RABIES

SAMPLING, SUBMISSION, SHIPMENT

DR. SHUBHAGATA DAS

Lecturer
Department of Pathology and Parasitology
Faculty of Veterinary Medicine
Chittagong Veterinary and Animal Sciences University, Khulshi, Chittagong 4202
Mob: +88 01717935112
E mail: shubho85@gmail.com

GENERAL ASPECTS:
Rabies is an acute viral infection causing encephalomyelitis in virtually all worm blooded
animal characterized by sign of abnormal behavior, nervous disturbance like hyperexitibility
and hyperirritability, impairment of consciousness, ascending paralysis and death Rabies
present on all continents and endemic in most African and Asian countries is a fatal and is
estimated to cause at least 55000 deaths per year world wide, about 56% of which occur in
Asia and 44% in Africa, particularly (84%) in rural areas

 Is the Oldest and Major Zoonotic Disease in the world; kills almost 55,000 human per
year world wide.

 Infects almost all worm blooded and dog, cattle, fox, jackal, cat, mongoose, bats acts
as potential reservoirs.

 In Bangladesh rabies death toll >2,000/year (Infectious Disease Hospital (IDH),

Dhaka reported cases only; in India:20,000/yr; Africa:24,000/yr–WHO estimates)

 300,000 people receive Post-exposure treatment / year in BD of which 85% from rural
areas.

 Large number of livestock die yearly (annual economic loss yet to Be Established)

RABIES IN DOMESTIC ANIMAL:

 All warm blooded animals are vulnerable to rabies infection

 Susceptibility is affected by factors such as viral variant, quantity of virus inoculated,
and the bite site.
 In addition, the degree of species susceptibility varies considerably. Younger animals
are usually more susceptible to rabies infection than older ones.
 Clinical observation may only lead to a suspicion of rabies because signs of the
disease are not characteristic and may vary greatly from one animal to another.
 The only way to perform a reliable diagnosis of rabies is to identify the virus or some
of its specific components using laboratory tests.
  Animal develops rabies most often when the bite of another animal transfers rabies-
virus-laden saliva to the wound. Rabies virus moves transneuronally from the site of
entry to the spinal cord and brain.

CLINICAL FEATURES OF SUSPECTED RABID ANIMAL:

Incubation period May vary: From 10 days to 6 months (may be upto 2 years)
Appears in two forms: Depending on the bite site and viral disposition in the CNS or PNS
• Furious or Maniacal form (By initial CNS manifestation; (mainly convulsions and
aggressive behavior with greatly exaggerated biting tendencies)
• Dumb or paralytic form (By initial PNS manifestation; predominantly paralytic
manifestation with docile behavior of animal)
In DOG: Drooping jaw, abnormal sound in barking, dry drooping tongue, Licking its own
urine, Regurgitation, Altered behavior, Biting and eating abnormal objects, Biting with no
provocation, Running without apparent reason, Stiffness upon running or walking,
Restlessness, sleepy appearance, Imbalance of gait etc. Pharyngeal paralysis and paralysis
leads to death with in 5-7 days on onset of disease

A. Furious form: It refers to syndrome in that excitation is the predominant changes and it
can be divided into stage of melancholy and stage of excitation.
Stage of melancholy:
• Lasts for 1-3 days.
• Behavioral change of the dogs showing tendency to bite animate or inanimate objects.
• Have continuous biting urge till death.
• Infected dog become unusually alert
• Gradually the signs of excitability increases.
• Pupil got dilated, appetite completely suppressed
• Drooling salivation and laryngeal muscle paralysis.
Stage of excitement:
• Lasts as long as 10 days.
• Maximum excitability and irritability.
• Dogs become very much aggressive.
• Photophobia, biting tendency and characteristics change of bark
• In coordination and muscle tremor
• Drooling salivation, unable to bark and increased libido

B. Dumb form: Also known as paralytic form characterized by-

• Paralysis at the lower jaw, tongue, larynx and hind quarters.
• The dogs become incapable to bite due to jaw muscle paralysis
• Appears sick in solitude, sluggish and morose.
• Progressive weakness and paralysis

