"> Your browser can't run Java!

RABIES IN ANIMALS
R. Jayakumar

Courtesy : Festschrift - Dr. S. Ramachandran

Introduction
The word ‘rabies ‘ comes from the Sanskrit word’ rabhas’, which means, “to do violence”. Rabies was mentioned in the legal documents of Mesopotamia in the 23rd century B.C. A number of excellent reviews have been published on several aspects of rabies history. Lise Wilkinson (1) has described an historical perspective to understand the nature of rabies. Galtier’s work influenced Louis Pasteur to develop ‘virus fixe’ in the rabbits. The first human case treated by Pasteur with dried rabbit spinal cord vaccine was the badly bitten Joseph Meister who in spite of the severity of his injuries survived without developing clinical rabies (2). Hundred years after Pasteur’s work at the Institute Pasteur, Paris, Noel Tordo et al. (1988) cloned and sequenced the rabies virus (3). 

Rabies virus
Taxonomically, rabies virus belongs to the Order Mononegavirales, Family Rhabdoviridae. Rhabdoviridae are characterized by a negative-sense genome of single stranded RNA and the family is divided into two genera, Vesiculovirus and Lyssavirus, the latter includes rabies and rabies-related viruses. The lyssaviruses are serologically distinct from other rhabdoviruses. Rabies virus is bullet-shaped with an average length of 180 nm and diameter of 75 nm. The rabies virus genomic RNA contains five genes, each of which codes for structural protein of the virus. The nucleocapsid core is formed from the RNA and three of the proteins: nucleoprotein (N), nonstructural (NS) transcriptase-associated phosphoprotein and large, virion-associated transcriptase (L). The others are lipid-containing envelope matrix protein (M) and the glycoprotein (G). The G protein spikes are anchored within the lipid containing bilayer. The total length of the genome RNA of Pasteur virus (PV strain) is 11932 nucleotides (4). This includes the 3' leader sequence, N, NS, M, G and L genes, the intergenic sequences and 5’ non-coding sequence. 

Rabies virus glycoprotein (G) is a type I membrane glycoprotein of molecular weight 35kDa. It is a trimer that forms a spike extending from the viral membrane (5). The complete mature glycoprotein molecule is 505 amino acids long (6). Rabies virus glycoprotein molecule (G) is composed of a cytoplasmic domain, a transmembrane domain, and an ectodomain, exposed as trimers at the virus surface (7). The ectodomain is involved in the induction of both VNAb production and protection after pre and post exposure vaccination (8).

Rabies epizootiology
Blancou, (9) presented an overall picture of the epizootiology of rabies in Eurasia and Africa. He concluded that the epidemiological rabies situations in the different countries of Europe, Asia and Africa reveal a great disparity depending on geographical location or country. The evolution of the epidemiological situations also varies depending on the region. Historically it is well established that canine rabies has attained an equilibrium between vector and virus and is no longer evolving. In contrast sylvatic rabies is rapidly evolving, and may expand or regress depending on variables that (despite the most sophisticated predictive mathematical or informational models) are not understood. 

Pathogenesis and pathology
A number of excellent reviews have been published on several aspects of rabies pathogenesis and pathology (10). The general scheme of the pathogenesis of rabies includes the following sequential steps (i) introduction of virus into a bite wound or laceration; (ii) migration via peripheral nerves to the central nervous system; (iii) spread through the CNS; (iv) centrifugal neural transport of virus, and (v) infection of non-nervous tissues. This general concept of the ‘flow” of infection is well substantiated and widely accepted. Recent and current research is centered on details of the infectious process at various sites and stages of the disease. The pathogenesis includes all the interactions of host and infectious agent. These include the sequential infection of cells, binding of virus to cellular receptors, endocytosis, translation and replication of viral RNA, assembly of virions, and cellular release of progeny virus. In most rabid animals the lesions are typical of a non-suppurative encephalomyelitis with ganglioneuritis and parotid adenitis, with the most significant lesions in the pons to hypothalamus and cervical spinal cord. Meningitis is present to some degree. Perivascular cuffs contain lymphocytes, plasma cells, macrophages and occasionally erythrocytes and the number of plasma cells present increases with the duration of clinical sign. 

In addition to centripetal viral movement by anterograde axoplasmic flow, virus moves centrifugally from neural perikaryon the CNS and cerebrospinal ganglia, later affecting almost all nerves of the body. Reports of pre-clinical periods of virus secretion in the saliva range from 3 days in cats; 7 days in dogs with a Mexican isolate and 13 days with an Ethiopian isolate; 29 days in foxes.

