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Spreading of West Nile virus infection in Croatia
Veterinary Microbiology 159 (2012) 504–508
Contents lists available at SciVerse ScienceDirect
Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Short communication
Spreading of West Nile virus infection in Croatia Ljubo Barbic´ a,*, Eddy Listesˇ b, Sanda Katic´ b, Vladimir Stevanovic´ a, Josip Madic´ a, Vilim Staresˇina a, Ankica Labrovic´ c, Annapia Di Gennaro d, Giovanni Savini d a
Faculty of Veterinary Medicine, University of Zagreb, Department of Microbiology and Infectious Diseases, Heinzelova 55, 10000 Zagreb, Croatia Croatian Veterinary Institute, Regional Veterinary Institute Split, Poljicˇka cesta 33, 21000 Split, Croatia c Veterinary Directorate, Ministry of Agriculture, Fisheries, and Rural Development of the Republic of Croatia, Ulica grada Vukovara 78, Zagreb, Croatia d Department of Virology, National Reference Center for West Nile disease, OIE Reference Laboratory for Bluetongue, Istituto G. Caporale Teramo, Teramo, Italy b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 30 January 2012 Received in revised form 20 April 2012 Accepted 30 April 2012
West Nile virus (WNV) is an emerging zoonotic pathogen with rapid global expansion. The virus circulation is confirmed in many countries of Mediterranean Basin and Southern and Central Europe. In our study detection of specific WNV antibodies was performed in horses and cattle sera samples collected from October 2010 to April 2011. Serum samples were randomly taken from different parts of Croatia and tested by IgG and IgM ELISA. Positive serological results were confirmed by virus neutralization assay (VN-assay) and plaque reduction neutralization test (PRNT). Results showed that WNV antibodies were present in 72 out of 2098 horse sera (3.43%) and 3 of 2695 cattle sera (0.11%). The highest seroprevalence was found in Eastern Croatia in counties next to Hungarian, Serbian and Bosnia and Herzegovinian state borders. In Adriatic part of Croatia positive animals were found only in the westernmost county, near Slovenian and Italian borders. Geographic distribution and number of positive horses indicated that WNV is highly present in Croatia and spreading from East to West. However, positive horses in westernmost part of country indicate possible second origin of spreading. Location of serological positive cattle supports the hypothesis that seropositive cattle could be indicators of high WNV activity in the respective geographic regions. ß 2012 Elsevier B.V. All rights reserved.
Keywords: West Nile Seroprevalence Spreading Horse Cattle Croatia
1. Introduction West Nile virus (WNV) is an arthropod-borne Flavivirus belonging to the Japanese encephalitis antigenic complex in the family Flaviviridae and is one of the most widely distributed encephalitic flaviviruses (Kramer et al., 2008). The virus is transmitted by mosquitoes (Culex genus mainly) and in its enzootic cycle primarily infects birds, which are reservoir hosts (Hubalek and Halouzka, 1999). WNV is affecting a wide range of other vertebrates, including reptiles, amphibians and mammals. In opposite, clinical manifestation of infection, except birds, generally occurs in humans and horses (Hayes et al., 2005;
* Corresponding author. Tel.: +385 1 2390 196; fax: +385 1 2390 214. E-mail address: [email protected] (L. Barbic´). 0378-1135/$ – see front matter ß 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetmic.2012.04.038
Castillo-Olivares and Wood, 2004). Human and horses are dead-end hosts because of their low grade of viremia (Komar, 2000). Virus pathogenicity could be influenced by several factors including host species and viral genotype (Petersen and Roehring, 2001; Solomon et al., 2003). Most of the isolates in Europe are grouped into lineage 1 within the European Mediterranean/Kenyan cluster however strains of different cluster (Israeli/American) and lineage (lineage 2) are found in Hungary, Austria and Italy (Bakonyi et al., 2006; Annon., 2008; Calistri et al., 2010; Zehender et al., 2011; Bagnarelli et al., 2011). Except for the surveillance of dead birds, the use of sentinel animals is the most common system of monitoring WNV circulation. Chickens and horses are commonly used as sentinel animals (Seidowski et al., 2010; Angelini et al., 2010). Because of their relatively high susceptibility
L. Barbic´ et al. / Veterinary Microbiology 159 (2012) 504–508
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and no signs of clinical disease were reported in the last 6 months. Serum samples from horses and cattle were examined for WNV IgG immunoglobulins using a commercial competitive enzyme-linked immunosorbent assay c-ELISA (ID screen West Nile competition ELISA kit, ID.VET, Montpellier, France). ELISA-positive samples were further tested by using the micro virus neutralization assay (VNassay) and plaque reduction neutralization test (PRNT). The PRNT titre was determined as the highest serum dilution with a 90% reduction of the number of plaques (OIE, 2008). Sera with a titre 1:10 were considered positive. Due to possibility of cross-reactions with other Flaviviruses, these samples were also tested for tick borne encephalitis virus (TBEV) by PRNT and Usutu virus by VN assay (Lelli et al., 2008). To determine the presence of possible recent infections the IgG-ELISA positive samples were also tested for WNV IgM by using commercial ELISA (IDEXX IgM WNV Ab Test, IDEXX-Istitut Pourquier, Montpellier, France). Differences in County’s seroprevalence rates were analysed by using the Fischer exact test.