In CATS: Rabid cats show extreme aggressiveness, great sensitivity to touch/noise, profuse
Salivation and may attempt to attack dog or even man.
In CATTLE: In cattle, rabies is manifested as abnormal movements of posterior extremity
foamy yellow froth and decrease in yield of milk. Drooping salivation, hyperesthesia, frenzy,
eating abnormal objects, paralysis or imbalanced of gait are common clinical manifestations.
COMMONLY USED DIAGNOSTIC TESTS FOR RABIES IN ANIMAL:

In presence of typical features diagnosis of rabies has been essentially clinical in both animals
and man. There are, however, instances both in man and animals when clinical diagnosis
becomes difficult. The definitive diagnosis of rabies can only be obtained by laboratory
investigations, otherwise it may result into a rabid animal remaining undiagnosed and hence a
greater risk to human beings. Similarly, persons bitten by animals who have died of diseases
presenting with similar clinical features can be unnecessarily vaccinated against rabies unless
diagnosis is established. Laboratory diagnosis may be required in human beings when the
clinical features are not unambiguous. It is of utmost importance in the era of organ
transplantation. Rabies though ante mortem as well as post-mortem diagnostic techniques are
available, the former do not have a sensitivity of more than 25% in best of hands. Battery of
tests should be performed. A positive test by one of the standard procedures overrides
negative reaction in the others. Negative tests do not rule out the possibility of rabies.
FACTS ABOUT COMMONLY EMPLOYED TESTS FOR RABIES DIAGNOSIS ARE
DESCRIBED AS FOLLOWS:

MORE RECENT AND RELIABLE DIAGNOSTIC TOOLS:

1. Reverse transcriptase polymerase Chain Reaction (RT-PCR)

2. Modified counterimmuno electrophoresis (CIEP) Serum
 3. Neutralization test
4. Enzyme Linked Immunosorbent Assay (ELISA)
5. Rapid Fluorescent Focus Inhibition Test (RFFIT)
6. Various Type Immunochromatoghrapic test:
a. Direct Rapid Immunochromatoghrapic Test (DRIT)
b. Lateral Flow Immunochromatoghrapic assay
7. Latex Agglutination tests

SPECIMEN FOR DIFFERENT DIAGNOSTIC TESTS FOR RABIES:

Test Specimen Test Type

Sellers Staining for Negri Brain Tissue Postmortem
Body Detection.
FAT Brian, Salivary Gland, Saliva, Postmortem
CSF
MIT Brain, Salivary Gland Postmortem
RTCIT Brain, CSF Postmortem, Ante mortem
RT-PCR Saliva, brain tissue, salivary Postmortem, Ante mortem
gland
CEIP Serum Ante mortem

ELISA Serum Ante mortem

RFFIT Serum Ante mortem

COLLECTION OF SAMPLES FOR DIAGNOSIS OF RABIES IN ANIMAL:

Saliva/Sputum:
Saliva is collected from under the tongue:
a) Wet a sterile cotton swab with tissue culture medium or physiological saline and remove
excess medium by squeezing on the sides of the vial.
b) Swab under the tongue, rinse in the tissue culture medium or physiological saline
containing 2% normal horse serum (NHS).
c) Take another swab similarly and make two smears each on clean labeled glass slides.
d) Air dry the glass slides for 10 minutes.
e) Discard the swabs in suitable disinfectant.
f) Treat the slides immediately with chilled acetone and process or wrap in paper and
dispatch to the laboratory.
Corneal Smear:
a) Retract the eye lids with thumb and one finger and press a clean marked slide against the
cornea.
b) Prepare two smears on each slide taking care to apply sufficient pressure to get the smear.
c) Avoid exerting too much pressure as it may damage the eye.
d) Air dry the smears for 10-15 minutes at room temperature.
e) Treat with chilled acetone and process further.

Skin Biopsy:
With very fine sharp scissors collect small pieces of skin from the site of bite and the face
near the mandible. Preserve in vial containing 50% glycerol saline (prepared by mixing equal
volume of glycerol and physiological saline and sterilized by autoclaving).
Cerebrospinal Fluid (CSF):
The CSF in acute phase of the disease is processed for isolation of the virus and in the later
phase for antibodies. It is collected by lumbar puncture. Usually no preservative is used but,
if required, 50% glycerol saline may be used.
Blood/Serum:
Acute phase venous blood specimen is collected as soon as possible with the usual aseptic
precautions. If the animal survives for several days, a second sample is taken. Serum should
be collected by using clot activator vials and kept freeze in sterile tube in -800C (for long
time) or in -200C for short time preservation.

