Results and Discussion
The three red-necked wallabies, aged 1.5 to seven years, showed sudden signs of a therapy-resistant neurological disease and an underlying non-suppurative meningoencephalitis was initially diagnosed histopathologically in all cases (Fig. 1a). Subsequently, the brains were analyzed for putative causative agents. Only RusV was consistently detected in the brain tissue from all animals via RT-qPCR (Table 1), whereas no other neuropathogens analyzed were detectable.
The animals originated from three different zoos in northeastern Germany (Fig. 2). Case 1 occurred in the zoo in Stralsund in which RusV had been originally discovered in encephalitic zoo animals, including a further red-necked wallaby that had died in 2018 (Bennett et al., 2020a, 2020b). Cases 2 and 3 originated from zoos in Perleberg and Paaren, respectively, in which RusV had not been detected before. Notably, a previous case of fatal neurological disease in a red-necked wallaby had been reported also for each of these two zoos but the cause had been unknown as no diagnostic material from these animals was available.
The three investigated wallabies of this study showed a sudden onset of disease with a wide spectrum of neurological signs affecting the locomotor system shortly before euthanasia (Table 1). In cases 2 and 3, the animals were euthanized within 24 h after the reported onset of disease (Table 1). The affected animal of case 1 exhibited hind leg paralysis and ataxia five months prior following a fox attack. It fully recovered until re-occurrence of more severe clinical signs a few hours before euthanasia (Table 1). The nutritional condition of the animals varied from very good (case 2) to cachectic (case 1; Table 1), which is consistent with previous cases (Bennett et al., 2020b; Pfaff et al., 2022). The reduced nutritional condition could indicate a prolonged course of disease, although obvious neurological signs had been observed only shortly before euthanasia. This observation needs to be assessed when data regarding the incubation period of RusV and further factors affecting the infection such as immune status are available.
The necropsies revealed only non-specific gross findings such as acute congestion of the liver, lungs and kidneys and acute, alveolar edema. However, a non-suppurative meningoencephalitis of varying degrees with perivascular lymphocytic cuffing and few microglial nodules was histopathologically detectable in the cerebrum, cerebellum and brain stem in all cases (Table 1 and Fig. 1a). RusV was consistently identified via RNA ISH in these brain localizations predominately in neuronal cell bodies and their processes (Fig 1b), as reported previously for other RusV-infected hosts (Bennett et al., 2020b; Pfaff et al., 2022). Although neurons seem to be the main target cell of the virus, no evidence of significant neuronal degeneration or necrosis was contemporaneously found. In addition, neither dystrophic mineralization was detectable by von Kossa stain, nor demyelination or loss of Nissl substance by LFBKV stain in any wallaby analyzed. Immunohistochemistry revealed that the perivascular cuffs consisted of numerous CD3-positive T cells and fewer iba-1 labelled microglia/macrophages (Fig. 1c-d). The microglial nodules largely comprised of iba-1 positive cells, with few interspersed T-cells (Fig. 1c-d). Remarkably, there was often no direct local connection between inflammatory reaction or microglial nodules and RusV RNA detection via ISH. Plump, perivascular astroglia were evident in only one localization of case 2 by GFAP labelling (Fig. 1e-f) indicating that an activation of astroglia seems not to be a prominent feature of this infection. Minimal iron deposits were only detected in a focal area in two out of three animals (cases 1 and 3) by Prussian Blue reaction. The pathogenesis of these minimal intravital hemorrhages remains unclear. A possible traumatic impact as a consequence of the neurological disorder could be speculated. Based on the focal, minimal occurrence we hypothesize that RusV-infection does not systematically affect blood vessels. These results are in line with the previously published reports of RusV infection (Bennett et al., 2020b; Pfaff et al., 2022).
In contrast to the striking inflammation of the brain and the detection of the virus in this organ, no other lesions were consistently traceable in the three animals and no RusV-specific RNA was found in other tissues tested by ISH, including heart, stomach, small and large intestine. The restriction of lesions and the viral distribution to the brain is typical for spill-over hosts of neuropathogens such as the Borna disease virus 1 (Schulze et al., 2020). These animals act as dead-end hosts and do not contribute to the spread of the virus. It seems likely that red-necked wallabies may play a similarly restricted role in the epidemiology of RusV. However, further investigations, including a comprehensive, systemic investigation of the route of entry, incubation period, potential further factors that may contribute to the disease, such as stress, viral load, time point of infection, preexisting conditions, such as trauma or parasite infestation, the viral tissue distribution and the analysis of secretions and excretions for the virus, are recommended to clearly address the pathogenesis of this new disease and the epidemiological significance of red-neck wallabies as hosts.
RusV seems to infect a wide range of mammalian hosts (Bennett et al., 2020a, 2020b; Pfaff et al., 2022) and our study raises the number of reported cases of RusV infections in red-necked wallabies to four (Bennett et al., 2020a, 2020b). This species, which is popular in zoological gardens as well as in private holdings, seems to be particularly susceptible to RusV infection, raising the question for putative preventive strategies. In addition to the previously published cases of encephalitic zoo animals and one wild Eurasian otter (Bennett et al., 2020a, 2020b; Pfaff et al., 2022), RusV was also detected in apparently healthy yellow-necked field mice (Bennett et al., 2020b). Certain small mammal species are known reservoir for infectious viruses, such as the Borna disease virus 1 (Hilbe et al., 2006; Puorger et al., 2010), and seem to play a role in the transmission of infections. Whether and how the RusV is transmitted from yellow-necked field mice to other animals is not clarified yet. At this point, pest control seems to be a putative preventive method and is recommended for zoological gardens and other holdings of red-necked wallabies in Northern Germany.
In addition to the first publications of this new disease (Bennett et al., 2020b; Pfaff et al., 2022), our data show that the virus seems to be broader distributed in northern Germany (Fig. 2), indicating a wider epizootic range. Partial RusV E1-encoding sequences of 715 bp lengths were generated for all three animals and revealed a minimal nucleotide identity of 92.7% among the available RusV sequences (Fig. 3). The sequence originating from case 1 from Stralsund was most closely related to the previously determined sequences from this location (≥98.9% nt identity), whereas the sequences from the two other wallabies were more distantly related to the sequences from Stralsund and among each other (92.9 to 93.7% nt identity), which is in line with their origin from separate locations (Fig. 2 and 3). Novel RusV genomes from this study are available under DDBJ/ENA/GenBank accession numbers: (in preparation).
Taken together, RusV infections should be included on the list of differential diagnoses for neurological disorders and non-suppurative meningoencephalitis in zoo and wild animals, especially red-necked wallabies, at least in Germany. The complete geographic distribution and the entire host range of the virus need to be further investigated.
Ethics statementThe authors confirm that the ethical policies of the journal, as noted on the journal’s author guidelines page, have been adhered to.