In vaccine production laboratories, 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 filtered air supply and laminar flow cabinets. In the absence of a filtered air supply, regular fumigation of laboratories and work cabinets should be carried out to keep airborne microbes to a minimum.
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.
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 a 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.
The level of airborne microbes should be regularly monitored in laboratories, cabinets and work stations. The cabinets and work stations should be regularly checked for their efficiency. This is carried out by opening blood agar Petri dishes for a specified time in a specified position. After appropriate incubation of the plates, bacterial counts are made.
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.
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, should be autoclaved 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.
Laboratory discipline is extremely important for the production of the vaccine. 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. 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. Laboratory animal breeding colony and experimental animals should be located in separate buildings away from the vaccine production unit. Animal houses should be designed and constructed in such a way that they can be easily cleaned and should be free from insects. Animal houses should be provided with facilities for food 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, leftoverfeed, fodder, urine, faeces etc., by disinfection and incineration. Experimental animal houses should be provided with birdproof netting 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 blackleg vaccine, guinea pigs, sheep and cattle are needed.
Guinea pigs used in vaccine production and testing should be obtained from a healthy colony. Animals weighing approximately 300 to 400 g are suitable for quality control tests. Guinea pigs used for testing of vaccine should be kept for a minimum period of three days to adapt to the new environment of sheds where the tests are to be carried out. Their daily morning and evening temperature should be recorded. Any guinea pig showing a temperature above 40°C should not be used and should be replaced.
One-year-old healthy unvaccinated sheep weighing not less than 20 kg are suitable. Sheep should be kept for one week to adapt to the new environment of sheds where the tests are to be carried out. Their daily morning and evening temperature should be recorded. Any sheep showing abnormal temperature should not be used and should be replaced.
One-year-old healthy unvaccinated cattle are suitable. These should be kept for one week to adapt to the new environment of sheds where the tests are to be carried out. Temperature of these animals should be recorded twice a day, and cattle showing a temperature above 40°C should not be used and should be replaced.
This is basically a blood agar medium in which liver extract and glucose are added to promote the growth of Clostridium chauvoei (Walker and Batty, 1979). This medium is recommended for isolation of Cl. chauvoei particularly from artificially infected animals for the preparation of seed. The formula and method of preparation are given in Appendix 1.
In this medium liver extract and sodium thioglycolate are added to promote anaerobiosis. This medium is used for production of vaccine. Vaccine produced in this medium gave good protection in animals. The formula and method of preparation are given in Appendix 1.
The main constituent of this medium is casein hydrolysate to which Lcysteine hydrochloride is added to promote anaerobiosis. Vaccine produced in fermenters by using this medium afforded a degree of protection which is noticeably above the minimum standard required (Cameron et al., 1986). The formula and method of preparation are given in Appendix 1.
For testing sterility, soybean-casein digest media and fluid thioglycolate broth are recommended (WHO, 1973). The formulae and methods of preparation are given in Appendix 1.
Blackleg is caused by Cl. chauvoei. There are reports of frequent isolation of Cl. septicum from blackleg lesions. For this reason, a few laboratories employ both these species for production of blackleg vaccine. However, difficulty in obtaining Cl. septicum from fresh specimens raises doubts whether this organism is the primary cause of blackleg or a post-mortem invader. Therefore, blackleg vaccine is prepared only the from Cl. chauvoei strain in most of the laboratories.
There are no reports of existence of different immunotypes of Cl. chauvoei, but the Australian strain CH3 is reported to be more immunogenic (Chandler and Gulasekharam, 1970). Although there is a marked degree of cross protection among strains, it has been observed that minor differences do occur (Kerry, 1967). In Australia, breakdown of immunity in blackleg vaccinated cattle has been reported and an additional strain was incorporated into the vaccine to obtain an adequate degree of protection (Reed and Reynolds, 1977).
For the production of blackleg vaccine a well characterized strain of Cl. chauvoei obtained from a known source should be used. However, a local isolate from a particular area or country can also be used. Before using a fresh isolate it is essential that its suitability be investigated. For the maintenance of antigenicity of the Cl. chauvoei strain, it is essential that it should be passaged in animals, properly cultivated and preserved. The methods of cultivation, isolation, identification and preservation are described.
Anaerobic organisms do not grow in the presence of oxygen. For the cultivation of anaerobic organisms on the surface of media, oxygen must be removed from the atmosphere either by using it for combustion or by replacing it with an inert gas. A suitable anaerobic jar is used for this purpose and a mixture of 90 percent hydrogen and 10 percent carbon dioxide is ideal to create anaerobic conditions. The Gaspak system is also useful for cultivation of anaerobes.
Special media are also available for the routine cultivation of anaerobic organisms in liquid media particularly for production of vaccines. Sodium thioglycolate or L-cysteine hydrochloride are incorporated in these to reduce oxygen potential and to provide suitable anaerobic conditions. A small amount of agar in media also helps development of anaerobic conditions. The composition of media is given in Appendix 1.
Immediately after death, Cl. chauvoei can be isolated in pure cultures from heart blood, liver or spleen. In addition, organisms can be also isolated from the lesion at the site of the inoculation.
Clean the surface of infected tissue with rectified spirit, make an incision in the centre of the lesion by a sterile scalpel and remove a small portion of skin. Sear the exposed portion of the tissue with a hot spatula and aspirate the oedematous fluid by inserting a Pasteur pipette. Remove a piece of muscle from the periphery of the lesion.
Although cultural methods are adequate for the isolation of Cl. chauvoei, in combination with biological method they give a higher recovery rate.
Cultural method. Inoculate liver extract-blood agar plates with oedematous fluid or blood or pieces of tissue ground in mortar, grow under anaerobic atmosphere in an anaerobic jar at 37°C for two to three days, and examine the colonies and subculture typical of Cl. chauvoei colonies.
Isolation can also be done directly in an anaerobic broth medium and pure cultures obtained by subculturing on blood agar plates under anaerobic conditions.