NB: INTERPRETATION OF ANTE MORTEM DIAGNOSIS IS-

• Difficult – require battery of tests
• No single test has been positive in every case
• Distribution of rabies antigen in nuchal biopsy or corneal impression may be
extremely irregular.
• False positive results have been obtained in fluorescent examination of corneal
epithelium
• Antibody response to infection on occasions may be absent (due to low
seroconversion)
• It is necessary to test repeated samples
• Samples should be tested using all currently available tests
• Ante-mortem diagnosis should be attempted only by experienced laboratories
• A negative diagnosis does not rule out rabies

SO, POSTMORTEM DIAGNOSIS IS MORE CONFIRMATORY AND RELIABLE

FOR RABIES DETECTION.
Rabies virus goes from the inoculation point to one nerve, and then it reaches the central
nervous system passively. Once the central nervous system is infected, rabies virus may
diffuse towards peripheral organs (i.e. salivary glands, eyes, peripheral nerves). Thus,
samples for postmortem diagnosis are collected from the brain and sometimes from other
organs for certain histological or cytological signs of viral replication or viral antigen or
virulence and pathogencity of non-inactivated virus or viral nucleic acid.
Factors for collecting brain samples:
• Rabies diagnosis requires fresh (not fixed) brain tissue sampled from the brainstem
and cerebellum
 • Patterns of virus spread within the central nervous system suggest that a thorough
examination of the brain stem is critical to rabies diagnosis.
• Viral antigen is widespread in the brain of most animals positive for rabies, but
because spread may also be unilateral, especially in larger animals.
• They are classically applied to brain tissue, but they can also be applied, though less
effectively, to other organs (e.g. salivary glands).
• In the brain, rabies virus is particularly abundant in the thalamus, pons and medulla.
The structure of choice is the thalamus as it was positive in all cases.
• Rabies virus antigen can localize unilaterally thus the entire cerebellum and
underlying brainstem is the optimal sample. A full cross-section of the mid-
cerebellum and subjacent rostral brain stem is needed for a valid test.

PROCESS TO COLLECT BRAIN TISSUE SAMPLES FORM SUSPECTED RABID

ANIMAL FOR POST MORTEM DIAGNOSIS:

HEAD REMOVAL:
Virus replication is not homogenous in the brain. The Ammon's horn (hippocampus) and
rachidian bulb are two areas where virus is easily found in high concentrations. The classical
way to collect brain samples is to remove the head from atlanto-occipital joint.

PREREQUISITES:
Instruments:
The instruments and materials to be used for sampling are then collected and taken into
the necropsy room:
• For every head or entire carcass, these consist of:
• Plate for the central nervous system,
• Scalpel and one toothless forceps (for brain removal after opening the skull),
• Microscope slides for the fluorescent antibody test,
• Plastic tubes, one for the cell culture test, to keep a sample of the brain and a for mice
inoculation test when needed
• Fixation vial when histological examination is performed,
• Blade (scalpel) to open the skull.
• Large scalpels used for cutting the skin and reclination of temporal muscles,
• Rat toothed forceps for holding the skin and muscle while cutting them,
• Hammer and chisel
• A pair of holding forceps to hold the head on the necropsy table.

Precautions:
1. Precautions should be taken when handling central nervous system tissues from
suspected rabies cases. Gloves should always be worn and precautions must be taken
to prevent aerosols.
2. The use of cutting tools, scissors and scalpels, should be used with care to prevent
injury and contamination.
3. Disposal of the carcasses and the disposable materials after the diagnostic procedures
should be done either by Incineration or burying in quicklime deep enough in a
protected place not to be unearthed by carnivores.
4. The different reusable materials are decontaminated chemically the same day.
 5. The necropsy room is washed and decontaminated every day.

COLLECTION OF THE BRIAN TISSUE:

It is recommended that a pool of brain tissues that includes the brain stem should be collected
and tested. To reach these parts of the brain, it is necessary to remove the entire organ after
having opened the skull in a necropsy room.

