNA from each sample was eluted in 40 μL RNase-free water. Nucleic acid concentration was assessed with the NanoDrop Spectrophotometer (Thermo Scientific, Waltham, MA). Complementary DNA (cDNA) was prepared in a 20 μL reaction using 5 μL extracted RNA and SuperScript II Reverse Transcriptase (Invitrogen, Carlsbad, CA).

Polymerase chain reaction.

MassTag PCR was performed using primers for detection of Lyme borreliosis–associated Borrelia spp., Bartonella spp., Ehrlichia spp., Anaplasma spp., F. tularensis, C. burnetii, Rickettsia spp., RF Borrelia spp., and Babesia spp. (Table 1).22,23,29 Of template DNA, 2 μL was used in all reactions. Reaction conditions were 94°C for 15 minutes; one cycle at 94°C for 30 seconds, 65°C for 30 seconds, and 72°C for 30 seconds, followed by 11 cycles with annealing temperature decreased by 1°C in each cycle. The final PCR was run for 38 cycles at 94°C for 30 seconds, 54°C for 30 seconds, and 72°C for 30 seconds.

Table 1

Primer sequences used for pathogen detection

PathogenGene targetPrimer sequenceReference
Lyme borreliosis–associated Borrelia spp.flaBFwd: GCAATGACAAAACATATTGRGGAASTTGA22
Rev: YACAATGACMGATGAGGTTGTRGC
Relapsing fever Borrelia spp.flaBFwd: GCTGAAGAGCTTGGAATGCAAC23
Rev: GCAATTGCYTCATCCTGATTTG
Bartonella spp.hbpAFwd: CTTCTGCRGCACAAGCTGCTGAT22
Rev: CCACCAATATARAAACCTGTCCAAGA
Ehrlichia spp.16SrRNAFwd: CGTAAAGGGCACGTAGGTGGACTA22
Rev: CACCTCAGTGTCAGTATCGARCCA
Anaplasma spp.16SrRNAFwd: GGGCATGTAGGCGGTTCGGT22
Rev: TCAGCGTCAGTACCGGACCA
Francisella tularensisfopAFwd: ATGTTTCGGCATGTGAATAGTTAA22
Rev: ACCACTGCTTTGTGTAGTAGCTGAA
Coxiella burnetiiIS1111Fwd: GCTCCTCCACACGCTTCCAT22
Rev: GGTTCAACTGTGTGGAATTGATGAGT
Rickettsia spp.ompBFwd: YAAAAGTCAGCGTTACTTCTTYGA22
Rev: YGCTTRTTTGCAACTGTTGTACC
Babesia spp.18SrRNAFwd: CGACTAGDGATTGGDGGTCGTCThis study
Rev: CTHTGTCTGGACCTGGTGAGKTT
Lymphocytic choriomeningitis virusRdRpFwd: CCACTYTTGTCTGCACTGTCTATThis study
Rev: CTTTTTGATGCGCAATGGAT
OrthopoxvirusVETFSFwd: ACCAACTATATTACCTCATCAGTTAThis study
Rev: TTAAACAAGTTCATAGCTACACCCA
FlavivirusNS5Fwd: ATGGCHATGACNGACACNACThis study
Rev: TTCTTYTCTCTYTTNCCCATCAT
Toxoplasma gondiiB1Fwd: GAAGAGATCCAGCAGATCTCGTThis study
Rev: TGAGAGGAGGCAGCACAAG
Puumala/Tula hantavirusNFwd1: TATGGIAATGTCCTTGATGT24
Rev1: GCACAIGCAAAIACCCA
Fwd2: CCIAGTGGICAIACAGC24
Rev2: AAICCIATIACICCCAT
Dobrava hantavirusGFwd1: GGACCAGGTGCAGCTTGTGAAGC25
Rev1: ACCTCACAAACCATTGAACC
Fwd2: ATGCCAGCGAGTCGACCAA26
Rev2: GAGCTATTATGTAAGATTGC
Leptospira spp.LA0322FwdA: CATTCATGTTTCGAATCATTTCAAA27
RevA: GCCCAAGTTCCTTCTAAAAG
secYFwdB: GAATTTCTCTTTTGATCTTCG28
RevB: GAGTTAGAGCTCAAATCTAAG
Bartonella spp.*gltAFwd: GGTCTATCAYGACTCKATTGATATTAThis study
Rev: GCACGTGGRTCATAATTTTTATARAC
16SrRNAFwd: ATAGCAAGTCGAGCGCRCTThis study
Rev: GCCCGACGGCTAACATTC
Borrelia spp.*flaBFwd: GGGGATGATYATMAATCATAATACThis study
Rev: TGCAATCATWGCCATTGC
Babesia spp.*18SrRNAFwd: TTCGACGGTAKGGTATTGGCThis study
Rev: GGGAATTTCACCTCTGACAGTYAA

