Dr.R.Jayakumar, M.V.Sc., Ph.D; Professor and Head, Department of Veterinary Epidemiology and Preventive Medicine, Madras Veterinary College, Chennai-600 007.

The Present Situation

Rabies is one of the most feared zoonotic diseases in the world. All warm-blooded animals are susceptible to infection by the virus, but the main vectors of human infection are dogs and cats. Development of rabies can be prevented by post exposure vaccination, and with a few exceptions, the exact time and source of human infection is usually known. However, the effective use of post exposure vaccination depends on the rapid and accurate detection of rabies virus in specimens obtained from the source of human infection. 96% of the human as well as animal rabies infection is due to dog bite in India. Among the pet animals, dogs are the most important transmitters of urban rabies to man.  Hence it is of paramount importance in the prevention of the spread of rabies to man and other domestic animals. Hence it is necessary to detect the disease as early as possible in a rabid dog or cat. The clinical manifestations of rabies is  easily recognized and clinically diagnosed by the veterinarians or even by the laymen. But in the case of dumb rabies the diagnosis is much more difficult and the laboratory diagnosis is the only way whereby rabies can be confirmed. In developing countries where rabies is endemic, not every laboratory can afford to be equipped with costly sophisticated equipments. Therefore, alternative methods which are rapid, reliable, economic and easy without depending upon sophistication have to be developed.

 Post-mortem Laboratory diagnosis of Rabies in pet Animals: 

After death, the CNS is the main source of specimens to be sent to the laboratory. If a complete necropsy has been performed, the brain is entirely taken. If for some reasons a necropsy is not possible a specimen of brain tissue may be obtained through the following methods.(Bourhy and Sureau, 1990).

(a). Occipital foramen route: Brain samples may be taken through this route by using a plastic pipette. Cut the skin and neck muscles over the joint between the occipital bone and atlas vertebrae bend the head forward to give access to the occipital foramen, pass a straw through the foramen, screw it into the brain stem, cerebellum, hippocampus and cortex.

(b). Retro orbital route: Push the eyeball to one side, use a trocar to make an entry through the posterior wall of the eye socket, introduce through this hole a straw or a 2 ml disposable plastic pipette, screwing it in the direction of the occipital foramen; the Straw or pipette will contain parts of cortex, hippocampus and cerebellum.( Montano Hirose et al. 1991).

 Preservation of Specimens:

A detailed  and simplified technique for the collection, storage and shipment of brain specimens for rabies diagnosis was described by  Barrat and Blancou (1988).

(a). Brain or skin biopsy: Refrigerate for 1-3 days; freeze at –200C or -700C or in liquid nitrogen for indefinite periods. For virus isolation the preservation is in 50% glycerol. Formalin inactivates rabies virus but diagnosis can be made by a modified fluorescent antibody test by histology and by immunoenzymatic techniques.

(b). Corneal smears for FAT: Corneal smears are best preserved on slides, fixed in cold acetone and sent to the laboratory refrigerated. 

(c). Saliva and throat swabs: Saliva can be refrigerated, frozen or preserved in 50% glycerol for virus isolation only. Saliva and throat swabs can be stored and frozen in a buffered transport medium.

Rapid Rabies Laboratory Diagnosis: 

The design of the methods to be applied to the laboratory diagnosis of rabies and the selection of the most suitable one for a rapid diagnosis are based on the knowledge of the replication and antigenic structure of the rabies virus including the most recent data obtained through the use of monoclonal antibodies and molecular virology.

Advantages of Rapid Diagnostic Tests: The advantages of these tests are that they require only little quantities of the sample and the results are available quickly ranging from 5-15 minutes like the latex agglutination test, to one hour with the Dot ELISA test. These tests are also reagent conservative, they do not require sophisticated equipment and are ideal for use in remote areas.  The sensitivity and specificity of these rapid tests correlate well with the standard FAT for rabies detection. 

