DE DE GRUYTER J Vet Res 60, 227-231, 2016

OPEN DOI:10.1515/jvetres-2016-0034
G

Apoptosis in natural rabies virus infection

in dogs

Sevil Atalay Vural1, Mehmet Fatih Bozkurt2, Ali Ozkara3,

Mehmet Eray Alcigir1, Fatma Sayin Ilhan4
1
Department of Pathology, Faculty of Veterinary Medicine,
Ankara University, Diskapi, 06110, Ankara, Turkey
2
Department of Pathology, Faculty of Veterinary Medicine,
Afyon Kocatepe University, 03200, Afyonkarahisar,
Turkey
3
Pendik Veterinary Control and Research Institute, Pendik, 34890, Istanbul, Turkey
4
Department of Pathology, Faculty of Veterinary Medicine,
Balıkesir University, 10145, Balıkesir, Turkey
sevilvural@yahoo.com

Received: October 30, 2015 Accepted: August 29, 2016

Abstract

Introduction: In the present study apoptosis was investigated in the cornu ammonis and cerebellum of 10 dogs naturally
infected with rabies virus. Diagnosis of rabies was based on the results of fluorescent antibody staining and experimental
inoculation. Material and Methods: The paraffin tissue sections were stained with haematoxylin and eosin, avidin-biotin
complex peroxidase (ABC-P), and terminal deoxynucleotidyl transferase biotin-dUTP nick end-labelling (TUNEL) methods.
Results: Histopathological examination revealed encephalomyelitis of varying severity and the presence of Negri bodies. Dense
rabies antigens were determined in the motor neurons with ABC-P method. On the other hand, Bcl-2 protein and Bax protein
gave positive reaction in seven and five cases, respectively. TUNEL staining demonstrated very marked apoptotic changes in the
nuclei of neurons localised deep in the substantia alba of the cerebellum. Similar changes were also determined in perivascular
mononuclear cells and glia cells within the substantia alba. No apoptopic changes were found in the motor neurons of the cornu
ammonis. Conclusion: The absence of apoptotic changes in the neurons was considered to be the consequence of the necrotic
changes that developed in these neurons.

Keywords: dogs, rabies, central nervous system, apoptosis, histopathology.

Introduction virus replicates and disseminates in the central nervous

system by fast axonal transport along neuroanatomical
Rabies is caused by rabies virus (RABV) connections (8, 18). Specific macroscopical lesions are
belonging to the genus Lyssavirus of Rhabdoviridae not present, but self-inflicted wounds and foreign
family, which affects all warm-blooded animals as well bodies in the stomach of a carnivore should raise
as humans (15, 24). Globally, around 3.9 billion people suspicion. Microscopically, pathognomonic findings
are at risk of contracting rabies and more than 150 are characterised by non-suppurative encephalomyelitis,
countries are affected, with billions of dollars being ganglioneuritis, and parotid adenitis (18, 21). For this
spent on protection (3, 15, 17). RABV has two biotypes reason the research projects aim to find the fastest and
known as “street virus” and “fixed virus”. Fixed safest methods of early diagnosis (25, 26).
RABV, vaccine strains, are laboratory biotypes and is RABV is a neurotrophic virus which kills or
not secreted in saliva. Street RABV is tropic for causes function disorders by infecting the neurons at
salivary glands (18). Animals and humans acquire the the central nervous system. There is a common opinion
infection by introduction of RABV-laden saliva that cell death occurs via apoptosis (5, 23). Apoptosis
through a bite of a rabid animal, or mucosal exposure. was firstly defined in 1972 as death of cells caused by
On gaining receptor-mediated entry into the neuron, the demolition of the genes that control the internal

