New
standard DFA protocol for rabies
Rabies
diagnosis in animals
The direct fluorescent antibody test (dFA) is the test most frequently
used to diagnose rabies. This test requires brain tissue from animals
suspected of being rabid. The test can only be performed post-mortem (after
the animal is dead).
Rabies diagnosis in humans
Several tests are necessary to diagnose rabies ante-mortem (before death)
in humans; no single test is sufficient. Tests are performed on samples
of saliva, serum, spinal fluid, and skin biopsies of hair follicles at
the nape of the neck. Saliva can be tested by virus isolation or reverse
transcription followed by polymerase chain reaction (RT-PCR). Serum and
spinal fluid are tested for antibodies to rabies virus. Skin biopsy specimens
are examined for rabies antigen in the cutaneous nerves at the base of
hair follicles.
The
importance of routine rabies tests
Rapid and accurate laboratory diagnosis of rabies in humans and other
animals are essential for timely administration of postexposure prophylaxis.
Within a few hours, a diagnostic laboratory can determine whether or not
an animal is rabid and inform the responsible medical personnel. The laboratory
results may save a patient from unnecessary physical and psychological
trauma, and financial burdens, if the animal is not rabid.
In addition,
laboratory identification of positive rabies cases may aid in defining
current epidemiologic patterns of disease and provide appropriate information
for the development of rabies control programs.
Essential characteristics for routine rabies test
The nature of rabies disease dictates that laboratory tests be standardized,
rapid, sensitive, specific, economical, and reliable.
Laboratory
tests for rabies
The standard test for rabies testing is dFA. This test has been thoroughly
evaluated for more than 40 years, and is recognized as the most rapid
and reliable of all the tests available for routine use. All rabies laboratories
in the United States perform this test (post-mortem) on animals suspected
of having rabies.
Other tests
for diagnosis and research, such as electron microscopy (EM), histologic
examination, immunohistochemistry (IHC), RT-PCR, and isolation in cell
culture are useful tools for studying the virus structure, histopathology,
molecular typing, and virulence of rabies viruses.
Direct
fluorescent antibody test (dFA)
The dFA
test is based on the observation that animals infected by rabies virus
have rabies virus proteins (antigen) present in their tissues. Because
rabies is present in nervous tissue (and not blood like many other viruses),
the ideal tissue to test for rabies antigen is brain. The most important
part of a dFA test is flouresecently-labelled anti-rabies antibody.
When labelled antibody is incubated with rabies-suspect brain tissue,
it will bind to rabies antigen. Unbound antibody can be washed away and
areas where antigen is present can be visualized as fluorescent-apple-green
areas using a fluorescence microscope. If rabies virus is absent there
will be no staining.
Antigen
detection by dFA
The rabies antibody used for the dFA test is primarily directed against
the nucleoprotein (antigen) of the virus (see The
Virus section on viral structure). Rabies virus replicates in the
cytoplasm of cells, and infected cells may contain large round or oval
inclusions containing collections of nucleoprotein (N) or smaller collections
of antigen that appear as dust-like fluorescent particles if stained by
the dFA procedure.
Positive dFA
Negative dFA
General
histopathology
Histologic examination of biopsy or autopsy tissues is occasionally useful
in diagnosing unsuspected cases of rabies that have not been tested by
routine methods. When brain tissue from rabies virus-infected animals
are stained with a histologic stain, such as hematoxylin and eosin, evidence
of encephalomyelitis may be recognized by a trained microscopist. This
method is nonspecific and not considered diagnostic for rabies.
Before current
diagnostic methods were available, rabies diagnosis was made using this
method and the clinical case history. In fact, most of the significant
histopathologic features (changes in tissue caused by disease) of rabies
infection were described in the last quarter of the 19th century.
After Louis Pasteur's successful experiments with rabies vaccination,
scientists were motivated to identify the pathologic lesions of rabies
virus.
Histopathologic
evidence of rabies encephalomyelitis (inflammation) in brain tissue and
meninges includes the following:
- Mononuclear
infiltration
- Perivascular
cuffing of lymphocytes or polymorphonuclear cells
- Lymphocytic
foci
- Babes
nodules consisting of glial cells
- Negri
bodies
| Perivascular
cuffing or inflammation around a blood vessel. Perivascular inflammatory
cell infiltrates in hematoxylin & eosin stained brain tissue.