to WNV infection (Castillo-Olivares and Wood, 2004), reports of equine cases might potentially provide early warning of corresponding WNV transmission in human population (Ward and Scheurmann, 2008; Laperriere et al., 2011). In the last decade many serological surveys have included cattle. Seroprevalence rates in cattle were ranging from 0.6 to 5.8%, but also absence of serological positive cattle even in outbreak regions was described (Samoilova et al., 2003; Ozkul et al., 2006; Jime´nez-Clavero et al., 2007; Papa et al., 2010). In Croatia WNV infection was reported in humans (Vesenjak-Hirjan et al., 1991), European brown bears (Madic´ et al., 1993) and horses (Madic´ et al., 2007). Despite infection confirmation and establishment of surveillance plan since 2010, until present time there is no reported bird mortality or human or equine clinical cases in Croatia due to WNV infection. Aim of our study was to determine the presence of West Nile virus in Croatia by testing horse and cattle sera from different parts of the country. 2. Materials and methods
3. Results Between October 2010 and April 2011 2098 horse and 2695 cattle sera were tested for WNV antibodies. The animals which were randomly selected, originated from all Croatian counties with the exception of Dubrovacˇko-neretvanska county. Criterion for selection of animals was that they were not moved in international transport or moved between counties. Age of animals was from eight months to 23 years. Horses included in this study had no WNV vaccination history. At the time of sampling all animals were asymptomatic
Out of 2098 horse sera samples 100 were IgG ELISA positive (Tables 1 and 2). Positive sera were further tested by confirmatory test PRNT. Out of 100 ELISA positive sera, 72 were PRNT positive. Specific antibodies titre range was from 1:10 to 1:160 (Table 2). Significant differences in titre were found between animals from the same county and even from the same stud farm. Positive animals were found in nine Croatian counties, eight of which are located in the Eastern and Central Croatia. The ninth county which
Table 1 Seroprevalence of West Nile virus infection in cattle and horses in Croatian counties (counties with evidence of WNV infection are shadowed). Croatian counties
Cattle sera Number of samples
Bjelovarsko-bilogorska Brodsko-posavska Dubrovacˇko-neretvanska Grad zagreb Istarska Karlovacˇka Koprivnicˇko-krizˇevacˇka Krapinsko-zagorska Licˇko-senjska Mepimurska Osjecˇko-baranjska Pozˇesˇko-slavonska Primorsko-goranska Sisacˇko-moslavacˇka Splitsko-dalmatinska Sˇibensko-kninska Varazˇdinska Viroviticˇko-podravska Vukovarsko-srijemska Zadarska Zagrebacˇka Total
Horse sera ELISA positive
PRNT VN-test confirmed
Seroprevalence (%)
Number of samples
207 276 0 0 107 56 126 57 46 131 310 234 30 149 127 38 37 135 135 73 421
0 2 0 0 0 0 0 1 1 0 3 0 0 0 0 0 0 0 0 0 0
0 1 0 0 0 0 0 0 0 0 2 0 0 0 0 0 0 0 0 0 0
0 0.36 0 0 0 0 0 0 0 0 0.65 0 0 0 0 0 0 0 0 0 0
53 213 0 16 108 8 4 1 4 0 500 22 21 563 12 0 0 26 119 2 426
2695
7
3
0.11
2098
ELISA positive
PRNT VN-test confirmed
Seroprevalence (%)
1 6 0 0 5 0 0 0 0 0 47 3 0 13 0 0 0 1 10 0 14
1 3 0 0 3 0 0 0 0 0 35 2 0 10 0 0 0 1 8 0 9
1.88 1.41 0 0 2.78 0 0 0 0 0 7.00 9.09 0 1.78 0 0 0 3.84 6.72 0 2.11
100
72
3.43
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Table 2 PRNT results of ELISA positive horse sera from nine Croatian counties with evidence of WNV activity. Croatian counties
ELISA positive
PRNT results Negative
Bjelovarsko-bilogorska Brodsko-posavska Istarska Osjecˇko-baranjska Pozˇesˇko-slavonska Sisacˇko-moslavacˇka Viroviticˇko-podravska Vukovarsko-srijemska Zagrebacˇka Total
Positive
Number of tested sera regarding titre 1:20 – 1 1 11 1 3 – 1 1
1:40 1 1 1 9 – 3 1 4 1
1:80 – – 1 7 – 1 – 2
1:160 – – – 3 – – – 1 1
19
21
11
5
1 6 5 47 3 13 1 10 14
0 3 2 12 1 3 0 2 5
1 3 3 35 2 10 1 8 9
1:10 – 1 – 5 1 3 – 2 4
100
28
72
16
evidenced animals with neutralising antibodies is located in the Western part of the country near to Slovenian and Italian borders (Fig. 1). All IgG ELISA positive samples were negative when tested by IgM ELISA. Also all ELISA positive samples were negative for TBEV and Usutu virus. The seroprevalence on state level was 3.43%. The highest seroprevalence was found in the Pozˇesˇko-slavonska county (9.09%) followed by the Osijecˇko-baranjska county (7.00%),
the Vukovarsko-srijemska county (6.72%) and the Viroviticˇko-podravska county (3.84%) (Table 1). Statistically significant higher seroprevalence was confirmed with Fisher exact test in Osijecˇko-baranjska county (p = 0.00036). Of 2695 tested cattle serum samples seven were WNV IgG ELISA positive. Out of these seven positive samples, using PRNT, WNV specific antibodies were confirmed in only three animals in Eastern part of Croatia (Fig. 1). Two of
Fig. 1. Seroprevalence of WNV in horses and geographic distribution of WNV serological positive cattle in Croatia (Croatian counties are marked as: 1, Osijecˇko-baranjska; 2, Vukovarsko-srijemska; 3, Pozˇesko-slavonska; 4, Viroviticˇko-podravska; 5, Brodsko-posavska; 6, Bjelovarsko-bilogorska; 7, Sisacˇkomoslavacˇka; 8, Zagrebacˇka; 9, Istarska).
