Anthrax spore vaccine is a living vaccine. To avoid contamination during its preparation, it is necessary to keep the bacterial population in the environment of the work area to a minimum. This can be best achieved by providing a filtered air supply and laminar flow cabinets. In the absence of filtered air supply, regular fumigation of laboratories and work cabinets should be carried out to keep airborne microbes to a minimum.
All the work surfaces of the laboratories should be kept clean and dust free at all times, the benches and work area should be swabbed regularly with a disinfectant such as methylated spirit or chloros at the beginning and at the end of each working day.
Formaline is commonly used for fumigation. It works best at temperatures above 24°C and at humidity above 65 percent. Fumigation should be carried out at the end of the working day. For one cubic metre space, a mixture of 13 ml of formaline (37 percent) and 6.5 g of potassium permanganate is placed in a Petri dish and the area should be sealed. Following overnight fumigation, seals are removed and the room can be used for work after removing the excess of formaline vapour by an exhaust fan.
Ultraviolet irradiation is often employed for the disinfection of the cabinets. The ultraviolet lamp should be kept clean and its bacteriocidal activity regularly checked, since its germicidal power deteriorates after some time, although the discharge of the rays continues. The cabinets and work stations should be regularly checked for their efficiency.
The level of airborne microbes should be regularly monitored in laboratories, cabinets and work stations. This is carried out by opening blood agar Petri dishes for a specified time in a specified position. After appropriate incubation of the plates, colony counts are made.
Adequate staff should be provided to avoid the necessity for the same staff to work in the production of other vaccines on the same working day. Staff must wear sterile protective clothing, gowns, head covers and face masks, and must change their shoes before entering the laboratory. Protective clothing should be changed daily, and should be autoclaved after use or discarded if disposable.
Visitors and persons not directly concerned with production should not be allowed to enter the vaccine production laboratories. Service personnel having occasional duties inside the laboratories should wear protective clothing before entering.
Pathological specimens for diagnosis should be processed only in separate areas not used for manufacturing biological substances.
Anthrax organisms are among the dangerous group of bacteria. There is a risk of contracting the infection particularly while handling virulent strains and artificially infected animals. However simple precautions will protect laboratory workers from infection with anthrax. Laboratory discipline is extremely important and it is recommended that:
For vaccine production and quality control, it is essential that arrangements should be made for the regular supply of good quality experimental animals from a known source. Specific pathogen free animals are ideal for the production and testing of vaccines. However, in their absence the animals reared in isolation obtained from disease free stocks can be used.
Laboratory animals or other animals used for production and quality control of vaccines should be provided with adequate housing. The breeding colony for healthy laboratory animals and experimental animals should be located in separate buildings away from the vaccine production unit. The animal house should be designed and constructed in such a way that it can be easily cleaned, and it should be free from insects. It should be provided with facilities for feed storage, washing and disinfection of cages, an isolation unit for quarantine, a post-mortem room and an incinerator for disposal of waste and dead animals.
In ideal conditions, experimental animal rooms should be provided with filtered air exhaust systems with absolute filters to prevent escape of virulent organisms, as well as facilities for effective sterilization of effluent water, leftover feed, fodder, urine and faeces by disinfection and incineration. In the absence of the above facilities, the experimental animal house should be provided with birdproof netting to prevent spreading of virulent organisms by free flying birds, and a drainage system where effluents should be properly disinfected. Animal sheds and equipment should be disinfected at regular intervals.
The efficacy of the vaccine depends upon the results of animal experiments.
Thus:
For the production and testing of anthrax spore vaccine, guinea pigs, sheep and goats are needed.
Guinea pigs used in vaccine production and testing should be obtained from a healthy colony. Animals weighing approximately 400 to 500 g are suitable for quality control tests. Guinea pigs utilized in testing of vaccine should be kept for a minimum period of three days to adapt to the new environment of shed where the tests are to be carried out. The temperature of guinea pigs should be recorded twice a day, morning and evening. Any guinea pig showing a temperature of above 40°C should not be used and replaced.
One to two year-old healthy unvaccinated sheep weighing approximately 20 kg are suitable for the quality control tests. Sheep should be kept for one week to adapt to the new environment of the sheds where the tests are to be carried out. Their daily morning and evening temperature should be recorded. Any sheep showing an abnormal temperature should not be used and should be replaced.
One-year-old healthy unvaccinated goats, weighing approximately 20 kg, are suitable. They should be kept for one week to adapt to the new environment of sheds where the tests are to be carried out. The temperature of these goats should be recorded twice a day. Any goat showing a temperature above 40°C should not be used and should be replaced.
For the production of anthrax spore vaccine solid medium is used. Two types of solid media, i.e. nutrient agar and casein digest agar are used for the vaccine production. Yield of viable spores in nutrient agar medium is satisfactory but comparatively lower than casein digest agar medium. Vaccine produced from casein digest agar medium is safe and yield of viable spores is also higher, and this medium has been recommended by WHO in requirements for production of anthrax spore vaccine, live (veterinary), 1967.
This is the simplest medium used for production of anthrax spore vaccine. The method of its preparation is given in Appendix 1.
The main constituent of this medium is a tryptic digest of casein. The medium is buffered and salts are added to promote sporulation. The formula and method of its preparation are given in Appendix 1.
For testing absence of contamination, fluid thioglycolate, soybean-casein digest and motility media are recommended. The formula and methods of their preparation are given in Appendixes 1 and 2.
