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RABIES VACCINE
By:J.S.K. NAGARAJAN,ASST. PROFESSOR,JSS UNIVERSITY,(OFF CAMPUS, JSS COLLEGE OF PHARMACY),OOTY- 643 001.E-mail: [email protected]: 09443149945
• Viral disease - cause acute encephalitis (inflammation of the
brain) in warm-blooded animals.
• Zoonotic (animals to humans).
• Bite from an infected animal.
• For a human, rabies is almost invariably fatal if post-exposure
prophylaxis is not administered prior to the onset of severe
symptoms.
• Virus infects the CNS, cause disease in the brain & death.
• Causative Organism – Lyssavirus genus(Family Rhabdoviridae)
Rabies -Introduction
Virus lifestyle
• Virus replicated in muscle cells near site of bite for most of incubation time.
– Incubation time 30-90 days.
• Then ascends along motor and sensory axons at rate of 12-100mm/day and has predilection for brainstem and medulla
• Enters salivary glands after replication in CNS.
Animal Behavior
• Classic Picture of rabid, mangy dog foaming at the mouth…not often seen, signs frequently more subtle.
• Animals - display aggressive behavior, ataxia, irritability, anorexia, lethargy/excessive salivation.
• Cats are more likely to be aggressive than dogs
• Animals exhibit change in instinctive behavior: nocturnal animal walking around in daylight (i.e. raccoons)
• Unprovoked bites
Transmission
• Almost all transmission is by bite
• 50 times greater risk than a scratch
• One human case may have been acquired in a laboratory transmitted by aerosol
• In wild animals: Rabies can be transmitted transplacentally
• Transplants in human- possible
• Human-to-human: Never has been confirmed
• Rabies virus never isolated from blood
Rabies virus
• Risk of developing rabies after a bite: 5-80%.
– Depends upon….
• Severity of exposure
• Location of the bite
• The biting animal
• Bites on head and neck have shorter incubation time (as short as 15 days) because of rich peripheral nerve supply
Encephalitic Form
• Hydrophobia: Patient can’t swallow because violent jerky contraction of diaphragm and accessory muscles of inspiration when pt attempts to swallow liquids
- Patients will be terrified during this reaction and may even experience this at the sight of water or if water touches their face.
• Aerophobia: an extreme fear of air in motion can be elicited from some patients. This can also cause violent muscle spasms in the neck and pharynx.
• Hallucinations, seizures, ataxia, focal weakness and arrhythmias can occur.
Paralytic Rabies
• Other form is “dumb” or paralytic rabies. Similar to Guillain-Barre.
– Prominent limb weakness. Consciousness initially spared
• Two forms can overlap or progress from one to the other
• Coma after one week of neuro symptoms with death a few days after.
Rabies vaccine
PRINCIPLE:
Potency of rabies vaccine is determined bycomparing a lethal intra-cerebral dose of arabies virus with the dose of the standardpreparation of rabies vaccines necessary togive for same protection.
Rabies Vaccine AssayTest Animals:
Mice – 3 -4 weeks old
11 – 15 gm
6 X 16 and 4 X 10
Calculate LD 50 For Virus suspension
Estimation Of Potency
Preparation of Standard Challenge Virus Suspension:Inject the Virus (0.3ml of 10 fold diln. -Intra Cerebrally)
Sacrifice the animal(after getting signs)
Harvest the brain aseptically
Wash with saline to remove blood clotsMethod 1 Method 2
10% suspension brain homogenised Brain homogenised with diluentgives 10% suspension
Centrifuge lightly & collect the supernatant liquid
Distribute into sterile vials Distribute in sterile ampoules/vials
Freeze dry and store @ -200 C Store @ -600 C ( maintain NLT 1 Yr.)(maintain for NLT 3yrs)
Rabies Vaccine AssayVirus Tire of the Challenge Virus:
Prepare 3-10 fold dilution of std.challenge virus suspension.↓
0.03ml inject intracerebrally to 4 X 10 mice.↓
Observe mice for 14 days.↓
Count the number of mice surviving in each group.↓
Calculate the virus titre of std challenge vurus suspension by statistical method.
Determination of Potency:
Potency of rabies vaccine is determined by comparing the dose necessary to protect mice against a lethal intracerebral dose of rabies vaccine necessary to provide the same protection.
Rabies Vaccine Assay cont’nPrepare 3-5 fold serial dilution of standard and test soln of vaccines
Separate mice in 6 groups of 16each
Inject the vaccine 0.03ml(Intra-Peritoneal)
After 7 days inject the same
After further 7 days inject 0.03ml Standard challenge virus suspension by 5 to 50 LD50
Observe for 14 days and record the number of survivals
Calculate the potency of the preparation under examination by standard statistical methods.
RABIES ANTI SERUM
• Rabies anti serum is a preparation containing the specific globulin [or] its derivatives obtaines by purification of hyper immune serum[or] plasma of healthy horses [or] other animals having the specific activity of neutralizing the rabies virus.
• BIOLOGICAL ASSAY OF RABIES ANTISERUM:
The potency of rabies antiserum is determined by comparing a lethal intracerebral dose of rabies virus with the dose of standard preparation of rabies antiserum necessary to give same protection.
PROCEDURE:
STANDARD PREPARATION:
Standard preparation is dried serum (or)
Other preparation , the potency of which has been determined in relation to international standard.
TEST ANIMALS:
Mice, 10g-14g-animal same sex.
TEST VIRUS:
Any suitable strain rabies virus of known potency, such as the CVS strain may be used.
TEST DOSE OF VIRUS:
20-1000 LD50 intracerebral injection to each mouse.
DET. OF TEST DOSE OF VIRUS:Virus dilutionequal quantity of 2%v/v solution of heat
inactivated horse serum in water .↓
Maintain at 37⁰c at one hour.
2) Prepare is 10 fold dilution in a 2%v/v solution of heat –inactivated normal horse serum
Inject into mouse.↓
The test is not valid unless the quantity of virus used lies between 20-1000 LD50.
DETERMINATION POTENCY OF RABIES ANTISERUM:
Prepare 2 fold dilution of std preparation and test preparation with 2% v/v heat inactivated
normal horse serum in water.↓
To each dilution add a quantity of suspension of test virus
↓Keep the mix at 37⁰c for 1hr
↓Inject 0.03ml intracerebrally to mice
↓
Observe mice for 14days↓
Mice dying before 5th day after inoculation of virus are eliminate from test, all the mice dying between 5th -14th day after
showing signs of rabies↓
Count the no of mice surviving↓
Calculate potency of the test preparation by std statisticalmethod
– The preparation pass the test it found to have 80units /ml– The preparation for the test also same as standard
Rabies Vaccine Assay cont’nVaccine complies with the test if the estimated potency is NLT 2.5 IU/single dose.
- The test is not valid unless (a) for both the ppn under examination and the std
ppn, the 50% protective dose lies between the largest and smallest doses given to the mice;
(b) there is not deviation from linearity or parallelism of the dose response lines, the confidence limit (P= 0.95) are NLT 25% & NMT 400% of the estimated potency;
(c) the titre of the challenge virus suspension lies between 5 to 50 LD50.