Several techniques may be used to detect rabies antigen: Demonstration of Negri bodies by Sellers staining, Direct Fluorescent antibody test (FAT), Rapid Rabies Enzyme Immunodiagnosis (RREID), Latex agglutination Test, Virus isolation in new born mice, virus isolation in cell cultures, Immunoperoxidase test (IPT), Peroxidase and antiperoxidase test (PAP), Avidin-biotin test, Dipstick dot ELISA, Dot ELISA and Electron Microscopy. Recently monoclonal antibodies are routinely used in many laboratories. Detection of rabies viral RNA by Dot and Slot Hybridization, In situ Hybridization and different types of Polymerase Chain Reaction (PCR) is a  recent advanced  technique.

1.      Negri body examination: These inclusions corresponding to aggregates of viral proteins, are specific for rabies virus infection, the staining techniques are not specific since they merely detect affinity for acidophilic stains. In the case of autolysed brain materials, interpretation of the result may  be difficult. Presence of Negri bodies in positive cases varies from 10 to 65% ( Sellers, 1927). Further more, there is a risk of false positive results due to other inclusions as seen in some viral infections as well as in healthy wild and domestic animals  ( Dekashan et al.1958 and Berkman et al. 1960). Negri bodies can be demonstrated on methanol fixed brain smears taken from hippocampus major and stained with Sellers stain. The result is obtained within one hour. The WHO has abandoned this test in its recommendations. 

2.      Direct Fluorescent antibody Test (FAT): The FAT is performed on fresh unfixed impression smears of brain, cornea, frozen sections of skin biopsies, smears with cells of centrifugation sediment of saliva, throat swab and cerebrospinal fluid. These smears are fixed in cold acetone. A rabies antinucleocapsid FITC conjugate is used. The result is obtained within two hours. The FAT provides a reliable diagnosis in 98-100% cases ( Dean and Abelseth ( 1973). 

3.      Rapid Rabies Enzyme Immuno Diagnosis: The RREID test is an ELISA test performed on the supernatant of brain or salivary gland suspensions. This test is based on the immunocapture of the rabies nucleocapsid antigen by an antinucleocapsid polyclonal globulin coated to the ELISA plates, followed by the addition of the same globulin conjugated to horse radish peroxidase enzyme. The results are obtained within three hours.  ( Perrin et al. 1986).The correlation between FAT and RREID is 96-99%(  Jayakumar et al.1989).

 4. Immunoperoxidase Test (IPT): Immunoperoxidase staining techniques have also been used, when good morphology is important in addition to requirement for the sensitive and specific detection of rabies antigen ( Bourgon and Charlton, 1987 and Fekadu et al.1988).Immunoperoxidase test may be used instead of FAT. Both techniques have the same sensitivity but peroxidase test takes longer time to perform. ( Jayakumar and Ramadass, 1991).The sensitivity of the immunoperoxidase test can be improved by the addition of peroxidase and antiperoxidase  or avidin biotin horseradish peroxidase conjugates.( Jayakumar et al., 1994).

5.      Dipstick / Dot ELISA : In the Dipstick / Dot ELISA nitrocellulose strips as solid support replaces the micro titer plates. This test is reagent conservative, does not require sophisticated equipment, and is ideal for use in geographically remote areas. This test can be improved by the addition of avidin-biotin horseradish peroxidase conjugate. The results can be preserved for longer time and this test can be used in retrospective epidemiological survey of rabies ( Jayakumar  and Padmanaban, 1994., Jayakumar et al.1995 ., Jayakumar et al.1995 .,Jayakumar et al.1995 and  Jayakumar et al. 1997). 

6.      Rapid Latex Agglutination Test: Latex beads sensitized with rabies immunoglobulins quickly agglutinate in the presence of rabies virus antigens. In the same manner latex beads sensitized with purified rabies antigen quickly agglutinates in the presence of rabies specific antibodies. This test could also be used as a quantitative test. This test requires less than 15 minutes to perform on clean microscopic slide of glass plate or VIDAL Test plates.( Jayakumar  et al.1995 and Kasempimolporn  et al 2000)

7.      Electron Microscopy Examination: Brain specimens may be fixed in osmic acid or in glutaraldehyde and formic acid or in formalin followed by the above mentioned fixatives. After inclusion in epon, ultra thin sections are stained with uranyl acetate and lead citrate.