In the past capsule forming Pasteur strains were used for the production of anthrax vaccines. These vaccines induced severe reactions and were not safe in all animals. During the last 40 years, capsule forming strains have been replaced by uncapsulated attenuated strains derived from a virulent strain. In most countries, anthrax spore vaccine is prepared from an uncapsulated avirulent strain 34 F 2 of B. anthracis. This strain was originally isolated by Sterne (1937) from a virulent strain. Its isolation and history are briefly described below:
The virulent strain of B. anthracis grown on nutrient agar at 37°C in normal atmospheric conditions produces frosted glass-type rough colonies of medusa head type, while the virulent strain grown on 50 percent horse serum agar and incubated at 37°C in an atmosphere of 30 to 50 percent carbon dioxide produces smooth raised mucoid colonies with clearly defined edges. These colonies are quite different from those grown in ordinary conditions on nutrient agar. The organisms from mucoid colonies are capsulated and are in short chains. After further incubation for 3 to 4 days, examination under a low power objective shows that some of these mucoid colonies have a fan-shaped outgrowth at their outer edges. These outgrowths have a filamentous appearance and bacilli are in long chains and do not show capsulation. These variants on subculture on ordinary or enriched nutrient agar produce typical anthrax colonies of frosted glass appearance and hairlike structure, irrespective of whether they are grown in an atmosphere containing 30 to 50 percent carbon dioxide or not. On repeated subcultures or passage through the animals, organisms do not change their characteristics and show no tendency to revert to the capsulated virulent form. In moderate doses these variants are not virulent to mice and guinea pigs. However, massive doses can kill guinea pigs and mice, but even then no trace of capsulation is observed.
The main advantages of the 34 F 2 strain are that it is stable, that it cannot produce capsule in vitro, that it is safe in all animals, that it induces solid immunity lasting for one year, that immunogenicity can be tested in laboratory animals.
This strain has been found to be safe and effective in protecting animals and is used in most countries of the world for the production of anthrax spore vaccine. The preservation of the strain is described later.
For testing the potency of anthrax spore vaccine, highly virulent challenge strains are required. Two types of challenge strains, i.e. guinea pig challenge strain 17 JB and a virulent strain are used. In most countries the potency of anthrax spore vaccine is carried in guinea pigs but in a few countries sheep and goats are used. It is recommended to carry out the potency test of anthrax spore vaccine in guinea pigs.
Guinea pig challenge strain was derived by Sterne by continuous passage in guinea pigs of the Pasteur II strain until virulence could not be further enhanced. This strain is consistently virulent for guinea pigs but not for rabbits, domestic animals or man. It is used as a challenge strain for testing the potency of anthrax spore vaccine in guinea pigs. This strain can be obtained from the Central Veterinary Laboratory, Weybridge, Surrey, UK.
It is recommended to preserve the 17 JB strain by freeze-drying. For the maintenance of maximum virulence, passage the strain at least once in guinea pigs before freeze-drying. Propagate the organisms on nutrient agar and inoculate the guinea pigs subcutaneously. Isolate the organisms from heart blood or spleen just before death.
Propagate the strain on nutrient agar. Harvest in physiological saline solution when there is maximum sporulation. Test the purity by smear examination and motility test. The detailed method of propagation and purity test is given later.
Mix the spore suspension with equal amounts of stabilizers (Appendix 3) and freeze-dry in 1 ml quantities in ampoules or vials. Carry out the primary drying for 18 hours and the secondary for four hours. Seal the ampoules or vials under vacuum. Test the viability of spores on media. Label the containers and store at -20°C in the quality control laboratory. Freeze-dried spores stored at -20°C retain viability for years.
Determine the minimum lethal dose (MLD) of the freeze-dried spores in guinea pigs weighing 400 to 500g. Prepare tenfold dilutions in physiological saline solution. Inoculate 1.0 ml subcutaneously in guinea pigs and observe for ten days. Record the death of guinea pigs due to anthrax and determine the MLD.
For testing the potency of anthrax spore vaccine, it is recommended to use at least 100 MLD of guinea pig challenge 17 JB strain as per recommendations of the British Pharmacopoeia (Veterinary), 1985.
In certain countries, potency testing of anthrax spore vaccine is carried out in sheep. For this purpose a highly virulent strain of B. anthracis is required. For the maintenance of maximum virulence, the strain should be given three quick passages in sheep by using blood. Blood from the third passage should be collected just before death and cultured on nutrient agar, spores freeze-dried and stored at -20°C or lower in the quality control laboratory. Determine the MLD of the freeze-dried virulent spores in sheep. Prepare tenfold dilutions in physiological saline solution. Inoculate 1 ml subcutaneously in sheep and observe for ten days. Record the death of sheep due to anthrax and calculate the MLD. One hundred MLD of virulent strain should be used as a challenge dose in sheep for estimating the potency of anthrax spore vaccine.
The preparation of good seed is a prerequisite for the production of a safe and effective vaccine. The strain used for the production of vaccine seed-lots must be a standard strain capable of yielding safe and immunogenic vaccine that meets all the manufacturing requirements as laid down by WHO for the anthrax spore vaccine, live (veterinary) 1967. It is therefore essential that the seed-lot be subjected to a full range of tests, i.e. identity, freedom from contamination, safety, potency and stability, prior to use in vaccine production. Strain 34 F 2 (Sterne) is a suitable strain and is being used in most countries. This strain can be procured from the Central Veterinary Laboratory, Weybridge Surrey, UK.