A. Opening of the skull:

This operation is performed by two persons. One person holds the head on a plastic butcher's
block using a forceps introduced into the eye sockets. The other person opens the head. After
having checked that the number on the bag containing the specimen is the same as that on the
plate, the opening of the skull is performed. The different steps are:
1. Cutting of the skin
2. Reclination of temporal muscle
3. Opening the skull with the blade dedicated to this sample
4. Brain removal
5. Using the plate, forceps and scalpel dedicated to this sample, meningeal membranes are
opened and the brain is put on the plate which is then passed to the person who performs the
different tests used for rabies diagnosis.

B. Sampling of isolated central nervous system

The most important areas in the central nervous system are the Ammon's horn and the
rachidian bulb. Ammon's horn is a white semi cylindrical body on the floor of the fourth
ventricle. It may be exposed using different techniques:

1. A longitudinal incision 1 to 2 cm lateral to the interhemispheric line

2. A cross section made at the posterior third of the cerebral hemisphere. When this section
has reached the Ammon's horn, a longitudinal one gives access to the entire organ
3. A longitudinal section with scissors beginning at the posterior third of the brain and at the
first third closed to the interhemispheric line.

N.B: In some conditions this step may be hazardous if laboratory technicians are not
fully trained, or under field conditions. In such cases, there are two possible methods of
collecting some brain samples without opening the skull:

C. Occipital foramen route brain sampling:

Sampling is done through the occipital foramen by using a drinking straw (5 mm in diameter) or a 2 ml
disposable plastic pipette The steps of this procedure are:
1. Cutting the skin and neck muscles over the joint between the occipital bone (condylus occipitalis) and the
atlas (atlas),
2. Bend the head to give access to the occipital foramen,
3. Screw the straw into the foramen in the direction of an eye. This route crosses the rachidian bulb, the basis of
cerebellum, Ammon's horn and cortex,
4. The straw is pinched between the fingers and then drawn back gent Occipital foramen route
Or,
D. Retro-orbital route brain sampling
This technique was developed by MONTANO HIROSE and coll. (1991).
The steps are successively:
1. Push the eyeball aside
2. Use a trocar to make an entry through the posterior wall of the eye socket
3. Introduce through this hole a straw or a 2 ml disposable plastic pipette toward the occipital foramen.
The cerebral tissues sampled here are the same as the ones sampled by occipital foramen, but they
are taken in the opposite order.

E. Salivary gland sampling:

Sometimes it may be useful to check for rabies excretion i.e. presence of rabies virus in the salivary glands of
the animal. Sub-mandibular salivary glands are easy to sample and may be tested by different techniques such as
fluorescent antibody test, cell culture test or mice inoculation test. The removal of the sub-mandibular salivary
glands is possible using the following technique:
1. The head is placed to one side
2. An incision is made in the skin covering the posterior angle of the lower jaw
3. The gland is superficial behind the posterior part of the jaw (it must not be confused with the sub maxillary
lymph nodes.)

GENERAL SAFETY PRECAUTIONS DURING COLLECTION OF SPECIMEN:

