GB2030451A - Anti-clostridium vaccine - Google Patents

Abstract

An anti-clostridium vaccine comprises cell-free toxoids of Clostridium perfringens types A, B, and D, Cl. oedematiens, and Cl.septicum, and an adsorbent adjuvant (e.g. alumina gel), in a physiologically acceptable injectable aqueous carrier. The antigenic activity of the toxoids, expressed in combining units per ml of the vaccine, is as follows:- toxoid of Cl.perfringens 2-100 type A toxoid of Cl.perfringens 2-100 type B toxoid of Cl.perfringens 1-80 type D toxoid of Cl.oedematiens 1-80 toxoid of Cl.septicum 2-100. Suitably there is 1-10 mg of adsorbent adjuvant per ml of vaccine.

Description

SPECIFICATION Vaccine for prophylaxis and treatment of clostridioses of animals and poultry The present invention relates to veterinary microbiology and more particularly it relates to a vaccine for prophylaxis and treatment of clostridiosis in animals and birds.

The present invention resides in a vaccine for prophylaxis and treatment of clostridiosis of animals and poultry which comprises toxoids of Clostridium perfringens types A, B, and D, Cl. oedematiens, Cl. septicum, separated from the bacterial mass, a sorbent and a solvent, the antigenic activity of said toxoids, expressed in binding units per ml of the vaccine, and the amounts of sorbent and solvent being as follows: toxoid of CI. perfringens type A 2-100 toxoid of CI. perfringens type B 2-100 toxoid of CI. perfringens type D 1-80 toxoid of CI. oedematiens 1-80 toxoid of CI. septicum 2-100 sorbent, preferred 1-10 mg solvent balance.

The vaccine is of use against the following diseases of cattle, horses, sheep, pigs, other commercial or husbanded animals and poultry that are produced by said causative agents, for example: gas gangrene, malignant edema, endometritis, vaginitis, mastitis, miscarriage, bradzot of sheep, and enterotoxemia, especially for younger livestock and newborns in particular.

It is not recommended to use a vaccine in which the antigenic activity of its components is below the minimum doses given since the vaccine will fail to produce antitoxic immunity in the vaccinated animals and birds at the protective level.

Nor is it recommended to use a vaccine in which the antigenic activity of its components is higher than the maximum doses given, since the production of antitoxins in the vaccinated animals is inhibited with excess component (antitoxic immunity).

It is recommended that the vaccine also contain a toxoid of Cl. tetani, separated from the bacterial mass, its antigenic activity, expressed in binding units per ml of the vaccine, being 1-80.

The proposed vaccine is, then, as noted above, of especial use against infectious enterotoxemia in young animals, principally newborns of all agricultural animals and poultry, against gynaecological diseases (endometritis, vaginitis, mastitis) in female adults of cattle, horses, sheep, and pigs, wound complications, gas gangrene, and malignant edema, of animals and poultry, and bradzot, of sheep, that are caused by the said combination of microorganisms of the genus Clostridium.

It is recommended that the vaccine also contain toxoids of CI. botulinum of at least two of the types A, B, C, D, E, and F, separated from the bacterial mass, their antigenic activity, expressed in combining units per ml of the vaccine, being as follows:toxoid of CI. botulinum type A 2-100 toxoid of Cl. botulinum type B 0.2-80 toxoid of Cl. botulinum type C 0.2-80 toxoid of Cl. botulinum type D 0.2-80 toxoid of Cl. botulinum type E 0.2-80 toxoid of CI. botulinum type F 0.2-80 Such a proposed vaccine is used for prophylaxis and treatment of forage toxicoinfection, anaerobic enterotoxemia, gynaecological diseases and wound complications.

The incorporation into the vaccine of toxoids of CI. tetani and CI. botulinum types A, B, C, D, E, and F is explained by the necessity for prophylaxis and treatment of clostridiosis caused by the said microorganisms.

