Ý ß  Detailed DETECTION and IDENTIFICATION TECHNIQUES with literature reports for Rabies virus (with special reference to raccoon rabies variant) Use sub-contents list below, or simply scroll down the page to view findings.

OVERALL DETECTION AND IDENTIFICATION TECHNIQUES - Editorial Comment

Sample Collection and Shipping

• For virus detection in dead animals, the whole body (for small animals), whole head, whole brain, or for large animals a complete cross section of the brain stem plus either cerebellum or hippocampus are collected. In living individuals saliva, CSF, swabs from the throat, nasal mucosa or eye, eyeball impression smears and nuchal or facial skin biopsies may be tested. Samples are usually sent refrigerated and should reach the laboratory within 48 hours. Samples may be frozen but repeated freeze-thaw cycles must be avoided. 
• For antibody detection, blood is usually collected for testing of serum. Serum is stored frozen until it is tested. CSF can also be tested.
• In Procyon lotor - Common Raccoon:
  • Testing of raccoons for rabies diagnosis should involve examination of the cerebrum, brain stem and cervical spinal cord. 

Antibody Detection

The mouse neutralisation test, serum neutralisation tests in cell culture, such as the rapid fluorescence focus inhibition test (RFFIT), or ELISA can be used for detection of antibodies to rabies virus, indicating either prior exposure to the virus, or vaccination.  Mouse neutralisation, RFFIT and ELISA generally provide concordant results, although there may be discrepancies with sera of low titre, close to the cut-off points of the tests. (J93.27.w1)

• Mouse protection test
  • The mouse protection test was the first serological test developed for rabies antibody detection but is now outdated. It involves mixing dilutions of serum and virus before inoculating the mixture into mice and noting whether or not the mice die from rabies.
  • In Procyon lotor - Common Raccoon:
    • A study comparing antibodies in raccoon sera as detected by mouse inoculation and the RFFIT found that more sera were positive with the RFFIT than with the mouse protection test. Another study found that in general the mouse protection test and RFFIT test results correlated well with one another.
• Indirect fluorescent antibody test (IFA)
  • The indirect fluorescent antibody test can be used for detection of rabies antibodies in serum samples.
  • In Procyon lotor - Common Raccoon:
    • An indirect fluorescent antibody test, has been used for detection of rabies virus antibodies in raccoons. 
• RFFIT
  • The RFFIT (rapid fluorescent focus inhibition test) can be used for determination of rabies virus neutralizing antibodies. It uses a standard dose of rabies virus, to be neutralised by test serum prior to inoculation of susceptible cells. Unfortunately, non-specific cytotoxicity may affect the results of this test.
  • In Procyon lotor - Common Raccoon:
    • The RFFIT has been used for detection of virus neutralizing antibodies in raccoons.
• FIMT
  • The fluorescence inhibition microtest (FIMT) can be used for determination of rabies antibodies in sera of various species. Unfortunately, non-specific cytotoxicity may affect the results of this test.
  • In Procyon lotor - Common Raccoon:
    • The FIMT can be used for the determination of virus neutralizing antibodies in raccoons. However, cytotoxicity of raccoon sera may interfere with the results of this assay.
• ELISA
  • ELISAs have been developed for the detection of antibodies to either whole rabies virus or the envelope glycoprotein (G) of rabies virus. An indirect ELISA, using anticanine immunoglobulin, has been shown to be useful for the measurement of rabies-specific antibody in sera of a variety of carnivores.
  • In Procyon lotor - Common Raccoon: 
    • Cross-reaction of the anticanine immunoglobulin in the indirect ELISA was demonstrated for raccoons. (J1.24.w6)

Antigen Detection

A variety of methods may be used to detect rabies virus or virus antigen.

• Light microscopy/Detection of Negri bodies
  • Histological examination of brain sections or impression smears by light microscopy, in particular detection of characteristic Negri bodies, was the standard method of rabies diagnosis for many years. This has been replaced by the direct fluorescent antibody test, but is still used routinely in some developing countries. It has the advantages of requiring little equipment and giving a rapid result (within a couple of hours) but it is much less sensitive than immunological methods, particularly for partially autolysed samples. Additionally, false positive results may occur if nonspecific inclusion bodies are present. The OIE no longer recommends histopathology for the diagnosis of rabies.
• Electron microscopy
  • Examination by electron microscopy can be used to detect viral inclusions (corresponding to Negri bodies) and virus particles. 
• Mouse inoculation test
  • For many years mouse inoculation was considered the most sensitive way to detect rabies virus. However, it has now been superseded by modern methods for routine diagnosis. Several days are required for a diagnosis by this method. It is still used occasionally to confirm a test result, for example following human exposure to a suspect rabid animal which is negative by other tests. 
• Virus isolation in cell culture
  • Cell culture is not commonly used for virus isolation but can be useful in cases where immunofluorescence is inconclusive. It can be used for testing saliva and cerebrospinal fluid samples from living individuals as well as for testing brain and salivary gland tissues post mortem. Murine blastoma cells are recommended for cultivation of street rabies strains. Immunofluorescence is used to detect virus antigen in the cultured calls. While a positive cell culture result in a sample from a living animal allows a definite diagnosis of rabies, a negative result in samples from a live animal is not conclusive. A plaque assay has been developed using chicken embryo cells.
• Fluorescent Antibody Test (FAT)
  • The direct fluorescent antibody test (FAT) is presently the standard test for rabies virus detection. It is fast and reliable (sensitive and specific) for use on fresh or frozen tissue, and may be used on tissue which is degraded. It can be carried out on histological sections or on impression smears. In large animals, distribution of virus within the CNS may vary considerably and it is important to examine the cerebellum, brain stem and hippocampus using the FAT. Recently, a procedure has been developed allowing the use of FAT on formalin-fixed tissues.
• Immunoperoxidase
  • An immunoperoxidase method, using the streptavidin-biotin complex and polyclonal or monoclonal antibodies may be used for detection of rabies in formalin fixed paraffin embedded tissue sections.
  • Raccoon rabies variant:
    • The monoclonal antibody (mAb) 802-2 was shown in a study to detect rabies antigen in paraffin embedded tissue sections from rabid raccoons. (J3.136.w4)
• Direct rapid immunohistochemical test (dRIT)
  • This test can be used on brain touch impressions and the reaction product is visible under an ordinary light microscope (magenta inclusions, while the neuronal background is blue). The test allows diagnosis within one hour. It can be used on frozen samples or samples preserved in glycerol, and has a sensitivity similar to that of the direct fluorescent antibody test.
• RT-PCR
  • RT-PCR to detect lyssavirus nucleic acid is not used in routine rabies diagnosis but is useful for confirmation of IFA test results in formalin-fixed or decomposed material unsuitable for virus isolation, and has been used in antemortem diagnosis. This method may be used on samples of brain tissue, saliva or CSF, and may allow detection of very small amounts of viral material in tissues which are fresh, degraded or fixed.
• NASBA
  • NASBA is able to detect very small amounts of rabies virus RNA and has been used to test the saliva and CSF in humans with clinical illness.