The attenuated strains loose their antigenicity gradually on repeated subcultures on artificial media. It is therefore essential that for vaccine production, culture should be kept within three passages from the original seed obtained from the reference laboratory. For this reason a large stock of freeze-dried seed should be prepared and stored at -20°C or lower. This can be used for several years. It is therefore necessary that the production of anthrax spore vaccine be based on seed-lot systems. Two types of seed-lots, i.e. master seed and working seed, should be prepared for the production of anthrax spore vaccine. The working seed-lot is prepared from master seed. This seed-lot system ensures that over a period of several years, the seed strains themselves are not subcultured. It is strongly recommended that the seed-lot should be prepared in the production laboratory and then submitted to the quality control laboratory for testing. Full records should be kept of the origin, passage and storage of seed-lots including the date of subculture and medium used for its propagation. The use of seed-strain for production lots should also be recorded. A new batch of seed-lot should be prepared before its complete exhaustion, because if the passage material is later found unsuitable, it will be necessary to revert to a previous passage level. Seed strain should be maintained both in freeze-dried form and on artificial media. In the event of damage of one, an alternative supply of culture will be available. Seed-lots should be properly labelled and stored in the production laboratory at 4°C in a freezer or refrigerator that does not contain contaminating materials.
The ampoule of vaccine seed obtained from the reference laboratory should be opened as follows:
Reconstitute the freeze-dried material in 1 ml sterile physiological saline solution, plate it on nutrient agar Petri dishes and incubate at 37°C for 24 hours. Fish out the typical anthrax colonies, plant on nutrient agar slants and incubate at 37°C for 24 hours. Check the purity of the slants by smear examination after staining with Gram's method and motility test. The procedure to carry out the motility test is described in Appendix 2.
Suspend the growth of the pure slants in 4 ml sterile physiological saline solution. Inoculate 2 ml in each nutrient agar Roux flask. Incubate the Roux flasks at 37°C for 72 hours and then for four days at room temperature, wash the growth of each flask with 10 ml physiological saline solution with the help of glass beads. Harvest the growth from each Roux flask separately. Check the purity of spore suspension by smear examination after staining with Gram's method and motility test.
Pool the pure spore suspensions in an Erlenmeyer flask, add an equal volume of sterilized stabilizer and freeze-dry. The preparation of the stabilizer is given in Appendix 3. Check the vacuum of the freeze-dried containers and discard those that do not have a vacuum. Check the purity of the freeze-dried spore by smear examination after staining with Gram's method and motility test. Lable each container and store at -20°C or lower. This is designated as the master seed-lot which can be used for several years.
The working seed-lot is prepared from the master seed-lot. Check the vacuum of the container of the freeze-dried master seed-lot and reconstitute in 1 ml sterile physiological saline solution, and inoculate several nutrient agar slants prepared in screw-capped tubes. Incubate at 37°C for 24 hours. Check the purity by smear examination and motility test. Store pure slants at +4°C. These culture slants are used for the preparation of inoculum for the production of vaccine up to six months.
It should be noted that the working seed-lot should not be more than three passages removed from the well characterized master seed-lot, and that vaccines should be made from the working seed-lot without additional passage.
Control of the seed-lot is carried out once in the beginning when seed-lots are prepared.
Test the purity of the seed-lot by morphological, cultural and motility tests.
Morphological test. Prepare a smear from five samples of the seed-lot cultured in nutrient broth and nutrient agar, stain by Gram's method and examine under the microscope. The seed-lot must contain only B. anthracis and must be free from contaminants.
Cultural tests. Streak five nutrient agar plates with five samples of seed-lot and incubate at 24 to 48 hours at 37°C. Examine the morphology of colonies by naked eye and through a magnifying glass. Prepare a smear and examine under the microscope after staining with Gram's stain. The seed-lot must not contain any bacteria except B. anthracis.
Inoculate five samples of seed-lot into five Erlenmeyer flasks containing 50 ml of nutrient broth, incubate at 37°C and observe up to seven days. Test the purity by smear examination from each flask daily up to seven days after staining with Gram's stain. The seed-lot must contain only B. anthracis. Inoculate five samples of seed-lot into five Erlenmeyer flasks containing 50 ml of thioglycolate fluid medium and incubate at 37°C and observe daily up to seven days. Test the purity from each flask by smear examination daily up to seven days after staining with Gram's method. There must not be any growth in the thioglycolate broth as B. anthracis is an aerobe and does not grow in fluid thioglycolate medium.
Motility test. Test the motility of five samples of seed-lot by inoculating in nutrient broth and fluid thioglycolate medium. The seed-lot must be free from motile organisms.
The seed-lot must be tested for its safety by preparing a batch of vaccine from it. The safety test should be carried out on sheep as per WHO requirements for anthrax spore vaccine, live (veterinary), 1967.
Inoculate three healthy, one- to two-year-old sheep which were not immunized against anthrax, with 5 000 million viable spores subcutaneously and observe for ten days. Record the temperature daily both in the morning and evening. The seed-lot is considered safe when none of the sheep show any severe reactions except for a 2–3°C rise in body temperature which persists four to five days. At the site of inoculation there is an oedematous swelling which subsides within six to seven days.
The seed-lot must be tested for its immunogenicity by preparing a batch of vaccine from this as per WHO requirements for anthrax spore vaccine live (veterinary), 1967.
As per recommendation of the British Pharmacopoeia (Veterinary), 1985, the kind of animals employed for testing the potency of anthrax vaccine depend on the strain used for production of vaccines. When the strain used for vaccine production is not lethal to guinea pig or mouse, carry out the potency test on guinea pigs. When the strain is lethal to the guinea pig but not to the rabbit, carry out the test in rabbits. When the strain is lethal to the rabbit, carry out the test in sheep. Strain 34 F 2 (Sterne) being not lethal to guinea pigs, it is recommended to carry out the test in guinea pigs. In some countries the potency test is carried out in sheep. Method of potency tests in guinea pigs and sheep are described.