The selection of the minimum and maximum doses of antigenic activity of toxoids of CI. botulinum and CI.

tetani in the proposed vaccine is governed by the same reasons as specified above for toxoids of Cl.

perfringens types A, B, and D, Cl. oedematiens, and CI. septicum.

It is recommended that the vaccine contain alumina gel as the sorbent. This sorbent ensures complete sorption of the toxoids and does not produce any harmful effect on animals.

It is recommended to use a mixture of a phosphate buffer and a physiological salt solution, taken in the weight ratio of 1:1, as the solvent.

The proposed vaccine is a liquid preparation that typically separates on standing into a colourless supernatant liquid and a white, pale-brown or yellowish precipitate. When shaken, the vaccine gives a homogeneous suspension free from flakes or extraneous impurities.

The vaccine has been tested on smaller laboratory animals and on agricultural animals such as cattle, sheep, pigs, camels, horses, etc., on newborn calves, gilts, lambs, poultry (e.g. hens), and commercial animals (minks), Observation of the animals has shown that the active formation of intense immunity stimulated the production of a high titre of toxoids to each component of the vaccine, and the vaccinated animals developed immunity (in primary immunization) in 10-25 days and (after re-vaccination) in 5-10 days. The length and activity of the immunity produced by the proposed vaccine were studied on 100 animals that were infected with the corresponding virulent culture or a toxin which is a constituent of the proposed vaccine, 1, 2,4, 6, 10, 12 months after the vaccination and 2,4, and 5 years after the vaccination.

Non-immunized animals, and also animals who had the corresponding disease in the past, were used as controls. The animals were selected into the control group according to the analogue principle.

The animals were immunized in accordance with immunization methods developed for each species of animals and poultry.

In cases where one or several of the causative agents were revealed, all profitable healthy cattle, calves, pigs, sheep, horses, camels, poultry, minks, and other animals were given a prophylactic vaccination irrespective of the pregnancy term. At affected farms, where the preparation was used for the first time, the cattle, horses, camels, pigs, and sheep were vaccinated from one to three times. The vaccine was given in doses from 0.5 to 5 ml. parenterally or in an aerosol form. The animals and poultry were re-vaccinated 3-6 months following the first vaccination, at the time of intense reproduction, or for special indications (outbreaks of forage poisoning, etc).

In cases of urgent prophylaxis of anaerobic enterotoxemia of newborns, when the vaccination of the mother is useless, the newborns of non-vaccinated mothers are given the vaccine. The vaccination is effected in two injections.

The vaccine we have tested does not produce any harmful effect on the vaccinated animal or the progeny.

It is harmless and areactogenic. The newborn animals are healthy, strong, viable, and gain weight quickly.

The vaccine proves quite effective against the diseases caused by Clostridium perfringens types A, B, and D, Cl. oedematiens, Cl. septicum Cl. titani, and Cl. botulinum. The efficacy is hundred per cent. At the same time, all animals in the control group (100 per cent) were affected by the disease and from 80 to 100 per cent animals perished..

The vaccine remains effective up to five years.

The proposed vaccine for prophylaxis and treatment of clostridiosis in animals and poultry can be prepared as follows.

Growth medium containing sources of nitrogen, carbon, phosphorus and mineral salts is inoculated with separate cultures of Clostridum peffringens types A, B, and D, Cl. oedematiens, Cl. septicum, Cl. tetani, and Cl. botulinum types A, B, C, E, and F. The cultures are grown under anaerobic conditions at a temperature of 35-38"C, the pH of the medium being 5-10. The time of cultivation depends on the species and type of the microorganism and averages from two hours to ten days. At the end of the cultivation period, a culture suspension of each species and type is separated into the culture filtrate and the microbial or bacterial mass.