Further details of various tests, and their references, are provided in the literature reports below.

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Sample Collection & Shipping

• For virus detection in dead animals, the whole body (for small animals), whole head, whole brain, or for large animals a complete cross section of the brainstem plus either cerebellum or hippocampus are collected. In living individuals saliva, CSF, swabs from the throat, nasal mucosa or eye, eyeball impression smears and nuchal or facial skin biopsies may be tested. Samples are usually sent refrigerated and should reach the laboratory within 48 hours. Samples may be frozen but repeated freeze-thaw cycles must be avoided. 
• For antibody detection, blood is usually collected for testing of serum. Serum is stored frozen until it is tested. CSF can also be tested.
• In Procyon lotor - Common Raccoon:
  • Testing of raccoons for rabies diagnosis should involve examination of the cerebrum, brainstem and cervical spinal cord. 

Samples

For virus detection

• Euthanasia of a rabies suspect animal should be by a means which does not damage the brain. (B209.1.w1)
• A whole carcass, intact head or brain tissue may be sent to a specialist laboratory. (D242)
  • The whole head should be sent. (B47, B58.1.w1)
  • The head, brain, or in small animals (e.g. bats) the whole carcass should be refrigerated and sent to a suitable diagnostic laboratory under refrigeration. (B209.1.w1)
  • Brainstem cores may be sent as an alternative to the whole brain for larger species. (B209.1.w1)
• If just brain tissue is sent, this must include a complete cross section of the brainstem plus either cerebellum or hippocampus. (D242)
  • Note: Most animals with rabies have widespread virus in the brain. However some individuals, particularly larger animals, have unilateral spread, therefore a complete cross-section is required to confirm that the animal is rabies-negative. (D242)
  • Examination of the brain stem is critical for rabies diagnosis. The brain may be examined at the pons, medulla or midbrain. Examination of cerebellar tissue as well as brainstem is advantageous since there are characteristic intracytoplasmic inclusions in large neurons of the foliar regions of the cerebellum, which are easily identified using DFA. Hippocampus is useful for testing (in addition to the brain stem) if the cerebellum is unavailable. (D242)
• Note: Salivary glands are not suitable for testing for rabies diagnosis; a negative test on saliva or salivary gland cannot rule out the possibility of rabies. (D242)
• In living individuals saliva and CSF may be tested for the presence of rabies virus by cell culture, RT-PCR or NASBA. (J21.73.w3, J93.36.w2, J98.358.w3)
• Saliva, throat swabs, eye swabs, swabs of nasal mucosa and CSF may be collected for intravitam diagnosis. Place throat swabs in 1 -2 ml of buffered saline solution containing 10% inactivated serum plus antibiotics. (B360.10.w10)
• For intravitam diagnosis, a smear can be taken directly from the eyeball, by firmly pressing a clean microscope slide against it. This can then be fixed, stained and read by the fluorescent antibody test. (B360.11.w11)
• Nuchal skin biopsy samples from living human patients can be used. (J93.36.w2)
• Biopsies from the neck in humans may used. Samples have also been taken from the neck of other species and from facial skin of mice. (B360.11.w11)
• In Procyon lotor - Common Raccoon:
  • Testing of raccoons for rabies diagnosis should involve examination of the cerebrum, brain stem and cervical spinal cord: areas of the CNS in which rabies lesions have been found consistently in raccoons with naturally acquired rabies. (J212.4.w1)
    • Note: CNS infection is usually bilateral, but not necessarily symmetrical. (J212.4.w1)

For antibody detection

• Blood/serum.
  • Serum is separated from clotted blood and stored frozen prior to testing. (J4.213.w4)
• CSF
  • Demonstration of rabies virus neutralizing antibodies in the CSF, in association with suggestive clinical signs, supports a diagnosis of rabies. (B209.1.w1)
    • Vaccination against rabies does not result in the presence of rabies virus neutralizing antibodies in the CSF. (B209.1.w1)
    • Antibodies are, rarely, found in the CSF of a healthy individual animal; this strongly suggests prior illness with and recovery from rabies. (B209.1.w1)
  • In humans, antibodies may be detected in only about 14% of individuals in the first eight days of infection, but positive in about 58% from day nine onward. (J98.358.w3)

Shipping

• Place specimens for virus isolation into sterile containers. (B360.10.w10)
• Shipment should be as soon as possible, preferably reaching the laboratory within 48 hours. (D242)
  • Brain samples will be preserved refrigerated for at least 48 hours. (D242)
• Ship on ice, or for longer journeys, on dry ice. (B360.10.w10)
  • Freezing once will not reduce test sensitivity but there may be additional delays in sample testing (until the samples have been thawed). (B209.1.w1, D242)
  • Repeated freeze-thaw cycles should be avoided as test sensitivity may be reduced. (D242)
• If refrigeration is not possible, such as in field surveillance programmes, particularly in the tropics, samples may be preserved by being placed in 50% saline-glycerine solution. (B209.1.w1, B360.10.w10)
• Samples must be packaged according to safety rules. (B360.10.w10)
• Samples must be correctly packaged for proper biocontainment, avoiding contamination of the outside of the package and also avoiding cross-contamination of samples in the package. (D242)

Mouse Protection Test

• The mouse protection test was the first serological test developed for rabies antibody detection but is now outdated. It involves mixing dilutions of serum and virus before inoculating the mixture into mice and noting whether or not the mice die from rabies.
• In Procyon lotor - Common Raccoon:
  • A study comparing antibodies in raccoon sera as detected by mouse inoculation and the RFFIT found that more sera were positive with the RFFIT than with the mouse protection test. Another study found that in general the mouse protection test and RFFIT test results correlated well with one another.

