Immunohistochemical and histopathological studies of fixed rabies virus in goats

 

S. ATALAY VURAL1* , G. ALCIGIR1 and S. BERKIN1

ABSTRACT The purpose of this study was to systematically demostrate and compare the development of pathological and immunohistochemical changes in goats which were infected by a fixed rabies virus that was used in vaccines’ production. The results are as follows:

In histopathological examinations, varying degrees of  inflammatory, degenerative, and necrotic changes were detected in the central nervous system; in the immunoperoxidase (IP) method, intra-and/or extracellular viral antigens were observed on the cerebellum, cornu ammonis, thalamus, pons cerebri, nucleus caudatus, spinal cord, medulla oblongata, Gasserian ganglion, eye and the retropharyngeal lymph nodes; in the immunofluorescence (IF) method, intra-and/or extracellular viral antigens were also seen in the same locations with the exception of retropharyngeal lymph nodes. It was also observed that the antigens were qualitatively and quantitatively well stained with both of methods. However the visibility of antigens in the retropharyngeal lymph nodes besides the central nervous system, and eye and the facilities of applying made the IP method much more advantageous than the IF method.

Keywords : Rabies virus, histopathology, immunoperoxidase, immunofluorescence

 

INTRODUCTION

            Rabies viruses include the street virus, the causative agent of rabies in humans and animals through natural transmission, and the fixed rabies virus, a laboratory adapted form. The latter virus was developed by Pasteur with serial intracerebral passages of the street virus (Sullivan1993). The virulence and incubation period of the fixed rabies virus are constant (Buxton & Fraser 1977; Jayakumar, Ramadass & Baghavan 1989). It is employed in many ways, such as in studying the replication of viruses and in the development of  vaccines (Tordo & Kouknetzoff 1986; Consales, Valentini, Albas, Mendonca, Fuches, Soares & Pereira 1988).

For the diagnosis of naturally occuring rabies by histopathological examination, it is important to demonstrate the presence of  Negri bodies in addition to the nonpurulent encephalomyelitis. 

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1   University of Ankara, Faculty of Veterinary Medicine, Department of  Pathology, 06110 Ankara, Turkey. e-mail : svural@veterinary.ankara.edu.tr

However, Negri bodies are found in only 50-80 % of the cases (Goldwasser & Kissling 1958; Atanasiu, Dragonas, Tsiang & Harbi 1971; Koprowski 1973). Demonstration of the viral antigen by the use of immunohistochemical methods greatly increases the chances of diagnosing the disease. There is an 87-98 % possibility of diagnosing rabies by using the immunoperoxidase (IP) method, and 87-100 % by the immunofluorescent (IF) method (Anjaria & Jhala 1985; Kotwal & Narayan 1985, 1987; Jayakumar et al. 1989; Zimmer, Weigand, Manz, Frost, Reinacher & Frese 1990). Since Negri bodies are not formed following inoculation of animals with fixed rabies virus, it is impossible to detect infection on histopathological examination as in the case of street virus infection. However, IP and IF methods are employed to detect viral antigens in the tissues and organs of animals experimentally inoculated with the fixed rabies virus (Madhusudana & Tripathi 1990; Jackson & Park 1998).

The purpose of this study was to systematically demostrate the development of pathological and immunohistochemical changes in goats which were infected by a fixed rabies virus that was used in vaccines’ production. The results were compared with each other.

 

MATERIALS AND METHODS

            The examined materials were obtained from ten goats, each of which was infected by the intracerebral inoculation of 0.2 ml of diluted Challenge Virus Standard (CVS) used for the production of a rabies vaccine by the Etlik Veterinary Control and Animal Diseases Research Institute, in Turkey. These animals were slaughtered when became agonized after the injection.

            Tissue samples after conducting a post mortem examination of each animal were taken from the cornu ammonis, nucleus caudatus, thalamus, pons cerebri, cerebellum, medulla oblongata, cervical spinal cord, Gasserian ganglion, parotid and submandibular salivary gland, retropharyngeal lymph node, vestibular region of the nose, an intact eye, skin around the ear and mouth, trachea, thymus, lung, heart, spleen, liver, kidney and adrenal gland. These samples were fixed in 10 % buffered formalin, and embedded in paraffin wax. Sections were stained with hematoxyline and eosin, and if deemed necessary, Periodic acid-Schiff, luxol fast blue, for histopathological examination (Luna 1968). Fixed rabies virus antigens in the tissues were demonstrated by using polyclonal rabbit antinucleocapsid protein sera (obtained from the Rabies Centre of Expertise OIE Reference Laboratory for Rabies WHO Collaborating Centre Animal Disease Research Institute, Canada) by means of a modified IP method (Strept-Avidin Biotin Peroxidase) and an indirect IF method (Heyderman 1979; Noorden & Polak 1983; Fekadu, Greer, Chandler & Sanderlin 1988; Vural 1997).

