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Sero-epidemiological study of the presence of hantaviruses in domestic dogs and cats from Belgium
Research in Veterinary Science 92 (2012) 221–224
Contents lists available at ScienceDirect
Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
Sero-epidemiological study of the presence of hantaviruses in domestic dogs and cats from Belgium A. Dobly ⇑, C. Cochez, E. Goossens, H. De Bosschere, P. Hansen, S. Roels, P. Heyman Research Laboratory for Vector-Borne Diseases, Queen Astrid Military Hospital, Bruynstraat, 1120 Brussels, Belgium, and Veterinary and Agrochemical Research Centre, Groeselenberg 99, 1180 Brussels, Belgium
a r t i c l e
i n f o
Article history: Received 22 January 2009 Accepted 9 February 2011
Keywords: Hantavirus Felis catus Canis familiaris Rodent Distribution
a b s t r a c t Hantaviruses are worldwide rodent-borne pathogens infecting humans and other animals mainly through inhalation of aerosols contaminated with rodent excreta. Few data are available on hantavirus serology and geographical distribution in dogs and cats. We therefore screened sera from pet dogs (N = 410) and cats (N = 124) in two regions of Belgium, using IgG ELISA and IFA. We analysed the effect of the owner’s address as well as pet gender and age on hantavirus status. Hantavirus antibodies were found in both species with a significantly higher seroprevalence in cats than in dogs (16.9% vs. 4.9%, P = 0.001). More dogs were infected in highly forested southern Belgium (harbouring more rodents) than in northern Belgium (10.5% vs. 3.0%, P = 0.002). In the south, hantavirus sero-positive cats were found in more densely forested localities than sero-negatives ones were (P = 0.033). These results are consistent with the ecological variations of hantavirus risks in humans. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Emerging infectious diseases represent an increasing concern worldwide (Jones et al., 2008). The screening for zoonotic pathogens in potential animal hosts and in domestic animals is a crucial step to be prepared for new emerging threats (Zeier et al., 2005). Pets are indeed a key issue as they are in close proximity to humans and so numerous. In 2004 in Belgium, the 10 106 inhabitants possessed more than 1 106 dogs and 2 106 cats (http:// statbel.fgov.be). These domestic dogs and cats can repeatedly be in contact with rodents or rodent contaminated areas, potential sources of hantaviruses. Rodent-borne hantaviruses belong to the Bunyaviridae family. They are carried worldwide by Muroid rodents (and insectivores, Arai et al., 2008. Each hantavirus is mainly associated with a distinct reservoir species but spillover infections in other rodent species are reported (Klingström et al., 2002). Bites represent one important means of intraspecific transmission, at least in rats (Glass et al., 1988). However indirect transmission is not negligible as, under the optimal conditions, Puumala hantavirus (PUUV) can survive for up to 2 weeks outside of its host (Kallio et al., 2006). In the natural reservoirs, hantaviruses establish a persistent infection, and the host develops a strong neutralizing antibody response
⇑ Corresponding author. Address: Interactions and Surveillance, Veterinary and Agrochemical Research Centre, Groeselenberg 99, 1180 Brussels, Belgium. Fax: +32 23790479. E-mail address: [email protected] (A. Dobly). 0034-5288/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2011.02.003
(Chu et al., 1994). Yet few detrimental effects have been described in the reservoir species (Kallio et al., 2007). Despite this, more than a third of the 43 reported serotypes can cause disease in humans: hemorrhagic fever with renal syndrome (HFRS) in Europe and Asia, and hantavirus pulmonary syndrome on the American continent (Lee and van der Groen, 1989). HFRS is caused by eight serotypes. Its symptoms include fever, abdominal and back pain as well as renal dysfunction, sometimes with hemorrhagic manifestations (Vapalahti et al., 2003). Nephropathia epidemica (NE), the milder form of HFRS, caused by PUUV hantavirus, is present in North-Western Europe (including Belgium). Humans are infected by inhalation of aerosols contaminated with excreta from infected rodents. Worldwide, about 150,000–200,000 people are annually hospitalised for HFRS, mainly in Asia (Vapalahti et al., 2003). In spite of this incidence, little is known about the presence of hantavirus in non-rodent mammals. Apart from insectivores, hantaviruses are known in chiropters (bats, Kim et al., 1994) and artiodactyls (even-toed ungulates, Ahlm et al., 2000). In 20 species of non-human primates, experimental infection leads to an antibody reaction (Yanagihara et al., 1988). As for carnivores, Felis catus (cat) was found positive (2.9–9.6%, Xu et al., 1987; Nowotny, 1994; Leighton et al., 2001) as well as Canis familiaris (dog, 4.7%, Malecki et al., 1998) and Vulpes vulpes (red fox, 2.4%, Escutenaire et al., 2000b). Non-rodent infections seem to remain rare as several studies report no infection: 2400 mammals, including dogs and cats (Groen et al., 1995); various mammal species (Jay et al., 1997). However Hantaan (HTNV) serotype was detected in a bird (Dzagurova et al., 1995).