• The first serological test for rabies antibody was the virus neutralisation test in mice (mouse protection test). (J212.11.w1)
• In this test, serum at various dilutions is mixed with virus, agitated and incubated for 90 minutes at 37°C before being inoculated intracerebrally into mice. The mice are then observed for at least 14 days; mice dying in the first four days are considered non-specific, those dying after this are considered to have died of rabies. The serum neutralizing antibody titre is determined by the highest dilution at which the inoculated mice do not succumb to rabies. (B357.21.w21)
• In Procyon lotor - Common Raccoon:
  • A study comparing antibody titres in the sera of 38 raccoons from Florida by mouse protection test and RFFIT found that 17 sera were positive by mouse protection test at titres of >1:2, while these plus a further six sera were positive by RFFIT at a titre of >1:5 and the remaining 14 sera were negative in both tests. "The average point prevalence of neutralizing antibody was 20.9% (range, 15.5% [1970] to 25.0% [1974]." It was suggested that the RFFIT may detect more individuals with rabies antibodies than detected by the mouse inoculation test. (J100.148.w1)
  • A study compared rabies antibodies in sera from Iowa raccoons, as detected using three serological tests: rapid fluorescent focus inhibition test (RFFIT), mouse serum neutralisation (MSN) and indirect fluorescent antibody (IFA) test. With the RFFIT test, 51 of 985 raccoons (5%) showed serum neutralising titres of  >3.0 (titres 3.0 - 24.2). Testing of 24 of these sera in the MSN test gave 23 as positive (titres 3.2 - 17.9); one raccoon with a RFFIT test titre of 4.8 was negative with the MSN; in general the results of the MSN correlated well with the RFFIT. With the IFA, six raccoons were positive; these animals had RFFIT titres of 4.3 - 17.6 and the three which had been tested by MSN had MSN titres of 5.2-6.0. It was noted that the IFA detects antibodies to the internal nucleocapsid proteins whereas the SN antibody tests detect antibody to surface glycoprotein. The fact that both types of antibodies were detected was considered to confirm that the wild raccoons had been exposed to rabies antigen. (J1.28.w9)

Indirect Fluorescent Antibody Test (IFA)

• The indirect fluorescent antibody test can be used for detection of rabies antibodies in serum samples.
• In Procyon lotor - Common Raccoon:
  • An indirect fluorescent antibody test, has been used for detection of rabies virus antibodies in raccoons. 

• The indirect fluorescent antibody test has been accepted as a "rapid, sensitive and reproducible method for measuring rabies antibody." (B357.21.w21)
• In Procyon lotor - Common Raccoon:
  • An indirect fluorescent antibody test, has been used for detection of rabies virus antibodies in raccoons. The test uses cells (Madin Darby canine kidney (MDCK)) infected with Street Alberta Dufferin (SAD) rabies virus incubated in eight-chamber cell culture slides to produce about 30% infected cells, then fixed and stored frozen. The test is carried out by adding dilutions of serum to the wells, incubating for 30 minutes, then adding fluorescein isothiocyanate conjugated goat origin anti-raccoon immunoglobulin G. Titres were expressed as "the reciprocal of the highest serum dilution still exhibiting specific fluorescence." (J1.28.w9)
  • A study compared rabies antibodies in sera from Iowa raccoons, as detected using three serological tests: rapid fluorescent focus inhibition test (RFFIT), mouse serum neutralisation (MSN) and indirect fluorescent antibody (IFA) test. With the RFFIT, 51 of 985 raccoons (5%) showed serum neutralising titres of  >3.0 (titres 3.0 - 24.2). Testing of 24 of these sera in the MSN test gave 23 as positive (titres 3.2 - 17.9); one raccoon with a RFFIT test titre of 4.8 was negative with the MSN. With the IFA, six raccoons were positive; these animals had RFFIT titres of 4.3 - 17.6 and the three which had been tested by MSN had MSN titres of 5.2-6.0. It was noted that the IFA detects antibodies to the internal nucleocapsid proteins whereas the SN antibody tests detect antibody to surface glycoprotein. The fact that both types of antibodies were detected was considered to confirm that the wild raccoons had been exposed to rabies antigen. (J1.28.w9)

Rapid Fluorescent Focus Inhibition Test (RFFIT)

• The RFFIT (rapid fluorescent focus inhibition test) can be used for determination of rabies virus neutralizing antibodies. It uses a standard dose of rabies virus, to be neutralised by test serum prior to inoculation of susceptible cells. Unfortunately, non-specific cytotoxicity may affect the results of this test.
• In Procyon lotor - Common Raccoon:
  • The RFFIT has been used for detection of virus neutralizing antibodies in raccoons.

