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-MEDVED
I
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UPANC
Hantaviruses, which are members of a genus within the family Bunyaviridae, can cause either of two human zoonoses: hemorrhagic fever with renal syndrome (HFRS) or hantavirus pulmonary syndrome (HPS).1 Leptospirosis, also a zoonotic infection, is caused by pathogenic members of the bacterial genus Leptospira.2 Both etiologic agents are rodent-borne and are maintained in persistently infected small rodents. Transmission from reservoir hosts to humans is possible through inhalation of virus-contaminated aerosol of rodent excreta (hantaviruses)1 or direct or indirect contact with contaminated environment (leptospira).3 Farmers, soldiers, hunters, campers, hikers, veterinarians, and laboratory workers have the highest risk of infection. The widespread geographic distributions of rodents harboring these pathogens indicate considerable disease-causing potential essentially worldwide. Dual infection of these two agents in humans has been described previously.4,5 We have reported dual infection with Dobrava virus (DOBV) and Leptospira interrogans serovar hardjo in one patient.5
During September 2002, we trapped rodents in northeastern Croatia at two sites: Oku
ani (45°22'28''N, 17°17'05''E) and Nova Gradi
ka (45°18'30''N, 17°17'10''E) (approximately 20 km apart) (Figure 1
), which were recently shown to be natural foci of HFRS.6 The same region in northeastern Croatia is also known as an area where human and animal leptospirosis are common.7 Sampling of small rodents was done at a hillside on Psunj mountain, 
400 meters above sea level, in forests where common beech (Fagus sylvatica L.) and sessile oak (Quercus petraea (Matt.) Liebl.) predominate. Rodents were captured using Sherman live traps (H.B. Sherman Traps, Tallahassee, FL) and snap traps. We followed animal experimentation guidelines approved by the American Society of Mammalogists.8 Rodents were identified to species, humanely killed, and dissected aseptically, with kidney and lung tissues collected for further studies. Small 50-mg pieces of frozen lung tissues were used for immunoblotting and kidney tissue was extracted for detection of hantaviral RNA. A Western blot was used to test for the presence of hantaviral nucleocapsid antigen. Homogenized specimens were separated by electrophoresis and immunoblotted with rabbit polyclonal antibodies against Puumala virus (PUUV) and DOBV recombinant antigen. A swine anti-rabbit antibody conjugated to horseradish peroxidase was used as the secondary antibody and bands were stained with diaminobenzidine. Total RNA was extracted from kidney tissue of antigen-positive rodent(s) with TRIzol reagent (GIBCO-BRL, Gaithersburg, MD) following the manufacturer’s instructions. Hantaviral RNA in antigen-positive rodents was detected in their tissues by reverse transcription–polymerase chain reaction (PCR) using the cross-reactive outer primers MOF103 and MOR204, which amplify a 490-basepair region from the M segment (encoding G1) of a number of different hantaviruses, as previously described.9 A second-round PCR (for nucleotides 1296–1620) was done using PUUV-specific primers G1F (5'-GTGTCCAGAGATTCCGTGGT-3') and G1R (5'-GAAC-ATAAGTATGCGAATGCGAATGCAA-3') previously described,10 which reside within the amplified region of the RT-PCR primers in a nested fashion. All PCR products were sequenced automatically using ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin Elmer Applied Biosystems, Foster City, CA).
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Rodents captured included bank voles (Clethrionomys glareolus, n = 57), yellow-necked field mice (Apodemus flavicollis, n = 35), and long-tailed field mice (Apodemus sylvaticus, n = 13). PUUV antigen was detected by Western blot in one female bank vole of a total 33 bank voles captured at Nova Gradika (Figure 1
). Hantaviral RNA was extracted from kidney tissue of the antigen positive rodent. The PCR product authenticity was confirmed by sequence analysis.
Leptospiral DNA also was obtained from the same vole. Serotyping by microscopic agglutination using rabbit antisera and genetic characterization by digestion with a restriction enzyme, followed by pulsed-field gel electrophoresis, showed that this leptospire was most closely related to the genotype L. interrogans sensu stricto serogroup Australis serovar lora.
Except for the Adriatic coastal and islands, all of Croatia has been found to be endemic for HFRS.11 During HFRS outbreaks in Croatia, small, focal, disease-endemic areas with a high prevalence of antibody to hantaviral antigens were reported at Mala Kapela mountain,12 Novska,13 and Dinara mountain.11,14 In the last 2002 HFRS outbreak, a newly recognized disease-endemic area was confirmed near Nova Gradika (in northeast Croatia).6 In addition, a small survey of rodents showed that the entire area of Posavina in northeastern Croatia was a wide natural focus of leptospires (Figure 1).7,15
The bank vole is considered to be the principal reservoir of PUUV in Croatia, with yellow-necked field mice, long-tailed field mice, and striped field mice (Apodemus agrarius) playing minor roles in maintenance and transmission of hantaviruses.11,12 Coincidentally, rodents most commonly found with leptospires are these same four species.15 Serovar lora has been isolated from yellow-necked field mice and genetically characterized in Croatia.7
We found one bank vole captured near Nova Gradika with a dual infection of PUUV and L. interrogans serovar lora (Figure 1). Since these pathogens share the same reservoir host, this suggests that their geographic distribution provides opportunity for individual bank voles to be infected with both pathogens.