Consensus genus primers used for species discrimination.

All samples that were positive on the MassTag PCR assay were retested in individual PCR assays for the microbe(s) of interest using the same primer pairs. The presence of relevant sequence in the resulting PCR product was required as confirmation before a sample was declared agent positive. In cases where species differentiation was required, we designed primers within a discriminatory region of the genome to amplify partial sequences that were then compared with existing genomic data from GenBank. To discriminate among Bartonella spp., we designed primer pairs that amplified 513 nucleotides (nts) of the gltA gene and 800 nts of the 16SrRNA gene. For Babesia spp., we used a primer pair that amplified 607 nts of the 18SrRNA gene, and for Borrelia spp., an assay targeting the flaB gene amplified a sequence of 966 nts (Table 1). Sequences for Baronella spp. and Borrelia miyamotoi were deposited in GenBank under accession nos. KT452901–KT452935. All alignments were generated in Geneious v 6.1.5 (Biomatters Ltd., Auckland, New Zealand). Maximum-likelihood phylogenetic tree for Bartonella was constructed using the Jukes–Cantor model with 1,000 bootstrap replicates in Mega 6.0.30

All samples were additionally tested for LCMV, orthopoxvirus, flaviviruses, hantaviruses, and Toxoplasma gondii in single-agent PCR assays. For detection of RNA viruses, 2 μL cDNA were used as template. For the detection of orthopoxviruses and T. gondii, 2 μL DNA was used. Nested PCR assays were used to detect the presence of hantaviruses (PUUV, DOBV, and TULV).2426 All primer sequences are listed in Table 1. PCR products were size-fractionated in 1% agarose gels and visualized using ethidium bromide before purification for dideoxy sequencing.

Detection of Leptospira by renal tissue culture and PCR.

Kidney tissue from each animal (N = 242) was available for Leptospira detection. Tissue was inoculated into Korthof's medium for isolation and stabilization of Leptospira isolates. Isolates were grown to a density of 2–4 × 108/mL and harvested by centrifugation at 14,000 rpm. Genomic DNA was extracted from each kidney tissue using the QIAamp DNA Mini Kit (Qiagen). Two PCR assays were used for species identification. In the first assay, real-time PCR was used as described by Merien and others27 with a primer set that amplified 331 nts of the locus LA0322 obtained from the complete genome sequence of Leptospira interrogans serovar Lai. All specimens were tested 10 times and mean melting temperatures were calculated to distinguish Leptospira genomic species. In a second assay, PCR was done with primers that amplified a 658-nt segment of secY that includes a 245-nt fragment suitable for rapid phylogenetic determination.28 Amplified products were visualized in a 1.5% agarose gel, and their sizes were estimated by comparison with a 100-bp ladder (Amersham Biosciences, Little Chalfont, Buckinghamshire, United Kingdom). PCR products were purified by QIAquick PCR Purification Kit (Qiagen) and sequenced. DNA sequence clustal alignments were done using the LaserGene software package (DNASTAR, LaserGene, Madison, WI). In addition, a primer set that amplified a 563-nt DNA fragment of Leptospira kirschneri was used to confirm the speciation of L. kirschneri isolates.31

Results

In our survey, 114 of 242 rodents (47%) were positive for at least one agent. Polymicrobial infections were detected in 32 rodents (13%). Dual infections were detected in 26 rodents (11%), triple infections in four rodents (3%), and quadruple infections in two rodents (0.8%) (Table 2).