 

RESULTS

            In the macroscopical examinations, the meningeal blood vessels were seen congested. In two cases, the retropharyngeal lymph nodes were swollen, and cut surface appeared to be more moist than usual and dark brownish in colour.

            In the histopathological examinations, many of the blood vessels in the central nervous system were hyperaemic and surrounded by lymphocytic cells in which there were small amounts of macrophages (Fig. 1a). Similar perivasculer infiltrations were  also observed in the meningeal blood vessels of the cerebral cortex, cerebellum, pons cerebri, thalamus, and nucleus caudatus. Some neurons were found to be degenerative, or necrotic (Fig. 1b-d). The Nissl bodies in the neurons of the pons cerebri, cerebellum, medulla oblangata, and spinal cord were distributed in an irregular rough granular form or located  at the periphery of the cytoplasm in the form of small granules. Some neurons of the medulla oblongata, pons cerebri, and spinal cord of the animals were atrophic. Neuronophagia was apparent in the cornu ammonis, cerebellum (Fig. 1a), thalamus (Fig. 1c), pons cerebri, medulla oblongata, spinal cord, and Gasserian ganglion. Babes’ nodules were also observed in the cerebellum, thalamus, and pons cerebri. Focally or diffusely distributed proliferation of glia cells was noticed in the cornu ammonis, cerebellum, thalamus, pons cerebri, medulla oblongata, and spinal cord. Focal areas of demyelination in the cerebellum was observed in some animals. Focal haemorrhages was noticed in the Gasserian ganglion (Fig. 1d), spinal cord, and cornu ammonis. Perivascular oedema was also present in  the pons cerebri of some animals. The extent of the varying degrees of nonpurulent encephalitis or meningoencephalitis observed in the animals is summarized in Table 1.

            In the kidneys, some glomerular capillaries appeared to have taken the form as wire loops and others had undergone atrophy. The parietal membrane of Bowman capsules and the tubular basal membranes were thickened. Fibrosis was observed in some intertubular areas of the medullary regions and the  areas surrounding the glomeruli where atrophy was present.

            Sinus catarrh was seen in the most of retropharyngeal lymph nodes. The lymphoid follicules were hyperplastic and lymphoblasts were present in the central regions. Thymus was also hyperplastic. Lymphocytic cells and sparsely distributed macrophages were noticed in the vestibular region of the nose and trachea particularly around some glands.

            In the preparations stained by the IP method, viral antigens were observed (Table 1) in the Purkinje cells, cerebellar peduncle neurons, and the stratum granulosum nerve cells of the cerebellum  (Fig. 2a), pyramidal and cerebral cortex neurons of cornu ammonis, thalamus (Fig. 2b), pons cerebri (Fig. 2c), nucleus caudatus, spinal cord, medulla oblongata (Fig. 2d), Gasserian ganglion, eye and retropharyngeal lymph nodes. They were seen as a fine dust or rough granules from brick-red to brown in colour in the perikaryon and extensions (axon and dendrites) of the neurons, glia cells and freely around them in the nervous system. They were also seen in the perivascular lymphocytic cells, endothelial cells and some ependimal cells of the pons cerebri, and the medulla oblongata.

            In IF stained preparations, the viral antigens were observed (Table 1) in the same regions of central nervous system (Fig. 3a-d), Gasserian ganglion, and eye. The presence of viral antigens was revealed by the presence of glistening, yellow, mostly fine granules and some coarser particles. The granules in some sections were so big that they could be mistaken for inclusion bodies.

 

DISCUSSION

            The necropsies of animals infected with fixed rabies viruses are generally reported not to reveal any pathological changes  (Burkhart, Jervis & Koprowski 1950) or as seen in this study, only hyperemia in brain vessels was reported (Sinchaisri, Nagata, Yoshikawa, Kai & Yamanauchi 1992).