The activity of toxins, expresses in MLD per ml for albino mice was as follows: toxin of Cl. perfringens type A 800-1200 toxin of Cl. peffringens type B 1000-3000 toxin of Cl. perfringens type D 1000-3000 toxin of Cl. oedematiens 12,000-20,000 toxin of Cl. septicum 800-1000 The culture filtrate containing the toxin is separated and passed through a Seitz filter. Then, the toxin is partially detoxicated by adding 0.4-05. per cent by volume of a 40 per cent formaldehyde solution into the culture filtrate at a temperature of 37-38"C, the pH of the medium being 7-7.2.Formaldehyde is added in portions, once or twice within 1-20 days, depending on the species and the type of the microorganism producing the toxin. The only exceptions are toxins of Cl. perfringens type B, Cl. oedamatiens and Cl.

septicum, which are attenuated with formaldehyde within 1 to 30 days to give the toxoid.

The partially attenuated toxin or toxoid is then concentrated and purified by precipitating with a 45-60 per cent solution of ammonium sulphate or sodium hexametaphosphate in the presence of IN hydrochloric acid.

The precipitated toxin or toxoid is dissolved in a mixture of a phosphate buffer and physiological salt solution taken in the weight ratio of 1:1. The obtained solution is sterilized by passing through Seitz filters.

Adding a 40 per cent formaldehyde solution to the partly attenuated toxin at a temperature of 37-38"C converts it into the toxoid.

The toxoid obtained is tested for specific harmlessness on guinea pigs, and its antigenic activity, expressed in binding units, is determined. The toxoid is also tested for sterility.

The thus obtained monotoxoids, separated from the microbial cells, are mixed together and adsorbed on aluminium hydroxide gel.

If necessary, a preservative may be added to the prepared vaccine, such as the preservative which is a solution of sodium merthiolate (1 :10,000).

The prepared vaccine is tested for sterility, toxicity, Al203 content, and immunogenecity.

The vaccine which we have prepared in this way possesses high antigenic and immunogenic properties and produces in vaccinated animals an effective antitoxic immunity to all the causative agents. The immunity persists from 1 to 2 years. The immunity to specific agents can last up to 5 years. The vaccine dose is reduced two times. The titres of antitoxins in animals toward all monotoxoids increase 8-10 times, and the antitoxins are produced twice as fast. Following the primary immunization, the animals are re-vaccinated only once in their lifetime or only for special epizootic indications (at intervals from 2 to 5 years). The intensity of the immunity is 15-20 times higher than that attained with the known polyvalent toxoid-vaccine.

The preparation is harmless and areactogenic for both vaccinated animals and man (milk and meat of vaccinated animals). The time of expiration of the vaccine has increased to five years.

Compared with the known preparations, the present vaccine can be made free from ballasts (microbial cells, nonspecific proteins). The activity of the vaccine is determined by the number of binding units and can be controlled at all stages in production and use, which guarantees efficacy of the vaccine. In addition, improved method of testing the vaccine, especially for immunogenecity and safety, can be used.

The use of the proposed vaccine for prophylactic purposes at affected farms, especially at large industrialized complexes, should prove its universal properties since it can effectively be used for vaccination of all animals or poultry against 5-11 widely spread natural causative agents, the vaccination dose being small. The vaccine protects females from forage poisoning, wound complications and complications following the delivery. The vaccine can also be used for prophylaxis and treatment of saplings from anaerobic enterotoxemia, through the agency of colostrum of their mothers, it should guarantee complete cure of diseased animals and specific prophylaxis of the mothers themselves against said causative agents, with the efficacy being 100 per cent. Rare cases of infections need not be serious and symptomatic treatment should ensure rapid cure.Animals borne from vaccinated mothers are viable, strong, and rapidly gain weight. The use of the proposed vaccine at affected animal farms should thus ensure rapid eradication of the diseases. At large animal breeding farms, the vaccine should ensure the formation of a herd free from 5-11 widely spread natural causative agents that otherwise are the cause of considerably loss.

For a better understanding of the invention, the following examples of its practical embodiment are given by way of illustration.