© 2016 S.A. Vural et al. This is an open access article distributed under the Creative Commons
Attribution- NonCommercial-NoDerivs license (http://creativecommons.org/licenses/by-nc-nd/3.0/)
22 S.A. Vural et al./J Vet Res/60 (2016) 227-
231
mechanism of cells and results in the fractures of the the samples were incubated with AEC and
DNA helix. It starts with the stimulation of some counterstained with Gill’s haematoxylin. As control,
internal and external factors. There are biochemical and the samples were treated with labelling solution instead
morphological changes in the stimulated cells (6). of TdT.
These reactions are formed directly or indirectly by an The severity of immunoreactivity was graded
increase in the level of substances that are under according to the density in 10 different microscopic
control of apoptotic genes (Bax, Bad, Bad protein- areas as + mild, ++ moderate, +++ severe, - no staining.
proapoptotic) or anti-apoptotic genes (Bcl-2, Bcl-xl,
Boa). All these substances and energy required for
apoptosis are supported by mitochondria (7, 20).
Results
In these days, the role of apoptosis has been
studied in regard to experimental infections of mice
Histopathologically, many of the vessels in the
with Sindbis virus, Crosse virus, and reovirus (4, 10,
cornu ammonis and cerebellum were hyperaemic and
14, 16). The present study describes the distribution of
surrounded by mononuclear cells. Similar perivascular
viral antigen and apoptosis in the central nervous
cells were also seen around meningeal vessels. Some
system of dogs infected with street rabies virus.
neurons were degenerative or necrotic. Neuronophagia
and Babes’ nodules were visible. The proliferation of
glia cells was focally observed and different numbers
Material and Methods of Negri bodies were seen in all samples (Figs 1-3).
Morphological changes related to apoptosis also drew
Animals. The study involved 10 dogs which had attention, especially in internal/external granular layer
been determined to be rabies-positive based on the (Fig. 4) and at cerebellar nucleus in the cerebellum, to
results of fluorescent antibody staining and a lesser extent in the neurons located in the cornu
experimental inoculation. ammonis. The most evident feature of these changes
Sample collection and histopathological was the phenomenon of neurons becoming smaller and
examinations. The cerebellum and cornu ammonis shrinking, cytoplasm getting narrow, and chromatins
were fixed in 10% buffered formalin, processed by coming together as rough particles near the nuclear
routine method, and embedded in paraffin. The 5 μm membrane.
sections were mounted on glass slides, and stained with Immunohistochemically, high levels of rabies
haematoxylin and eosin (HE). Selected sections were virus antigen were detected in motor neurons of the
also stained with cresyl violet. cornu ammonis, Purkinje cells, external layer, and
Immunoperoxidase staining. The sections were cerebellar nucleus (Fig. 5) in all animals. Staining of
stained for rabies virus antigen by avidin-biotin Bcl-2 protein was positive in seven dogs and Bax
complex peroxidase (ABC-P) method using polyclonal protein in five animals (Figs 6-9). Bax protein was
rabbit anti-rabies virus nucleoprotein sera (1:500, detected in the Purkinje cells, cerebellar nucleus, and
Animal Disease Research Institute, Canada) and external granular layer of the cerebellum contrary to the
polyclonal rabbit anti-Bax and Bcl-2 antibodies (1:500, cornu ammonis. On the other hand, Bcl-2 protein was
Santa Cruz Biotechnology, USA) according to the localised in the Purkinje cells, cerebellar nucleus, and
procedure provided by the producer (LSAB Kit, Dako). motor neurons of the cornu ammonis.
The colour was developed by a final incubation with 3- At TUNEL staining, the most evident reaction was
amino-9 ethylcarbazole (AEC). As a control group, the found in external/internal granular layer and cerebellar
samples were treated with normal rabbit sera instead of nucleus of the cerebellum. Similar changes were seen
primary antibody. in perivascular mononuclear cells (Fig. 10) and glia
In-situ detection of apoptotic cells by TUNEL cells which were located in substantia alba. At the same
assay. DNA fragmentation was assessed in situ in time, the differences in localisation and degree of
sections using the terminal deoxynucleotidyl positive reaction were not significant in the cerebellum
transferase (TdT)-mediated dUTP-digoxigenin nick of all dogs. No positive TUNEL reactions were seen in
end-labelling (TUNEL) kit as recommended by the the cornu ammonis.
manufacturer (In Situ Cell Death Detection Kit, POD, The results are summarised in Tables 1 and 2.
Roche, Germany). To visualise the reaction products,

Table 1. Changes in cerebellum cells affected with rabies virus

Affected group of cells Pathomorphological and immunohistochemical findings

Morphological TUNEL Immunohistochemical examinations
changes positivity Viral antigen Bcl-2 protein Bax protein
Purkinje cells + - +/+++ + +
External granular layer + + ++/+++ - +
Internal granular layer + + + - +
Cerebellar nucleus + + ++/+++ + +
Fig. 1. Neuronophagia (N) and perivascular mononuclear cell infiltration (star), cerebellum, HE, 100×
Fig. 2. Negri bodies in Purkinje cells (arrows), dog, HE, 400×
Fig. 3. Negri bodies in motor neuron (arrows), cornu ammonis, dog, HE, 400×
Fig. 4 Apoptotic changes and dense chromatins in nucleus, dog, Cresyl violet, 1250×

Fig. 5. Viral antigen at cerebellar nucleus, dog, ABC-P, 400×

Fig. 6. Bcl-2 protein in cerebellar nucleus, dog, ABC-P, 400×
Fig. 7. Bcl-2 protein in Purkinje cells, dog, ABC-P, 400×
Fig. 8. Bax protein in cerebellar nucleus, dog, ABC-P, 400×
Fig. 9. Apoptosis of cerebellar nucleus, dog, TUNEL, 400×
Fig. 10. Apoptosis of perivascular mononuclear cells, dog, TUNEL, 100×
Table 2. Changes in cornu ammonis cells affected with rabies virus