Antimortem specimens: In Hydrophobia case: The person collecting the samples like saliva
and corneal smears should stand on the side or back of the patient. The cerebrospinal fluid
should also be collected with precaution. b) Rabid Animals: The animals must be restrained
before collection of antimortem specimens to avoid any chances of a bite.
Postmortem specimens: These include brain, spinal cord, salivary glands etc. - all of which
should be handled carefully and treated as highly infected material. Protective clothes like
laboratory coats, rubber aprons, thick rubber gloves, mask and plain goggles should be used
at the time of collection of samples, autopsy as well as handling of infectious material.
5. Equipment and Glassware: Instruments like scissors, forceps etc. used for collection and
processing of specimens should be sharp to avoid undue pressure on gloves. No niched
glassware is permitted. Any leaking mixer should be rectified.
6. Aerosols: Air borne rabies infection has been demonstrated hence all procedures which
generate aerosols (high speed mixing, centrifugation, pipetting etc.) should be carried out in
hoods with laminar flow of air under negative pressure. Centrifugation and mixing should be
carried out in tightly closed containers. Laminar hoods should have UV light for disinfection.
When not in use mouth pipetting should be strictly avoided.
7. Handling of experimental animal: The worker must use thick rubber gloves for handling
of animals. For daily examination of small animals forceps should be used and at no stage the
animals are to be handled with bare hands.
8. Safe handling of hazardous chemicals: Hazardous chemicals like Beta-propiolactone
used for inactivation of rabies virus etc. which are highly carcinogenic should always be
handled with care under hood. Transfer should be carried out by use of propipettes.
9. Disposal of equipment, glassware and protective cloths: The used equipment, glassware
etc. should be placed in the properly marked autoclavable containers containing suitable
disinfectants. The used gloves before taking off are dipped several times in quarter nary
ammonium compound. It any doubt of spillage on to apron coat is apprehended it is packed
separately for decontamination. The used disposable plastic material is also placed in specific
marked container.
10. Decontamination: It is done by boiling for sufficient in time in sterilizer or preferably
autoclaving. The small animals may be put in formalin jar pending disposal.
11. Disposal: The carcass of the small animals should be placed in plastic leak proof bags,
sealed and incinerated. The bedding of animals and disposable plastic ware may also be
incinerated.
12. Pre exposure immunoprophylexis
All the persons working in rabies laboratory and handling rabies virus or rabid animals
should be administered pre exposure immunoprophylaxis to protect them against contracting
the disease. The availability of safer anti rabies vaccines has made this task very easy. The
details of pre exposure immunization have been discussed.
13. Post exposure management
Every effort should be made to avoid occurring of mishaps in the laboratory due to
negligence by strictly following the safety precautions. However, in case of accident where
the spillage or inoculation of virus occurs following action may be initiated:
a) Working area should be immediately and meticulously cleaned by using proper
disinfectants.
b) The broken glass pieces should be picked up with the help of forceps and discarded in
appropriate containers for disinfection. One must be cautious about picking of fine pieces of
1
glass which can sometimes penetrate even thick rubber gloves.
c) Any accidental inoculation or even suspicion of the same onto a person must be
immediately reported to the appropriate authorities.
d) The soiled area on the body must be immediately washed with plenty of soap and water to
wash off as much of the virus as is possible. Care must be taken not to increase the existing
trauma. However, if the spillage is only in the eyes, wash the eyes thoroughly and repeatedly
with plenty of water.
e) On exposed sites other than eyes, apply an antiseptic which is readily available in the
laboratory. These include any quaternary ammonium compound, spirit, ethanol, tincture,
iodine, savlon, dettol etc.
f) As far as possible suturing of the wound be avoided. If it is inevitable, minimum number of
stitches should be applied to bring the skin in apposition. Local and parental administration of
antibiotics shall depend upon the condition of the wound.
g) The post exposure immunization shall depend upon the immunization status of the person.
Combined serum and vaccine therapy shall be indicated in persons who are not immunized or
who have not responded satisfactorily to previous immunization against rabies. Two booster
doses of anti rabies vaccine shall be needed by those who have been previously immunized
satisfactorily.

GENERAL GUIDELINE FOR SHIPMENT OF THE COLLECTED SAMPLES:

1. During the shipment of suspect material for diagnosis (animal heads, brain or other tissue
samples), no risk of human contamination should arise: brains must be placed in a leak-proof
rigid container (animal heads will be wrapped in absorbent material) as prescribed in the
International Air Transport Association (IATA) Dangerous Goods Regulations must be
followed.

2. When it is not possible to send refrigerated samples, other preservation techniques may be
used. The choice of the preservative is closely linked to the tests to be used for diagnosis:
Formalin inactivates the virus, thus the isolation tests cannot be used and diagnosis depends
on using a modified and less sensitive direct fluorescent antibody test (FAT),
immunohistochemistry or histology.
3. Infectivity at room temperature may be extended for several days if brain material is kept
in a mixture of 50% glycerol in phosphate buffered saline (PBS). It does not protect against
titer decline due to thermal conditions and therefore, because rabies is thermo labile, the virus
titer will decline during glycerol/PBS storage. Under normal transport conditions in the
tropics, this protection may only be effective for a matter of several days. Therefore,
whenever possible samples in glycerol/saline should be kept refrigerated. As the virus is not
inactivated by glycerol/PBS, all laboratory tests can be used on these samples

Transport without preservation:

1
DR. SHUBHAGATA DAS, Lecturer DPP, CVASU
Transport without preservation is the classical way to send a specimen to the diagnostic
laboratory. The veterinary authorities alone are authorized to rabies samples by post. Brains
are placed in a hermetically sealed, rigid container which is then identified.
1. Remove the head from the body of the animal (except bats) and place the head in a small
plastic bag. Cool specimen in a refrigerator or freezer before packaging, to enhance
preservation.