222
A. Dobly et al. / Research in Veterinary Science 92 (2012) 221–224
The presence of hantavirus in dogs or cats has never been reported in Belgium. This study first supposed that antibodies against hantavirus were detectable in dog and cat sera in Belgium. In the case of a positive result, this work would test two subsequent hypotheses. Firstly it is known that human PUUV-seroprevalence is linked to the quantity of forests, a good indicator of bank vole abundance, the PUUV reservoir (Heyman et al., 2002). Will the geographical pattern of hantavirus in dogs and cats be the same than in humans? We therefore first hypothesised that a higher hantavirus seroprevalence would be observed in pets from forested southern Belgium. Secondly, in bank voles, hantavirus presence increases with the age of the rodents (Escutenaire et al., 2000a). Will this age effect observed in companion animals? We hypothesised higher seroprevalence in older pets. 2. Materials and methods We retrospectively tested 534 pet sera (410 dogs and 124 cats), initially sent to private laboratories for routine veterinary analyses. We favoured samples from two regions isolated from each other: north-west Belgium (hereafter called northern Belgium), known to harbour few cases of human hantavirus infections, and southeast Belgium (called southern Belgium), location of numerous human hantavirus infections. For 360 dogs and 120 cats, the owner address, pet’s age and/or gender were available. We compared our results to the number of human hantavirus infections in Belgium in 2003 (data from the Scientific Institute of Public Health: www.iph.fgov.be/epidemio/labo). We analysed the sera using an IgG PUUV and HTNV enzymelinked immunosorbent assay (ELISA: Progen, Heidelberg, Germany). Samples that proved to be positive as well as an equal number of randomised samples that tested negative were tested again using an immuno-fluorescent assay (IFA-IgG) directed against various hantaviruses including PUUV (Euroimmun, Lubeck, Germany). We followed the manufacturer’s instructions. The sensitivity and the specificity of both tests are 97–99.5%. It should be noted that serological testing does not enable the definite identification of the infecting hantaviral serotype due to cross-reactions between serotypes. As, to our knowledge, positive and negative control sera for dogs and cats are not available, the cut-off (CO) for ELISA was calculated as in previous studies (e.g. Heyman et al., 2002: average optical density (OD) of 10 known negative sera plus 3 times their standard deviation. We compared series by calculating the ratio: observed OD/CO. A sample was considered as positive if this ratio was at least to 1.5. Ratios between 1.0 and 1.5 were considered ‘‘grey zone’’ values. This avoided overestimating the prevalence. All statistical tests were two-tailed with a level of significance at 0.05 except when indicated otherwise (e.g. age effect on seroprevalence because of our initial hypothesis). We used nonparametric statistics when normality was not expected. 3. Results We detected hantavirus antibodies in both regions with a higher seroprevalence in cats than in dogs (16.9% vs. 4.9%, Mann– Whitney U test, N = 534, P = 0.0012, Fig. 1 and Table 1). In IFA, all samples showed a higher titre for PUUV (cross-reacting with Tula, TULV) than for Seoul (SEOV). For both species, the average ELISA ratios were higher in more forested south than in northern Belgium (Mann–Whitney U test: dogs, 0.838 vs. 0.581, N = 410, P = 0.000; cats, 1.005 vs. 0.899, N = 124, P = 0.010). Moreover more dogs were infected in southern Belgium than in northern Belgium (10.5% vs. 3.0%, Mann–Whitney U test, N = 410, P = 0.002, Table 1). This geographical difference in dog seroprevalence is consistent
with the number of human cases reported in 2003 (North: 1.9 vs. South: 30.0 individuals per million). Moreover the cat prevalence was influenced by the forest cover in each studied administrative localities (http://www.statbel.fgov.be). Indeed, in the South, PUUV sero-positive cats were found in more densely forested localities than PUUV sero-negative ones (30.5% vs. 15.2%, one-tailed Mann–Whitney U test, N = 52, P = 0.033). We did not observe such a relationship for dogs. The mean pets’ age was 8.6 years for dogs (SD = 3.7, N = 105) and 7.7 years for cats (SD = 5.2, N = 90, Table 1). The dogs’ age was not linked to the serological status towards hantavirus (onetailed two-sample T test, N = 105, P = 0.19). Older cat’s showed an almost-significant tendency of higher seroprevalence (N = 90, P = 0.065, Table 1). Finally, in both species, we observed a non-significant tendency to have higher seroprevalence in females than in males (Table 1).