Biological method. The guinea pig is most susceptible and commonly used as an experimental animal. Grind the tissue in mortar and prepare a 10 percent suspension in physiological saline solution, and inoculate 0.5 ml by intramuscular route in the leg. Within 14 hours after inoculation, the guinea pig becomes dull, depressed, and a soft swelling develops at the site of the inoculation. After 24 hours inflammation spreads to the neighbouring muscular area which fills with gas. The animal dies within 48 hours. Upon incision tissues are haemorrhagic, oedematous and fluid oozes out. Muscle and subcutaneous tissues are filled with gas. Isolate the organisms immediately after death from heart blood, spleen, liver, oedematous fluid and tissues from the lesion.
Identification of Cl. chauvoei can be carried out by morphological, cultural, biological and fluorescent antibody tests.
The differentiation of Cl. chauvoei and Cl. septicum is often necessary. These two are similar morphologically, and both are pleomorphic. Cl. chauvoei is shorter, plumper and more ovoid than Cl. septicum, the former grows singly, whereas the latter frequently forms long chains. The impression smears prepared from the peritoneal surface of the liver of a guinea pig inoculated with Cl. chauvoei reveals citron, short forms which occur singly. On the other hand, with Cl. septicum elongated filaments are observed.
Cl. chauvoei is a more fastidious organism than Cl. septicum and to ensure growth a special medium containing liver extract and glucose is required.
The majority of the colonies of Cl. chauvoei when grown on 1.8 percent agar are umbonate with raised lips, whereas the majority of Cl. septicum colonies are usually spreading rhizoidal types.
Cl. chauvoei ferments sucrose but usually not salicin and Cl. septicum ferments salicin but usually not sucrose.
Both Cl. chauvoei and Cl. septicum can be readily differentiated by fluorescent antibody tests.
The Guinea pig is an excellent laboratory model of Cl. chauvoei and is used for the propagation of the strain. However, it is essential to passage the strain periodically either in sheep or also cattle. Guinea pigs weighing 300 to 400 g and one-year-old susceptible healthy cattle and sheep are suitable.
Inoculate guinea pigs with 0.2 ml, and sheep and cattle with 1 ml of suitable dilution of virulent organisms by intramuscular route. When spores are used, activate by diluting in solution of 2 percent of CaCl2. 2H2O. Inoculated animals show oedematous swelling at the site of inoculation. Cattle and sheep die within four to five days and guinea pigs within two days after inoculation.
Collect heart blood of the animals preferably when moribund or immediately after the death under aseptic conditions using a pasteur pipette. Isolate the organisms by seeding on the media. Organisms can also be isolated in pure cultures from oedematous fluid, tissues from site of inoculation, spleen and liver of the animals.
Preparation of spores
Glycerination of spores
Freeze-drying of spores. Cl. chauvoei spores are best preserved by freezedrying. Suspend the concentrated spore suspensions prepared as above in stabilizers (Appendix 2) and freeze-dried in 1 ml quantities in ampoules or vials. Carry out the primary drying for 18 hours and secondary four hours. vials. Carry out the primary drying for 18 hours and secondary four hours. Seal the ampoules or vials under vacuum. Test the viability of spores on media. Label the containers and store at -20°C. Freeze-dried spores stored at -20°C retain viability for years.
Determine the LD 50 of the freeze-dried spores in guinea pigs. When challenge tests shall be carried out in sheep or cattle, it is advantageous to determine LD 50 in these animals, however, because of economic considerations it is recommended to determine the LD 50 in guinea pigs.
Prepare tenfold dilutions of freeze-dried spores in a physiological saline solution containing 2 percent CaCl2.2H2O. Inoculate 1 ml intramuscularly in guinea pigs weighing 300 to 400g. Observe the guinea pigs for five days. Record the death of guinea pigs due to Cl.chauvoei and calculate the LD 50 by the Spearman-Karber method.
For testing the potency of Cl.chauvoei vaccine either virulent vegetative cultures or virulent spores activated with calcium chloride are used.
There is no uniformity of the challenge dose employed for testing the potency of blackleg vaccine. It ranges from ten to 100 guinea pig LD 50 (Joubert and Valette, 1976, Crichton et al., 1986 and Cameron et al., 1986). A 0.25 to 1 ml of 1:10 dilution of frozen virulent vegetative cultures was used (Knight and Kent, 1976). Recently in Australia detailed studies were carried out and standards for Cl. chauvoei vaccine were laid down in the Therapeutic Goods Order No. 13 (1984), wherein it was specified to use 10 LD 50 guinea pig challenge dose of spores of CSL strain CH4-C 6207 for testing the potency of blackleg vaccine in guinea pigs (Crichton et al., 1986). It is recommended to use at least 10 LD 50 guinea pig challenge dose of spores for testing the potency of Cl. chauvoei vaccine in guinea pigs (Crichton et al., 1986).
For the production of Cl. chauvoei vaccine, it is essential that a well characterized seed strain capable of yielding safe and immunogenic vaccine that meets the requirements as laid down in the British Pharmacopoeia (Veterinary), 1985, should be used. Organisms which grow during the course of infection in animals possess full antigenic components. This full antigenic pattern should be maintained as nearly as possible in any seed strain to be used for production of vaccine. Repeated passage of organisms in an artificial medium results in loss of both virulence and antigenicity. Therefore, Cl.chauvoei cultures should be kept within three passages in artificial media from the original seed obtained from infected cattle, sheep or guinea pigs. For this reason, a large stock of freeze-dried seed should be prepared and stored at -20°C or lower. It is necessary that the seed lots should be subjected to a full range of control tests, i.e. identity, safety and potency, prior to use in vaccine production.
It is necessary that production of Cl.chauvoei 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 Cl.chauvoei vaccine. This seed-lot system ensures that over a period of years. the seed strain is not subcultured on artificial media.