L. Barbic´ et al. / Veterinary Microbiology 159 (2012) 504–508
them were located in Osijecˇko-baranjska county with antibody titre 1:10 and one in Brodsko-posavska county with antibody titre 1:20. All three animals were negative to TBEV and Usutu virus. Seroprevalence on state level was 0.11%. Seroprevalence in Osijecˇko-baranjska county was 0.65% and in Brodsko-posavska county 0.36% (Table 1). 4. Discussion In presented study seroprevalence of WNV in Croatia was determinate by testing of 2098 horse and 2695 cattle sera by ELISA test and confirmation of positive results with PRNT method. Out of 2098 horse sera ELISA positive were 100 and 72 were confirmed as positive with PRNT. A finding of 28% false positive results is in accordance with specificity of ELISA described in other studies (Soteloa et al., 2011; Barros et al., 2011). Also four out of seven cattle sera were false positive using ELISA. Results of this study confirmed presence of WNV infection in Croatia with seroprevalence in horses on state level of 3.43% what is around 8.5 times higher than seroprevalence between 2001 and 2002 (Madic´ et al., 2007). State level seroprevalence is generally in accordance with recent studies in other countries (Angelini et al., 2010; Papa et al., 2010; Barros et al., 2011). In the study performed 2001–2002 by Madic´ et al. (2007) all positive animals were located on one stud farm in Osijecˇko-baranjska county in Eastern part of Croatia with farm seroprevalence of 4.9%. In our study, on the same farm positive horses were detected with seroprevalence of 8.57% (data not showed). In opposite to former study, positive animals were found on different locations in Osijecˇko-baranjska county with overall seroprevalence of 7.0% on county level (Table 1). These result confirmed presence of WNV for at least 10 years and spreading of disease in county where seroprevalence was statistically higher than on the state level. Circulation of the WNV infection is also supported by five positive animals in mentioned part of country aged eight to eleven months which confirmed recent circulation of WNV and with significant differences between titre in animals from same counties (Table 2) and even in animals from the same stud farm. Absence of IgM positive animals could be explained by sampling season between end of October and April. Positive animals were also found in other counties in Eastern and Central part of Croatia which confirmed WNV spreading on state level. The highest seroprevalence were detected in Eastern counties and decline to central part of Croatia (Fig. 1). As all tested animals had not been moved in international transport or between counties, seroprevalence rates, on counties level, were taken as indicator of possible way of spreading of WNV in Croatia. There is a strong possibility of multiple introduction of WNV infection to eastern part of Croatia. Even so there is possibility of endemistaion of WNV in Croatia. Hypothesis of endemic cycle in Eastern part of Croatia is in accordance with endemisation of the infection in other countries in Mediterranean and Sothern European region (Calistri et al., 2010; Monaco et al., 2011). It is possible that Eastern part of Croatia is part of bigger endemic region. This sentence is supported by evidence of WNV continuing
507
circulation in Hungary, Romania and recently confirmation of WNV circulation in Serbia (Calistri et al., 2010; Lupulovic´ et al., 2011). The four counties, with seroprevalence rate higher than 3%, were located in Eastern Croatia, next to the state borders with Hungary and Serbia. Seroprevalence of 12% in horses in Northern Serbia (Lupulovic´ et al., 2011) is in accordance with decline of seroprevalence from Eastern to Central part of Croatia and implicate possible way of WNV spreading is in this part of Europe from east to west. Positive horses were also found in westernmost part of Croatia, apart from geographic region with confirmed WNV activity in Eastern and Central Croatia (Fig. 1). In previous study of Madic´ et al. (2007) all tested horse sera, from this region, were negative. This result could be in accordance with endemisation and overwintering of WNV infection in Italy (Calistri et al., 2010; Monaco et al., 2011) which implicate second way of WNV infection introduction and spreading in Croatia from second WNV endemic part of Europe. As co-circulation of different viral strain was demonstrated in Hungary (Bakonyi et al., 2006) and Italy (Savini et al., 2008; Monaco et al., 2011; Zehender et al., 2011; Bagnarelli et al., 2011) possible two ways of WNV spreading in Croatia must be further confirmed with molecular characterization of WNV isolates. In our WNV study, serological survey also included cattle from different parts of Croatia. Detected WNV seroprevalence based on randomly sampled 2695 tested cattle were 0.11% on state level. This seroprevalence is slightly lower than in other studies (Samoilova et al., 2003; Ozkul et al., 2006; Papa et al., 2010). Significant differences in WNV seroprevalence on state and counties level in cattle than in horses presented in this study is in accordance with study of other authors (Jime´nez-Clavero et al., 2007). Lower seroprevalence could be explained by risk factors as species dependent susceptibility and immunological response to infection and difference in husbandry between cattle and horses (Jime´nez-Clavero et al., 2007). All the positive cattle were detected in Eastern part of Croatia with possible endemic cycle and highest WNV seroprevalence in horses. This finding implicate that evidence of positive cattle could be indicator of high WNV activity in some geographic regions. In conclusions, WNV infection is widely spread in different regions in Croatia. Geographical distribution and different seroprevalence in horses indicating possibility of two different ways of WNV spreading in Croatia. Eastern Croatia could be a part of larger WNV endemic area in Europe. Also location where positive cattle were detected supports that including of cattle in surveillance system could be appropriate to measure level of viral activity in some region. Absence of reported bird mortality or human and equine clinical cases in Croatia despite high seroprevalence of WNV infection in horses could be explained by silent circulation of low virulence viral strain or unrecognizing of disease due to insufficient surveillance system. Conflicts of interest None.