• The RFFIT involves incubation of a serum-virus mixture in multi-chamber slides followed by addition of a suspension of BHK-21 13S cells to the chambers. The chambers are then stained and examined. The total incubation time is 24 hours. The test measures the ability of serum to block infection of the BHK cells by a tissue culture adapted CVS rabies strain (i.e. it appears to measure neutralizing antibody). (B357.21.w21)
• The RFFIT (rapid fluorescent focus inhibition test) uses a standard dose of virus, to be neutralised by test serum prior to inoculation of susceptible cells (e.g. baby hamster kidney (BHK) cells) on a multichamber slide. After a period of incubation, the cells are stained with a fluorescein isothiocyanate-conjugated anti-rabies serum and examined under a microscope, equipped to detect fluorescein isothiocyanate fluorescence) at x320, observing 20 fields per slide chamber. This method requires highly trained, proficient staff to read the slides. (J212.11.w1)
• The modified RFFIT uses serial twofold dilutions of test serum to which a standard dose of virus is added. After a period of incubation, BHK-21 cells are added and mixed; samples of the mixture from each well are then transferred in duplicate into wells of plates, further incubated, rinsed, fixed, stained with fluorescein-conjugated anti-rabies nucleocapsid antibody and examined. In control wells with virus but no antibody, rabies virus-specific inclusions were visible in about 40% of cells. The endpoint of virus neutralization was defined as "the reciprocal of the highest serum dilution capable of reducing the number of rabies virus-infected cells by 50%. (J80.48.w1)
• Note: unfortunately, non-specific cytotoxicity interfering with viral growth may affect results in tests such as the rapid fluorescent focus inhibition test (RFFIT) or the fluorescence inhibition microtest (FIMT). (J1.24.w6)
• In Procyon lotor - Common Raccoon:
  • RFFIT was used to detect virus neutralizing antibodies in raccoons following experimental oral vaccination with an attenuated (SAD-B19) rabies virus vaccine and rabies virus challenge. (J1.25.w1)
  • The modified RFFIT was used to detect virus neutralizing antibodies in raccoons following intramuscular vaccination of free-ranging raccoons in Pennsylvania using inactivated rabies virus vaccine. (J1.26.w8)
  • The RFFIT was used to used to detect virus neutralizing antibodies in raccoons following intramuscular vaccination. (J1.26.w9)
  • RFFIT was used to detect virus neutralizing antibodies in raccoons following experimental oral vaccination and rabies virus challenge. (J1.38.w2)
  • RFFIT was used to detect virus neutralizing antibodies in raccoons following experimental oral vaccination with raccoon poxvirus recombinant expressing rabies virus glycoprotein. (J20.165.w1) 
  • RFFIT was used to detect virus neutralizing antibodies in raccoons following distribution of oral vaccines in Massachusetts. (J4.213.w4)
  • RFFIT was used to detect antibodies of raccoons trapped in the National Zoological Park, Washington, D.C. from June 1981 to October 1983 and in raccoons trapped at the National Zoological Park's Conservation and Research Center, Front Royal, Virginia, August 1981 to August 1983. (P103.1983.w1)
  • A study comparing antibody titres in the sera of 38 raccoons from Florida by mouse inoculation and RFFIT found that 17 sera were positive by mouse inoculation at titres of >1:2, while these plus a further six sera were positive by RFFIT at a titre of >1:5 and the remaining 14 sera were negative in both tests. "The average point prevalence of neutralizing antibody was 20.9% (range, 15.5% [1970] to 25.0% [1974]." It was suggested that the RFFIT may detect more individuals with rabies antibodies than detected by the mouse inoculation test. (J100.148.w1)
  • A study compared rabies antibodies in sera from Iowa raccoons, as detected using three serological tests: rapid fluorescent focus inhibition test (RFFIT), mouse serum neutralisation (MSN) and indirect fluorescent antibody (IFA) test. With the RFFIT, 51 of 985 raccoons (5%) showed serum neutralising titres of  >3.0 (titres 3.0 - 24.2). Testing of 24 of these sera in the MSN test gave 23 as positive (titres 3.2 - 17.9); one raccoon with a RFFIT titre of 4.8 was negative with the MSN. With the IFA, six raccoons were positive; these animals had RFFIT titres of 4.3 - 17.6 and the three which had been tested by MSN had MSN titres of 5.2-6.0. It was noted that the IFA detects antibodies to the internal nucleocapsid proteins whereas the SN antibody tests detect antibody to surface glycoprotein. The fact that both types of antibodies were detected was considered to confirm that the wild raccoons had been exposed to rabies antigen. (J1.28.w9)

Fluorescent Inhibition Microtest (FIMT)

• The fluorescence inhibition microtest (FIMT) can be used for determination of rabies antibodies in sera of various species. Unfortunately, non-specific cytotoxicity may affect the results of this test.
• In Procyon lotor - Common Raccoon:
  • The FIMT can be used for the determination of virus neutralizing antibodies in raccoons. However, cytotoxicity of raccoon sera may interfere with the results of this assay.

• The fluorescence inhibition microtest (FIMT), a micro modification of the RFFIT can be used for determination of rabies antibodies in sera. (J1.24.w6)
• Note: unfortunately, non-specific cytotoxicity interfering with viral growth may affect results in tests such as the rapid fluorescent focus inhibition test (RFFIT) or the fluorescence inhibition microtest (FIMT). (J1.24.w6)
• In Procyon lotor - Common Raccoon:
  • The fluorescence inhibition microtest (FIMT), a micro modification of the RFFIT, can be used for determination of rabies antibodies in sera of raccoons and other species. (J1.24.w6)
  • The FIMT was used to used to detect virus neutralizing antibodies in raccoons following intramuscular vaccination. (J1.26.w9)
  • The modified rapid fluorescent focus-forming microtest was used to detect virus neutralizing antibodies in raccoons following experimental vaccination. (J1.28.w12)
  • Raccoon sera have been found often to be cytotoxic in the FIMT, interfering with results of this assay. (J1.24.w6)

ELISA Techniques

• ELISAs have been developed for the detection of antibodies to either whole rabies virus or the envelope glycoprotein (G) of rabies virus. An indirect ELISA, using anticanine immunoglobulin, has been shown to be useful for the measurement of rabies-specific antibody in sera of a variety of carnivores.
• In Procyon lotor - Common Raccoon: 
  • Cross-reaction of the anticanine immunoglobulin in the indirect ELISA was demonstrated for raccoons. (J1.24.w6)