It is strongly recommended that the seed-lot be prepared in the production laboratory and submitted to the quality control laboratory for testing its efficacy. Full records should be kept of its origin, passage and date of subculture and medium used and storage. The use of seed-lots for the production of vaccine should also be recorded. A new batch of seed-lots should be prepared before its complete exhaustion, because if later on the passage material is found unsuitable it will be necessary to revert to a previous passage level. Seed-lots should be properly labelled and stored in the vaccine production laboratory in a freezer that does not contain contaminated material or at 4°C. The strain used for challenge should be stored in the quality control laboratory.
For the preparation of the seed-lot, either a standard strain of Cl. chauvoei obtained from a reference laboratory or a well characterized local isolate, properly evaluated to yield safe and immunogenic vaccine, can be used.
The ampoule of a freeze-dried Cl.chauvoei strain should be opened as follows:
Reconstitute the freeze-dried material in 1 ml of sterile physiological saline solution, inoculate 0.1 ml in test tubes containing 10 ml of anaerobic media and add 0.1 ml of 50 percent glucose. Incubate at 37°C for 24 to 48 hours. Growth is evidenced by production of gas, turbidity in the medium and change of colour of meat particles to brown black. Test the purity by staining the smear with Gram's stain and aerobic contamination on blood agar slants.
Passage the culture once in sheep or cattle, culture the organisms in an anaerobic medium and test the purity as described earlier. Pool the pure culture in an Erlenmeyer flask and filter it with gauze to remove meat particles if any. Add an equal volume of sterilized stabilizer and freeze-dry. Composition of the stabilizer is given in Appendix 2. Check the vacuum of the freeze-dried containers and discard those that do not have a vacuum.
Check the purity of the freeze-dried culture by culturing on both anaerobic broth and blood agar slants. Label each container and store at -20°C or lower. This is designated as the master seed-lot and can be used for years.
Working seed-lot is prepared from the master seed-lot at least once or twice a year. Check the vacuum of the container of the freeze-dried master seed-lot, reconstitute in 1 ml sterile physiological saline solution and inoculate 0.1 ml of culture in anaerobic broth prepared in screw capped tubes, and add 0.1 ml of 50 percent glucose solution to each tube. Incubate at 37°C for 24 hours. Growth is evidenced by production of gas, turbidity in the medium and meat particles turning brown-black. Test the purity of the culture and store pure cultures after tightly closing the caps at 4°C. These cultures are used for the preparation of inoculum for production of vaccine up to six to 12 months.
It should be noted that the working seed-lot should not be more than two passages from a well characterized master seed-lot, and that vaccine should be made from the working seed-lot without additional passage.
Control of seed-lot is done once in the beginning when seed-lots are prepared.
Test the bacterial contamination of seed-lots by morphological and cultural tests.
Morphological test. Prepare smears from samples of seed-lot, stain by Gram's stain and examine under the microscope. The seed-lot must not contain any other bacteria except Cl. chauvoei.
Cultural test. Streak blood agar plates with the seed-lot and incubate for 48 hours under anaerobic conditions 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 other bacteria except Cl. chauvoei.
Test the seed-lot on blood agar slants and incubate under aerobic conditions at 37°C for seven days. Examine the slants carefully for any growth. There must not be any growth on blood agar slants as Cl. chauvoei is a strict anaerobe and shall not grow under aerobic conditions.
Inoculate samples of seed-lot into Erlenmeyer flasks containing 50 ml thioglycolate fluid medium, incubate at 37°C for seven days. Cl.chauvoei grows under anaerobic conditions. Test the purity by smear examination daily up to seven days after staining with Gram's method. The seed-lot must not contain any other bacteria except Cl. chauvoei.
The seed-lot must be tested for its safety by preparing a batch of vaccine from it. The safety test should be carried out either on sheep or cattle as per the British Pharmacopoeia (Veterinary), 1985 for Cl. chauvoei vaccine.
Sheep. Inoculate three one-year-old sheep not vaccinated against blackleg with 4 ml of the concentrated vaccine or 10 ml unconcentrated vaccine subcutaneously at the inner face of the thigh. Observe for ten days and record their temperature in the morning and evening. The seed-lot passes the test if there is no untoward reaction except slight swelling at the site of inoculation which subsides in four to five days.
Cattle. Inoculate three one-year-old cattle not vaccinated against blackleg with 4 ml of the concentrated vaccine or 10 ml unconcentrated vaccine in the neck by subcutaneous route. Observe the inoculated animals for ten days and record their temperature twice a day in the morning and evening. The seed-lot passes the test if there is no untoward reaction except slight swelling at the site of inoculation which subsides in four to five days.
The seed-lot must be tested for its immunogenicity by preparing a batch of vaccine from it. The test shall be carried out on guinea pigs as per recommendations of the British Pharmacopoeia (Veterinary), 1985.
Inoculate ten guinea pigs subcutaneously with 2 ml of the vaccine as a primary dose or a quantity of the vaccine not greater than the minimum dose stated on the label. After 28 days re-inoculate these guinea pigs with 2 ml of the vaccine as a secondary dose. Record the temperature of the guinea pigs daily. Fourteen days after the secondary dose, challenge all vaccinated guinea pigs along with five controls by intramuscular route with 10 guinea pig LD 50 of virulent Cl. chauvoei spores diluted in a 2 percent solution of CaCl2.2H2O. In place of virulent spores, virulent vegetative cultures can be used for challenge and in this case there is no need for activation with CaCl2.2H2O.
The seed-lot passes the test if none of the vaccinated guinea pigs dies from Cl. chauvoei infection within five days of challenge, while all the controls die of Cl. chauvoei infection in 48 hours if virulent vegetative cultures are used, and in 72 hours if virulent spores are used.
Repeat the test if one of the vaccinated guinea pigs dies. The seed-lot passes the test if none of the vaccinated guinea pigs of the second group dies from Cl. chauvoei infection within five days of challenge, while all the controls die within 72 hours when virulent spores are used for challenge and within 48 hours when virulent vegetative cultures are used.
Both liver-meat extract anaerobic broth and semi-synthetic medium are suitable for vaccine production. For small-scale production prepare media in 8-litre volumes in 9-litre aspirator bottles and sterilize in an autoclave at 121°C for 45 minutes. For large scale production, fermenters are employed.