Guinea pigs. Inoculate ten healthy guinea pigs weighing approximately 500 g with 5 million viable spores subcutaneously and observe for 21 days, and record their body temperature daily. None of the animals should show untoward reactions except for a 1–1.5°C rise in temperature. If more than two animals die from non-specific causes, retest the vaccine. Challenge all the immunized guinea pigs along with three controls with 100 MLD and three controls with 10 MLD of 17 JB virulent guinea pig strain of B. anthracis. Observe these animals for 10 days and record their body temperature. All the vaccinated guinea pigs should survive and show no untoward reactions except slight rise in their body temperature, while all the controls must die from anthrax. Repeat the test if one of the vaccinated animals dies. The seed-lot passes the test if there is a 100 percent protection of the vaccinated guinea pigs and 100 percent death of the controls from anthrax.
Sheep. Inoculate ten healthy two-year-old sheep weighing approximately 20 kg with 5 million viable spores subcutaneously and observe for 21 days and record their temperature daily. None of the animals should show any untoward reaction except for a 1–3°C rise in their temperature. If more than two animals die from non-specific causes, retest the vaccine. Challenge all the immunized sheep along with three controls with 100 MLD of virulent strain of B. anthracis. Observe the animals for ten days and record their temperature daily. The vaccinated sheep should not show any untoward reaction except for a slight rise in their temperature and should survive the challenge, while all the controls must die from anthrax. Repeat the test if one of the vaccinated sheep dies. The seed-lot passes the test if there is 100 percent protection of the vaccinated sheep and 100 percent death of the controls from anthrax.
Prepare casein digest agar medium in Roux flasks. From 50 standard Roux flasks approximately 100 000 doses of anthrax spore vaccine can be obtained. Incubate the Roux flasks containing the medium for 48 hours at 37°C before inoculation to ensure sterility. Place all the Roux flasks in the inoculation room, start the ultraviolet lamp and sterilize for 30 minutes. Remove the condensation water from each Roux flask under strict sterile conditions using a pipette. The medium should be dry before inoculation.
Prepare working seed from the master seed-lot before initiating production of anthrax spore vaccine. Place 10 ml of physiological saline solution in each tube of the working seed. Suspend the bacterial growth with a pipette. Inoculate 2 ml of bacterial suspension in each production flask very carefully by placing the mouth of the flask as near as possible to the burner with the assistance of a technician. Spread the inoculum evently on the surface of the agar by to and fro movements of flasks. Cover the mouth of each Roux flask with a paper cap. Incubate the flasks at 37°C for three days. Thereafter keep the flasks at room temperature for seven days. Check the smear of randomly selected flasks to find out the degree of sporulation. Harvest the growth from the Roux flasks when 90 percent of the organisms are in sporulated forms.
Pipette 20 ml of physiological saline solution and place a few glass beads in each Roux flask. Wash off the growth by to and fro movements of the flasks. Place the flasks on the working bench in an upright position. Harvest the spore suspension using a pipette under strict sterile conditions. Harvest the growth from ten Roux flasks in one Erlenmeyer flask of 250 ml capacity.
Inoculate 0.1 ml of spore suspension from each Erlenmeyer flask in 100 ml of nutrient broth and incubate at 37°C overnight. Check the purity by staining the smear with Gram's staining and motility test. Discard the contaminated smears.
Determine the weight of a sterilized Erlenmeyer flask. Pool the pure spore suspension in the above flask and estimate the weight of the spore suspension. Add twice the weight of the bacterial suspension of sterile, pure and neutral glycerol. Add sufficient glass beads for mixing the glycerinated suspension. Shake the flask vigorously to ensure thorough mixing of the suspension. This suspension is referred to as “vaccine concentrate”.
Check the purity of vaccine concentrate as described earlier and keep it at 20°C for three weeks to destroy vegetative bacteria and weak spores, and store at 4°C. Alternatively to destroy the vegetative bacteria, heat the suspension at 65°C for one hour in a water bath and store the vaccine concentrate at 4°C for three weeks before carrying out the control tests.
Carry out the tests for bacterial contamination of vaccine concentrate by morphological, cultural and motility tests as described before. The vaccine concentrate must not contain any bacteria other than B. anthracis.
Determine the number of live spores in the vaccine concentrate by plating suitable dilutions on nutrient agar plates. The procedure is described in Appendix 4.
To avoid the possibilities of gross error, it is advisable to carry out the spore counts on at least three samples of the vaccine concentrate, and an average of three counts should be calculated. There is a tendency for the spores to clump; deposit them gradually in the vaccine concentrate and shake vigorously before taking samples for counting.
Carry out the safety test on the species for which the vaccine is going to be used. If the vaccine is intended for several species, carry out the safety test on sheep or goats.
Inoculate two healthy sheep or goats subcutaneously with twice the dose recommended for vaccination, i.e. 20 million spores. Observe the animals for ten days and record their temperature both in the morning and evening. The vaccine concentrate passes the test if no abnormal systemic reaction is observed and none of the animals show severe reactions except a transient oedema at the site of inoculation which subsides in 3 to 5 days. Some animals show dullness and a 1–2°C rise in body temperature for three to five days, and become normal afterwards.
The development of progressive oedema at the site of inoculation indicates that the vaccine may be unsuitable for goats. The severity of the local reactions may vary according to the strain and adjuvant if any was used in the vaccine, but necrosis should not develop.
Carry out the potency test in guinea pigs in parallel with the international reference vaccine procured from an international reference centre. The procedure is described in the section on control of the seed-lot. The vaccine passes the test if all the vaccinated animals survive and all controls succumb to the challenge from anthrax. Repeat the test if one of the vaccinated animals dies after the challenge.
After determining the number of culturable spores in the vaccine concentrate, calculate the dilution factor depending upon the number of spores to be employed per dose.
It is recommended that the vaccine should contain not less than 10 million culturable spores per dose for cattle, buffaloes and horses and not less than 5 million for sheep, goats and pigs.