4. Discussion and conclusions This study showed that domestic carnivores are susceptible to hantavirus infection in Belgium. This is consistent with the airborne infection route and the pets’ behaviour. According to our test results, the observed hantavirus probably correspond to the Puumala serotype. However Tula and Seoul serotypes cannot be ruled out. Indeed only neutralization tests are able to clearly identify the causal serotype. Neutralization tests however are difficult; they require a BSL3 environment and an extensive hantavirus library in culture. Only a few labs worldwide are able to perform such tests and for this study we decided not to seek identification as in Belgium PUUV, TULV and SEOV circulate (Heyman et al., 2002). It is not known whether carnivores develop any hantavirus disease but low infection rates in dogs and cats suggest dead-end infections, as with humans. In humans, hantaviral RNA is only present during the few days of the acute phase of the disease (Pettersson et al., 2008). We can hypothesise that, as with humans, carnivores neutralise hantaviruses shortly after infection. This very short viraemia hampers in most cases virus isolation from the serum. It is important to mention that our study involves a bias as all tested pets were sampled in veterinary laboratories for a health problem or a routine health check. This absence of randomised sampling is difficult to avoid and is typical in such epidemiological studies. We can indeed compare our results to those of previous works. We observed a seroprevalence of 4.9% in dogs and 16.9% in cats. This is similar to what was previously reported for dogs in America (4.7%, Malecki et al., 1998 but our results concerning cats were higher than any previous observations (2.9–9.6%, China, Austria and Canada: (Xu et al., 1987; Nowotny, 1994; Leighton et al., 2001). The difference in cats cannot be attributed to our method, as we defined a high cut-off for being positive. The high cat seroprevalence could be partly explained by a bank vole demographic peak in 2005, accompanied by a steep increase of the number of human cases (Heyman et al., 2007). Finally, it is interesting to note that, in one study, 23% of chronically ill cats in the UK were found to be hantavirus-seropositive (Bennett et al., 1990). Nevertheless, the higher dog PUUV-seroprevalence observed in southern than in northern Belgium allowed us to demonstrate that the same geographical difference exists in dogs and in humans. The higher hantavirus infection rate of cats compared to dogs can be linked to the cats’ specialisation in catching small rodents (30% of domestic cat preys (Woods et al., 2003). This is consistent with the fact that sero-positive cats from southern Belgium are found in more forested environments than sero-negative ones. Contrary to what is observed in the rodent reservoirs (Escutenaire et al., 2000a), we did not detect an age-dependency
A. Dobly et al. / Research in Veterinary Science 92 (2012) 221–224
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Fig. 1. Belgium maps with the number of (a) dogs and (b) cats tested in this study per administrative locality. The legend gives the category ranges and, in brackets, the numbers of animals in each category. The positive animals are indicated by circles.
in pets’ seroprevalence (only a tendency of higher seroprevalence in older cats). It is possible that the high age of tested pets in comparison to small rodents had masked an age effect. About the gender difference, the PUUV reservoir shows a higher seroprevalence in males than in females (Escutenaire et al.,
2000a). This is explained by the very different life histories of males and (territorial) females, which is not the case in pets. However, if a gender difference exists in hantavirus prevalence within domestic carnivores, it seems to favour infection in females. This is surprising. However, female cats might be more
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A. Dobly et al. / Research in Veterinary Science 92 (2012) 221–224
Table 1 Hantavirus seroprevalence in dogs and cats according to region of origin, age and gender. North Belgium
Dogs
N
%
305
Cats
77
3.0 ⁄ 18.2
Total
382
6.0
⁄
South Belgium
Belgium
Young
Old
N
%
N
%
N
%
105
410
18.4
124
4.9 ⁄ 16.9
49
47
10.5 ⁄ 14.9
43
16.3
47
27.7
36
16.7
33
30.3
152
11.8
534
7.7
92
17.4
103
18.4
68
13.2
86
22.1
N 56
Males %
N
10.7
32
Females % 9.4
N
%
53
17.0
N = number tested, % = seroprevalence, which was compared with Mann–Whitney U tests (⁄P < 0.002). Dogs were considered old from 9 years old; cats were considered old from 7 years old.