Prepare inoculum from the working seed-lot. Boil the anaerobic broth for ten minutes to drive off the oxygen and cool at 37°C before inoculation. Inoculate 2 ml of working seed in 200 ml of anaerobic broth. Add 2 ml of sterile 50-percent glucose solution and incubate at 37°C for 24 hours. Growth of Cl. chauvoei is evidenced by gas formation and turbidity in the medium. Test the purity by smear examination and aerobic contamination on blood agar slants. Store the inoculum at 4°C. Use the pure seed cultures as inoculum.
Cool the production media to 37°C and seed with 200 ml of the inoculum in each aspirator bottle containing 8 litres of medium. Add 160 ml of 50-percent glucose solution to each flask to make a final concentration of 1 percent. Incubate at 37°C for three to five days. Growth of Cl. chauvoei is evidenced by gas formation and turbidity in the medium. Shake the aspirator bottles, test the purity by smear examination and aerobic sterility on blood agar slants. Incubate the slants at 37°C for 48 hours. Discard the contaminated bottles.
In the fermenter, production of Cl. chauvoei vaccine is carried out under controlled conditions. Seed the medium with 5 percent of inoculum and maintain the fermenter temperature at 37°C. Feed 50-percent glucose solution at regular intervals to give a final concentration of 1 percent and stir the medium slowly. Adjust the pH to 7.2 during the growth of organisms. The growth is completed between 18 and 24 hours. Test the purity as above.
Prepare a 20-percent solution of formalin by adding 200 ml of formalin (37 percent) to 800 ml distilled water and add to each aspirator bottle in sufficient quantity to give 0.7 percent formalin in the final concentration. For 8-litre medium, 280 ml of 20-percent formalin solution is sufficient. Shake the bottles and keep at 37°C for six days. Store the bottles at 4°C. Formalize the Cl. chauvoei organisms grown in the fermenter by adding sufficient quantity to give 0.7 percent formaline in the final concentration. Maintain the temperature of the fermenter 37°C for six days.
Boil the Erlenmeyer flasks containing 100 ml of anaerobic broth. Cool it to 37°C. Inoculate 2 ml samples into the anaerobic broth. Add 2 ml of 50 percent of glucose solution. Incubate at 37°C for five days. Cl. chauvoei organisms are considered inactivated when there is no growth in the anaerobic medium. If growth is observed, incubate the bottles once more at 37°C and retest as above.
Pool the inactivated cultures from bottles into a stainless steel tank fitted with magnetic drive and thoroughly mix by stirring for 30 minutes. Adjust the pH to 6.8 by sodium hydroxide solution.
Alum and aluminium hydroxide gel adjuvants are commonly used for the production of Cl. chauvoei vaccine.
Prepare a 20-percent solution of alum in distilled water, sterilize in the autoclave at 121°C for 30 minutes and add to the inactivated Cl. chauvoei culture in sufficient quantity to give 1 percent alum in final concentration. For one litre of inactivated culture, add 50 ml of 20-percent potash alum solution. Slowly stir the vaccine at 20°C for four hours and store it at 4°C.
Allow the precipitated vaccine to settle at 4°C for three days, siphon off the supernatant from the alum-precipitated culture to give 2.5 times concentration, i.e. from one litre of precipitated vaccine, siphon off 600 ml supernatant. Thus one litre of precipitated vaccine is concentrated to 400 ml. Mix the concentrated vaccine by slowly stirring for one hour at 20°C and store at 4°C.
Sterilize aluminium hydroxide gel at 121°C for 60 minutes. When cool, pump into the tank containing inactivated Cl. chauvoei culture in sufficient quantity to give 10 percent gel in final concentration. Slowly stir the adjuvanted vaccine at 30-minute intervals for five minutes at 20°C. Carry out the adsorption in this manner for ten hours. Store the adjuvanted vaccine at 4°C. Allow it to settle for three days at 4°C. Siphon off the supernatant from the adsorbed vaccine to give 2.5 times concentration, i.e. from one litre of final adsorbed vaccine siphon off 600 ml supernatant. Thus one litre of aluminium gel adsorbed vaccine is concentrated to 400 ml. Mix the concentrated vaccine by slowly stirring for 30 minutes at 20°C.
Thiomersal is added in the adjuvanted vaccine as a preservative.
Prepare a 10-percent solution of thiomersal in physiological saline solution and add in the concentrated vaccine in sufficient quantity to give 0.01 percent thiomersal in the final concentration. Mix the vaccine slowly for 30 minutes at 20°C and store at 4°C.
For the preparation of mixed bacterins of Cl. chauvoei and Cl. septicum, add equal amounts of double strength of concentrated bacterins and mix thoroughly at 20°C for 30 minutes.
For the preparation of mixed bacterins of Cl. chauvoei and P. multocida, add equal amounts of double strength of concentrated bacterins and mix thoroughly at 20°C for 30 minutes.
Transfer the required filling equipment, vials and stoppers after sterilization in the filling room which has been prepared before, and start the ultraviolet light. Filling operations should be conducted in such a way as to avoid any contamination or alteration of the vaccine.
Transfer the processed vaccine into a sterile vaccine filling tank fitted with magnetic drive in the filling room by using sterile compressed air. Slowly mix the vaccine for one hour at room temperature. Fill the Cl. chauvoei vaccine in multidose containers preferably in 100 ml vials by a pipetting machine, transferring measured quantities of vaccine from the bulk container to the final containers. Close the vials by sterilized rubber stoppers with the help of a forceps. Seal the vials with aluminium collars immediately. 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 individually labelled. If the filling is carried out directly from final bulk containers, the number of vials filled in one session without stoppage is referred as final lot or filling lot.
It is necessary to check the filling process at least twice a year. Fill about 500 vials with a nutrient broth at the end of the working day and incubate them at 37°C. If more than 1 percent of the vials are contaminated, the filling room, filling machines and filling procedures must be properly checked and the fault rectified.