Dilute the vaccine concentrate in 50 percent glycerine saline solution pH 7.0. Prepare glycerine saline by mixing four parts neutral glycerine with six parts of physiological saline solution and sterilize in the autoclave at 121°C for 45 minutes.
In some laboratories 0.1 percent saponin is incorporated as an adjuvant in the vaccine. This is added during the dilution of the vaccine concentrate by glycerine saline.
Transfer the required filling equipment, vials and stoppers after sterilization to the filling room which has been prepared before. Start the ultraviolet light and carry out the sterilization for 30 minutes. Filling operations should be conducted in such a way to avoid any contamination or alteration of the vaccine.
Transfer the vaccine concentrate into a sterile vaccine filling tank fitted with magnetic drive in the vaccine filling room. Add the required quantity of sterilized glycerine saline solution. Add saponin if required. Mix the vaccine slowly at room temperature for two hours.
Fill the vaccine in a multidose container in 10 or 20 ml vials using a pipetting machine under sterile conditions.
Close the vials with sterilized rubber stoppers and seal the vials with aluminium collars immediately, then place the vials in aluminium boxes and store at 4°C.
The quantity present in a single bulk container determines the batch size. If the final bulk is distributed in smaller containers, each of them is referred to as filling lot and is labelled individually. If the filling is carried out directly from the final bulk container, the number of vials filled in one session without stoppage is referred to as final lot or filling lot.
In certain laboratories anthrax spore vaccine is prepared in freeze-dried form. For this purpose mix the spore suspension after harvesting with an equal volume of stabilizer. The composition of the stabilizer is given in Appendix 3. Distribute 1 ml in vials. Carry out the primary drying for 18 hours and secondary for four hours. Seal the freeze-dried containers under vacuum or under dry oxygen-free nitrogen. Test all the sealed containers for leaks, discard defective ones and store at 4°C or lower.
Apply the following control tests on each filling lot from the samples collected by random selection. If the vaccine is in freeze-dried form, apply hese tests on the vaccine reconstituted to the form in which it is to be used.
Inspect every container visually under proper illumination to detect the presence of foreign particles. Discard vials showing foreign particles, clumps and defects in closures.
Carry out the tests for identity and for absence of contamination on every final lot. According to the British Pharmacopoeia (1985), 1 percent of containers in a batch with a minimum of three or maximum of ten is considered suitable for carrying out the test. Carry out the tests by:
The methods of the above tests are described in the section dealing with control of seed-lot, and motility test in Appendix 2. Vaccine should not contain any bacteria other than B. anthracis.
Determine the number of culturable spores by plating suitable dilutions on nutrient agar. The procedure is described in Appendix 4.
The vaccine should not contain less than 10 million culturable spores per dose for cattle, buffaloes and horses and not less than 5 million per dose for sheep, goats and pigs.
In any case as per the British Pharmacopoeia (Veterinary) 1985, the number of live spores determined by a plate count method must not differ more than 20 percent from the standard numbers for a particular animal.
Determine the pH of each final lot of the vaccine. Standardize the pH meter with standard buffer solution of pH 5.0 and 7.0. Rinse the electrodes with distilled water, dry and test the pH of the vaccine. The pH value of anthrax spore vaccine should be 7.0±0.3.
Carry out the innocuity test on each final lot of the vaccine. It is not necessary to carry out this test when the safety test is done on animals of the species for which the vaccine is intended to be used.
Inoculate two guinea pigs intraperitoneally with 0.2 ml of vaccine (reconstituted if freeze-dried) and two guinea pigs subcutaneously with 1 ml. Observe these animals for ten days. Vaccine passes the test if none of the animals shows signs of illness. If one of the animals dies or shows signs of ill health during observation, repeat the test. The vaccine lot passes the test if none of the animals in the second group dies or shows signs of ill health during observation.
Determine the moisture content of each filling lot of freeze-dried anthrax spore vaccine. Dry the freeze-dried vaccine over phosphorous pentoxide under vacuum (0.01–0.03 mm Hg) at 56°C until a constant weight is obtained. Test the moisture content of five samples of freeze-dried vaccine. Moisture levels of freeze-dried anthrax spore vaccine should be less than 2 percent.
Test the stability of every filling lot of freeze-dried anthrax spore vaccine by using the accelerated degradation test. Store five vials of vaccine at 37°C for four weeks. Determine the number of culturable spores at the end of the storage period in parallel with unstored vaccine and with the reference preparation. There should not be a drop in the number of spores below the prescribed limits required for immunization on storage at 37°C for four weeks.
Carry out the long-term stability test by storing liquid vaccine at 4°C for 180 days or more. Determine the number of culturable spores before and after samples were held at 4°C in parallel with the reference preparation. The vaccine should contain the required number of culturable spores prescribed for immunization of animals.
Maintain a complete passage history of the seed-lots and cultures used in production and quality control of anthrax spore vaccine. Label all cultures properly and store them at the appropriate temperature in an orderly manner. Keep a complete and accurate inventory of all stocks and of all the vials and ampoules issued or used.
Keep the records of production and control protocols in such a way that it is possible to trace all steps in the manufacture and testing of a batch of vaccine. Keep the detailed records of sterilization of all apparatus and materials used in its manufacture. Keep the written records clearly indicating all steps in processing and filling including both the process controls and control test of the final product. Keep the written records of all the tests irrespective of their results throughout the dating period of each lot of vaccine and have them available at all times for inspection by the control authority.
Keep the distribution record in such a manner that it permits rapid recall of any particular batch.
A sample of a suitable summary protocol for production and control of anthrax spore vaccine is given in Appendix 5. The tests specified are based on the WHO requirements for anthrax spore vaccine, live (veterinary). The protocol is intended for the reporting of the data to the national control authority or to the authorities in other countries to whom the vaccine may be exported.