Example 1 Ten litres of a casein growth medium sterilized at a temperature of 1 1 2"C for thirty minutes are inoculated with the culture Clostridium peffringens type A, taken in a quantity of 2 per cent of the growth medium volume. The cultivation is carried out under anaerobic conditions at a temperature of 38"C for 2-10 hours at a pH of the medium of 5-10. The same casein medium is used for parallel cultivation Clos.perfringens types B, D, CI. oedematiens, CI. septicum, CI. tetani, and CL botulinum types A, B, C, D, E, and F. All cultures are grown separately by the described method; the time of cultivation for each culture is from 2 hours to 10 days.

At the end of cultivation, 12 litres of the suspension of each culture are separated into the culture filtrate and the microbial mass. The culture filtrate (11.5 litres) containing the toxin is separated and passed through a Seitz filtre. The toxin is then (unless otherwise specified below) detoxicated with a 40 per cent formaldehyde solution.

The toxin of Clostridium perfringens type A is partly detoxicated with a 40 per cent formaldehyde solution at a temperature of 37-38"C for 14 days. Formaldehyde is added in two portions (0.2 per cent by volume in each portion) at a 24-hour interval.

The toxin of C. perfringens type B is detoxicated and converted into the corresponding toxoid with a 40 per cent solution of formaldehyde at pH 7.0-7.2, at a temperature of 37-38"C, for 1-3 days. The entire portion of formaldehyde (0.4-0.5 per cent by volume) is added at a time.

The toxin CL peffringens type D is detoxicated partly with a 40 per cent solution of formaldehyde at pH of the medium of 7-7.2, at a temperature of 37-38"C, for 1-30 days, the required quantity of formaldehyde being added in two portions (0.2 per cent by volume in each portion), at a 1-2 day interval.

The toxins of C. oedematiens and CL septicum are detoxicated and converted into the corresponding toxoids by the same procedure as described for C. perfringens type B.

The attenuated toxin or toxoid obtained from the culture filtrate (11.6 litres) is purified and concentrated by precipitation with a 25 per cent solution of sodium hexametaphosphate, taken in a quantity of 0.5-1 per cent by volume, in the presence of 1 N hydrochloric and each at pH of 3.0-3.8.

The precipitated toxin or toxoid (100 g) is dissolved in three litres of a mixture of a phosphate buffer and a physiological salt solution (1:1), and the obtained solution is sterilized by passing it through Seitz filtres.

The resultant products are concentrated solutions (separated from microbial cells) of toxoids of CL perfringens type B, CL oedamatiens, Cl. septicum, and concentrated solutions of partly detoxicated toxins of CI. perfringens types A and D.

The concentrated solution of partly detoxicated C. perfringens type A is converted into the corresponding toxoid by adding 0.2 per cent by volume of a 40 per cent formaldehyde solution in the course of 14-20 days.

The partly detoxicated toxin CL perfringens type D is converted into the toxoid by a procedure similar to that described for CL perfringens type A.

The monotoxoids obtained, as separated from microbial cells, are tested for sterility, toxicity, specific properties, and antigen it activity.

The antigenic activity of the concentrated solution of each toxoid, separated from microbial cells, expressed in binding or combining units per ml of solution, is as follows: toxoid of CL perfringens type A 350 toxoid of CL perfringens type B 300 toxoid of C. perfringens type D 500 toxoid of C. oedematiens 500 toxoid of CL septicum 200 The concentrated solutions separated from microbial cells, prepared as described above, are diluted with a physiological salt solution (0.85 per cent sodium chloride solution) to obtain solutions having the following antigenic activities expressed in combining units per ml of the vaccine: toxoid of CL perfringens type A 2 toxoid of CL peffringens type B 2 toxoid of C. perfringens type D 1 toxoid of CL oedematiens 1 toxoid of CL septicum 2 To prepare 2.5 1 of vaccine according to the invention, 200 ml of dilute solutions of each monotoxoid, having said antigenic activity, are mixed together, and alumina gel is added (400 ml as Al203).