Affected group of cells Pathomorphological and immunohistochemical findings

Morphological TUNEL Immunohistochemical examinations
changes positivity Viral antigen Bcl-2 protein Bax protein
CA-C1 + - ++/+++ + -
CA-C2 - - ++/+++ + -
CA-C3 - - ++/+++ - -
Dentate gyrus + - ++/+++ - -

Discussion changes related to apoptosis and TUNEL positive

reactions are investigated (9). These reactions are
Apoptosis, defined as the programmed cell death, mostly observed in the neurons (10, 12). TUNEL
is a widespread cell biological process. Apoptosis positive neurons were seen in all layers of the cerebral
occurs normally during development and aging and as cortex, cingulate gyrus, Ammon’s horn in the
a homeostatic mechanism maintaining cell populations hippocampus, and neurons in the thalamic and
in tissues. It also occurs as a defence mechanism, e.g. hypothalamic regions after infection with CVS strain of
in immune reactions or when cells are damaged by rabies virus. In addition to neurons, the inflammatory
disease or toxic agents (7, 14, 19). cells lining the meninges, ependymal cells lining the
Apoptosis is tightly regulated and can be induced ventricle, cells of the choroids plexus, and rare glial
by a variety of stimuli and conditions, both and microglial cells in the cortex and white matter also
physiological and pathological, such as viruses and showed labelling (21). In spite of the presence of viral
other factors causing disruption of cellular functions antigen, TUNEL reaction does not occur in the
(7). Apoptosis classically occurs via either the intrinsic Purkinje cells of the cerebellum (12). Immuno-
or extrinsic pathways. In brief, the intrinsic pathway histochemically, rabies virus antigen was marked,
occurs in the mitochondrion when apoptosis is together with morphological changes, both in the motor
triggered by a variety of intracellular signals, where neurons of the cornu ammonis, Purkinje cells, and
cytochrome c is released from the mitochondrial cerebellar nucleus. However, TUNEL positive
membrane to the cytoplasm and then activates caspase- reactions were not seen in the cornu ammonis contrary
9 which subsequently activates caspase-3 and DNA to the cerebellum. This situation may be interpreted as
cleavage (2, 14, 22). By contrast, the extrinsic necrosis of neurons or may suggest that these neurons
pathway can be induced by members of the tumour are more resistant to apoptosis than other neuronal cell
necrosis factor (TNF) family of cytokine receptors, types, although it seems less probable. Similar changes
such as TNFR1 and Fas Ligand (FasL). They recruit were observed in perivascular mononuclear cells and
adapter proteins to their cytosolic death domains glia cells which were located in the substantia alba.
(DDs), which bind procaspases containing death- Yan et al (27) inoculated wild type virus into
effector domains (DED), particularly procaspase-8 experimental mice, and reported very few TUNEL
(2, 14). positive neurons despite the presence of clinical signs
Apoptosis has typical morphological and of the disease, suggesting that apoptosis may not be an
biochemical characteristics. The morphological essential neuropathogenic mechanism. Street-virus-
changes of infected cells are evidenced by smaller infected suckling mice revealed TUNEL labelling of
nucleus, denser chromatin, fragmentation of nuclei, and occasional microglial cells in the cerebral cortex and
separation of DNA from internucleosomal space. brain stem (21). On the other hand, evaluation of
Genomic DNA internucleosomal fragmentation is the morphological features of neuronal apoptosis in
most significant biochemical sign of apoptosis. The postmortem brain tissue from human rabies victims
energy consumed by apoptotic processes leads cell to collected from different countries suggested that
death. Caspase family proteases, Bcl-2 family proteins, neuronal apoptosis does not play an important role in
and p53 gene product play a central role in the process human rabies encephalitis (11).
of apoptotic cell death. The Bcl-2 family of proteins Jackson and Park (10) found Bax protein
governs mitochondrial membrane permeability and can associated with apoptosis in the cerebral cortex and
be either pro-apoptotic or anti-apoptotic (1, 13). pyramidal neurons of the cornu ammonis, indicating
In the recent years, neurotropic viruses in that Bcl-2 family proteins play an important role in
particular have been shown to induce apoptosis within experimental rabies. In the present study, Bax-positive
the CNS, and depletion of these non-regenerating staining was observed especially in cerebellar nucleus
neuronal cells by apoptosis may result in neurological and external granular layers of cerebellum, but no
morbidity (1, 5, 7, 10, 12, 14, 27). This association positive reaction was noted in the cornu ammonis. In
between viral infection of the CNS and apoptosis has the same tissues, more significant positive reactions
spawned a new area of rabies virus research. For this were observed in the case of Bcl-2 protein. These
purpose, the effect of laboratory adapted rabies virus results suggested that there was no apoptotic effect in
strains is researched experimentally, and morphological tissues, and this has been also interpreted as an early
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