At the time of release of a lot of anthrax spore vaccine sufficient quantities of samples from each final lot should be retained. Collect the samples by random selection, so that they are representative of the lot and store at 4°C. These samples are mainly required for the following essential purposes:
Discard the retained samples when it is known that the complete lot has been distributed, used or destroyed at the end of the expiry period. To check the keeping quality particularly as regards potency of anthrax spore vaccine, it is beneficial to keep some samples from time to time beyond the expiry period.
Identify the anthrax spore vaccine, living, by label. The label on the container should show:
The label on the packing or leaflet in the package should, in addition to the information shown on the label of the container, include the following details:
RELEASE FOR DISTRIBUTION
Release the anthrax spore vaccine lot when all the required quality control tests have been performed, summarized, reviewed, and all the official control requirements are satisfied.
SHIPPING
Ship the anthrax spore vaccine under cold conditions over ice in insulated boxes. Do not freeze the liquid vaccine. Freeze-dried vaccine can be shipped in freezers.
Transportation from the manufacturing laboratories to distribution points should be as rapid as possible. Well-packed vials in insulated boxes can be expected to maintain temperatures of 4–15°C for 24 to 48 hours during transport, provided they are not exposed to direct sunlight. Vehicles should go directly from the point of collection to their destination without long stops en route. Place a temperature indicator in each box during shipment to know the temperature of the vaccine during shipment.
At distribution point, the vaccine should be stored in a cold room at +4°C. Users of the vaccine who do not have adequate storage facilities should obtain it directly from distribution point. In the field, when maintenance of low temperature is impracticable, the vaccine should not be exposed to direct sunlight and utilized within a short time.
STORAGE
Store anthrax spore vaccine, living, in a dark and dry place at +4°C in a cold room. Storage at room temperature is not recommended. In field conditions, it is difficult to maintain vaccine at low temperature; all efforts should be made that vaccine is exposed to room temperature for a short period only. During vaccination, store the vaccine in insulated boxes covered with ice. It is necessary to protect the vaccine from direct sunlight.
EXPIRY DATE
Expiry date of the anthrax spore vaccine depends upon the type of vaccine. Generally, liquid vaccine can be expected to retain its potency for a period between six to 12 months from the date of the completion of the potency test at +4°C. There are reports that some liquid anthrax spore vaccines are stable up to 24 months. Stability of freeze-dried vaccine is more than 24 months at +4°C.
DESCRIPTION
The vaccine is a suspension of living spores prepared from an uncapsulated strain (34 F 2) of B. anthracis preserved in 50 percent glycerine saline.
It has a syrup-like consistency of brownish colour, with a small deposit on the bottom of the vial which is evenly homogenized in the fluid mass when shaken.
USE
It is used to prevent anthrax in cattle, buffaloes, horses, sheep, goats and pigs. It can also be used in camels and elephants.
It should be inoculated subcutaneously: in cattle, buffaloes and horses, in the middle of the neck; in sheep, goat and swine on the inner face of the thigh.
DOSE
| Cattle, buffaloes, horses | 1.0 ml |
| Sheep, goats and pigs | 0.5 ml |
REACTION
The vaccinated animals may have a mild local oedema at the site of inoculation and also a febrile reaction usually lasting for two or three days. In lactating animals there may be a decrease in milk production for two to three days.
IMMUNITY
The immunity is established in ten days following inoculation. The animals are protected against natural infection for about one year.
STORAGE
Stored in a dark and dry place at +4°C, it maintains its potency for 24 months.
PRECAUTIONS
It should not be used by persons who have abrased skin on their hands. After use syringes and needles should be thoroughly sterilized in boiling water for one hour. Do not vaccinate pregnant and young animals under three months of age. Animals suffering from pleuropneumonia should not be vaccinated. Shake the vials before use.
PACKING
The vaccine is issued in 10 and 20 ml vials.
NUTRIENT BROTH
It can be prepared from media available from commercial sources as per manufacturer's instructions or can be prepared in laboratories as follows:
| Meat extract (Difco) | 3 g |
| Peptone | 10 g |
| Sodium chloride | 5 g |
| Distilled water | 1 000 ml |
Weigh all the ingredients in a flask, add 1 000 ml of distilled water and dissolve by boiling in autoclave for 20 minutes. Adjust the pH to 8.2 and autoclave at 110°C for ten minutes to precipitate the salts. Finally adjust the pH to 7.4, filter and distribute as desired and sterilize at 121°C for 30 minutes in the autoclave.
NUTRIENT AGAR
Mix 1.5 to 2 percent of agar with the nutrient broth, melt in autoclave at 110°C for 20 minutes. Adjust the pH to 7.6 when cool. Dispense 5 ml molten agar in tubes for making the slant, 20 ml in tubes for making the stab agar and 120 ml in Roux flasks for production of vaccine. Sterilize by autoclaving at 121°C for 30 minutes. For making the slants, tubes are placed in a slanting position while the medium is hot. Roux flasks are placed on the flat surface of a working bench. When the agar solidifies, incubate at 37°C for 48 hours to detect contamination. Store the tubes at +4°C. Roux flasks are used for production of vaccine immediately.