• ELISAs have been developed for the detection of antibodies to either whole rabies virus or the envelope glycoprotein (G) of rabies virus. (J93.27.w1)
  • An ELISA for detection of the envelope glycoprotein (G) is advantageous since neutralizing antibodies are considered to be the most reliable for indicating protection against rabies virus. (J93.27.w1)
  • ELISA is advantageous because it does not require cell culture techniques (which the RFFIT requires) or facilities for keeping live mice (required for the mouse neutralization test). (J93.27.w1)
  • Plates using G protein can be stored for several months at -20 °C and, because they do not contain whole virus, they may be used by laboratories which do not have the safety facilities required for working with whole rabies virus. (J93.27.w1)
• An indirect ELISA, using anticanine immunoglobulin, has been shown to be useful for the measurement of rabies-specific antibody in sera of carnivores. Cross-reaction of the anticanine immunoglobulin was demonstrated for various carnivores. A comparison of the ELISA with the FIMT on fox and skunk sera showed good correlation. (J1.24.w6)
• In Procyon lotor - Common Raccoon: 
  • Cross-reaction of the anticanine immunoglobulin in the indirect ELISA was demonstrated for raccoons. (J1.24.w6)
  • ELISA was used to used to detect virus neutralizing antibodies in some field serum samples from raccoons following intramuscular vaccination. (J1.26.w9)

Light Microscopy/Detection of Negri Bodies

• Histological examination of brain sections or impression smears by light microscopy, in particular detection of characteristic Negri bodies, was the standard method of rabies diagnosis for many years. This has been replaced by the direct fluorescent antibody test, but is still used routinely in some developing countries. It has the advantages of requiring little equipment and giving a rapid result (within a couple of hours) but it is much less sensitive than immunological methods, particularly for partially autolysed samples. Additionally, false positive results may occur if nonspecific inclusion bodies are present. The OIE no longer recommends histopathology for the diagnosis of rabies.

• Histological examination by light microscopy allows the detection of lesions of non-suppurative encephalitis, and Negri bodies may be detected. (B209.1.w1)
• Negri bodies are viral inclusions, as seen under electron microscopy. They consist of "a reticulogranular matrix containing tubular structures contiguous with maturing virus particles". Under light microscopy these are round or oval, acidophilic and strongly refringent, staining pink with haematoxylin-eosin and red with Mann's stain. They may often be found in different areas of the brain from where inflammatory lesions are evident. (B360.10.w10)
  • Negri bodies vary in frequency according to the animal species, whether the infection is natural or experimental (more with experimental) and with rabies virus strain. They are larger and more numerous when the incubation period and clinical disease phase are longer. (B360.10.w10)
  • Negri bodies may be distinguished from other inclusions by fluorescence, as well as by shape and size, intracytoplasmic distribution, colour and localization within the brain. (B360.10.w10)
• Negri bodies can be detected in either histological sections or impression smears, looking at bilateral samples from the hippocampus, brain stem and cerebellum. (J21.73.w3)
• The presence of Negri bodies (acidophilic cytoplasmic inclusions) within neurons on histopathological examination of the CNS allows definitive diagnosis. (B47, B58.1.w1, B417.2.2.5.w1, J13.23.w2, P65.1.w1)
• False negatives occur result because not all individuals develop Negri bodies. (B47, B209.1.w1, J15.23.w4)
  • Negri bodies are detected with standard stains in about 60-80% of rabies cases. (P65.1.w1)
• False positive results may occur if nonspecific inclusion bodies are present. (B209.1.w1)
  • It is not always easy to distinguish Negri bodies from other inclusion bodies. (J15.23.w4)
• Light microscopy does not require special equipment, is inexpensive and is rapid (a result can be obtained within two hours). (J15.23.w4)
  • Staining of an unfixed tissue smear by Seller's method allows diagnosis in less than one hour; other histopathological methods generally involve fixing the tissue and embedding it in paraffin, with test results within three days. (B417.2.2.5.w1)
• This is still [2001] the standard diagnostic technique for rabies in some developing countries. (J15.23.w4)
• This has gradually been replaced by use of the FAT (see below). (J21.73.w3)
• Laboratories in the USA which usually use immunofluorescent antibody staining for diagnosis of rabies may still occasionally make a diagnosis of rabies on the presence of Negri bodies. [1992] (J1.28.w10)
• This is no longer used for diagnosis in the USA: it detects only about 50 - 80% of cases which would be detected with the immunofluorescent antibody test. [1996](J128.9.w1)
• In an Ursus maritimus - Polar bear, no Negri bodies were found in the spinal cord; no histological lesions were found in the brain. [Infection was confirmed by other tests](J1.27.w9)
• Note: The sensitivity of histopathological examination of brain specimens for Negri bodies is much lower than the sensitivity of immunological methods, particularly when the specimen being tested is partially autolysed. The OIE no longer recommends histopathology for diagnosis of rabies. (B417.2.2.5.w1)

Electron Microscopy

• Examination by electron microscopy can be used to detect viral inclusions (corresponding to Negri bodies) and virus particles. 

• Electron microscopy can be used on fresh specimens, also on formalin-fixes or paraffin-embedded specimens. Virus particles and virus inclusions (corresponding to Negri bodies) may be seen. The viral inclusion is "a compacted mass of viral nucleocapsid, surrounded by numerous bullet-shaped viral particles budding from intracyoplasmic membranes." (B360.10.w10)

Mouse Inoculation Techniques

• For many years, mouse inoculation was considered the most sensitive way to detect rabies virus. However, it has now been superseded by modern methods for routine diagnosis. Several days are required for a diagnosis by this method. It is still used occasionally to confirm a test result, for example following human exposure to a suspect rabid animal which is negative by other tests. 