            Perivascular mononuclear cell infiltration, glia cell proliferation, neuronal degeneration, neuronophagia and demyelination noticed in this study have also been reported as a histopathological changes found most frequently in the cerebral and cerebellar cortices, pons cerebri, spinal cord, and Gasserian ganglion (Burkhart et al. 1950; Field 1951; Johnson 1965; Jackson & Park 1998). Inflammatory reactions reported in the meninges (Burkhart et al. 1950; Field 1951; Johnson 1965; Jackson & Park 1998), were also observed around the blood vessels on the meninges covering the cerebral cortex, cerebellum, thalamus, pons cerebri and nucleus caudatus in this study. In addition to these, perivascular oedema was seen in the meninges of the pons cerebri. Field (1951), has described the rough appearance of the Nissl granules in the neurons, and has stated that this is often encountered in the pons cerebri. Similar observations were seen in the neurons of the pons cerebri, medulla oblongata, spinal cord and cerebellum in this study. Neuronal degeneration and necrosis result from infection with fixed rabies virus, as in other studies (Miyamoto & Matsumoto 1966; Sullivan1993) except for the primary forms of the inclusions observed under electron microscopes, no inclusion bodies are encountered.

The wire loops formation of the glomerular blood vessels and the thickening of  the parietal membrane of Bowman capsules and the tubular basal membranes in the kidneys of the vaccinated animals were thought to be the result of antigen-antibody complexes.

Fixed rabies viral antigens in IP preparations mostly concentrate in the cerebrum, cerebellum, pons cerebri, brain stem and the dorsal root ganglion of this region, spinal, and trigeminal nerves (Jackson, Reimer & Ludwin 1989; Jackson & Wunner 1991; Sinchaisri et al.1992; Jackson & Park 1998). In this study, IP positivity was encountered in the cornu ammonis, cerebellum, thalamus, pons cerebri, spinal cord, medulla oblongata, nucleus caudatus, Gasserian ganglion, eye, and retropharyngeal lymph nodes. On the other hand, the antigens were mostly found in substantia grizea besides in substantia alba, is similar to the findings in this study in which either that relatively higher chances of finding antigens in this region or that antigens are never found there (Jackson et al. 1989; Jackson & Wunner 1991).

The viral antigens in IF preparations have mostly shown on the cerebrum, cerebellum, brain stem, and the dorsal root ganglion of this region, spinal cord, trigeminal nerves, eye, heart, nasal mucosa, trachea, lung, kidneys, urinary bladder, adrenal gland, oral, stomach, the Averbach and Meissner plexus of the intestines, hair follicle, and muscle (Correa-Giron, Allen & Sulkin 1970; Ravaioli, Palliola, Pestalozza, Granieri  & Ciucnini 1970; Fischman & Schaeffer 1971; Johnson & Mercer 1964; Kucera, Doliva, Coulon & Flamand 1985; Coulon, Derbin, Kucera,  Lafay, Prehaud & Flamand 1989; Tsiang, Lycke, Ceccaldi, Ermine  & Hirardot 1989; Mathusudana  & Tripathi 1990; Tsiang, Ceccaldi & Lycke 1991). In this study, the positiveness of  IF is almost in similar in central nervous system. Although retropharyngeal lymph nodes have not examined in the literatures, it was examined. But no antigens were seen. Johnson & Mercer (1964) reported that viral antigens become localised only in the neurons, but not in glia, meningeal, ependymal, or vascular cells and they tend to be concentrated in the neurons close to the ependymal cells and in the axons on the substantia alba, that IF positivity tends to increase especially in the perikaryons and extensions of the Purkinje cells, and that viral antigens show irregular distribution on the cornu ammonis, cerebrum, brain stem, cerebellum and the anterior regions of the spinal cord. In this study, viral antigens were encountered not only in neurons, but they also in extraneuronal regions close to the neurons and in some glia cells.

Despite the same tissues were examined as being mentioned in several articles (Fischman & Schaeffer 1971; Madhusudana & Tripathi 1990; Jackson & Wunner 1991), in this study fixed rabies viral antigens were not observed in the IF and IP preparations except central nervous system, eye, and retropharyngeal lymph nodes. In addition, contrary to the literature, viral antigens could not be traced in the IF and IP preparations in two of the cases necropsied despite the inflammation changes observed in all the animals. This insignificant variations obtained in this study is considered to be the result of the result of the viral antigen being denatured during a time period, differences in inoculation methods, and concentrations and strains of the viruses used. During the study it was found that organs from different animals yielded different results for the two methods. This has been ascribed to practical errors has. This was noticed in the qualitative and quantitative evaluation in the tissues and cells of the viral antigens.

