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.