The finished vaccine contains the following components the toxoids being given in terms of the antigenic activity expressed in combining units per ml of the vaccine:

toxoid of Cl. perfringens type A 2 toxoid of Cl. perfringens type B 2 toxoid of C. peifringens type D 1 toxoid of Cl. oedematiens 1 toxoid of C. septicum 2 alumina gel (as A1203) 1 ml mixture of phosphate buffer and physiological salt solution (1:1) balance The finished vaccine is tested by known methods for completeness of sorption, sterility, toxicity, and immunogenecity (on mice). The vaccine is sterile, harmless, and immunogenic.

In order to prevent development of enterotoxemia in young pigs and calves at farms where 100 per cent of the animals were affected with the disease (against the background of dyspepsia), the mortality rate being 50-80 per cent, all adult female cattle and pigs were immunized with the vaccine.

The development of the disease in the young stock was as follows: profuse diarrhoea on the 2nd or 3rd day following the birth, sometimes with blood i n the feces, the animals abstained from food, lost weight quickly and died within 2-3 days. In female animals, endometrites and mastitis were observed.

The vaccine was used to immunize 100 pregnant cows and 150 pigs. The vaccine was given parenterally, 5 ml in two injections. The control group contained 20 cows and 50 pigs. The immunized animals delivered 95 calves and 1200 gilts. Fifty young calves had the disease in a mild form and ten of them perished. Of 50 affected gilts, 25 of them perished.

All animals in the control group were affected by the disease, and a hundred percent mortality was observed with the calves and gilts born from the control animals.

Example 2 The vaccine contains the following components, the toxoids being given in terms of their antigenic activity, expressed in combining units per ml:

toxoid of CL perfringens type A 10 toxoid of CL perfringens type B 10 toxoid of Cl. perfringens type D 5 toxoid of Cl. oedematiens 4 toxoid of CL septicum 10 alumina gel (as Al2O3) 5 mg mixture of phosphate buffer and physiological salt solution (1::1) balance The vaccine is prepared by a procedure similar to that described in Example 1.

The finished preparation is sterile, harmless, and innumogenic. It is used for prophylaxis and treatment of calves and gilts with anaerobic enterotoxemia against the background of dyspepsia. The clinical history of the disease is similar to that described in Example 1.

The vaccine was used to immunize 100 cows and 230 pigs in pregnancy. The vaccine was given parenterally in a dose of 5 ml in two injections. The control group contained 20 cows and 30 pigs. The immunized animals delivered 97 calves and 1796 gilts. The disease did not developrand none of the young stock perished. The non-immunized cows gave 18 calves, all of them had the grave form of the disease and 20 12 calves perished. The non-immunized pigs gave 205 gilts, 200 of them had the disease and 150 perished.

Example 3 The vaccine contains the following components, the toxoids being given in terms of their antigenic activity, expressed in combining unit per ml:

toxoid of C. perfringens type A 100 toxoid of C. perfringens type B 100 toxoid of C. perfringens type D 80 toxoid of C. oedematiens 80 toxoid of C1. septicum 100 alumina gel (as A1203) 10 mg mixture of phosphate buffer and physiological salt solution (1::1) balance The proposed vaccine is prepared by the method described in Example 1.

The preparation is sterile, harmless, immunogenic and can be used for prophylaxis and treatment of young animals (calves, pigs) with anaerobic enterotoxemia against the background of dyspepsia, the clinical history of the disease being similar to that described in Example 1.

The proposed vaccine was used to immunize 50 cows and 100 pigs in pregnancy. The vaccine was given in two injections in a dose of 5 ml. The control group contained 20 cows and 50 pigs. The immunized animals produced 50 calves and 960 gilts. 25 gilts developed the disease and 10 of them perished. 25 calves developed the disease in the mild form and ten of them perished. 200 young pigs were slightly affected and 50 of them perished.

One hundred per cent of the animals in the control group developed the disease.

All animals delivered by the non-immunized cows and pigs in the control groups developed the disease and perished.