2. When shipping samples consisting of only cerebellum and brain-stem; first place the brain
tissue in a small plastic container, then place in the small plastic bag. If sharp objects protrude
from the specimen (e.g., bone fragments, porcupine quills); wrap specimen in several layers
of newspaper prior to putting head into plastic bag.

3. Heads or small animals are wrapped in absorbing paper and then placed in a resistant
plastic bag which is also identified. This first package is then introduced into a second one,
which is tightly closed and placed in an insulated box made of expanded polystyrene.
4. This box contains absorbing and cooling material and it is sealed with an adhesive tape. An
envelope containing all the information available on the samples is attached to the outside of
the box.
The box is then placed in a closed carton box where a label indicates clearly “BEWARE!
BIOLOGICAL SPECIMEN FOR RABIES DIAGNOSIS. INFECTIOUS HAZARD!"

Transport using preservative solutions:

If rapid transport with refrigerants is not possible, preservative solutions may be used.
• The choice of preservative depends on the laboratory techniques that will be used for
diagnosis.
• Formalin solution inactivates rabies virus. So transport is safe but the different
inoculation tests cannot then be used. Diagnosis can then still be done by FAT and
histology.
• Glycerin solution does not inactivate the rabies virus rapidly. It is a preservative
which inhibits temporarily the growth of contaminants. As rabies virus is not
inactivated, all laboratory techniques (including inoculation tests) may be used on
samples submitted in glycerin.

Preservative solutions for diagnosis:

If possible the samples of brain and salivary glands may be sent in wide mouth leak proof
containers preserved on ice. However, if the samples are to be sent long distance these may
be preserved by use of following:
a) 10% formal saline/Zenker’s fluid for half of the brain
b) 50% glycerol saline for other half of the brain and salivary glands.
c) Tissue culture medium 2% NHS saline for saliva, CSF,urine etc.
d) 10% Nutral buffer formalin or Bouins solution for Histopathological diagnosis or
cytological test.

NB: Do not use glass, wire or other materials capable of causing wounds for any purpose,
including use as containers, tag fasteners, etc.

Labeling:
All the specimens e.g. slides, vials must be labelled with number of specimens, name of the
patient, or species of the animal, type of preservative used etc. Permanent markers should be
used. The parcels should also be labelled properly.
Information to be enclosed
The species and breed of animal, contact with other animal, symptoms, mode and date of
death, vaccination status etc.

Transportation
a) By courier
b) By air/by post.
Utmost urgency should be exhibited in transportation of these specimens because any undue
delay, especially in tropical climates, shall wither away the cooling effect of ice and result
into putrefaction of the sample making it unsuitable for the diagnosis.

SUGGESTED MATERIALS FOR USE AND PROPER HANDLING:

1) Primary Container: Ziplock bags or heavy-duty garbage bags appropriately sized for the
specimens with an absorbent material (absorbent pads, paper towels, etc) placed in the bag to
prevent blood and body fluid from leaking. If sharp objects protrude from the specimen such
as shattered bone, wrap the specimen in several layers of newspaper. Always tightly seal or
fasten the primary container to contain the specimen.

2) Secondary Container: A metal can, heavy plastic pail with a lid or a heavy-duty Plastic
garbage bag may be used as the secondary container. These must also be sealed to help
prevent leakage of blood or body fluid. The Rabies Submission Form must be enclosed in a
plastic bag (ziplock) and taped to the outside of the secondary container.

3) Rigid Shipping Container: A tightly sealed, thick-walled Styrofoam container with or

without an exterior fiberboard liner may be used as the shipping container. It should also be
clearly labeled “Rabies” with permanent marker. The secondary container is placed inside the
shipping container along with sufficient frozen cool packs and cushion material to prevent
damage to the specimen during transport.
FIG: A STANDARD PACKING METHOD FOR RABIES SAMPLES:
A MODEL RABIES SUBMISSION FORM:
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Recommended Web Sites:

• www.who.int
• www.who.int/rabies/rabnet/en
• www.apcri.org
• www.rabiescontrol.org
http://apps.who.int/globalatlas/default.asp
http://www.who.int/rabies/en/
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