Tsiang, Koulakoff, Bizzini & Berwald-Netter (1983) in IF examinations of neuroblastoma and dorsal root ganglion cell cultures infected with CVS reported the finding one to two cytoplasmic inclusions in each cell. However, Sinchaisri et al. (1992) could not detect inclusion bodies formed by viral nucleoprotein in cytoplasm and they attributed that these results to the fact that either the animals died before the viral antigens could accumulate in sufficient quantity to form inclusion bodies or they related it to the experimental conditions that they used. Although inclusion bodies were not seen, it could not be determined whether or not the large granules observed in the IP and IF preparations were inclusion bodies in this study.

In the histopathological examinations of this study, the extent of the varying degrees of nonpurulent encephalitis or meningoencephalitis were observed. But, they were usually seen in central nervous system caused by many viral diseases. For doing a differentiative diagnosis, the immunohistochemical examinations had to be done. Thus, it was shown that the fixed rabies virus antigen in formalin-fixed and paraffin-embedded tissue sections were seen clearly by using the IP and IF methods in this study. But, the IF method requires fluorescent microscopy and fresh preparation of procedure each time. On the other hand, the IP method could be able to use with ordinary light microscope and it has practical importance for the laboratories. All the slide preparations in IP method were permanent which means they were available for storing and rechecking. As a result, the antigens were also well-stained by these methods in both tissue sections but they were more qualitatively and quantitatively in IF method than IP method. But, the IP method proved to be much more advantageous for a viral antigens to be in different tissue as being retropharyngeal lymph nodes and the positiveness of results obtained from eye was much more than the IF method.

 

 

 

REFERENCES

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Acknowledge: I thank Alexander Wandaler for hyperimmun sera and Ismet Yoruk for obtained materials. This work was supported University of Ankara, Research Fund.

 

 

 

FIG. 1a Hyperaemia, perivascular infiltration (thin arrow) and neuronophagia (thick arrow) in the cerebellum HE X100

FIG. 1b Neuronal necrosis (arrow) in the pons cerebri HE X320

FIG. 1c Neuronal necrosis (arrow) and neuronophagia (thick arrow) in the thalamus HE, X200.

FIG. 1d Neuronal necrosis (thin arrows) and haemorrhagia (thick arrow) in the Gasserian ganglion HE X400

 

FIG. 2a Granular and/or diffuse IP positive staining in Purkinje cells (thin arrows) and extensions (thick arrows) X250

FIG. 2b IP positive staining in a neuron and extensions (thin arrows) and neuropil (thick arrow) of thalamus X250

FIG. 2c IP positive staining in neurone and extensions (thin arrows) and neuropil (thick arrows) of the pons cerebri X250

FIG. 2d IP positive staining in the medulla oblongata (thin arrows) X320

 

FIG. 3a IF positive staining in neurone and extensions (thin arrows) and neuropil (thick arrow) of cornu ammonis X320

FIG. 3b IF positive staining in a neuron (thick arrows) and extensions (thin arrows) of the pons cerebri X320

FIG. 3c IF positive staining in the thalamus (thin arrows) X320

FIG. 3d IF positive staining in the nucleus caudatus (thick arrows) X320

 

 

 

 

 

Dr. Sevil Atalay Vural

University of Ankara, Faculty of Veterinary Medicine

Department of Pathology,

06110 Dıskapı, Ankara/TURKEY

e-mail: svural@veterinary.ankara.edu.tr

Fax: +(90) 312. 317 83 81 / +(90) 312. 316 44 72

 

 

 

 

November 24, 2000

                               

 

Prof J. BOOMKER

ARC-Onderstepoort Veterinary Institute

Private Bag X05, Onderstepoort 0110, South Africa

 

 

 

 

Dear Prof Boomker,

 

Thank you very much for referees’s advice and help dated September 12, 2000. I met the requirements of the referee and sent manuscripts, typed on Microsoft Word 95 together with a diskette.

 

Yours sincerely.

 

 

Dr. Sevil Atalay Vural

 

 

 

 

Enclosed:

1.   Two copies of the manuscripts

2.   Six figures

3.   One diskette