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L. Barbic´ et al. / Veterinary Microbiology 159 (2012) 504–508
Acknowledgements Funding for this project was provided by Ministry of Agriculture, Fisheries and Rural Development of Republic of Croatia and by grants No. 053-0481153-1138 from the Ministry of Science, Education and Sports, Republic of Croatia. The authors thank to all the laboratory personnel in Croatia and Italy for their help during the study. References Angelini, P., Tamba, M., Finarelli, A.C., Bellini, R., Albieri, A., Bonilauri, P., Cavrini, F., Dottori, M., Gaibani, P., Martini, E., Mattivi, A., Pierro, A.M., Rugna, G., Sambri, V., Squintani, G., Macini, P., 2010. West Nile virus circulation in Emilia-Romagna, Italy: the integrated surveillance system 2009. Euro Surveill. 15 (16) pii=19547http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId=19547. Annon., 2008. World organization for Animal Health – OIE. West Nile fever, Austria. Immmediate Notification 2008. http://web.oie.int/ wahis/public.php?page=single_report&pop=1&reportid=7424. Bagnarelli, P., Marinelli, K., Trotta, D., Monachetti, A., Tavio, M., Del Gobbo, R., Capobianchi, M.R., Menzo, S., Nicoletti, L., Magurano, F., Varaldo, P.E., 2011. Human case of autochthonous West Nile virus lineage 2 infection in Italy September 2011. Euro Surveill. 16 (43) pii=20002In: http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId=20002. Bakonyi, T., Ivanics, E., Erdelyi, K., Ursu, K., Ferenczi, E., Weissenbock, H., Nowotny, N., 2006. Lineage 1 and 2 strains of encephalitic West Nile virus, Central Europe. Emerg. Infect. Dis. 12, 618–623. Barros, S.C., Ramos, F., Fagulha, T., Duarte, M., Henriques, M., Luı´s, T., Fevereiro, M., 2011. Serological evidence of West Nile virus circulation in Portugal. Vet. Microbiol. 152, 407–410. Calistri, P., Giovannini, A., Hubalek, Z., Ionescu, A., Monaco, F., Savini, G., Lelli, R., 2010. Epidemiology of West Nile in Europe and in the Mediterranean Basin. Open Virol. J. 4, 29–37. Castillo-Olivares, J., Wood, J., 2004. West Nile virus infection of horses. Vet. Res. 35, 467–483. Hayes, E.B., Sejvar, J.J., Zaki, S.R., Lanciotti, R.S., Bode, A.V., Campbell, G.L., 2005. Virology, pathology, and clinical manifestations of West Nile virus disease. Emerg. Infect. Dis. 11, 1174–1179. Hubalek, Z., Halouzka, J., 1999. West Nile fever – a reemerging mosquitoborne viral disease in Europe. Emerg. Infect. Dis. 5, 643–650. Jime´nez-Clavero, M.A., Tejedor, C.G., Rojo, G., Soriguer, R., Figuerola, J., 2007. Serosurvey of West Nile virus in equids and bovids in Spain. Vet. Rec. 161, 212. Komar, N., 2000. West Nile viral encephalitis. Rev. Sci. Tech. 19, 166–176. Kramer, L.D., Styer, L.M., Ebel, G.D., 2008. A global perspective on the epidemiology of West Nile virus. Annu. Rev. Entomol. 53, 61–81. Laperriere, V., Brugger, K., Rubel, F., 2011. Simulation of the seasonal cycles of bird, equine and human West Nile virus cases. Prev. Vet. Med. 98, 99–110. Lelli, R., Savini, G., Teodori, L., Filipponi, G., Di Gennaro, A., Leone, A., Di Gialleonardo, L., Venturi, L., Caporale, V., 2008. Serological evidence of Usutu virus occurrence in north-eastern Italy. Zoonoses Public Health 55, 361–367.
