RABIES SAMPLING, SUBMISSION, SHIPMENT

DR. SHUBHAGATA DASLecturerDepartment of Pathology and ParasitologyFaculty of Veterinary MedicineChittagong Veterinary and Animal Sciences University, Khulshi, Chittagong 4202Mob: +88 01717935112E 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) Serum3. Neutralization test4. 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 Body Detection.

Brain Tissue Postmortem

FAT Brian, Salivary Gland, Saliva, CSF

Postmortem

MIT Brain, Salivary Gland Postmortem

RTCIT Brain, CSF Postmortem, Ante mortem

RT-PCR Saliva, brain tissue, salivary gland

Postmortem, Ante mortem

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 skin2. Reclination of temporal muscle3. Opening the skull with the blade dedicated to this sample4. Brain removal5. 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 systemThe 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 line2. 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 organ3. 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 samplingThis technique was developed by MONTANO HIROSE and coll. (1991).The steps are successively:1. Push the eyeball aside2. Use a trocar to make an entry through the posterior wall of the eye socket3. 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 theyare 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 side2. An incision is made in the skin covering the posterior angle of the lower jaw3. 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 immunoprophylexisAll 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 glass which can sometimes penetrate even thick rubber gloves. 1

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:

1DR. 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 brainb) 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 enclosedThe species and breed of animal, contact with other animal, symptoms, mode and date of death, vaccination status etc.

Transportationa) By courierb) 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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