• Mouse inoculation may be used for virus isolation. (B209.1.w1)
• For many years, mouse inoculation was considered the most sensitive way to detect rabies virus. (J13.23.w2)
• Following intracerebral inoculation of (preferably) weanling mice (six per specimen), death within 48 hours is considered non-specific. Mice should be observed for at least 21 days, preferably 28 days. Paralysis occurs within 5-10 days in newborn mice, 8-15 days in adult mice. Diagnosis is confirmed using the fluorescent antibody test if available, otherwise by detection of Negri bodies and by virus neutralisation index test. For a faster detection, individual mice in a group are sacrificed every day or every second day, starting from three days (for newborn) or five days (for adult) mice, the brain being tested using the fluorescent antibody test. (B360.10.w10)
• Mouse inoculation tests involve intracerebral inoculation of Swiss mice with test suspensions (e.g. pooled bilateral samples of hippocampus, cerebral cortex, cerebellum and pons-medulla) and observation of the mice for development of neurological signs, followed by examination of their brains by histopathology for the presence of Negri bodies or by immunofluorescence microscopy for the presence of rabies virus antigen. (B47, J63.5.w1, P65.1.w1)
  • Laboratory mice have a relatively short incubation period, typical clinical signs, and consistently develop Negri bodies. [1959](J63.5.w1)
  • With positive samples, inoculated mice die at four to 28 days post inoculation and their brains are positive with the FAT. (J15.23.w4)
    • Deaths in the first week may be due to trauma, bacterial infection or other problems, while survival past three weeks indicates a negative result. (P65.1.w1)
• This is now used only infrequently. (B209.1.w1)
• Mouse inoculation is slightly less sensitive than immunofluorescence microscopy. (B47)
• Mouse inoculation is still used sometimes to confirm rabies in surveillance in the USA. [2005](J4.227.w1)
• The mouse inoculation test may still be used to confirm a negative result, for example when an animal which is suspected to have exposed a human to rabies is negative for Negri bodies and negative with the fluorescent antibody test (FAT). (B47, J15.23.w4, J270.10S4.w1)
• This system sometimes allows detection of rabies when the amount of virus in the samples is too small for detection by FAT. (J15.23.w4)
• A major disadvantage is the delay in diagnosis: several days are required for a positive diagnosis. (B47, P65.1.w1)
• The presence of antibodies late in infection may interfere with this test. (B360.11.w11)
• This test is less sensitive in specimens where decay is present. (B360.11.w11)
• Virus isolation via mouse inoculation may be attempted for results which remain equivocal with the FAT after repeated testing.  (N7.48.w5)
• In an Ursus maritimus - Polar bear, a positive mouse inoculation test was one way by which rabies was confirmed. (J1.27.w9)

Cell Culture Techniques

• Cell culture is not commonly used for virus isolation but can be useful in  cases where immunofluorescence is inconclusive. It can be used for testing saliva and cerebrospinal fluid samples from living individuals as well as for testing brain and salivary gland tissues post mortem. Murine blastoma cells are recommended for cultivation of street rabies strains. Immunofluorescence is used to detect virus antigen in the cultured calls. While a positive cell culture result in a sample from a living animal allows a definite diagnosis of rabies, a negative result in samples from a live animal is not conclusive. A plaque assay has been developed using chicken embryo cells.

• This may be used for virus isolation. (B209.1.w1, J21.73.w3)
• Cell culture is now used only infrequently. (B209.1.w1)
• Virus isolation in cell culture can be used in cases where immunofluorescence is inconclusive. This involves infection of mouse neuroblastoma cells or baby hamster kidney cells, followed by immunofluorescence for detection of virus antigen indicating growth of the (non-cytopathic) virus. (J128.9.w1)
• Murine blastoma cells, BHK-21 cells or the CER cell line may be used. Cells are set up in chamber slides and allowed to settle, then inoculated with test supernatants and incubated overnight. The supernatant is aspirated off, the cells air dried, acetone fixed and stained using an anti-nucleocapsid FITC conjugate, then examined using a fluorescent microscope. Intra-cytoplasmic fluorescent inclusions are present in positive but not in negative specimens. (B360.10.w10)
• Murine blastoma (NA C1300) cell lines have been reported most susceptible to rabies virus; many cell lines, such as BHK-21, used routinely for cultivation of "fixed" rabies strains are not ideal for isolation of "street" strains of the virus. (J21.73.w3)
• Virus isolation in cell culture can be used for testing samples of brain tissue and salivary glands post-mortem. (J21.73.w3)
• Virus isolation in cell culture can be used for testing saliva and cerebrospinal fluid samples from living individuals. (J21.73.w3)
• Virus isolation in murine neuroblastoma cells is used sometimes to confirm rabies cases in surveillance in the USA. (J4.227.w1)
• NOTE: A negative result in samples from a live animal is not conclusive: many animals have been found to be rabies virus positive in the brain but negative in saliva samples; even clinical patients with rabies may be negative for virus in biopsy, saliva or CSF. (J21.73.w3)
• A plaque assay has been developed for detection of rabies virus, using confluent monolayers of chicken embryo cells, infected at 37°C and followed by application of agarose or Sephadex G-100 overlay and incubation for 96 hours. [2004](J238.103.w1)
• Virus isolation via cell culture may be attempted for results which remain equivocal with the FAT after repeated testing.  (N7.48.w5)

Fluorescent Antibody Test (FAT)

• The direct fluorescent antibody test (FAT) is presently the standard test for rabies virus detection. It is fast and reliable (sensitive and specific) for use on fresh or frozen tissue, and may be used on tissue which is degraded. It can be carried out on histological sections or on impression smears. In large animals, distribution of virus within the CNS may vary considerably and it is important to examine the cerebellum, brain stem and hippocampus using the FAT. Recently, a procedure has been developed allowing the use of FAT on formalin-fixed tissues.