Diagnosis
Dogs are the principal transmitters of rabies virus in most of Asia. Transmission is almost exclusively via infected saliva. Virus appears in the saliva of dogs before and during the appearance of clinical signs. Rapid and accurate laboratory diagnosis of rabies is essential for timely administration of post-exposure prophylaxis. Reliable results may save a patient from unnecessary psychological trauma and financial burdens. The clinical manifestations of rabies in animals especially dogs and cats with furious form are easily recognized and clinically diagnosed by the veterinarians or even by the laymen. But in the case of dumb form of rabies, the diagnosis is much more difficult and the laboratory diagnosis is the only way whereby rabies can be confirmed. Negri bodies are regarded as pathognomonic of rabies in man and animals, although their absence does not exclude the disease. The Babes granules or nodules have been mentioned as histological elements of neuronophagic activity (11). The fluorescent antibody test (FAT) introduced by Goldwasser and Kissling is now regarded as superior to other diagnostic assays in speed and accuracy (12). Following digestion with trypsin or pepsin to unmask the immunoreactive sites, formalin-fixed brain tissue is stained by the FA technique (13).

Animal inoculation: The isolation of rabies virus by intracerebral inoculation of animals is feasible in several laboratory animal species: rabbits, guinea pigs, hamsters or mice. Suckling mice are more susceptible to some strains of street rabies virus than older mice but for diagnostic purpose newly weaned mice are used and it remains a standard confirmatory test for the laboratory diagnosis of rabies. Although the clinical disease in mice is brief, the incubation periods of street rabies virus are typically long and may vary from 7 to 28 days. The observation period may be reduced if sufficient numbers of mice are inoculated so that one mouse can be killed at daily intervals and the brain examined by the FAT. In this way a diagnosis of rabies may be made well in advance of the appearance of clinical signs and/or death. 

Virus isolation in cell culture: Fixed rabies virus can be grown in various cell cultures and these systems have been used for experimental studies. Numerous studies have compared the infection rate of BHK and murine neuroblastoma cells with street rabies virus and have found that murine neuroblastoma cells are more susceptible to infection (14).

Enzyme-linked techniques: Atanasiu et al. (15) proposed the use of an enzyme immunoassay for detection of rabies antigen in tissue impression. This has been modified for the detection of rabies antigen in tissue sections and impressions. This immuno-enzymatic system has been further developed into an enzyme-linked immunosorbent assay (ELISA) type test, designated Rapid Rabies Immuno-enzymatic Diagnosis (RRIED) for which microscope and fluorescence are not necessary. RRIED is an ELISA test performed on the supernatant of brain or salivary gland suspensions. The test is based on the immunocapture of the rabies nucleocapsid antigen by an antinucleocapsid polyclonal globulin coated to ELISA plates, followed by the addition of the same globulin conjugated to peroxidase. In comparison with the standard FAT it was equally sensitive with efficacy levels of 100%. Jayakumar et al. (16) developed a Dipstick Dot ELISA for the detection of rabies antigen in animal brain specimens. This test employed the nitrocellulose membrane as solid support replacing the microtitre plates and was compared well with FAT. Subsequently, modifications have been developed for the detection of rabies antigen in tissue suspensions using the nitrocellulose membrane as solid support in the enzyme immunotechniques (17). A simple and rapid latex agglutination test for the detection of rabies antigen was described (18).

Polymerase chain reaction (PCR): Since the first use of PCR to detect rabies RNA in 1990, many techniques have been published. Some were concerned with diagnosis, typing of the virus using restriction fragment length polymorphism (RFLP), with molecular epidemiology, by correlating the genome variability with its geographical location, or with its host. Several reviews compare these different approaches (19).

Dot hybridization: Dot hybridization is used to detect specific rabies RNA in brains, either from experimental or from brain materials to be processed for routine diagnosis. Radioisotope or biotin or DIG labeled CDNA probes can be employed to identify minute amounts of specific viral RNA (20). A sensitive and specific non-radioactive DNA probe for detection and identification of rabies virus was developed to show 98.43 % positive results by dot-blot hybridization method (21).