exposed to hantavirus infection because of their higher hunting activities linked to pregnancy. This survey first showed that domestic carnivores in Belgium are infected by hantavirus. Furthermore this work demonstrated that, in dogs, the seroprevalence was not uniform but was higher in forested southern Belgium than in northern part of the country. This pattern is similar to what is observed in humans and can be explained by the presence of more rodents in the forested South. This finding stresses the importance of ecological factors on hantavirus prevalence in domestic species, as observed in humans. Acknowledgements No conflict of interest existed. Thanks are due to Flavien Riocreux for the maps, to Geneviève Ducoffre for the human data and to Kato Van Wassenhove for her technical help. The laboratory testing was sponsored by the Belgian Ministry of Defence. References Ahlm, C., Wallin, K., Lundkvist, A., Elgh, F., Juto, P., Merza, M., Tärnvik, A., 2000. Serologic evidence of Puumala virus infection in wild moose in northern Sweden. American Journal of Tropical Medicine and Hygiene 62, 106–111. Arai, S., Bennett, S.N., Sumibcay, L., Cook, J.A., Song, J.-W., Hope, A., Parmenter, C., Nerurkar, V.R., Yates, T.L., Yanagihara, R., 2008. Phylogenetically Distinct Hantaviruses in the Masked Shrew (Sorex cinereus) and Dusky Shrew (Sorex monticolus) in the United States. American Journal of Tropical Medicine and Hygiene 78, 348–351. Bennett, M., Lloyd, G., Jones, N., Brown, A., Trees, A.J., McCracken, C., Smyth, N.R., Gakell, C.J., Gaskell, R.M., 1990. Prevalence of antibody to hantavirus in some cat populations in Britain. Veterinary Record 127, 548–549. Chu, Y.K., Rossi, C., LeDuc, J.W., Lee, H.W., Schmaljohn, C.S., Dalrymple, J.M., 1994. Serological relationships among viruses in the Hantavirus genus, family Bunyaviridae. Virology 198, 196–204. Dzagurova, T., Tkachenko, E., Slonova, R., Ivanov, L., Ivanidze, E., Markeshin, S., Dekonenko, A., Niklasson, B., Lundkvist, Å., 1995. Antigenic relationships of hantavirus strains analysed by monoclonal antibodies. Archives of Virology 140, 1763–1773. Escutenaire, S., Chalon, P., Verhagen, R., Heyman, P., Thomas, I., Karelle-Bui, L., Avsic-Zupanc, T., Lundkvist, Å., Plyusnin, A., Pastoret, P.P., 2000a. Spatial and temporal dynamics of Puumala hantavirus infection in red bank vole (Clethrionomys glareolus) populations in Belgium. Virus Research 67, 91–107. Escutenaire, S., Pastoret, P.P., Sjölander, K.B., Heyman, P., Brochier, B., Lundkvist, Å., 2000b. Evidence of Puumala hantavirus infection in red foxes (Vulpes vulpes) in Belgium. Veterinary Record 147, 365–366. Glass, G.E., Childs, J.E., Korch, G.W., LeDuc, J.W., 1988. Association of intraspecific wounding with hantaviral infection in wild rats (Rattus norvegicus). Epidemiology and Infection 101, 459–472. Groen, J., Gerding, M.N., Jordans, J.G., Clement, J.P., Nieuwenhuijs, J.H., Osterhaus, A.D., 1995. Hantavirus infections in The Netherlands: epidemiology and disease. Epidemiology and Infection 114, 373–383.
Heyman, P., Van Mele, R., De Jaegere, F., Klingström, J., Vandenvelde, C., Lundkvist, Å., Rozenfeld, F., Zizi, M., 2002. Distribution of hantavirus foci in Belgium. Acta Tropica 84, 183–188. Heyman, P., Cochez, C., Ducoffre, G., Mailles, A., Zeller, H., Abu Sin, M., Koch, J., van Doornum, G., Koopmans, M., Mossong, J., Schneider, F., 2007. Haemorrhagic Fever with Renal Syndrome: an analysis of the outbreaks in Belgium, France, Germany, the Netherlands and Luxembourg in 2005. Euro Surveillance 12:pii=712. Jay, M., Ascher, M.S., Chomel, B.B., Madon, M., Sesline, D., Enge, B.A., Hjelle, B., Ksiazek, T.G., Rollin, P.E., Kass, P.H., Reilly, K., 1997. Seroepidemiologic studies of hantavirus infection among wild rodents in California. Emerging Infectious Diseases 3, 183–190. Jones, K.E., Patel, N.G., Levy, M.A., Storeygard, A., Balk, D., Gittleman, J.L., Daszak, P., 2008. Global trends in emerging infectious diseases. Nature 451, 990–994. Kallio, E.R., Klingström, J., Gustafsson, E., Manni, T., Vaheri, A., Henttonen, H., Vapalahti, O., Lundkvist, Å., 2006. Prolonged survival of Puumala hantavirus outside the host: evidence for indirect transmission via the environment. J. General Virology 87, 2127–2134. Kallio, E.R., Voutilainen, L., Vapalahti, O., Vaheri, A., Henttonen, H., Koskela, E., Mappes, T., 2007. Endemic Hantavirus Infection Impairs the Winter Survival of its Rodent Host. Ecology 88, 1911–1916. Kim, G.R., Lee, Y.T., Park, C.H., 1994. A new natural reservoir of hantavirus: Isolation of hantaviruses from lung tissues of bats. Archives of Virology 134, 85–95. Klingström, J., Heyman, P., Escutenaire, S., Brus Sjölander, K., De Jaegere, F., Henttonen, H., Lundkvist, Å., 2002. Rodent host specificity of European hantaviruses: evidence of Puumala virus interspecific spillover. Journal of Medical Virology 68, 581–588. Lee, H., van der Groen, G., 1989. Hemorrhagic fever with