Table 2

Summary of all agents and coinfections detected in different rodent species

 NApodemus flavicollisApodemus agrariusApodemus sylvaticusMyodes glareolusSorex araneusMicrotus arvalisMicrotus agrestisMuscardinus avellanarius
Total242131533436411
Borrelia afzelii5213
Bartonella spp.2916751
Babesia microti8431
Ehrlichia spp.211
Borrelia miyamotoi9171
Anaplasma spp.431
Francisella tularensis22
Leptospira spp.523712111
 Hantavirus4336241
 DOBV38362
 PUUV44
 TULV11
 Orthopoxvirus11
Infections with two agents
Leptospira, B. afzelii11
Leptospira, Ehrlichia11
Leptospira, B. microti11
Leptospira, Bartonella33
Leptospira, Anaplasma11
Leptospira, B. miyamotoi312
Leptospira, TULV11
Leptospira, DOBV1010
Bartonella, DOBV33
B. microti, B. miyamotoi11
Anaplasma, DOBV11
Infections with three agents
Leptospira, B. afzelii, B. microti11
Bartonella, Anaplasma, DOBV11
B. microti, Leptospira, DOBV22
Infections with four agents
Bartonella, B. afzelii, Leptospira, DOBV11
Bartonella, F. tularensis, Leptospira, DOBV11

DOBV = Dobrava virus; PUUV = Puumala virus; TULV = Tula virus.

Leptospira.

Leptospira species were the most frequently detected agents with 52 Leptospira-positive rodents (21%). Apodemus spp. have previously been implicated as reservoirs of Leptospira in Croatia.32,33 Consistent with this earlier work, we found that 49 of 52 Leptospira-positive rodents represented Apodemus spp. Bacterial isolation from kidney tissue was successful in 40 PCR-positive rodents, and speciation assays indicated that 22 were L. interrogans, 16 L. kirschneri, and two Leptospira borgpetersenii.

Borrelia.

Five rodents were positive for Lyme borreliosis–associated Borrelia spp. All were identified as Borrelia afzelii (> 99% identity with B. afzelii PKo strain, accession no. CP002933). The presence of this species in Croatian rodents has been reported previously.34 Although Borrelia garinii is known to circulate in Croatia, we did not detect any rodents infected with this species. RF Borrelia was detected in nine rodents (4%) from four different localities. All RF Borrelia were identified as B. miyamotoi (> 99% identity with LB-2001 strain, accession no. CP006647) with the majority (7/9) detected in A. agrarius. Two B. miyamotoi-positive rodents originated from Ivanić-Grad in the central part of Croatia, and the remaining seven were trapped in the eastern end of the country.

Bartonella.

Bartonella spp. were detected in 29 animals (12%) in six different localities with the highest prevalence in Ilok and Draganić (26.7% and 25.0%, respectively). Although A. flavicollis was the most frequent reservoir of Bartonella in this study (9 of 29 Bartonella-positive rodents) and in earlier studies,35 A. agrarius as well as M. glareolus and S. araneus were also positive for Bartonella. To determine the phylogeny of Bartonella in these rodents, we obtained partial sequences of 16SrRNA and gltA genes by PCR. Previous reports have indicated that sequence analysis of 16SrRNA gene alone is inadequate for species/genotype demarcation in Bartonella, and an additional loci need to be analyzed, with gltA the preferred gene used to establish Bartonella phylogeny.36 Our analysis based on a 388-nt gltA sequence indicated that representatives of six putative Bartonella species were present in the these rodents (Figure 2). Five rodents were infected with strains representative of Bartonella grahamii and nine rodents were infected with strains of Bartonella taylorii. Both species are distributed throughout Eurasia and are frequently detected in Apodemus and Myodes rodents.37,38 A single A. flavicollis was infected with a strain that clustered with Bartonella elizabethae species group. Seven A. flavicollis were infected with a genotype similar to strain N40 isolated from A. sylvaticus from the United Kingdom.39 On the basis of high sequence divergence relative to other strains and a distinct gltA phylogenic clade formed by this group, we speculate that this genotype constitutes a new Bartonella species. Sequences obtained from six A. agrarius formed a monophyletic clade and likely constitute another potential novel Bartonella species. We also detected a single Bartonella strain (ARH567) in A. agrarius that clustered with AR15-3 strain from the United States (FN645480) and strains from Sweden (AY454539), Siberia (EF682090). Analysis of the 16SrRNA sequence indicated that ARH567 was > 99% identical to B. rochalimae (strain BAA-1498, accession no. FN645459) and thus may constitute a B. rochalimae subspecies.