Hantaviruses are principally associated with single rodent hosts.16 There are no data on the prevalence hantavirus/leptospira dual infections among rodents. It is also not known what would be the risk for humans to encounter both infections at the same time. To the best of our knowledge, our study is the first to demonstrate co-infection of a hantavirus and a leptospira in a rodent reservoir host. From previous studies it is known that northeastern Croatia has a status of a combined focus of leptospirosis and hantavirus infection (Figure 1).6,7,15 We showed that dual infection with these two pathogens may occur in bank voles under natural conditions. Therefore, determining the presence and spread of these etiologic agents of human illnesses by testing local rodent populations might predict the potential for disease emergence. We suggest that tests for both pathogens be used for diagnosis of illnesses in patients suspected to have HFRS or leptospirosis in Croatia and elsewhere in the region.
Received July 14, 2005. Accepted for publication November 28, 2005.
Acknowledgments: We thank Professor Charles H. Calisher (Colorado State University, Fort Collins, CO) for editorial advice.
Financial support: This work was supported by grants TP-01/0021-06 (P. I. A. Markoti
) and 0021005 (P. I. A. Markoti) from the Croatian Ministry of Science, Education and Sport, and by the Pasteur Institute (Paris, France).
* Address correspondence to Lidija Cvetko, Institute of Immunology, Department for Research and Development, Cellular Immunology Unit, Rockefellerova 10, HR-10 000 Zagreb, Croatia. E-mail: lcvetko{at}imz.hr 
Authors’ addresses: Lidija Cvetko and Alemka Markoti
, Institute of Immunology, Department for Research and Development, Cellular Immunology Unit, Rockefellerova 10, HR-10 000 Zagreb, Croatia, Telephone: 385-1-468-4500, Fax: 385-1-468-4303, E-mails: lcvetko{at}imz.hr and amarkotic{at}net.hr. Nenad Turk and Zoran Milas, Department of Microbiology and Infectious Diseases, Faculty of Veterinary Medicine, University of Zagreb, Heinzelova 55, HR-10 000 Zagreb, Croatia, Telephone: 385-1-239-0207 and 385-1-239-0200. Fax: 385-1- 244-1390, E-mails: turk{at}vef.hr and zoran.milas{at}vef.hr Josip Margaleti, Department of Forest Protection and Hunting, Faculty of Forestry, University of Zagreb, Svetoimunska 25, HR-10 000 Zagreb, Croatia, Telephone: 385-1-235-2555, Fax: 385-1-231-8616, E-mail: josip.margaletic{at}inet.hr. Marica Mileti-Medved, Institute of Public Health, Vladimira Nazora bb, HR-35 000 Slavonski Brod, Croatia, Telephone: 385-35-444-796, Fax: 385-35-440-244, E-mail: zzjzsb{at}sb.htnet.hr. Alexander Plyusnin, Haartman Institute, Department of Virology, PO Box 21, FIN-00014, University of Helsinki, Helsinki, Finland, Telephone: 358-9-1912-6486, Fax: 358-9-1912-6491, E-mail: Alexander.Plyusnin{at}Helsinki.Fi. Guy Baranton, and Daniele Postic, Institut Pasteur, Unite de Bacteriologie Medicale et Moleculaire, 25–28 Rue de Dr. Roux, Paris Cedex 15, France, Telephone: 33-1-45-68-83-37, Fax: 33-1-40-61-30-01, E-mails: gbaran{at}pasteur.fr and dpostic{at}pasteur.fr. Tatjana Avi-upanc, Institute of Microbiology and Immunology, Medical Faculty, University of Ljubljana, Zaloka 4, 1000 Ljubljana, Slovenia, Telephone: 386-1-543-7450, Fax: 386-1-5437401, E-mail: tatjana.avsic{at}MF.UNI-LJ.SI.
A, Kuzman I, Babi K, Gagro A, Nichol ST, Ksiazek TG, Rabati S, Dekaris D, 2002. Double trouble: hemorrhagic fever with renal syndrome and leptospirosis. Scand J Infect Dis 34: 221–224.[Web of Science][Medline]-Medved M, Markoti A, Cebalo LJ, Turkovi B, Avi-upanc T, 2002. Hemorrhagic fever with renal syndrome in Croatia. Lancet 360: 415–416.[Web of Science][Medline] J, Stareina V, Slavica A, Riquelme-Sertour N, Bellenger E, Baranton G, Postic D, 2003. Molecular characterisation of Leptospira spp. strains isolated from small rodents in Croatia. Epidemiol Infect 130: 159–166.[Medline]i-upanc T, Nemirov K, Petrovec M, Trilar T, Poljak M, Vaheri A, Plyusnin A, 2000. Genetic analysis of wild-type Do-brava hantavirus in Slovenia: co-existence of two distinct genetic lineages within the same natural focus. J Gen Virol 81: 1747–1755. A, Nichol ST, Kuzman I, Sanchez AJ, Ksiazek TG, Gagro A, Rabati S, Zgorelec R, Avi-upanc T, Beus I, Dekaris D, 2002. Characteristics of Puumala and Dobrava infections in Croatia. J Med Virol 66: 542–551.[Web of Science][Medline]i B, Turkovi B, Aleraj B, Tvrtkovi N, 1991. Hemorrhagic fever with renal syndrome (HFRS) in Croatia. Incidence of human infections and wild animal reservoirs. Lijec Vjesn 113: 320–323.[Medline]i B, Puntari D, Turkovi B, Aleraj B, Tvrtkovi N, 1996. New natural focus of hemorrhagic fever with renal syndrome in Novska. Croat Med J 37: 115–118. N, Bori B, Turkovi B, Ivi I, Baki J, Erceg M, Tvrtkovi N, 2002. Dinara – new natural focus of hemorrhagic fever with renal syndrome in Croatia. Croat Med J 43: 576–580.[Web of Science][Medline]i B, Kovai H, Sebek Z, Aleraj B, Tvrtkovi N, 1982. Small terrestrial mammals as reservoir of leptospires in the Sava Valley (Croatia). Folia Parasitol 29: 177–182.[Medline]
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