renal syndrome. Progress in Medical Virology 36, 62–102. Leighton, F.A., Artsob, H.A., Chu, M.C., Olson, J.G., 2001. A serological survey of rural dogs and cats on the southwestern Canadian prairie for zoonotic pathogens. Canadian Journal of Public Health 92, 67–71. Malecki, T.M., Jillson, G.P., Thilsted, J.P., Elrod, J., Torrezmartinez, N., Hjelle, B., 1998. Serologic survey for Hantavirus infection in domestic animals and coyotes from New Mexico and northeastern Arizona. Journal of the American Veterinary Medical Association 212, 970–973. Nowotny, N., 1994. The domestic cat: a possible transmitter of viruses from rodents to man. The Lancet 343, 921. Pettersson, L., Klingström, J., Hardestam, J., Lundkvist, Å., Ahlm, C., Evander, M., 2008. Hantavirus RNA in saliva from patients with hemorrhagic fever with renal syndrome. Emerging Infectious Diseases 14, 406–411. Vapalahti, O., Mustonen, J., Lundkvist, Å., Henttonen, H., Plyusnin, A., Vaheri, A., 2003. Hantavirus infections in Europe. The Lancet Infectious Diseases 3, 653– 661. Woods, M., McDonald, R.A., Harris, S., 2003. Predation of wildlife by domestic cats Felis catus in Great Britain. Mammal Review 33, 174–188. Xu, Z.Y., Tang, Y.W., Kan, L.Y., Tsai, T.F., 1987. Cats - source of protection or infection? A case-control study of hemorrhagic fever with renal syndrome. American Journal of Epidemiology 126, 942–948. Yanagihara, R., Amyx, H.L., Lee, P.-W., Asher, D.M., Gibbs, C.J., Gajdusek, D.C., 1988. Experimental hantavirus infection in nonhuman primates. Archives of Virology 101, 125–130. Zeier, M., Handermann, M., Bahr, U., Rensch, B., Müller, S., Kehm, R., Muranyi, W., Darai, G., 2005. New ecological aspects of hantavirus infection: a change of a paradigm and a challenge of prevention - a review. Virus Genes 30, 157–180.
Contents lists available at ScienceDirect
Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
Sero-epidemiological study of the presence of hantaviruses in domestic dogs and cats from Belgium A. Dobly ⇑, C. Cochez, E. Goossens, H. De Bosschere, P. Hansen, S. Roels, P. Heyman Research Laboratory for Vector-Borne Diseases, Queen Astrid Military Hospital, Bruynstraat, 1120 Brussels, Belgium, and Veterinary and Agrochemical Research Centre, Groeselenberg 99, 1180 Brussels, Belgium
a r t i c l e
i n f o
Article history: Received 22 January 2009 Accepted 9 February 2011
Keywords: Hantavirus Felis catus Canis familiaris Rodent Distribution
a b s t r a c t Hantaviruses are worldwide rodent-borne pathogens infecting humans and other animals mainly through inhalation of aerosols contaminated with rodent excreta. Few data are available on hantavirus serology and geographical distribution in dogs and cats. We therefore screened sera from pet dogs (N = 410) and cats (N = 124) in two regions of Belgium, using IgG ELISA and IFA. We analysed the effect of the owner’s address as well as pet gender and age on hantavirus status. Hantavirus antibodies were found in both species with a significantly higher seroprevalence in cats than in dogs (16.9% vs. 4.9%, P = 0.001). More dogs were infected in highly forested southern Belgium (harbouring more rodents) than in northern Belgium (10.5% vs. 3.0%, P = 0.002). In the south, hantavirus sero-positive cats were found in more densely forested localities than sero-negatives ones were (P = 0.033). These results are consistent with the ecological variations of hantavirus risks in humans. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Emerging infectious diseases represent an increasing concern worldwide (Jones et al., 2008). The screening for zoonotic pathogens in potential animal hosts and in domestic animals is a crucial step to be prepared for new emerging threats (Zeier et al., 2005). Pets are indeed a key issue as they are in close proximity to humans and so numerous. In 2004 in Belgium, the 10 106 inhabitants possessed more than 1 106 dogs and 2 106 cats (http:// statbel.fgov.be). These domestic dogs and cats can repeatedly be in contact with rodents or rodent contaminated areas, potential sources of hantaviruses. Rodent-borne hantaviruses belong to the Bunyaviridae family. They are carried worldwide by Muroid rodents (and insectivores, Arai et al., 2008. Each hantavirus is mainly associated with a distinct reservoir species but spillover infections in other rodent species are reported (Klingström et al., 2002). Bites represent one important means of intraspecific transmission, at least in rats (Glass et al., 1988). However indirect transmission is not negligible as, under the optimal conditions, Puumala hantavirus (PUUV) can survive for up to 2 weeks outside of its host (Kallio et al., 2006). In the natural reservoirs, hantaviruses establish a persistent infection, and the host develops a strong neutralizing antibody response