Lupulovic´, D., Martı´n-Acebes, M.A., Lazic, S., Alonso-Padilla, J., Bla´zquez, A.B., Escribano-Romero, E., Petrovic´, T., Saiz, J.C., 2011. First serological evidence of West Nile virus activity in horses in Serbia. Vector Borne Zoonotic Dis. 11, 1303–1305. Madic´, J., Huber, D., Lugovic´, B., 1993. Serological survey for selected viral and rickettsial agents of brown bears (Ursus arctos) in Croatia. J. Wildl. Dis. 29, 572–576. Madic´, J., Savini, G., Di Gennaro, A., Monaco, F., Jukic´, B., Kovacˇ, S., Rudan, N., Listesˇ, E., 2007. Serological evidence for West Nile Virus infection in horses in Croatia. Vet. Rec. 160, 772–773. Monaco, F., Savini, G., Calistri, P., Polci, C., Pinoni, C., Bruno, R., Lelli, R., 2011. West Nile disease epidemic in Italy: first evidence of overwintering in Europe? Res. Vet. Sci. 91, 321–326. World Organisation for Animal Health (Office International des E´pizooties: OIE), 2008. West Nile fever, Chapter 2.1.20.In: Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, OIE, Paris, France, pp. 377–385. Ozkul, A., Yildirim, Y., Pinar, D., Akcali, A., Yilmaz, V., Colak, D., 2006. Serological evidence of West Nile Virus (WNV) in mammalian species in Turkey. Epidemiol. Infect. 134, 826–829. Papa, A., Danis, K., Baka, A., Bakas, A., Dougas, G., Lytras, T., Theocharopoulos, G., Chrysagis, D., Vassiliadou, E., Kamaria, F., Liona, A., Mellou, K., Sarogiou, G., Panagiotopoulos, T., 2010. Ongoing outbreak of West Nile virus infections in humans in Greece. Euro Surveill. 15 (34) pii=19644In: http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId=19644. Petersen, L.R., Roehring, J.T., 2001. West Nile virus: a reemerging global pathogen. Emerg. Infect. Dis. 7, 611–614. Savini, G., Monaco, F., Calistri, P., Lelli, R., 2008. Phylogenetic analysis of West Nile virus isolated in Italy in 2008. Euro Surveill. 13 (48) pii=19048In: http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId=19048. Seidowski, D., Ziegler, U., von Ro¨nn, J.A., Mu¨ller, K., Hu¨ppop, K., Mu¨ller, T., Freuling, C., Mu¨hle, R.U., Nowotny, N., Ulrich, R.G., Niedrig, M., Groschup, M.H., 2010. West Nile virus monitoring of migratory and resident birds in Germany. Vector Borne Zoonotic Dis. 10, 639–647. Samoilova, T.I., Votiakov, V.I., Titov, L.P., 2003. Virologic and serologic investigations of West Nile circulation in Belarus. Cent. Eur. J. Public Health 11, 55–62. Solomon, T., Ooi, M.H., Beasley, D.W., Mallewa, M., 2003. West Nile encephalitis. BMJ 326, 865–869. ˜ asb, A., Venteob, A., Carmina, Soteloa, E., Llorentea, F., Rebollob, B., Camun G., Lubisic, A., Rodrı´guez, M.J., Sanzb, A.J., Figuerolad, J., Jime´nezClavero, M.A., 2011. Development and evaluation of a new epitope-blocking ELISA for universal detection of antibodies to West Nile virus. J. Virol. Methods 174, 35–41. Vesenjak-Hirjan, J., Punda-Polic´, V., Dobec, M., 1991. Geographical distribution of arboviruses in Yugoslavia. J. Hyg. Epidemiol. Microbiol. Immunol. 35, 129–140. Ward, M.P., Scheurmann, J.A., 2008. The relationship between equine and human West Nile virus disease occurrence. Vet. Microbiol. 129, 378–383. Zehender, G., Ebranati, E., Bernini, F., Lo Presti, A., Rezza, G., Delogu, M., Galli, M., Ciccozzi, M., 2011. Phylogeography and epidemiological history of West Nile virus genotype 1a in Europe and the Mediterranean basin. Infect. Genet. Evol. 11, 646–653.