Inspect every container visually under proper illumination to detect the presence of foreign particles. Examine the vials against both white and black background. Discard vials showing foreign particles, clumps and defects in closures.
Perform the identity test on every final lot of the vaccine. The potency test of Cl. chauvoei vaccine serves as an identity test. The method is described in the section dealing with control of seed-lots. The vaccine lot passes the identification test if it protects susceptible animals against infection with Cl. chauvoei.
Carry out the sterility test on every final lot of the vaccine. According to the British Pharmacopoeia, Veterinary (1985), 1 percent container of a lot with a minimum of three or a maximum of ten is considered a suitable sample size for carrying out the test. Collect the sample by random selection including samples at the beginning and at the end of the filling operation. Shake the vials thoroughly before withdrawing the vaccine. Sterilize the rubber stopper with rectified spirit before taking out the samples from the vial. Carry out the test as follows:
Examine all the containers on the third, fifth, seventh and on the last day. The vaccine passes the test if there is no evidence of microbial growth. If any container or plate shows growth, the test has to be repeated with double the number of containers. The test is considered satisfactory when there is no evidence of microbial growth.
Determine the pH of each final lot of the vaccine. Standardize the pH meter with standard buffer solution at pH 5 and 7. Rinse the electrodes with distilled water, dry and test the pH of the vaccine. The pH of Cl. chauvoei 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 a safety test is carried out on animals of the species for which the vaccine is intended to be used.
Inoculate two mice weighing approximately 20 g each with 0.4 ml of concentrated or 1 ml of unconcentrated vaccine by subcutaneous injection, and two guinea pigs weighing approximately 300 to 400 g with 0.8 ml of concentrated or 2 ml unconcentrated vaccine by subcutaneous injection. Observe these animals for seven days. The vaccine is acceptable if none of the animals shows signs of ill health during observation. If one of the animals dies or shows signs of illness 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.
Carry out the safety test on every lot of the vaccine. According to the British Pharmacopoeia Veterinary, 1985, safety tests should be carried out in susceptible animals of at least one of the species, i.e.either sheep or cattle, in which vaccine is intended to be used. The procedure of the test is described in the section dealing with control of seed-lots.
The potency test of the vaccine is generally carried out in the species in which the vaccine is intended to be used. However, it has been proved that guinea pig laboratory model is a valid indicator of field performance of Cl. chauvoei vaccine, thus the potency test is carried out in guinea pigs. The procedure of the test is described in the section dealing with control of seed-lots.
Maintain a complete passage history of the seed-lots and cultures used in production and quality control of Cl. chauvoei vaccine. Label all the cultures properly and store them at the appropriate temperature in an orderly manner. Keep a complete and accurate inventory of all the stocks of seed-lot, i.e. vials or ampoules of cultures 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 the steps in processing and filling including both process controls and control test of the final product. Keep the written records of all tests, irrespective of their results throughout the dating period of each lot of vaccine and in such a manner that they will be 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, if necessary.
A sample of a suitable summary protocol for production and control of Cl. chauvoei (blackleg) vaccine is given in Appendix 3. The protocol is intended for the reporting of the data to the national control authorities or other countries to whom vaccine may be exported.
At the time of release of a lot of Cl. chauvoei (blackleg) 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 the samples at the 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 the Cl. chauvoei (blackleg) vaccine, it is beneficial to keep samples from time to time beyond the expiry period.
Identify the Cl. chauvoei (blackleg) vaccine 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 Cl. chauvoei (blackleg) 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 Cl. chauvoei (blackleg) vaccine under cold conditions over ice in insulated boxes. Do not freeze the vaccine.
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 48 to 72 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 points, 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 Cl. chauvoei (blackleg) vaccine 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 Cl. chauvoei (blackleg) vaccine depends upon the type of vaccine. Generally this vaccine can be expected to retain its potency for three years at +4°C from the date of the completion of the potency test.
DESCRIPTION
Blackleg vaccine is a culture of a suitable strain of Cl. chauvoei grown in an anaerobic fluid medium which is inactivated and rendered atoxic in such a way that it retains its immunogenicity to which alum/aluminium hydroxide gel is added.
The vaccine has a brownish colour with a milky white deposit which disappears upon shaking.
USE
The vaccine is used to control blackleg in cattle and sheep. Calves should be vaccinated at four month of age. In endemic areas when a high degree of immunity is desired, a booster dose should be administered one month after the first vaccination. The vaccination should be repeated annually. Blackleg vaccination should generally be carried out after pasteurellosis vaccination.
DOSE (CONCENTRATED VACCINE)
| Cattle | 2.0 ml |
| Sheep | 1.0 ml |
Inoculate by subcutaneous route in the neck of cattle and on inner side of the thigh in sheep.
REACTION
Vaccinated animals may have a slight swelling at the site of inoculation which lasts for two to three days.
IMMUNITY
Immunity is established in 15 to 21 days after vaccination and persists for one year.
STORAGE
In a dark, dry place at +4°C, the vaccine retains its potency up to three years.
PRECAUTIONS
Vials once opened should be used on the same day and not be reused on subsequent days. If storage and reuse are absolutely unavoidable the vial must be kept in a refrigerator at +4°C and never for more than one working week.
Do not freeze the vaccine.
NUTRIENT BROTH
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, place 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 autoclave.
BLOOD AGAR
Blood agar is used in the form of slants and plates. Add 1.5-2 percent agar to the nutrient broth, melt it, sterilize in autoclave at 121°C for 30 minutes and cool to 50°C. Add 10 percent of defibrinated sheep's blood and mix thoroughly with the medium. Distribute 5 ml in sterile test tubes and keep in a slanting position for making the slants, and 20 ml in 10-cm diameter sterile Petri dishes for making blood agar plates. When the agar solidifies, incubate the medium at 37°C for 24 hours. Check the medium for contamination and store at +4°C.