⇑ Corresponding author. Address: Interactions and Surveillance, Veterinary and Agrochemical Research Centre, Groeselenberg 99, 1180 Brussels, Belgium. Fax: +32 23790479. E-mail address: [email protected] (A. Dobly). 0034-5288/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2011.02.003
(Chu et al., 1994). Yet few detrimental effects have been described in the reservoir species (Kallio et al., 2007). Despite this, more than a third of the 43 reported serotypes can cause disease in humans: hemorrhagic fever with renal syndrome (HFRS) in Europe and Asia, and hantavirus pulmonary syndrome on the American continent (Lee and van der Groen, 1989). HFRS is caused by eight serotypes. Its symptoms include fever, abdominal and back pain as well as renal dysfunction, sometimes with hemorrhagic manifestations (Vapalahti et al., 2003). Nephropathia epidemica (NE), the milder form of HFRS, caused by PUUV hantavirus, is present in North-Western Europe (including Belgium). Humans are infected by inhalation of aerosols contaminated with excreta from infected rodents. Worldwide, about 150,000–200,000 people are annually hospitalised for HFRS, mainly in Asia (Vapalahti et al., 2003). In spite of this incidence, little is known about the presence of hantavirus in non-rodent mammals. Apart from insectivores, hantaviruses are known in chiropters (bats, Kim et al., 1994) and artiodactyls (even-toed ungulates, Ahlm et al., 2000). In 20 species of non-human primates, experimental infection leads to an antibody reaction (Yanagihara et al., 1988). As for carnivores, Felis catus (cat) was found positive (2.9–9.6%, Xu et al., 1987; Nowotny, 1994; Leighton et al., 2001) as well as Canis familiaris (dog, 4.7%, Malecki et al., 1998) and Vulpes vulpes (red fox, 2.4%, Escutenaire et al., 2000b). Non-rodent infections seem to remain rare as several studies report no infection: 2400 mammals, including dogs and cats (Groen et al., 1995); various mammal species (Jay et al., 1997). However Hantaan (HTNV) serotype was detected in a bird (Dzagurova et al., 1995).
222
A. Dobly et al. / Research in Veterinary Science 92 (2012) 221–224
The presence of hantavirus in dogs or cats has never been reported in Belgium. This study first supposed that antibodies against hantavirus were detectable in dog and cat sera in Belgium. In the case of a positive result, this work would test two subsequent hypotheses. Firstly it is known that human PUUV-seroprevalence is linked to the quantity of forests, a good indicator of bank vole abundance, the PUUV reservoir (Heyman et al., 2002). Will the geographical pattern of hantavirus in dogs and cats be the same than in humans? We therefore first hypothesised that a higher hantavirus seroprevalence would be observed in pets from forested southern Belgium. Secondly, in bank voles, hantavirus presence increases with the age of the rodents (Escutenaire et al., 2000a). Will this age effect observed in companion animals? We hypothesised higher seroprevalence in older pets. 2. Materials and methods We retrospectively tested 534 pet sera (410 dogs and 124 cats), initially sent to private laboratories for routine veterinary analyses. We favoured samples from two regions isolated from each other: north-west Belgium (hereafter called northern Belgium), known to harbour few cases of human hantavirus infections, and southeast Belgium (called southern Belgium), location of numerous human hantavirus infections. For 360 dogs and 120 cats, the owner address, pet’s age and/or gender were available. We compared our results to the number of human hantavirus infections in Belgium in 2003 (data from the Scientific Institute of Public Health: www.iph.fgov.be/epidemio/labo). We analysed the sera using an IgG PUUV and HTNV enzymelinked immunosorbent assay (ELISA: Progen, Heidelberg, Germany). Samples that proved to be positive as well as an equal number of randomised samples that tested negative were tested again using an immuno-fluorescent assay (IFA-IgG) directed against various hantaviruses including PUUV (Euroimmun, Lubeck, Germany). We followed the manufacturer’s instructions. The sensitivity and the specificity of both tests are 97–99.5%. It should be noted that serological testing does not enable the definite identification of the infecting hantaviral serotype due to cross-reactions between serotypes. As, to our knowledge, positive and negative control sera for dogs and cats are not available, the cut-off (CO) for ELISA was calculated as in previous studies (e.g. Heyman et al., 2002: average optical density (OD) of 10 known negative sera plus 3 times their standard deviation. We compared series by calculating the ratio: observed OD/CO. A sample was considered as positive if this ratio was at least to 1.5. Ratios between 1.0 and 1.5 were considered ‘‘grey zone’’ values. This avoided overestimating the prevalence. All statistical tests were two-tailed with a level of significance at 0.05 except when indicated otherwise (e.g. age effect on seroprevalence because of our initial hypothesis). We used nonparametric statistics when normality was not expected. 