Contents lists available at SciVerse ScienceDirect
Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Short communication
Spreading of West Nile virus infection in Croatia Ljubo Barbic´ a,*, Eddy Listesˇ b, Sanda Katic´ b, Vladimir Stevanovic´ a, Josip Madic´ a, Vilim Staresˇina a, Ankica Labrovic´ c, Annapia Di Gennaro d, Giovanni Savini d a
Faculty of Veterinary Medicine, University of Zagreb, Department of Microbiology and Infectious Diseases, Heinzelova 55, 10000 Zagreb, Croatia Croatian Veterinary Institute, Regional Veterinary Institute Split, Poljicˇka cesta 33, 21000 Split, Croatia c Veterinary Directorate, Ministry of Agriculture, Fisheries, and Rural Development of the Republic of Croatia, Ulica grada Vukovara 78, Zagreb, Croatia d Department of Virology, National Reference Center for West Nile disease, OIE Reference Laboratory for Bluetongue, Istituto G. Caporale Teramo, Teramo, Italy b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 30 January 2012 Received in revised form 20 April 2012 Accepted 30 April 2012
West Nile virus (WNV) is an emerging zoonotic pathogen with rapid global expansion. The virus circulation is confirmed in many countries of Mediterranean Basin and Southern and Central Europe. In our study detection of specific WNV antibodies was performed in horses and cattle sera samples collected from October 2010 to April 2011. Serum samples were randomly taken from different parts of Croatia and tested by IgG and IgM ELISA. Positive serological results were confirmed by virus neutralization assay (VN-assay) and plaque reduction neutralization test (PRNT). Results showed that WNV antibodies were present in 72 out of 2098 horse sera (3.43%) and 3 of 2695 cattle sera (0.11%). The highest seroprevalence was found in Eastern Croatia in counties next to Hungarian, Serbian and Bosnia and Herzegovinian state borders. In Adriatic part of Croatia positive animals were found only in the westernmost county, near Slovenian and Italian borders. Geographic distribution and number of positive horses indicated that WNV is highly present in Croatia and spreading from East to West. However, positive horses in westernmost part of country indicate possible second origin of spreading. Location of serological positive cattle supports the hypothesis that seropositive cattle could be indicators of high WNV activity in the respective geographic regions. ß 2012 Elsevier B.V. All rights reserved.
Keywords: West Nile Seroprevalence Spreading Horse Cattle Croatia
1. Introduction West Nile virus (WNV) is an arthropod-borne Flavivirus belonging to the Japanese encephalitis antigenic complex in the family Flaviviridae and is one of the most widely distributed encephalitic flaviviruses (Kramer et al., 2008). The virus is transmitted by mosquitoes (Culex genus mainly) and in its enzootic cycle primarily infects birds, which are reservoir hosts (Hubalek and Halouzka, 1999). WNV is affecting a wide range of other vertebrates, including reptiles, amphibians and mammals. In opposite, clinical manifestation of infection, except birds, generally occurs in humans and horses (Hayes et al., 2005;
* Corresponding author. Tel.: +385 1 2390 196; fax: +385 1 2390 214. E-mail address: [email protected] (L. Barbic´). 0378-1135/$ – see front matter ß 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetmic.2012.04.038
Castillo-Olivares and Wood, 2004). Human and horses are dead-end hosts because of their low grade of viremia (Komar, 2000). Virus pathogenicity could be influenced by several factors including host species and viral genotype (Petersen and Roehring, 2001; Solomon et al., 2003). Most of the isolates in Europe are grouped into lineage 1 within the European Mediterranean/Kenyan cluster however strains of different cluster (Israeli/American) and lineage (lineage 2) are found in Hungary, Austria and Italy (Bakonyi et al., 2006; Annon., 2008; Calistri et al., 2010; Zehender et al., 2011; Bagnarelli et al., 2011). Except for the surveillance of dead birds, the use of sentinel animals is the most common system of monitoring WNV circulation. Chickens and horses are commonly used as sentinel animals (Seidowski et al., 2010; Angelini et al., 2010). Because of their relatively high susceptibility
L. Barbic´ et al. / Veterinary Microbiology 159 (2012) 504–508
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and no signs of clinical disease were reported in the last 6 months. Serum samples from horses and cattle were examined for WNV IgG immunoglobulins using a commercial competitive enzyme-linked immunosorbent assay c-ELISA (ID screen West Nile competition ELISA kit, ID.VET, Montpellier, France). ELISA-positive samples were further tested by using the micro virus neutralization assay (VNassay) and plaque reduction neutralization test (PRNT). The PRNT titre was determined as the highest serum dilution with a 90% reduction of the number of plaques (OIE, 2008). Sera with a titre 1:10 were considered positive. Due to possibility of cross-reactions with other Flaviviruses, these samples were also tested for tick borne encephalitis virus (TBEV) by PRNT and Usutu virus by VN assay (Lelli et al., 2008). To determine the presence of possible recent infections the IgG-ELISA positive samples were also tested for WNV IgM by using commercial ELISA (IDEXX IgM WNV Ab Test, IDEXX-Istitut Pourquier, Montpellier, France). Differences in County’s seroprevalence rates were analysed by using the Fischer exact test.