• Rabies diagnosis may be made using fluorescent rabies antibody test (FAT) (immunofluorescence microscopy using fluorescein-tagged conjugates to test for the presence of rabies virus antigen in sections of brain tissue). (B47, B58.1.w1, J15.23.w4)
• This uses fluorescein isothiocyanate (FITC), which emits a characteristic apple-green fluorescence. Rabies antibodies (from immune serum, tissue culture supernatant or ascitic fluid) are labelled with this marker then layered over a tissue section or smear containing rabies antigen. The antibody and antigen form a complex and the fluorescent compound becomes fixed to the antigen sites. The non-fixed antibodies are washed away and the remaining fixed compound is visible under a fluorescent microscope. Sensitivity approaches 100%. (B360.11.w11)
  • Staining is reduced when specimens which have been preserved in 50% glycerol saline are tested. (B360.11.w11)
  • Sensitivity is reduced when used on formalin-fixed paraffin embedded tissues; tissues may be treated by trypsin digestion, or better, trypsin-pepsin digestion, before the FAT is applied. (B360.11.w11)
• This is rapid, sensitive, and used worldwide for rabies diagnosis. (B209.1.w1)
• This is presently the standard test for rabies virus detection. It is fast and reliable (sensitive and specific) for use on fresh or frozen tissue. (J3.136.w4, J4.215.w3) 
• The standard FAT cannot be used on formalin fixed tissues. (D242)
• A standard protocol for carrying out the direct FAT on impression smears of brain samples from animals suspected of rabies has been developed, and is described on the CDC website, for use in diagnosis of rabies in animals in the USA. (D242, W170.05Sept04.R1)
• Examination of cerebellar tissue as well as brainstem is advantageous since there are characteristic intracytoplasmic inclusions in large neurons of the foliar regions of the cerebellum, which are easily identified using DFA. Hippocampus is useful for testing (in addition to the brain stem) if the cerebellum is unavailable. (D242)
• Note: "Modifications or short cuts in procedures often lead to false positive and false negative results and non-specific or uninterpretable reactions." (D242)
• This is the most common diagnostic method for rabies. (J15.23.w4)
• The direct immunofluorescent antibody test on thin touch impressions of brain tissue (medulla, cerebellum and hippocampus) is preferred for rabies diagnosis. (J128.9.w1)
  • Thin touch impression smears are fixed in cold acetone for one to four hours, air dried then stained using fluorescein isothiocyanate-labelled anti-rabies antibody. Examination at 400x to 1,000x magnification reveals dustlike particles less than 1 µm diameter or intracytoplasmic large round/oval masses and strings (2.0 - 10 µm diameter), smooth, with bright edges. While large amounts of material may stain, in other cases there may only be a few small inclusions in one or two microscope fields. (J128.9.w1)
• In large animals it is important to examine the cerebellum, brain stem and hippocampus using the FAT; distribution of virus within the CNS may vary considerably. (B362.w7)
• This method is rapid, sensitive, specific, easy to perform and relatively inexpensive. (J128.9.w1)
• Immunohistochemistry is highly sensitive and may be used on samples which have been frozen, or are degraded. (J15.23.w4)
• Immunohistochemistry is slower and more costly than standard light microscopy. (J15.23.w4)
• Rabies-specific inclusions are detected by light microscopy following application of the fluorescent labelled antibody . (B209.1.w1) Direct or indirect fluorescent antibody test can be used. (J21.73.w3)
• The FAT may be carried out on either histological sections or impression smears, looking at bilateral samples from the hippocampus, brainstem and cerebellum. (J21.73.w3)
• The FAT is recommended by the OIE for rabies diagnosis. If a potent conjugate is used, this test is 98-100% reliable for detection of all seven Lyssavirus genotypes. (B417.2.2.5.w1)
• The immunofluorescent antibody (IFA) test is fast and reliable (sensitive and specific) for use on fresh or frozen tissue. (B209.1.w1, J3.136.w4, J4.215.w3, J212.6.w1)
• The IFA test has a sensitivity approaching 100%, but false positives can occur, due to e.g. nonspecific reactions and contamination. (N7.48.w5)
• Slide impressions or smears are made from brainstem, hippocampus and cerebellum and fixed, usually with cold acetone. (B209.1.w1)
• Impression smears are fixed with acetone. (J15.23.w4)
• The cerebrum, cerebellum, hippocampus, medulla, thalamus and brainstem should all be examined. (J15.23.w4)
• Labelled-labelled monoclonal or polyclonal antirabies virus reagent is added and the sample is examined by direct fluorescence microscopy. (B209.1.w1, J15.23.w4) 
  • Equine hyperimmune serum may be used, labelled with fluorescein isothiocyanate. (P65.1.w1)
• Reliable results are usually available in two to four hours. (B209.1.w1)
• The sensitivity of the test is affected by variables such as the degree of decomposition of the samples tested. (B209.1.w1)
• FAT may allow detection of viral antigen in relatively decomposed tissue from which the mouse inoculation test will not be effective. (J15.23.w4)
• Salivary glands are not recommended as an alternative to brain samples for detection of rabies viral antigen. (B209.1.w1)
• This test can also be used on tissue samples which have been fixed, for example in formalin; special techniques are needed for testing of paraffin-embedded sections. (B209.1.w1)
• Direct FAT can also be used on frozen nuchal skin biopsy samples from living human patients, but does not detect all cases (e.g. five of seven individuals in one study, generally 60-80% detection rate). (J93.36.w2)
• Disadvantages of the FAT are that:
  • Specialist laboratories and properly immunised personnel are required; there may be difficulties in getting samples to such laboratories and appropriate packaging and handling of suspect diagnostic samples is needed. (J212.6.w1)
  • It is not easily used on tissues which have been routinely formalin-fixed and paraffin embedded. (J212.6.w1)
  • This test may occasionally be negative when mouse inoculation is positive, and vice versa. (P65.1.w1)
• Note: The direct fluorescent antibody test is considered to have a sensitivity approaching 100%. However, false positive reactions can occur: if antigen staining is weak, or sparse or focal inclusions are detected, this may be due to nonspecific antibody binding, or sub-optimum test conditions. Additionally, sporadic staining in a rabies-negative sample may occur due to cross-contamination with material from strong positive samples tested previously. If the result is not clearly positive or negative, new slides should be made from the brain tissue, and the test repeated using reagents from two different commercial sources and using additional controls for specificity. For results which remain equivocal, virus isolation may be attempted via cell culture or mouse inoculation, or PCR assays may be used. (N7.48.w5)
  • False positive diagnoses, or test results not confirmable by multiple laboratories testing the same sample "are not uncommon" (J67.71.w1)
• A procedure has been developed, involving a digestion stage using proteinase K to expose the target molecule, allowing use of a fluorescent antibody test on formalin-fixed tissues. (J217.67.w1. J217.95.w1)
• In an Ursus maritimus - Polar bear the fluorescent antibody test was negative (infection was confirmed by other tests). (J1.27.w9)