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100x Magnification
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200x Magnification
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Babes Nodules
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Blood vessel without inflammatory cells (200x magnification)
A. Red blood cells
B. Squamous epithelial cells
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Negri
bodies
In 1903, most of the histopathologic signs of rabies were recognized,
but rabies inclusions had not yet been detected. At this time, Dr. Adelchi
Negri reported the identification of what he believed to be the etiologic
agent of rabies, the Negri body. In his report, he described Negri bodies
as round or oval inclusions within the cytoplasm of nerve cells of animals
infected with rabies. Negri bodies may vary in size from 0.25 to 27 µm.
They are found most frequently in the pyramidal cells of Ammon's horn,
and the Purkinje cells of the cerebellum. They are also found in the cells
of the medulla and various other ganglia. Negri bodies can also be found
in the neurons of the salivary glands, tongue, or other organs. Staining
with Mann's, giemsa, or Sellers stains can permit differentiation of rabies
inclusions from other intracellular inclusions. With these stains, Negri
bodies appear magenta in color and have small (0.2 µm to 0.5 µm), dark-blue
interior basophilic granules.
The presence
of Negri bodies is variable. Histologic staining for Negri bodies is neither
as sensitive nor as specific as other tests. Some experimentally-infected
cases of rabies display Negri bodies in brain tissue; others do not. Histologic
examination of tissues from clinically rabid animals show Negri bodies
in about 50% of the samples; in contrast, the dFA test shows rabies antigen
in nearly 100% of the samples. In other cases, non-rabid tissues have
shown inclusions indistinquishable from Negri bodies. Because of these
problems, the presence of Negri bodies should not be considered diagnostic
for rabies.
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Neuron without Negri bodies
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Negri body in infected neuron. Click on the image to see
a larger version.
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Enlargement
of a Negri body in Sellers stained brain tissue. Note the basophilic
(dark blue granules in the inclusion).
Click on the image to see a larger version.
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Immunohistochemistry
(IHC)
IHC methods for rabies detection provide sensitive and specific means
to detect rabies in formalin-fixed tissues. These methods are more sensitive
than histologic staining methods, such as H&E and Sellers stains.
Like the dFA test, these procedures use specific antibodies to detect
rabies virus inclusions. The techniques use enzyme-labeling systems that
increase sensitivity. In addition, monoclonal antibodies may be used to
detect rabies virus variants.
| Histologic
section of brain from a rabid animal |
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Click on the image to see a larger version.
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The
slide shows a rabies virus-infected neuronal cell with intracytoplasmic
inclusions. The red stain indicates areas of rabies viral antigen
by using IHC or avidin-biotin complex (ABC) technique.
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Ultrastructure
The ultrastructure of viruses can be examined by electron microscopy.
Using this method, the structural components of viruses and their
inclusions can be observed in detail. Rabies virus is in the family
of Rhabdoviruses. When viewed with an electron microscope Rhabdoviruses
are seen as bullet-shaped particles.
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Negatively
stained Rhabdovirus as seen through an electron microscope.
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Click on the image to see a larger version.
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A.
Notice the bullet shape of the virus.
B.
See the "bee hive" like striations of the RNP.
C.
Notice the glycoprotein spikes in the outer membrane bilayer.
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| Rabies
virus budding from an inclusion (Negri body) into the endoplasmic
reticulum in a nerve cell. |
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A.
Negri body.
B.
Notice the abundant RNP in the inclusion.
C.
Budding rabies virus.
Click
on the image to see a larger version.
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Click on the image to see a larger version. |
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Notice
the abundant strands of coiled RNP (almost everything in the image
is RNP).
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Amplification
methods
Samples containing small amounts of rabies virus may be difficult to confirm
as rabies-postive by routine methods. Virus isolation in cell cultures
increases the virus concentration because the virus replicates in cell
cultures. Mouse neuroblastoma cells (MNA) and baby hamster kidney (BHK)
cells provide an excellent environment for amplification of rabies virus
without the use of animals.
Another method
for amplifying the nucleic acid portion of rabies virus uses biochemical
methods. With this procedure, rabies virus RNA can be enzymatically
amplified as DNA copies. Rabies RNA can be copied into a DNA molecule
using reverse transcriptase (RT). The DNA copy of rabies can then
be amplified using polymerase chain reaction (PCR). This technique can
confirm dFA results and can detect rabies virus in saliva and skin biopsy
samples.
The figure
below shows PCR test results for rabies virus. The arrows indicate positions
of positive bands.
For
more information about rabies diagnosis, see:
Smith, J.S. (1995). Rabies Virus. In P.R. Murray, E.J.
Baron, M.A. Pfaller, F.C. Tenover, & R.H Yolken (Eds.) Manual of
Clinical Microbiology (997-1003), Washington, DC: American Society
for Microbiology.
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