to WNV infection (Castillo-Olivares and Wood, 2004), reports of equine cases might potentially provide early warning of corresponding WNV transmission in human population (Ward and Scheurmann, 2008; Laperriere et al., 2011). In the last decade many serological surveys have included cattle. Seroprevalence rates in cattle were ranging from 0.6 to 5.8%, but also absence of serological positive cattle even in outbreak regions was described (Samoilova et al., 2003; Ozkul et al., 2006; Jime´nez-Clavero et al., 2007; Papa et al., 2010). In Croatia WNV infection was reported in humans (Vesenjak-Hirjan et al., 1991), European brown bears (Madic´ et al., 1993) and horses (Madic´ et al., 2007). Despite infection confirmation and establishment of surveillance plan since 2010, until present time there is no reported bird mortality or human or equine clinical cases in Croatia due to WNV infection. Aim of our study was to determine the presence of West Nile virus in Croatia by testing horse and cattle sera from different parts of the country. 2. Materials and methods
3. Results Between October 2010 and April 2011 2098 horse and 2695 cattle sera were tested for WNV antibodies. The animals which were randomly selected, originated from all Croatian counties with the exception of Dubrovacˇko-neretvanska county. Criterion for selection of animals was that they were not moved in international transport or moved between counties. Age of animals was from eight months to 23 years. Horses included in this study had no WNV vaccination history. At the time of sampling all animals were asymptomatic
Out of 2098 horse sera samples 100 were IgG ELISA positive (Tables 1 and 2). Positive sera were further tested by confirmatory test PRNT. Out of 100 ELISA positive sera, 72 were PRNT positive. Specific antibodies titre range was from 1:10 to 1:160 (Table 2). Significant differences in titre were found between animals from the same county and even from the same stud farm. Positive animals were found in nine Croatian counties, eight of which are located in the Eastern and Central Croatia. The ninth county which
Table 1 Seroprevalence of West Nile virus infection in cattle and horses in Croatian counties (counties with evidence of WNV infection are shadowed). Croatian counties
Cattle sera Number of samples
Bjelovarsko-bilogorska Brodsko-posavska Dubrovacˇko-neretvanska Grad zagreb Istarska Karlovacˇka Koprivnicˇko-krizˇevacˇka Krapinsko-zagorska Licˇko-senjska Mepimurska Osjecˇko-baranjska Pozˇesˇko-slavonska Primorsko-goranska Sisacˇko-moslavacˇka Splitsko-dalmatinska Sˇibensko-kninska Varazˇdinska Viroviticˇko-podravska Vukovarsko-srijemska Zadarska Zagrebacˇka Total
Horse sera ELISA positive
PRNT VN-test confirmed
Seroprevalence (%)
Number of samples
207 276 0 0 107 56 126 57 46 131 310 234 30 149 127 38 37 135 135 73 421
0 2 0 0 0 0 0 1 1 0 3 0 0 0 0 0 0 0 0 0 0
0 1 0 0 0 0 0 0 0 0 2 0 0 0 0 0 0 0 0 0 0
0 0.36 0 0 0 0 0 0 0 0 0.65 0 0 0 0 0 0 0 0 0 0
53 213 0 16 108 8 4 1 4 0 500 22 21 563 12 0 0 26 119 2 426
2695
7
3
0.11
2098
ELISA positive
PRNT VN-test confirmed
Seroprevalence (%)
1 6 0 0 5 0 0 0 0 0 47 3 0 13 0 0 0 1 10 0 14
1 3 0 0 3 0 0 0 0 0 35 2 0 10 0 0 0 1 8 0 9
1.88 1.41 0 0 2.78 0 0 0 0 0 7.00 9.09 0 1.78 0 0 0 3.84 6.72 0 2.11
100
72
3.43
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Table 2 PRNT results of ELISA positive horse sera from nine Croatian counties with evidence of WNV activity. Croatian counties
ELISA positive
PRNT results Negative
Bjelovarsko-bilogorska Brodsko-posavska Istarska Osjecˇko-baranjska Pozˇesˇko-slavonska Sisacˇko-moslavacˇka Viroviticˇko-podravska Vukovarsko-srijemska Zagrebacˇka Total
Positive
Number of tested sera regarding titre 1:20 – 1 1 11 1 3 – 1 1
1:40 1 1 1 9 – 3 1 4 1
1:80 – – 1 7 – 1 – 2
1:160 – – – 3 – – – 1 1
19
21
11
5
1 6 5 47 3 13 1 10 14
0 3 2 12 1 3 0 2 5
1 3 3 35 2 10 1 8 9
1:10 – 1 – 5 1 3 – 2 4
100
28
72
16
evidenced animals with neutralising antibodies is located in the Western part of the country near to Slovenian and Italian borders (Fig. 1). All IgG ELISA positive samples were negative when tested by IgM ELISA. Also all ELISA positive samples were negative for TBEV and Usutu virus. The seroprevalence on state level was 3.43%. The highest seroprevalence was found in the Pozˇesˇko-slavonska county (9.09%) followed by the Osijecˇko-baranjska county (7.00%),
the Vukovarsko-srijemska county (6.72%) and the Viroviticˇko-podravska county (3.84%) (Table 1). Statistically significant higher seroprevalence was confirmed with Fisher exact test in Osijecˇko-baranjska county (p = 0.00036). Of 2695 tested cattle serum samples seven were WNV IgG ELISA positive. Out of these seven positive samples, using PRNT, WNV specific antibodies were confirmed in only three animals in Eastern part of Croatia (Fig. 1). Two of
Fig. 1. Seroprevalence of WNV in horses and geographic distribution of WNV serological positive cattle in Croatia (Croatian counties are marked as: 1, Osijecˇko-baranjska; 2, Vukovarsko-srijemska; 3, Pozˇesko-slavonska; 4, Viroviticˇko-podravska; 5, Brodsko-posavska; 6, Bjelovarsko-bilogorska; 7, Sisacˇkomoslavacˇka; 8, Zagrebacˇka; 9, Istarska).