In situ hybridization: Rabies virus RNA can be demonstrated in paraffin-embedded tissues using in situ hybridization. Negative (-) Sense 35S- and 3H- labelled RNA probes, specific for rabies virus nucleocapsid protein mRNA, are used for the detection of rabies virus RNA in the nervous system (22). A non- isotopic method of in situ hybridization (ISH) was developed for the detection of rabies virus RNA in paraffin-embedded tissues. Digoxigenin-labelled RNA probes for rabies virus glycoprotein mRNA were used. This method is more convenient than the radiolabelled method (23).

Vaccines and vaccinations
The work of Galtier, a veterinarian during 1879 and Louis Pasteur during 1884 led to the development of immunization procedures against rabies. This was followed by the development of attenuated vaccine made in rabbits that was used by Pasteur with success as a curative vaccine in man. In 1908, Fermi developed a phenolised vaccine that was much more stable than the dried spinal cord preparation. Semple, in 1911, introduced phenolised vaccine prepared using infected sheep, goat and rabbit brain. This vaccine is still being used globally. The Pasteur Institute of India, Coonoor, Tamil Nadu introduced in 1970, BPL inactivated 5% sheep brain vaccine which was an improvement over the earlier phenolised vaccine and this effectively reduced the vaccine dosage in animals and human. Its immune response and protective capacity was tested in dogs and was found that all the vaccinated dogs survived a lethal challenge with a local street rabies virus (24)

The first modified rabies vaccine for animal use was the low egg passage (LEP) vaccine using Flury strain isolated and adapted through 138 serial intracerebral passages in day-old chicks (25) and further modified by 40-50 serial intra-yolk sac passages in embryonating hen’s eggs (26). The basic LEP vaccine virus was further attenuated to the 183rd passage level in embryonating hen’s eggs to form Flury high egg passage (HEP) vaccine for use in cats and cattle (27). Petermann et al. (28) used the NIL line of hamster fibroblasts to prepare a rabies vaccine inactivated with BPL and used in cats, dogs and cattle. Several tissue culture vaccines are now available for domestic animals. For post exposure treatment potency of inactivated chick embryo cell culture adapted virus was determined in unvaccinated Indian stray dogs previously challenged with a high dose of virulent street rabies virus by intramuscular route. The vaccine was protective in dogs with 5 or 6 doses given post exposure (29). Animal rabies control has been frustrated in many countries by the existence of multiple wildlife reservoirs and the lack of efficacious oral vaccines in the past. Several approaches have been developed for vaccine production and delivery by oral route. The first report of oral vaccination appeared during 1971 (30), followed by several reports starting from the use of conventional tissue culture vaccine to naked nucleic acid vaccines for immunization of dogs and wild carnivores (31). The first live recombinant rabies vaccine was used as oral vaccine in foxes during 1986 (32). This vaccine, a vaccinia-rabies recombinant, was introduced in many European countries to control and in some cases eradicate rabies in wildlife. The recombinants expressing rabies virus surface spike glycoprotein (G) were produced by homologous recombination and used as an oral rabies vaccine (33). A new recombinant rabies vaccine (human adenovirus 5 containing the rabies glycoprotein) has been developed and tested in striped skunks (Mephitis mephitis) and red foxes (Vulpes vulpes). The results indicate that this vaccine has considerable potential as oral rabies vaccine in wildlife. The glycoprotein (G) of ERA strain of rabies virus was abundantly expressed in a baculovirus expression system and oral vaccination of raccoons with this protein resulted in the production of rabies virus neutralizing antibodies and protection (34,35). 

Genetic immunization, the latest addition to the field of vaccinology has shown in a number of animal models, to be an efficacious approach to induce protective immunity to infectious diseases. The advantages of DNA vaccines are their ease of construction, the low expense of mass production, their high temperature stability and their ability to induce a full spectrum of exceptionally long lasting immune responses including development of specific cytolytic T-cells. A plasmid vector expressing the full-length rabies virus glycoprotein (G) under the control of the SV40 promotor was found to induce long-lasting immunity to rabies virus without apparent negative side effects. The effect of genetic immunization of neonatal mice was tested with a plasmid vector expressing the rabies virus glycoprotein. The result shows that the immune system known to be prone to induction of immunological tolerance to some antigens applied during the early neonatal period, can readily respond to rabies virus glycoprotein induced by a plasmid vector. Antirabies virus neutralizing antibody elicited by plasmid DNA vaccination cross-neutralized a global spectrum of rabies virus variants. These results indicate that DNA vaccines could be a solution for providing developing countries with an inexpensive vaccine that is simple to prepare, highly efficacious and with excellent stability (36). A gene gunparticle-mediated vaccine with plasmid DNA confers protective immunity against rabies virus infection (37). DNA vaccine induced protection against rabies virus in dogs and all the vaccinated dogs were protected against a lethal challenge with a wild-type dog rabies strain (38).