LIVER EXTRACT
Prepare the liver extract either from liver powder available from commercial sources as per manufacturer's instructions, or from fresh liver. The preparation of liver extract from fresh liver is given below.
| Liver | 500 g |
| Distilled water | 1 000 ml |
Finely mince the liver, transfer to an Erlenmeyer flask and add the distilled water. Boil in autoclave for 20 minutes, filter and store at 4°C. Prepare the extract fresh. In place of fresh liver, powder available from commercial sources can also be used for the preparation of liver extract.
LIVER EXTRACT-BLOOD AGAR
This medium is recommended for isolation of Cl. chauvoei.
| Nutrient broth containing 1.8% agar | 75 ml |
| 50-percent glucose | 2 ml |
| Liver extract | 3 ml |
| Sheep's blood | 5 ml |
Distribute 20 ml in 10-cm diameter sterile Petri dishes for making blood agar plates.
LIVER-MEAT ANAEROBIC BROTH
This medium is recommended for the production of vaccine.
| Meat extract | 1 000 ml |
| Liver extract | 1 000 ml |
| Peptone | 20 g |
| Sodium thioglycolate | 2 g |
| Sodium chloride | 10 g |
| Dipotassium hydrogen phosphate | 8 g |
Dissolve peptone and sodium chloride in meat and liver extract. Adjust the pH to 8.2. Add dipotassium hydrogen phosphate and sodium thioglycolate. Distribute 5 ml in test tubes and 50 ml in 100-ml Erlenmeyer flasks and add pieces of meat. For production of vaccine in flasks transfer 8 litres in 9-litre aspirator bottles. Do not add meat particles in production flasks. Sterilize at 121°C in autoclave for 45 minutes.
SEMI-SYNTHETIC ANAEROBIC MEDIUM
This medium is recommended for production of vaccine in fermenters.
| Casein hydrolysate | 6 g |
| Meat extract | 2 g |
| Tryptone | 8 g |
| Peptone | 4.0 g |
| Yeast extract 2 g L-cysteine hydrochloride | 1.0 g |
| Na2HPO4.12H2O | 0.4 g |
| Distilled water | 1 000 ml |
| pH | 7.2 |
Add 50 percent glucose solution, 20 ml per litre, during inoculation.
Thoroughly grind the ingredients in a mortar. Stir in small amounts of warm water, add the remaining water, dissolve by heating in a steam jacketed media vessel taking special care to ensure complete solution of the L-cysteine. Adjust the pH to 7.4. Reheat the solution, filter, distribute into suitable containers and sterilize by autoclaving at 121°C for 45 minutes.
SOYBEAN-CASEIN DIGEST MEDIUM
This medium is recommended for testing sterility of biological substances.
| 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 | 7.01–7.5 |
Dissolve the ingredients in warm water, then cool to room temperature. Adjust the pH to 7.5 with 1 N sodium hydroxide. Filter, distribute into suitable vessels and sterilize in autoclave at 121°C for 20 minutes.
FLUID THIOGLYCOLATE MEDIUM
This medium is recommended for testing sterility of biological substances.
| 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 distilled water, stir and transfer to a suitable container, add the remaining distilled water. Dissolve by boiling in a 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 into a pink colour and this exceeds one-third of the depth 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.
SKIMMED MILK
Mix an equal volume of 5-percent sterile skimmed milk with spore suspension of Cl. chauvoei and freeze-dry.
SUCROSE-GLUTAMATE-DEXTRAN
| Sucrose | 5.0 g |
| Sodium glutamate | 1.0 g |
| Dextran | 5.0 g |
| Physiological saline solution | 100 ml |
Sterilize the solution by filtration. Suspend the spores of Cl. chauvoei in the above solution and freeze-dry.
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 |
Clostridium chauvoei strain
| 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 isolation and master seed-lot | |
| 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 OF VACCINE
Media
| Type of medium used | |
| Date of preparation | |
| Type of cultivation | Flasks/fermenter |
| Ingredients (flasks) | |
| Liver extract | |
| Meat extract | |
| Peptone | |
| Sodium chloride | |
| Dipotassium hydrogen phosphate K2HPO4 | |
| Sodium thioglycolate | |
| pH | |
| Quantity filled in each flask | |
| Number of flasks filled | |
| Sterilization | |
| Ingredients (fermenter) | |
| Casein hydrolysate | |
| Meat extract | |
| Tryptone | |
| Peptone | |
| Yeast extract | |
| L-cysteine hydrochloride | |
| Na2HPO4. 12H2O | |
| Distilled water | |
| pH | |
| Quantity prepared | |
| Sterilization |
Inoculum
| Date | |
| Quantity of media in each flask | |
| Quantity of inoculum | |
| No. of seed flasks inoculated | |
| Tests for bacterial contamination | |
| Methods used | |
| Results |
Inoculation
| Date | |
| Quantity inoculated in each production flask/fermenter | |
| Quantity of 50% glucose added in each flask/fermenter | |
| Number of flasks inoculated | |
| Duration of incubation flasks/fermenter |
Formalization
| Date | |
| Tests for bacterial contamination | |
| Methods used | |
| Results | |
| Quantity of formaline added in each flask/fermenter | |
| Duration of formalization at 37°C |
Inactivation test
| Test in media | |
| Date | |
| Duration of observation | |
| Results |
Pooling
| Date | |
| No. of flasks pooled |
Adjuvant
| Date | |
| Amount of inactivated culture of Cl. chauvoei | |
| Nature of adjuvant | |
| Quantity of adjuvant | |
| Duration of absorption by mixing |
Concentration
| Date | |
| Quantity of adjuvanted vaccine concentrated | |
| Period adjuvanted vaccine allowed to stand at 4°C | |
| Times concentration | |
| Quantity of concentrated vaccine |
Preservation
| Date | |
| Type of preservative added | |
| Concentration of preservative |
FILLING AND CONTAINERS
| Date | |
| Quantity of vaccine per container | |
| Quantity of containers |
CONTROL TESTS ON FINAL PRODUCT
Inspection of final containers
| Date | |
| Results |
Identity test
| Date | |
| Methods | |
| Results |
Sterility test
| Date | |
| No. of containers tested | |
| Media used | |
| Results |
Determination of pH
| Date | |
| Results |
Innocuity test
| Animal species used | |
| No. of animals | |
| Route and dose | |
| Date of inoculation | |
| Date of end of test | |
| Result |
Safety test
| Animal species used | |
| No. of animals | |
| Route and dose of inoculation | |
| Date of inoculation | |
| Date of end of test | |
| No. of animals showed post-inoculation reactions | |
| No. of animals died | |
| Results |
Potency test
| Animal species used | |
| No. of animals | |
| Date of primary inoculation | |
| Dose and route of inoculation | |
| Date of secondary inoculation | |
| No. of animals died before challenge | |
| Date of challenge | |
| Challenge strain | |
| Dose and route of challenge | |
| No. of controls | |
| No. of vaccinated animals challenged | |
| No. of vaccinated animals died | |
| No. of control animals died | |
| 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 Clostridium chauvoei (blackleg) vaccine satisfies all the national requirements.