CASEIN DIGEST AGAR MEDIUM
| Tryptic digest of casein | 50 g |
| Yeast extract (Difco) | 10 g |
| Calcium chloride (CaCl2, 6H2O) | 0.1 g |
| Ferrous sulphate (FeSO4, 7H2O) | 0.01 g |
| Magnesium sulphate (MgSO4, 7H2O) | 0.05 g |
| Manganese sulphate (MnSO4, 4H2O) | 0.03 g |
| Dipotassium hydrogen phosphate (K2HPO4) | 5.0 g |
| Potassium dihydrogen phosphate (KH2PO4) | 1.0 g |
| Agar | 22.0 g |
| Distilled water | 1 000 ml |
Weigh the six chemicals and dissolve in distilled water, adjust the pH to 7.6. Add agar and dissolve by autoclaving at 110°C for 30 minutes. Adjust the pH to 7.4 and add the potassium buffer salts. Dissolve and filter the medium. Distribute 120 ml in each Roux flask and sterilize in autoclave at 121°C for 30 minutes. After sterilization, flasks are placed on the table on a flat surface at room temperature. After the solidification of agar, incubate the flasks at 37°C for 48 hours to detect contamination. Examine each flask carefully after incubation and discard the contaminated ones.
SOYBEAN-CASEIN DIGEST MEDIUM
| Pancreatic digest of casein | 17.0 g |
| Papaic digest of soybean meal | 3.0 g |
| Sodium chloride | 5.0 g |
| Dipotassium hydrogen phosphate | 2.5 g |
| Glucose | 2.5 g |
| Distilled water | 1 000 ml |
| Final pH | 7.1–7.5 |
Dissolve the ingredients in warm water, then cool to room temperature. Adjust the pH to 7.6 with 1 N sodium hydroxide. Filter, distribute into suitable vessels and sterilize in autoclave at 121°C for 20 minutes.
FLUID THIOGLYCOLATE MEDIUM
| L-cysteine | 0.5 g |
| Sodium chloride | 2.5 g |
| Glucose | 5.5 g |
| Agar | 0.75 g |
| Yeast extract (Difco) | 5.0 g |
| Pancreatic digest of casein | 15.0 g |
| Distilled water | 1 000 ml |
| Sodium thioglycolate | 0.5 g |
| Resazurin sodium (0.1% fresh solution) | 1.0 ml |
| Final pH | 7.0–7.2 |
Grind the first six ingredients in a mortar. Add 200 ml of warm water, stir and transfer to a suitable container, add the remaining distilled water. Dissolve by boiling in water bath, taking special care to ensure complete solution of the L-cysteine. Add sodium thioglycolate, adjust the pH to 7.4, filter and add resazurin solution. Distribute into suitable vessels and sterilize by autoclaving at 121°C for 20 minutes. Cool to 25°C immediately and store at 20–30°C avoiding excessive light. If the uppermost portion of the medium has changed to a pink colour and this exceeds one third of the depths of the medium, it is unsuitable for use but may be restored once by heating in steam. Medium more than three weeks old should not be used.
The motility of the bacteria can be examined by the following methods:
HANGING DROP METHOD
The motility of bacteria can be observed by examining a hanging drop in which the organisms are able to move about freely. A special glass cavity slide with a hollow depression in the centre is used for this purpose. The method of detection of motility is described below:
It is not advisable to use an oil-immersion objective because due to the oil, the cover slip moves during focusing and false motility is produced.
During examination of motility it is essential to distinguish between true motility and Brownian movements. In true motility, organisms change their position in the field and cross the field. The Brownian movement is an oscillatory movement possessed by all small bodies, either living or dead, suspended in fluid. In Brownian movement neither is the position of the organism changed nor do organisms cross the field.
CULTIVATION IN SEMI-SOLID AGAR
In semi-solid agar, motile bacteria “swarm” and give a diffuse spreading growth which can be easily seen by naked eye. Motility thus can be detected more easily than by the “hanging drop” method.
Dissolve 0.2 percent of “Difco” agar in nutrient broth. It is important that the medium should be quite clear and transparent. Dispense 10 ml in test tubes and sterilize at 121°C for 30 minutes. Keep the medium to set in vertical position. Inoculate the culture under test with a straight platinum wire, making a single stab down from the centre of the tube to half the depth of the medium. Examine after incubation at 37°C for 18 hours.
The growth of non-motile bacteria is confined to the stab line and has sharply defined margins, leaving the surrounding medium clearly transparent. Motile bacteria typically give diffuse, hazy growths that spread throughout the medium rendering it slightly opaque.
HORSE SERUM
Collect the horse blood under aseptic conditions and allow it to clot. Store the container at 4°C. Take out the clear serum with a pipette. Filter it by EK Seitz pad and inactivate at 56°C for 30 minutes in a water bath. Store it at 4°C.
Mix an equal volume of sterile inactivated horse serum with spore suspension of B. anthracis and freeze-dry.
SKIMMED MILK
Mix an equal volume of 5 percent of sterile skimmed milk with spore suspension of B. anthracis and freeze-dry.
SUCROSE-GLUTAMATE-DEXTRAN
| Sucrose | 5.0 g |
| Sodium glutamate | 1.0 g |
| Dextran | 5.0 g |
| Physiological saline solution | 1 000 ml |
Sterilize the solution by filtration. Suspend the spores in the above solution and freeze-dry.
There are many procedures to carry out the viable spore count. The commonly used “pour plate” method is described below.
To avoid the possibilities of error, it is advisable to carry out spore counts in not less than three samples of spore concentrate. Spores have the tendency to clump together and settle to the bottom. These clumps should be dispersed by thorough shaking before drawing samples for counting.
Count the colonies in all five plates of a particular dilution. Multiply the average number per plate by the dilution factor to obtain the viable count per ml in original suspension. Calculate the average count for each dilution from 10-6 to 10-8. To obtain the average viable count in original stock suspension, take the average of three dilutions.