Figure 2.
Figure 2.

Maximum likelihood phylogenetic tree based on a 388-nucleotide fragment of Bartonella gltA gene. All sequences obtained in this study are indicated by * and the six species they cluster into are shown in gray. Only select relevant sequences from GenBank are shown in the tree.

Citation: The American Society of Tropical Medicine and Hygiene 94, 2; 10.4269/ajtmh.15-0517

Babesia.

Eight rodents from five localities were infected with Babesia, all identified as Babesia microti (100% identity to strain Jena, accession no. EF413181). Previous work has implicated A. flavicollis and M. glareolus as the primary reservoirs for B. microti in Europe.40 Of the eight positive rodents in our study, three were A. agrarius and one M. agrestis, implicating these species as additional reservoirs of Babesia spp.

Hantaviruses.

Hantaviruses were the second most frequently detected pathogens. Of 43 hantavirus-positive rodents, 38 were infected with DOBV, four with PUUV, and a single rodent was infected with TULV. Hantavirus-positive rodents originated from seven different locations with the highest prevalence in Žutica (34.5%) and Ilok (20%) (Table 3). Among Apodemus species, 28% of A. flavicollis and 2% of A. agrarius were DOBV positive. The highest prevalence of DOBV-positive rodents (30%) was in Žutica (Table 3). PUUV was detected in M. glareolus and TULV was detected in M. arvalis, similar to previous reports.12

Table 3

Number of agent-positive rodents at each trapping site

 Ivanić-GradMikanovciIlokCernaPapukŽuticaDraganićSušicaTotal
N = 39N = 28N = 30N = 9N = 18N = 87N = 16N = 15242
Borrelia afzelii13015
Bartonella418210429
Babesia microti121318
Ehrlichia112
Borrelia miyamotoi24129
Anaplasma314
Francisella tularensis0022
Leptospira41191124152
Hantaviruses1163301143
DOBV163261138
PUUV44
TULV11
Orthopoxvirus11

DOBV = Dobrava virus; PUUV = Puumala virus; TULV = Tula virus.

Other agents.

Francisella tularensis was detected in two A. agrarius from the Draganić site. One rodent was positive for orthopoxvirus. This is the first report of F. tularensis and orthopoxvirus infection in rodents in Croatia. Four rodents were positive for Anaplasma and two for Ehrlichia. Rickettsia, C. burnetii, LCMV, flaviviruses, and T. gondii were not detected. Among the agents detected, RF Borrelia, Bartonella, Babesia, Ehrlichia, and hantaviruses were detected in nucleic acids isolated from both lung and kidney tissues; Anaplasma, Francisella, and B. afzelii were detected in nucleic acids isolated only from lung tissue.

Discussion

In this study, we demonstrate the presence of multiple zoonotic pathogens in rodent species present in Croatia. Although this study was not designed to demonstrate a linkage between the presence of these agents and human disease, the data presented here may provide insight into assessing the risk of rodent-borne zoonoses in Croatia.