| 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 Clostridium chauvoei (blackleg) vaccine produced by (name of producer) meets all national requirements. 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.
Cameron, C.M., Botha, W.J.S. & Schoeman, J.M. 1986. Immunization of guinea pigs and cattle with a reduced dose Cl. chauvoei vaccine produced in a semi-synthetic medium. Onderstepoort J. Vet. Res., 53: 51–53.
Chander, H.M. & Gulasekharam, J. 1970. An evaluation of characteristics of Cl. chauvoei which possibly indicate a highly protective strain. Australian J. Exptl. Biol. and Med. Sci., 48: 187–197.
Crichton, R., Harris, D.A. & McKay D.J. 1986. Standards of Cl. chauvoei vaccine. The relationship between the response of guinea pigs and sheep following vaccination and challenge with virulent Cl. chauvoei. Australian Vet. J., 63(3): 68–70.
Joubert, L. & Valette, L.R. 1976. Les échecs de la vaccination antisymptomatique et les méthodes de contrôle d'activité des vaccins. Development Biol. Stand., 32: 151– 155.
Kerry, J.B. 1967. Immunological differences between strains of Cl. chauvoei. Vet. Sci., 8: 89–97.
Knight, P.A. & Kent, J. 1976. Some problems concerning the assay of Cl. chauvoei vaccine in laboratory animals. Development Biol. Stand. 32: 143–150.
Reed, E.A. & Reynolds, L. 1977. Failure of Cl. chauvoei vaccines to protect against blackleg. Australian Vet. J., 53: 393.
Walker, P.D. & Batty, I. 1979. Colonial morphology, fluorescent labelled antibody staining in the identification of species of genus Clostridium. Identification method for microbiologist (second edition). Technical series 14. New York, Academic Press.
WHO. 1973. Technical Report Series No. 530. General requirements for the sterility of biological substances. Geneva, World Health Organization.
FAO TECHNICAL PAPERS
FAO ANIMAL PRODUCTION AND HEALTH PAPERS:
| 1. | Animal breeding: selected articles from World Animal Review, 1977 (C* E* F* S*) |
| 2. | Eradication of hog cholera and African swine fever, 1976 (E* F* S*) |
| 3. | Insecticides and application equipment for tsetse control, 1977 (E* F*) |
| 4. | New feed resources, 1977 (E/F/S*) |
| 5. | Bibliography of the criollo cattle of the Americas, 1977 (E/S*) |
| 6. | Mediterranean cattle and sheep in crossbreeding, 1977 (E* F*) |
| 7. | Environmental impact of tsetse chemical control, 1977 (E* F*) |
| 7Rev. | Environmental impact of tsetse chemical control, 1980 (E* F*) |
| 8. | Declining breeds of Mediterranean sheep, 1978 (E* F*) |
| 9. | Slaughterhouse and slaughterslab design and construction, 1978 (E* F*) |
| 10. | Treating straw for animal feeding, 1978 (C* E* F* S*) |
| 11. | Packaging, storage and distribution of processed milk, 1978 (E*) |
| 12. | Ruminant nutrition: selected articles from World Animal Review, 1978 (C* E* F* S*) |
| 13. | Buffalo reproduction and artifical insemination, 1979 (E**) |
| 14. | The African trypanosomiases, 1979 (E* F*) |
| 15. | Establishment of dairy training centres, 1979 (E*) |
| 16. | Open yard housing for young cattle, 1981 (E* F* S*) |
| 17. | Prolific tropical sheep, 1980 (E* F* S*) |
| 18. | Feed from animal wastes: state of knowledge, 1980 (E*) |
| 19. | East Coast fever and related tick-borne diseases, 1980 (E* S*) |
| 20/1. | Trypanotolerant livestock in West and Central Africa, 1980 - Vol. 1 - General study (E* F*) |
| 20/2. | Trypanotolerant livestock in West and Central Africa, 1980 - Vol. 2 - Country studies (E* F*) |
| 20/3. | Le bétail trypanotolétant en Afrique occidentale et centrale Vol. 3 - Bilan d'une décennie, 1988 (F*) |
| 21. | Guideline for dairy accounting, 1980 (E*) |
| 22. | Recoursos genéticos animales en América Latina, 1981 (S*) |
| 23. | Disease control in semen and embryos, 1982 (E* F* S*) |
| 24. | Animal genetic resources - conservation and management, 1981 (E*) |
| 25. | Reproductive efficiency in cattle, 1982 (E* F* S*) |
| 26. | Camels and camel milk, 1982 (E*) |
| 27. | Deer farming, 1982 (E*) |
| 28. | Feed from animal wastes: feeding manual, 1982 (E*) |
| 29. | Echinococcosis/hydatidosis surveillance, prevention and control: FAO/UNEP/WHO guidelines, 1982 (E*) |
| 30. | Sheep and goat breeds of India, 1982 (E*) |
| 31. | Hormones in animal production, 1982 (E*) |
| 32. | Crop residues and agro-industrial by-products in animal feeding, 1982 (E/F*) |
| 33. | Haemorrhagic septicaemia, 1982 (E* F*) |
| 34. | Breeding plans for ruminant livestock in the tropics, 1982 (E* F* S*) |
| 35. | Off-tastes in raw and reconstitued milk, 1983 (E* F* S*) |
| 36. | Ticks and tick-borne diseases: selected articles from World Animal Review, 1983 (E* F* S*) |
| 37. | African animal trypanosomiasis: selected articles from World Animal Review, 1983 (E* F*) |
| 38. | Diagnosis and vaccination for the control of brucellosis in the Near East, 1983 (E* Ar*) |