IDENTIFICATION OF FINAL LOT
| Name and address of manufacturer | |
| Lot number of final product | |
| Date of manufacture of final lot | |
| Date of filling containers | |
| Number of containers and nature | |
| Date of last potency test | |
| Number of doses in each container | |
| Volume of single dose | |
| Expiry date |
Strains of Bacillus Anthracis
| Strain for vaccine production | |
| Name, history and origin | |
| Strain for testing | |
| Name, history and origin |
Preparation and control of vaccine seed-lots
Master seed-lot
| Date of preparation of master seed-lot | |
| Number of passages between master and seed-lot and strain obtained from reference centre | |
| Working seed-lot | |
| Date of preparation of working seed-lot | |
| Number of passages between working | |
| seed-lot and production |
Control of seed-lots
| Tests for bacterial contamination | |
Methods used Results |
PREPARATION AND CONTROL OF VACCINE CONCENTRATE
Preparation of vaccine concentrate
| Media | |
| Type of medium used | |
Date of preparation | |
Ingredients | |
Tryptic digest of casein | |
Yeast extract | |
Calcium chloride (CaCl2, 6H2O) | |
Ferrous sulphate (FeSO4, 7H2O) | |
Magnesium sulphate (MgSO4, 7H2O) | |
Manganese sulphate (MgSO4, 4H2O) | |
Dipotassium hydrogen phosphate (K2HPO4) | |
Potassium dihydrogen phosphate (KH2PO4) | |
Agar | |
Distilled water | |
pH | |
Number of Roux flasks prepared | |
Sterilization | |
Duration media incubated at 37°C | |
Results of incubation | |
| Inoculation | |
Date | |
Volume of inoculum | |
Number of Roux flasks inoculated | |
Duration of incubation at 37°C | |
Duration of incubation at room temperature | |
Date of checking of sporulation | |
| Harvesting | |
Date | |
Number of Roux flasks harvested | |
| Purity test | |
Date | |
Number of Erlenmeyer flasks containing spore suspension tested | |
Test methods | |
Results | |
| Glycerination | |
Date | |
Weight of empty flask | |
Number of Erlenmeyer flasks containing spore suspension pooled | |
Weight of spore suspension | |
Weight of neutral glycerine added | |
Duration of storage glycerinated | |
suspension at room temperature |
Control tests on vaccine concentrate
| Tests for bacterial contamination | |
Date | |
Test methods | |
Results | |
Test for number of culturable spores | |
First spore count | |
Date | |
Results | |
Second spore count | |
Date | |
Results | |
Third spore count | |
Date | |
Result | |
Average spore count | |
Dilution factor | |
| Safety test | |
Animal species used | |
Number of animals | |
Route and dose of inoculation | |
Date of inoculation | |
Date of end of test | |
Number of animals that showed postinoculation reaction | |
Number of animals died | |
Results | |
| Immunogenicity test | |
Animal species used | |
Number of animals | |
Date of inoculation | |
Dose and route of inoculation | |
Number of animals died before challenge | |
Date of challenge | |
Challenge strain used | |
Number of controls | |
Number of vaccinated challenged | |
Dose and route of challenge | |
Number of vaccinated died | |
Number of controls died | |
Results |
Dilution of vaccine concentrate
| Diluent | |
Type of diluent | |
Date of preparation | |
Weight of glycerine | |
Quantity of saline | |
pH | |
Quantity prepared | |
Sterilization | |
| Dilution | |
Date | |
Dilution factor | |
Quantity of vaccine concentrate | |
Quantity of glycerine saline | |
Total quantity of vaccine |
Adjuvant
| Type | |
| Date | |
| Concentration of adjuvant used | |
| Total quantity of adjuvant added in vaccine |
FILLING AND CONTAINERS
Liquid vaccine
| Date | |
| Quantity of containers filled | |
| Volume of vaccine per container |
Freeze-dried vaccine
| Date | |
| Type of stabilizer | |
| Quantity of vaccine mixed with stabilizer | |
| Quantity of vaccine filled per container | |
| Quantity of container freeze-dried |
CONTROL TESTS ON FINAL PRODUCT
Inspection of final containers
| Date | |
| Results |
Identity test and tests for absence of bacterial contamination
| Date | |
| Methods | |
| Results |
Test for number of culturable spores
| Date | |
| Average results of three spore counts |
Determination of pH
| Date | |
| Results |
Innocuity test
| Animal species used | |
| Number of animals | |
| Route and dose of inoculation | |
| Date of inoculation | |
| Results |
Residual moisture test for freeze-dried vaccine
| Date | |
| Method used | |
| Results |
Stability test
| Date | |
| Exposure temperature and duration | |
| Results |
INTERNAL CERTIFICATION
This certificate should be issued by the person taking entire responsibility for production of the vaccine.
I certify that lot No. of anthrax spore vaccine, living, satisfies Part A of the WHO Requirements for Anthrax Spore Vaccine, Live (Veterinary).
| Signature | |
| Name | |
| Date |
The protocol must be accompanied by a sample of the label and copy of the leaflet.
RELEASE CERTIFICATION BY NATIONAL CONTROL AUTHORITY FOR EXPORT OF VACCINE
I hereby certify that batch No. of anthrax spore vaccine, living, produced by (name of the producer) meets all national requirements as well as part A of the WHO Requirements for Anthrax Spore Vaccine, Live (Veterinary). The date of the last satisfactory potency test carried out by the National Control Authority is .
The final lot has been released by us under number .
The number appearing on the label of the containers is .
| Signature | |
| Name | |
| Date |
1985. British Pharmacopoeia (Veterinary). London, Her Majesty's Stationery Office.
WHO. 1967. Requirements for anthrax spore vaccine (Live for veterinary use). Technical Report Series, 1967, No. 361. (Requirements for biological substances No. 13). Geneva, World Health Organization.
Sterne, M. 1937. Onderstepoort J. Vet. Res., 8(271):9, 49.