8.      Virus Isolation Techniques:
(a).Virus isolation in mice: The mouse inoculation test does not give rapid results and the  observation period is at least for 28 days but it has the advantage of being a simple and   reliable method for further testing. ( Webster and Dawson , 1935). The correlation between FAT and mouse inoculations is 92- 99%.

(b).Virus isolation in cell cultures: Virus isolation in cell cultures is recommended for all  laboratories where tissue culture work is a common practice. ( Rudd et al.1980). Neuroblatoma cell lines are particularly susceptible to rabies virus, which will Produce  specific inclusions detectable by FAT in less than 24 hrs( Portnoi et al, 1982). This test  detects 96-97% of rabies positive samples.  One cycle of virus replication in this cell take  at least 18 hours.  (Bourhy et al.1989).

Use of molecular biological techniques:

Dot and slot blot hybridization’s: Viral RNAs are extracted from brain or salivary gland specimens after filtration on nylon membranes, hybridization is performed with 32p DNA probes or non-radioactive probes complementary to rabies genome RNA and mRNAs.  ( Ganesh and Jayakumar, 1999). Autoradigraphy or ELISA detects hybridizing labeled probes.  The results are obtained in 48hours  ( Chakaravarthy Reddy et al. 2002).

Polymerase chain reaction: The PCR techniques now a days play an important not only in the diagnosis of rabies in pet animals as well as in rabies epidemiological survey to differentiate between the rabies and non-rabies-related viruses. ( Ermine et al. 1990 ).  The process consists of sample preparation of extraction of total RNA from brain materials by employing TRIZOL or Guanidium isothiocyanate, reverse transcryptase with oligo (dT),  followed by PCR using gene specific primers.  The PCR amplification techniques of rabies virus nucleic acids are an alternative protocol for diagnosis and epidemiological studies of rabies virus ( Sacramento et al.1991).   A primer set mapping in the nucleocapsid gene of rabies virus allowed a specific and sensitive amplification of infected brain material fulfilling the diagnosis requirements.  Further PCR products can be checked by southern or Dot blot analysis using both radioactive and non-radioactive probes showed identical results in parallel with routine techniques.  For molecular epidemiological studies selection of another set of conserved primers flanking the niches evaluative pseudogene region.   This test was found to be efficient for all tested fixed rabies virus strains or rabies related virus isolates ( Heaton et al.1997).

In situ Hybridization: This procedure allows the reproducible in situ detection of rabies virus antigen and both genome and messenger RNAs in formalin fixed tissues. Ezhil Praveena et al .2006).These procedures can be used on sequential tissue sections and thereby permit comparison of results from tests detecting both antigen and RNA in the some tissue.  This antigen detecting procedures has also been used to identify both the phylogenetically distinct rabies viruses like rabies and rabies related viruses (Jackson et al  1989 and  Warner et al.1997 )

Reverse Transcriptase PCR : A rapid detection method for the rabies and rabies related viral RNA using  in-situ RT-PCR and RT-PCR ELISA has been available.  The detection of digoxigenin – labeled amplified products is performed by solution hybridization to two specific biotin labeled capture probes, which are complementary to the inner region of the amplification products.  The capture probe and amplified product hybrid are immobilized on a streptavidin-coated micro plate and products are detected by an anti-DIG fab  fragment  conjugated  to  peroxidase  and colorimetric  reaction  automatically  measured.  This method has  up to  100 fold  more sensitive  than southern  blot  and FAT.  The   complete  detection methodology  from RT-PCR  to  PCR ELISA  detection could be completed  within 10 hours.( Jayakumar et al.2003 ).

Demonstration of  Rabies  antibody: A  number of  serological  procedures have  been  described  for   measuring rabies antibody.  These include  the Complement  fixation test(CFT), Passive Heamagglutination  test (PHA), Heamagglutination inhibition test,  Plaque  reduction  test, Gel diffusion test, Counter immunoelectrophoresis test, Mouse neutralization test, indirect fluorescent antibody test, Radio immunoassay (RIA),  Rapid fluorescent focus inhibition  test (RFFIT) and ELISA test.