3. Results We detected hantavirus antibodies in both regions with a higher seroprevalence in cats than in dogs (16.9% vs. 4.9%, Mann– Whitney U test, N = 534, P = 0.0012, Fig. 1 and Table 1). In IFA, all samples showed a higher titre for PUUV (cross-reacting with Tula, TULV) than for Seoul (SEOV). For both species, the average ELISA ratios were higher in more forested south than in northern Belgium (Mann–Whitney U test: dogs, 0.838 vs. 0.581, N = 410, P = 0.000; cats, 1.005 vs. 0.899, N = 124, P = 0.010). Moreover more dogs were infected in southern Belgium than in northern Belgium (10.5% vs. 3.0%, Mann–Whitney U test, N = 410, P = 0.002, Table 1). This geographical difference in dog seroprevalence is consistent
with the number of human cases reported in 2003 (North: 1.9 vs. South: 30.0 individuals per million). Moreover the cat prevalence was influenced by the forest cover in each studied administrative localities (http://www.statbel.fgov.be). Indeed, in the South, PUUV sero-positive cats were found in more densely forested localities than PUUV sero-negative ones (30.5% vs. 15.2%, one-tailed Mann–Whitney U test, N = 52, P = 0.033). We did not observe such a relationship for dogs. The mean pets’ age was 8.6 years for dogs (SD = 3.7, N = 105) and 7.7 years for cats (SD = 5.2, N = 90, Table 1). The dogs’ age was not linked to the serological status towards hantavirus (onetailed two-sample T test, N = 105, P = 0.19). Older cat’s showed an almost-significant tendency of higher seroprevalence (N = 90, P = 0.065, Table 1). Finally, in both species, we observed a non-significant tendency to have higher seroprevalence in females than in males (Table 1).
4. Discussion and conclusions This study showed that domestic carnivores are susceptible to hantavirus infection in Belgium. This is consistent with the airborne infection route and the pets’ behaviour. According to our test results, the observed hantavirus probably correspond to the Puumala serotype. However Tula and Seoul serotypes cannot be ruled out. Indeed only neutralization tests are able to clearly identify the causal serotype. Neutralization tests however are difficult; they require a BSL3 environment and an extensive hantavirus library in culture. Only a few labs worldwide are able to perform such tests and for this study we decided not to seek identification as in Belgium PUUV, TULV and SEOV circulate (Heyman et al., 2002). It is not known whether carnivores develop any hantavirus disease but low infection rates in dogs and cats suggest dead-end infections, as with humans. In humans, hantaviral RNA is only present during the few days of the acute phase of the disease (Pettersson et al., 2008). We can hypothesise that, as with humans, carnivores neutralise hantaviruses shortly after infection. This very short viraemia hampers in most cases virus isolation from the serum. It is important to mention that our study involves a bias as all tested pets were sampled in veterinary laboratories for a health problem or a routine health check. This absence of randomised sampling is difficult to avoid and is typical in such epidemiological studies. We can indeed compare our results to those of previous works. We observed a seroprevalence of 4.9% in dogs and 16.9% in cats. This is similar to what was previously reported for dogs in America (4.7%, Malecki et al., 1998 but our results concerning cats were higher than any previous observations (2.9–9.6%, China, Austria and Canada: (Xu et al., 1987; Nowotny, 1994; Leighton et al., 2001). The difference in cats cannot be attributed to our method, as we defined a high cut-off for being positive. The high cat seroprevalence could be partly explained by a bank vole demographic peak in 2005, accompanied by a steep increase of the number of human cases (Heyman et al., 2007). Finally, it is interesting to note that, in one study, 23% of chronically ill cats in the UK were found to be hantavirus-seropositive (Bennett et al., 1990). Nevertheless, the higher dog PUUV-seroprevalence observed in southern than in northern Belgium allowed us to demonstrate that the same geographical difference exists in dogs and in humans. The higher hantavirus infection rate of cats compared to dogs can be linked to the cats’ specialisation in catching small rodents (30% of domestic cat preys (Woods et al., 2003). This is consistent with the fact that sero-positive cats from southern Belgium are found in more forested environments than sero-negative ones. Contrary to what is observed in the rodent reservoirs (Escutenaire et al., 2000a), we did not detect an age-dependency
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Fig. 1. Belgium maps with the number of (a) dogs and (b) cats tested in this study per administrative locality. The legend gives the category ranges and, in brackets, the numbers of animals in each category. The positive animals are indicated by circles.