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them were located in Osijecˇko-baranjska county with antibody titre 1:10 and one in Brodsko-posavska county with antibody titre 1:20. All three animals were negative to TBEV and Usutu virus. Seroprevalence on state level was 0.11%. Seroprevalence in Osijecˇko-baranjska county was 0.65% and in Brodsko-posavska county 0.36% (Table 1). 4. Discussion In presented study seroprevalence of WNV in Croatia was determinate by testing of 2098 horse and 2695 cattle sera by ELISA test and confirmation of positive results with PRNT method. Out of 2098 horse sera ELISA positive were 100 and 72 were confirmed as positive with PRNT. A finding of 28% false positive results is in accordance with specificity of ELISA described in other studies (Soteloa et al., 2011; Barros et al., 2011). Also four out of seven cattle sera were false positive using ELISA. Results of this study confirmed presence of WNV infection in Croatia with seroprevalence in horses on state level of 3.43% what is around 8.5 times higher than seroprevalence between 2001 and 2002 (Madic´ et al., 2007). State level seroprevalence is generally in accordance with recent studies in other countries (Angelini et al., 2010; Papa et al., 2010; Barros et al., 2011). In the study performed 2001–2002 by Madic´ et al. (2007) all positive animals were located on one stud farm in Osijecˇko-baranjska county in Eastern part of Croatia with farm seroprevalence of 4.9%. In our study, on the same farm positive horses were detected with seroprevalence of 8.57% (data not showed). In opposite to former study, positive animals were found on different locations in Osijecˇko-baranjska county with overall seroprevalence of 7.0% on county level (Table 1). These result confirmed presence of WNV for at least 10 years and spreading of disease in county where seroprevalence was statistically higher than on the state level. Circulation of the WNV infection is also supported by five positive animals in mentioned part of country aged eight to eleven months which confirmed recent circulation of WNV and with significant differences between titre in animals from same counties (Table 2) and even in animals from the same stud farm. Absence of IgM positive animals could be explained by sampling season between end of October and April. Positive animals were also found in other counties in Eastern and Central part of Croatia which confirmed WNV spreading on state level. The highest seroprevalence were detected in Eastern counties and decline to central part of Croatia (Fig. 1). As all tested animals had not been moved in international transport or between counties, seroprevalence rates, on counties level, were taken as indicator of possible way of spreading of WNV in Croatia. There is a strong possibility of multiple introduction of WNV infection to eastern part of Croatia. Even so there is possibility of endemistaion of WNV in Croatia. Hypothesis of endemic cycle in Eastern part of Croatia is in accordance with endemisation of the infection in other countries in Mediterranean and Sothern European region (Calistri et al., 2010; Monaco et al., 2011). It is possible that Eastern part of Croatia is part of bigger endemic region. This sentence is supported by evidence of WNV continuing
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circulation in Hungary, Romania and recently confirmation of WNV circulation in Serbia (Calistri et al., 2010; Lupulovic´ et al., 2011). The four counties, with seroprevalence rate higher than 3%, were located in Eastern Croatia, next to the state borders with Hungary and Serbia. Seroprevalence of 12% in horses in Northern Serbia (Lupulovic´ et al., 2011) is in accordance with decline of seroprevalence from Eastern to Central part of Croatia and implicate possible way of WNV spreading is in this part of Europe from east to west. Positive horses were also found in westernmost part of Croatia, apart from geographic region with confirmed WNV activity in Eastern and Central Croatia (Fig. 1). In previous study of Madic´ et al. (2007) all tested horse sera, from this region, were negative. This result could be in accordance with endemisation and overwintering of WNV infection in Italy (Calistri et al., 2010; Monaco et al., 2011) which implicate second way of WNV infection introduction and spreading in Croatia from second WNV endemic part of Europe. As co-circulation of different viral strain was demonstrated in Hungary (Bakonyi et al., 2006) and Italy (Savini et al., 2008; Monaco et al., 2011; Zehender et al., 2011; Bagnarelli et al., 2011) possible two ways of WNV spreading in Croatia must be further confirmed with molecular characterization of WNV isolates. In our WNV study, serological survey also included cattle from different parts of Croatia. Detected WNV seroprevalence based on randomly sampled 2695 tested cattle were 0.11% on state level. This seroprevalence is slightly lower than in other studies (Samoilova et al., 2003; Ozkul et al., 2006; Papa et al., 2010). Significant differences in WNV seroprevalence on state and counties level in cattle than in horses presented in this study is in accordance with study of other authors (Jime´nez-Clavero et al., 2007). Lower seroprevalence could be explained by risk factors as species dependent susceptibility and immunological response to infection and difference in husbandry between cattle and horses (Jime´nez-Clavero et al., 2007). All the positive cattle were detected in Eastern part of Croatia with possible endemic cycle and highest WNV seroprevalence in horses. This finding implicate that evidence of positive cattle could be indicator of high WNV activity in some geographic regions. In conclusions, WNV infection is widely spread in different regions in Croatia. Geographical distribution and different seroprevalence in horses indicating possibility of two different ways of WNV spreading in Croatia. Eastern Croatia could be a part of larger WNV endemic area in Europe. Also location where positive cattle were detected supports that including of cattle in surveillance system could be appropriate to measure level of viral activity in some region. Absence of reported bird mortality or human and equine clinical cases in Croatia despite high seroprevalence of WNV infection in horses could be explained by silent circulation of low virulence viral strain or unrecognizing of disease due to insufficient surveillance system. Conflicts of interest None.
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