| 39. | Solar energy in small-scale milk collection and processing, 1983 (E* F*) |
| 40. | Intensive sheep production in the Near East, 1983 (E* Ar*) |
| 41. | Integrating crops and livestock in West Africa, 1983 (E* F*) |
| 42. | Animal energy in agriculture in Africa and Asia, 1984 (E/F*) |
| 43. | Olive by-products for animal feed, 1985 (Ar* E* F* S*) |
| 44/1. | Animal genetic resources conservation by management, data banks and training, 1984 (E*) |
| 44/2. | Animal genetic resources: cryogenic storage of germplasm and molecular engineering, 1984 (E*) |
| 45. | Maintenance systems for the dairy plant, 1984 (E*) |
| 46. | Livestock breeds of China, 1985 (E*) |
| 47. | Réfrigération du lait à la ferme et organisation des transports, 1985 (F*) |
| 48. | La fromagerie et les variétés de fromages du bassin méditerranéen, 1985 (F*) |
| 49. | Manual for the slaughter of small ruminants in developing countries, 1985 (E*) |
| 50. | Better utilization of crop residues and by-products in animal feeding: research guidelines - 1. State of knowledge, 1985 (E*) |
| 50/2. | Better utilization of crop residues and by-products in animal feeding: research guidelines - 2. A practical manual for research workers, 1986 (E*) |
| 51. | Dried salted meats: charque and carne-de sol, 1985 (E*) |
| 52. | Small-scale sausage production, 1985 (E*) |
| 53. | Slaughterhouse, cleaning and sanitation, 1985 (E*) |
| 54. | Small ruminants in the Near East: Vol. 1, 1986 (E*) Selected papers presented at Tunis Expert Consultation |
| 55. | Small ruminants in the Near East: Vol. II, 1986 (E* Ar*) Selected papers from World Animal Review |
| 56. | Sheep and goats in Pakistan, 1985 (E*) |
| 57. | Awassi Sheep, 1985 (E*) |
| 58. | Small ruminant production in the developing countries, 1986 (E*) |
| 59/1. | Animal genetic resources data banks, 1986 (E*) 1 - Computer systems study for regional data banks |
| 59/2. | Animal genetic resources data banks, 1986 (E*) 2 - Descriptor lists for cattle, buffalo, pigs, sheep and goats |
| 59/3. | Animal genetic resources data banks, 1986 (E*) 3 - Descriptor lists for poultry |
| 60. | Sheep and goats in Turkey, 1986 (E*) |
| 61. | The Przewalski horse and restoration on its natural habitat in Mongolia, 1986 (E*) |
| 62. | Milk and dairy products: production and processing costs, 1988 (E* F* S*) |
| 63. | Proceedings of the FAO expert consultation on the substitution of imported concentrate feeds in animal production systems in developing countries, 1987 (E*) |
| 64. | Poultry management and diseases in the Near East, 1987 (Ar*) |
| 65. | Animal genetic resources of the USSR, 1989 (E*) |
| 66. | Animal genetic resources - Strategies for improved use and conservation, 1987 (E*) |
| 67/1. | Trypanotolerant cattle and livestock development in West and Central Africa - Vol. I, 1987 (E*) |
| 67/2. | Trypanotolerant cattle and livestock development in West and Central Africa - Vol. II, 1987 (E*) |
| 68. | Crossbreeding bos indicus and bos taurus for milk production in the tropics, 1987 (E*) |
| 69. | Village milk processing, 1988 (E* F*) |
| 70. | Sheep and goat meat production in the humid tropics of West Africa, 1988 (E/F*) |
| 71. | The development of village based sheep production in West Africa, 1988 (E* F* S*) |
| 72. | Sugarcane as feed, 1988 (E/S*) |
| 73. | Standard design for small-scale modular slaughterhouses, 1988 (E*) |
| 74. | Small ruminants in the Near East, Volume III: North Africa, 1988 (E*) |
| 75. | The eradication of ticks, 1989 (E/F*) |
| 76. | Ex situ cryoconservation of genomes and genes of endangered cattle breeds by means of modern biotechnological methods, 1989 (E*) |
| 77. | A training manual for embryo transfer in cattle, 1989 (E*) |
| 78. | Milking, milk production hygiene and udder health, 1989 (E*) |
| 79. | Manual of simple methods of meat preservation, 1989 (E*) |
| 80. | Animal genetic resources - A global programme for sustainable development, 1990 (E*) |
| 81. | Veterinary diagnostic bacteriology - A manual of laboratory procedures of selected diseases of livestock, 1990 (E*) |
| 82. | Reproduction in camels - a review, 1990 (E*) |
| 83. | Training manual on artificial insemination in sheep and goats, 1991 (E*) |
| 84. | Training manual for embryo transfer in water buffaloes, 1991 (E*) |
| 85. | The technology of traditional milk products in developing countries, 1990 (E*) |
| 86. | Feeding dairy cows in the tropics, 1990 (E*) |
| 87. | Manual for the production of anthrax and blackleg vaccines, 1991 (E*) |
Availability: March 1991
Ar — Arabic
C — Chinese
E — English
F — French
S — Spanish
*** In preparation
The FAO Technical Papers are available through the authorized FAO Sales Agents or directly from Distribution and Sales Section, FAO, Via delle Terme di Caracalla, 00100 Rome, Italy