in pets’ seroprevalence (only a tendency of higher seroprevalence in older cats). It is possible that the high age of tested pets in comparison to small rodents had masked an age effect. About the gender difference, the PUUV reservoir shows a higher seroprevalence in males than in females (Escutenaire et al.,
2000a). This is explained by the very different life histories of males and (territorial) females, which is not the case in pets. However, if a gender difference exists in hantavirus prevalence within domestic carnivores, it seems to favour infection in females. This is surprising. However, female cats might be more
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A. Dobly et al. / Research in Veterinary Science 92 (2012) 221–224
Table 1 Hantavirus seroprevalence in dogs and cats according to region of origin, age and gender. North Belgium
Dogs
N
%
305
Cats
77
3.0 ⁄ 18.2
Total
382
6.0
⁄
South Belgium
Belgium
Young
Old
N
%
N
%
N
%
105
410
18.4
124
4.9 ⁄ 16.9
49
47
10.5 ⁄ 14.9
43
16.3
47
27.7
36
16.7
33
30.3
152
11.8
534
7.7
92
17.4
103
18.4
68
13.2
86
22.1
N 56
Males %
N
10.7
32
Females % 9.4
N
%
53
17.0
N = number tested, % = seroprevalence, which was compared with Mann–Whitney U tests (⁄P < 0.002). Dogs were considered old from 9 years old; cats were considered old from 7 years old.
exposed to hantavirus infection because of their higher hunting activities linked to pregnancy. This survey first showed that domestic carnivores in Belgium are infected by hantavirus. Furthermore this work demonstrated that, in dogs, the seroprevalence was not uniform but was higher in forested southern Belgium than in northern part of the country. This pattern is similar to what is observed in humans and can be explained by the presence of more rodents in the forested South. This finding stresses the importance of ecological factors on hantavirus prevalence in domestic species, as observed in humans. Acknowledgements No conflict of interest existed. Thanks are due to Flavien Riocreux for the maps, to Geneviève Ducoffre for the human data and to Kato Van Wassenhove for her technical help. The laboratory testing was sponsored by the Belgian Ministry of Defence. References Ahlm, C., Wallin, K., Lundkvist, A., Elgh, F., Juto, P., Merza, M., Tärnvik, A., 2000. Serologic evidence of Puumala virus infection in wild moose in northern Sweden. American Journal of Tropical Medicine and Hygiene 62, 106–111. Arai, S., Bennett, S.N., Sumibcay, L., Cook, J.A., Song, J.-W., Hope, A., Parmenter, C., Nerurkar, V.R., Yates, T.L., Yanagihara, R., 2008. Phylogenetically Distinct Hantaviruses in the Masked Shrew (Sorex cinereus) and Dusky Shrew (Sorex monticolus) in the United States. American Journal of Tropical Medicine and Hygiene 78, 348–351. Bennett, M., Lloyd, G., Jones, N., Brown, A., Trees, A.J., McCracken, C., Smyth, N.R., Gakell, C.J., Gaskell, R.M., 1990. Prevalence of antibody to hantavirus in some cat populations in Britain. Veterinary Record 127, 548–549. Chu, Y.K., Rossi, C., LeDuc, J.W., Lee, H.W., Schmaljohn, C.S., Dalrymple, J.M., 1994. Serological relationships among viruses in the Hantavirus genus, family Bunyaviridae. Virology 198, 196–204. Dzagurova, T., Tkachenko, E., Slonova, R., Ivanov, L., Ivanidze, E., Markeshin, S., Dekonenko, A., Niklasson, B., Lundkvist, Å., 1995. Antigenic relationships of hantavirus strains analysed by monoclonal antibodies. Archives of Virology 140, 1763–1773. Escutenaire, S., Chalon, P., Verhagen, R., Heyman, P., Thomas, I., Karelle-Bui, L., Avsic-Zupanc, T., Lundkvist, Å., Plyusnin, A., Pastoret, P.P., 2000a. Spatial and temporal dynamics of Puumala hantavirus infection in red bank vole (Clethrionomys glareolus) populations in Belgium. Virus Research 67, 91–107. Escutenaire, S., Pastoret, P.P., Sjölander, K.B., Heyman, P., Brochier, B., Lundkvist, Å., 2000b. Evidence of Puumala hantavirus infection in red foxes (Vulpes vulpes) in Belgium. Veterinary Record 147, 365–366. Glass, G.E., Childs, J.E., Korch, G.W., LeDuc, J.W., 1988. Association of intraspecific wounding with hantaviral infection in wild rats (Rattus norvegicus). Epidemiology and Infection 101, 459–472. Groen, J., Gerding, M.N., Jordans, J.G., Clement, J.P., Nieuwenhuijs, J.H., Osterhaus, A.D., 1995. Hantavirus infections in The Netherlands: epidemiology and disease. Epidemiology and Infection 114, 373–383.
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