References

1. Lise Wilkinson. (1988). Understanding the nature of rabies: A historical perspective. In Rabies. p. 1. Eds. J.B. Campbell, and K.M. Charlton, Kluwer Academic, Boston.
2. Hoenig, L.J. (1985). Medical Times, 113: 35.
3. Tordo, N. et al. (1988). Virology, 165: 565.
4. Tordo, N. and Poch, O. (1988). Structure of rabies virus. In Rabies. p. 25. Eds. J.B. Campbell and K.M. Charlton, Kluwer Academic, Boston.
5. Tordo, N. et al. (1986). Nucleic Acids Research, 14: 2671.
6. Gaudin, Y. et al. (1992), Virology, 187: 627.
7. Anilionis, A. et al. (1981). Nature, 294: 275.
8. Delagneau, J.F. et al. (1981). Inst. Pasteur Virol., 132E: 473.
9. Blancou, J. (1988). Epizootiology of rabies: Eurasia and Africa. In Rabies. p. 243. Eds. J.B. Campbell, K.M. Charlton, Kluwer Acadamic, Boston.
10. Charlton, K.M. (1988). The pathogenesis of rabies. In Rabies. p. 105. Eds. J.B. Campbell and K.M. Charlton. Kluwer Acadamic, Boston.
11. Bedford, P.G.C. (1976). Vet. Rec., 99: 160.
12. Goldwasser, R.A. and Kissling, R.E.(1958). Proc. Soc. exp. Biol. Med., 98: 219.
13. Zalan, E. et al. (1979). J. Biol. stand., 7: 213.
14. Webster, W.A. and Casey, G.A. (1988). Diagnosis of rabies infection. In Rabies. p. 201. Eds. J.B.Campbell and K.M. Charlton, Kluwer Acadamic, Boston.
15. Atanasiu, P. et al. (1980). Dev. Biol. Stand., 46: 207.
16. Jayakumar, R. et al. (1994). Indian vet. J., 71: 866.
17. Jayakumar, R. et al. (1997). Indian J. Anim. Sci., 67: 40.
18. Jayakumar, R. et al. (1995). Indian J. Anim. Sci., 65: 414.
19. Ermine, A. et al. (1990). Molecular and Cellular Probes, 4: 189.
20. Heaton, P.R. et al. (1997). J. Clin. Microbiol., 35: 2762.
21. Ganesh, V. and Jayakumar, R. (1999). Indian J. Virol., 15: 77.
22. Jackson, A.C. and Wunner, W. (1991). J. Virol., 65: 2839.
23. Jackson, A.C. (1992). Molecular and Cellular Probes., 6: 131.
24. Jayakumar, R. et al. (1989). Revue Sci. Tech. Off. int. Epizoot., 199: 208.
25. Koprowski, H. and Cox, H.R. (1948). J. Immunol., 60: 533.
26. Koprowski, H. and Black, J. (1952). Proc. Soc. exp. Biol. Med., 80: 410.
27. Koprowski, H. et al. (1955). J. Am. vet. med. Ass., 127: 363.
28. Petermann, H.G. et al. (1967). C.R. Acad. Sci., (Paris). 265D: 2143.
29. Jayakumar, R. et al. (1998). Indian J.Virol., 14: 147.
30. Baer, G.M. et al. (1971). Am. J. Epidem., 93: 487.
31. Mayer, A. et al. (1972). Zent. Vet., (B), 19: 615.
32. Baer, G.M. (1976). Dev. Biol. Stand. 33: 417.
33. Pastoret, P.P. et al. (1988). Parassitologia, 30: 149.
34. Esposito, J.J. et al. (1988). Virology, 165: 313.
35. Charlton, J.B. et al. (1992). Arch. Virol., 123: 169.
36. Fu, Z.F. et al. (1993). Vaccine, 11: 925.
37. Lodmell, D.L. et al. (1998). Vaccine, 16: 115.
38. Perrin, P. et al. (1999). Vaccine, 18: 479.

Authors Corresponding address: 

Dr. R. Jayakumar
Assoc. Professor, 

Rabies Unit, Department of Animal Biotechnology, Madras Veterinary College, Chennai - 600 007, India 

The views expressed in this article are of the author(s), and any clarifications can be obtained from the author(s).