References: 

Bourhy,H and Sureau,P. (1990). Laboratory Methods for rabies diagnosis. Institute Pasteur,Paris, France.

Barrat, J and Blancou, J (1988). Simplified technique for the collection , storage and shipment of brain specimens for rabies diagnosis. WHO/Rab.Res./88.27. World Health Organization,Geneva.

Dean, D.J. and Abelseth,M.K. (1973). The fluorescent antibody test. In: Kaplan, M.M. and Koprowski, H., de., Laboratory techniques in rabies, pp73-84 (3 rd edition). Geneva, World Health Organization ( Monograph Series,No.23).

Sellers, T.F. (1927). A new method for staining negribodies  of rabies. Amer.J.Publ.Hlth 17:1080.

Berkman, R.N., Barner,R.D., Morill, C.C and Langham, R.F. (1960). Bovine malignant catarrhal fever in Michigan. II.Pathology.Am.J.Vet.Res. 21: 1015-1027.

Derkashan, ,I., Bhamayar,M., Noorsalchi,S., Fayaz, A and Mohammed, M and Abonraji,R. (1978). Light microscope diagnosis of rabies, a reappraisal. Lancet I: 302-303.

Perrin, P., Rollin,P and Sureau,P (1986). A rapid rabies enzyme immunodiagosis (RREID). A useful and simple technique for the routine diagnosis of rabies. J.Biol.Standard.14:217-222.

Jayakumar, R, Ramadass, P and Raghavan,N. (1989). Comparison of Enzyme Immunodiagnosis with Immunofluorescence for rapid Diagnosis of rabies in dogs. Zbl.Bakt.271:501-503.

Montano Hirose, J., Bourhy, H and Sureau, P. (1991). Retro-orbital route for brain specimen collection for rabies diagnosis. Vet.Record. 129: 291-292. 

Jayakumar,R and Ramadass, P.(1991). Evaluation of diagnostic tests for rabies in dogs. Indian Vet.J68:765-768.

Jayakumar, R., Nachimuthu, K and Padmanaban, V.D. (1994). A comparison between avidin-biotin peroxidase complex (ABC) and unlabelled antibody (PAP) procedures in rabies diagnosis. Indian Vet.J.71:866-869.

Jayakumar, R and Padnmanaban, V.D. (1994). A Dipstick Dot Enzyme Immunoassays for detection of rabies antigen. Zbl.Bakt.280:382-385.

Jayakumar, R., Shaw, A.M., Kumanan, K., Nachimuthu, K and Padmanaba, V.D. (1995). A modified Dot ELISA for the detection of rabies virus antigen. Indian J.Virol.11:51-53.

Jayakumar, R., Nachimuthu, K and Padmanaban, V.D. (1995). A Dot Enzyme Linked Immunosorb\ent assay (DOT ELISA): Comparison with standard fluorescent antibody test (FAT) for the diagnosis of rabies in animals. Comp.Immun.Microbiol.infect.Dis.18:269-273.

Jayakumar, R., Thirumurugan, G., Nachimuthu, K and Padmanaban, V.D. (1995).Detection of rabies virus antigen in animals by avidin-biotin Dot ELISA. Zbl.Bakt.285: 82-85.

Jayakumar , R., Thirumurugan,G., Nachimuthu, K and Padmanaban, V.D. ( 1997). Protein A Dot enzyme –linked immunosorbent assay (dot –ELISA): Comparison with fluorescent antibody test (FAT) in the diagnosis of rabies in animals. Ind.J Anim.Scie. 67:497-498.

Jayakumar, R., Chandran, N.D.J., Nachimuthu,K and Padmanaban, V.D. (1995). A rapid latex agglutination test for the detection of rabies antigen. Ind.J.Anim.Scie.65:414-416.

Rudd, R.J., Trimarchi,C.V and Abelseth, M.K.(1980). Tissue culture techniques for routine isolation of street rabies virus. J.Clin.Microbiol.12:590-593.

Portnoi,D., Favre, S and Sureau, P.(1982). Use of neuroblastoma cells (MNB) for the isolation of street rabies virus from field specimens. Rabies Infor.Exch., CDC, 6:35-36.