Immunoperoxidase Technique

• An immunoperoxidase method, using the streptavidin-biotin complex and polyclonal or monoclonal antibodies may be used for detection of rabies in formalin fixed paraffin embedded tissue sections.
• Raccoon rabies variant:
  • The monoclonal antibody (mAb) 802-2 was shown in a study to detect rabies antigen in paraffin embedded tissue sections from rabid raccoons. (J3.136.w4)

• Immunoperoxidase methods may be used to diagnose rabies in formalin fixed paraffin embedded tissues. (J3.136.w4, J212.6.w1)
  • The ABC (streptavidin-biotin complex) immunoperoxidase method successfully detected rabies in 39/40 individuals from a variety of species. Only one brain sample, cerebellum, was available for the single animal (cattle) in which the disease was not detected successfully. The test was effective on fixed tissues, in which the FAT cannot easily be used. Monoclonal antibodies (a mixture of three MAbs against rabies virus nucleocapsid antigen) produced more intense and discrete positive staining than did polyclonal antibodies of rabbit origin, against whole rabies virus. (J212.6.w1)
  • An immunoperoxidase method using the streptavidin-biotin complex (ABC) and polyclonal or monoclonal antibodies may be used for detection of rabies in formalin fixed paraffin embedded tissue sections. (J3.136.w4)
  • In an Ursus maritimus - Polar bear the immunoperoxidase test was positive for the virus antigen in the lumbar spinal cord and Gasserian ganglion sections. (J1.27.w9)
  • Raccoon rabies variant:
    • The monoclonal antibody (mAb) 802-2 was shown in a study to detect rabies antigen in paraffin embedded tissue sections from rabid raccoons at 1:40,000 dilution (and also, but fainter, at 1:80,000 dilution). (J3.136.w4)

Direct Rapid Immunohistochemical Test (dRIT)

• This test can be used on brain touch impressions and the reaction product is visible under an ordinary light microscope (magenta inclusions, while the neuronal background is blue). The test allows diagnosis within one hour. It can be used on frozen samples or samples preserved in glycerol, and has a sensitivity similar to that of the direct fluorescent antibody test.

• The direct rapid immunohistochemical test (dRIT) was developed recently and validated as a field test for rabies surveillance. This test can be used on brain touch impressions and the reaction product is visible under an ordinary light microscope (magenta inclusions, while the neuronal background is blue). The test allows diagnosis within one hour. It can be used on frozen samples or samples preserved in glycerol, and has a sensitivity similar to that of the direct fluorescent antibody test. (J84.12.w1)

RT-PCR Techniques

• RT-PCR to detect lyssavirus nucleic acid is not used in routine rabies diagnosis but is useful for confirmation of IFA test results in formalin-fixed or decomposed material unsuitable for virus isolation, and has been used in antemortem diagnosis. This method may be used on samples of brain tissue, saliva or CSF, and may allow detection of very small amounts of viral material, in tissues which are fresh, degraded or fixed.

• RT-PCR be used for the detection of lyssavirus nucleic acid. (B209.1.w1)
• RT-PCR may be used on samples of brain tissue, saliva or CSF. (J21.73.w3)
• This may allow detection of very small amounts of viral material, in tissues which are fresh, degraded or fixed. (J15.23.w4)
• PCR can be useful for confirmation of results of direct immunofluorescence in formalin-fixed or decomposed tissues, for which virus isolation is not possible. (J128.9.w1)
• This may be used as a confirmatory test following routine rabies diagnostics but is not recommended by WHO for routine primary diagnosis. (B209.1.w1)
  • False-positives may result from contamination or technical error. (B209.1.w1)
  • False-negatives may result, due to the diversity of lyssaviruses, if the initial primer  cannot allow for sequence heterogeneity. (B209.1.w1)
  • There are not yet universal primers for all known lyssaviruses. (B209.1.w1)
• RT-PCR is used sometimes to confirm rabies cases in surveillance in the USA.  (J4.227.w1)
• One advantage is that RT-PCR may give a result in just a few hours. (J21.73.w3)
• A major disadvantage is that not many laboratories are set up to use this test. (J21.73.w3)
• PCR can be used to detect which rabies virus variant is present. (J128.9.w1) 
  • See: Rabies virus - Viral Type Diversity (Viral Reports)
• PCR may be used for rabies virus detection in vivo: In one study, RT-PCR detected rabies virus in five of nine rabies-positive (confirmed at post mortem examination) patients, including detection in one of five patients at zero to four days after the onset of clinical signs and in three of eight patients at five to eight days after onset. (J93.36.w2)
• PCR assays may be used for results which remain equivocal with the FAT after repeated testing. (N7.48.w5)

NASBA Techniques

• NASBA is able to detect very small amounts of rabies virus RNA and has been used to test the saliva and CSF in humans with clinical illness.

• Nucleic-acid sequence based amplification (NASBA) has been described for diagnosis of rabies in human patients, using saliva and CSF samples. It was noted that rabies RNA was detected in the saliva and/or CSF of all patients from as early as two days after the onset of clinical signs, but that not all samples were positive. Repeated sampling and testing of saliva was recommended. Results could be obtained in about four hours. NASBA allowed detection of rabies RNA at about 100th of the amount required for detection by RT-PCR. (J98.358.w3)