Leptospira spp. and hantaviruses were the human pathogens most frequently detected in our study. The high prevalence of Leptospira-positive rodents parallels recent surveys in Croatia that revealed Leptospira infection in > 20% of rodents. The majority of Leptospira-positive rodents reported here were Apodemus species. In our previous work, Leptospira spp. were isolated from 13 of 28 A. flavicollis captured in Žutica forest.41 Leptospira interrogans, L. kirschneri, and L. borgpetersenii were identified as the Leptospira species in our study, similar to previous reports from Croatian rodents.32,33 Hantavirus and Leptospira also represented the most frequent coinfections. We previously showed coinfections with both agents in humans10 and rodents41,42 highlighting the potential risk for simultaneous transmission of these pathogens. The clinical underestimation of hantavirus and Leptospira coinfections was recently emphasized.43

The genus Bartonella contains over 30 named species. Approximately 20 are adapted to wild rodents and six of these have caused documented human infections.37 We detected the presence of six putative species present in Croatian rodents. Our data provide insight into the diversity of Bartonella in Croatia and the high overall genetic diversity of this genus worldwide. The extent of human infections associated with rodent-borne Bartonella is unknown as data regarding the incidence of zoonotic human Bartonella infections in Europe are not available. However, despite a relatively high prevalence of infected rodents, based on existing reports of infections due to rodent-borne Bartonella, we anticipate that the risk of human infection is low.

In this study, we report the first evidence of Borrelia miyamotoi in Croatia and have implicated Apodemus mice as reservoirs, similar to recent report from Switzerland.44 Borrelia miyamotoi is an emerging tick-borne pathogen phylogenetically classified within the RF Borrelia group. Originally detected in Asia, it was subsequently detected in North America and Europe.23,45,46 Recent work has also implicated it as a potential cause of meningoencephalitis.47,48

The prevalence of Anaplasma spp. infection in our study was 1.7%. Anaplasma spp. were detected in only two locations in central Croatia (Papuk and Žutica) among A. flavicollis and M. glareolus. Anaplasma spp. were previously reported in small rodents elsewhere in Europe,49 but not in Croatia. Ehrlichia spp. were detected in only two rodents (0.8%) of genus Apodemus in two locations (Ivanić-Grad and Mikanovci).

A limitation of this study was the unavailability of organs other than lungs and kidneys for analysis. This is because we used samples banked during the course of a hantavirus and Leptospira surveillance project wherein only lungs and kidneys were collected. Thus, we may have missed some bacteria and viruses that concentrate in other organs. Furthermore, we did not screen for all rodent-associated agents, nor did we pursue unbiased sequencing. Nonetheless, our data indicate that small rodents in Croatia are reservoirs for a wide range of viral, bacterial, and protozoan pathogens. We also demonstrated a high rate of coinfections that may have important implications for simultaneous transmission to humans.

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Author Notes

* Address correspondence to Rafal Tokarz, Center for Infection and Immunity, Mailman School of Public Health, Columbia University, 722 West 168th Street, New York, NY 10032. E-mail: rt2249@cumc.columbia.edu† These authors contributed equally to this work.

Financial support: Work reported here was supported by awards from the NIH (AI057158, Northeast Biodefense Center-Lipkin), USAID-PREDICT, DTRA, and by the grants 143-1430115-0103, 068-1430115-2119, 053-1430115-2116 from the Ministry of Science, Education and Sports of Republic Croatia and the project “The role of biotic agents on vitality of narrow-leafed ash in Croatian floodplain forests” from Croatian Science Foundation.

Authors' addresses: Ante Tadin, Alemka Markotić, and Petra Svoboda, University Hospital for Infectious Diseases “Dr. Fran Mihaljević,” Zagreb, Croatia, E-mails: ante.tadin@yahoo.com, alemka.markotic@gmail.com, and petra.petrasvoboda@gmail.com. Rafal Tokarz, Aaloki Desai, Komal Jain, and W. Ian Lipkin, Center for Infection and Immunity, Mailman School of Public Health, Columbia University, New York, NY, E-mails: rt2249@cumc.columbia.edu, as3745@cumc.columbia.edu, kj2230@cumc.columbia.edu, and wil2001@cumc.columbia.edu. Josip Margaletić and Marko Vucelja, Faculty of Forestry, University of Zagreb, Zagreb, Croatia, E-mails: josip.margaletic@sumfak.hr and vucelja.marko@gmail.com. Nenad Turk and Josipa Habuš, Faculty of Veterinary Medicine, University of Zagreb, Zagreb, Croatia, E-mails: turk@vef.hr and jhabus@vef.hr.

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