The bicolored white-toothed shrew Crocidura leucodon (HERMANN 1780) is an indigenous host of mammalian Borna disease virus

Abstract

Borna disease (BD) is a sporadic neurologic disease of horses and sheep caused by mammalian Borna disease virus (BDV). Its unique epidemiological features include: limited occurrence in certain endemic regions of central Europe, yearly varying disease peaks, and a seasonal pattern with higher disease frequencies in spring and a disease nadir in autumn. It is most probably not directly transmitted between horses and sheep. All these features led to the assumption that an indigenous virus reservoir of BDV other than horses and sheep may exist. The search for such a reservoir had been unsuccessful until a few years ago five BDV-infected shrews were found in a BD-endemic area in Switzerland. So far, these data lacked further confirmation. We therefore initiated a study in shrews in endemic areas of Germany. Within five years 107 shrews of five different species were collected. BDV infections were identified in 14 individuals of the species bicolored white-toothed shrew (Crocidura leucodon, HERMANN 1780), all originating from BD-endemic territories. Immunohistological analysis showed widespread distribution of BDV antigen both in the nervous system and in epithelial and mesenchymal tissues without pathological alterations. Large amounts of virus, demonstrated by presence of viral antigen in epithelial cells of the oral cavity and in keratinocytes of the skin, may be a source of infection for natural and spill-over hosts. Genetic analyses reflected a close relationship of the BDV sequences obtained from the shrews with the regional BDV cluster. At one location a high percentage of BDV-positive shrews was identified in four consecutive years, which points towards a self-sustaining infection cycle in bicolored white-toothed shrews. Analyses of behavioral and population features of this shrew species revealed that the bicolored white-toothed shrew may indeed play an important role as an indigenous host of BDV.

Figure 1. Collection sites of shrews and locations where BDV-positive white-toothed bicolored shrews were found.

White symbols, specimens collected; red circles, BDV-positive C. leucodon.

Figure 2. Relative trapping frequency (%) of the bicolored white-toothed shrews collected in this study (2005–2010).

Number of shrews: 57 (from one shrew no exact collection date was available).

Figure 3. Bicolored white-toothed shrew (C. leucodon, HERMANN 1780) and its taxonomic identification.

A. Bicolored body. B. White teeth of Crocidura species. C. Eyelashes-like hairs on tail of Crocidura species.

Figure 4. Phylogenetic analysis of BDV sequences obtained from bicolored white-toothed shrews.

A. 1824(P), C. 1110 bp nucleic acid sequences within the gene coding for p40 (N) of 14 BDVs obtained from naturally infected bicolored white-toothed shrews from Germany determined in this study (red diamonds), three Swiss , (blue circles) and one Bavarian (purple triangle) Crocidura leucodon-derived BDVs, and other representative BDVs mainly from spill-over hosts, which succumbed to BD. Evolutionary analyses were conducted in MEGA5 . For tree 4A (unrooted tree, 63 nucleotide sequences) the Neighbor-Joining method was used. For trees 4B (76 nucleotide sequences, outgroup BDV He/80, oldest available BDV sequence of the region where BDV was first reported) and 4C (69 nucleotide sequences, outgroup BDV No/98, most distant mammalian BDV known so far originating from a location far away from the BDV endemic regions) the Maximum Likelihood method was performed. The percentage of replicates in the bootstrap test (1000 replicates) is shown next to the branches. Values less than 70% are hidden. Cluster 1: Southwest Germany and Southern Rhine valley group, 1A. Baden-Wurttemberg and Bavaria west of Augsburg (Swabia) as well as central Bavaria around Munich, Germany, 1B. Graubuenden and Sankt Gallen, Switzerland, The Principality of Liechtenstein, and Austria (south of Lake Constance); Cluster 2: South German group (major parts of Bavaria), 3. Southern Saxony-Anhalt and Saxony (mainly along and between rivers Saale and Elbe south of Magdeburg); Cluster 4: Central German group: Lower Saxony, northern Saxony-Anhalt, Thuringia and bordering Saxony-Anhalt (mainly west of the river Saale), northern parts of Bavaria, Germany. BW, Baden-Wurttemberg; HE, Hesse; LS, Lower Saxony; SA, Saxony-Anhalt; SX, Saxony; TH, Thuringia (Germany); GB, Graubuenden; SG, Sankt Gallen (Switzerland); L, Liechtenstein (The Principality of Liechtenstein).

Figure 5. Locations of BDV-positive bicolored white-toothed shrews within BD endemic regions.

Endemic regions of BD (green circles, main endemic areas; green dots, BD occurs but is rare), locations at which positive bicolored white-toothed shrews were found in this study (orange triangles) and were reported in Switzerland , (red triangles) and Bavaria (brown triangles), genetic cluster groups (1A,B; 2; 3; 4), and movements of BD into new territories (blue arrows); BD was first described by Authenrieth in the Swabian Alb ; there was never an epidemic in the city of Borna but the disease occurred frequently in horses on farms in the district of Borna from the late 1880s until the 1960s but it is rare there now ; BDV-positive shrews from three major clusters (1, 3, 4) have been found so far. The distribution of BD in the scheme is based on reference data , , , , , , – and report data from Berichte über das Veterinärwesen im Königreich(e)/Freistaat Sachsen – for details see reference page 23, references 285 and 286, and page 24, references 309 and 310.

Figure 6. Immunohistochemical staining patterns of different organs with the polyclonal anti-P antibody and the monoclonal antibody Bo18.

A. Diffuse immunostaining in the area of the dentate gyrus, brain, anti-P antibody; shrew CL72; B. Diffuse immunostaining in the area of the dentate gyrus, brain, Bo18 Ab; Note slightly different immunostaining with presence of nuclear signals with anti-P antibody and absence of nuclear signals with Bo18 Ab. C. Absence of immunostaining in the dentate gyrus of a BDV-negative shrew; brain, CL61; D. Abundant immunostaining of olfactory epithelium with presence of strongly stained intranuclear bodies, anti-P antibody, shrew CL73; E. Lack of immunostaining in the olfactory epithelium of a BDV-negative animal, anti-P antibody, shrew CL61; F. Immunostaining of entire bronchial epithelium and underlying smooth muscle layer, lung, anti-P antibody, shrew CL62; G. Immunostaining of a group of glandular epithelial cells, salivary gland, anti-P antibody, shrew CL62; H. Immunostaining of keratinocytes and epithelia of hair follicles, skin; shrew CL62; I. Complete absence of immunostaining in the skin of a BDV-negative animal; anti-P antibody, shrew CL61; J. Immunostaining of myocardial cells; heart; Bo18 Ab; shrew CL76; K. Abundant immunostaining of adipocytes in abdominal fat tissue; anti-P antibody; shrew CL73; L. Complete absence of immunostaining in the fat tissue of a BDV-negative animal; anti-P antibody.