Example 4 The vaccine contains the following components, the toxoids being given in terms of their antigenic activity, expressed in combining units per ml: toxoid of C1. peffringens type A 2 toxoid of C1. perfringens type B 2 toxoid of C1. perfringens type D 1 toxoid of C1. oedamatiens 1 toxoid of C1. septicum 2 toxoid of CL tetani 1 alumina gel (as Awl203) 1 mg mixture of phosphate buffer and physiological salt solution (1 :1) balance The finished preparation is tested by the known methods for sterility, toxicity, and immunogenecity.

The vaccine is sterile, harmless, and immunogenic.

In order to prevent and treat anaerobic enterotoxemia of young cattle, pigs, sheep, horses, of endometritis vaginitis, mastitis, wound complications, malignant edema, bradzot of sheep, etc., the animals at farms affected with the diseases were immunized with the proposed vaccine. The disease among females was characterized by purulent discharges from the uterus and vagina, mastitis, miscarriages, malignant edema, about 50 to 70 per cent animals being affected by the disease. The disease in the young animals manifested itself as grave anaerobic enterotoxemia, the clinical history being the same as described in Example 1, associated with grave nerve affections and resulting in 100 per cent mortality among the newborns who perished within the first 3 days.

The vaccine was given to 200 cows, 219 pigs, 120 sheep and 80 horses in pregnancy. The preparation was given in two injections in a dose of 5 ml which was injected from one to three times, depending on the epizootic situation. The control group contained 20 pigs, 20 sheep, 20 horses and 20 cows. The immunized 150 pigs delivered 1350 gilts, the horses gave 60 foals, the sheep gave 150 lambs, and the cows gave 198 calves. The mild form of the disease was observed in 79 gilts, and the grave form in 70 gilts; 28 of them perished. The mild form developed in 30 foals and two of them perished. 60 lambs got diseased and 5 of them perished. The prophylactic use of the proposed vaccine in female animals to prevent endometritis, vaginitis, miscarriages and wound complications was effective in 75 per cent cases. The diseases were not eradicated in the control group.

One hundred per cent of the young animals born from the non-immunized mothers, developed the disease and perished.

Example 5 The vaccine contains the following components, with the toxoids being given in terms of their antigenic activity, expressed in combining units per ml:

toxoid of Cl. peffringens type A -10 toxoid of C. perfringens type B 10 toxoid of CL perfringens type D 5 toxoid of CL oedematiens 4 toxoid of Cl. septicum 10 toxcoid of CL tetani 5 -alumina gel (as Awl203) 5 mg mixture of phosphate buffer and physiological salt solution (1:1) balance The vaccine is prepared by a procedure similar to that described in Example 1. The finished preparation is sterile, harmless, and immunogenic, and it can be used for prophylaxis and treatment of young livestock (lambs, foals, calves, gilts, ete ) and female adults. The vaccine is effective against the diseases indicated in Example 4.

The proposed vaccine was used to immunize 242 pigs, 200 sheeps, 150 horses, and 400 cattle in pregnancy. The preparation was given parenterally in a dose of 5 ml (from 1 to 3 times), depending on the epizootic situation. The control group contained 24 animals of each species. The immunized animals gave 1630 gilts, 180 lambs, 80 foals and 389 calves. All young animals survived and none of them got the disease.

The same picture was observed with the adults immunized with the vaccine. The disease were not eradicated among the animals in the control group. One hundred percent of the animals born from the non-immunized mothers had the disease and perished Example 6 The vaccine contains the following components, the toxoids given as their antigenic activity, expressed in combining units per ml::

toxoid of CL perfringens type A 10 toxoid of CL perfringens type B 10 toxoid of C. peffringens type D 5 toxoid of C. oedematiens 4 toxoid of CI. septicum 10 toxoid of CL tetani 5 toxoid of CL botulinum type A 10 toxoid of CL botulinum type B 2 toxoid of CL botulinum type C 2 toxoid of Cl. botulinum type D 2 toxoid of CI. botulinum type E 2 toxoid of CL botulinum type F 2 alumina gel (as Al203) 5 mg mixture of phosphate buffer and physiological salt solution (1:1) balance The vaccine is prepared by a procedure similar to that described in Example 1.

The finished preparation is sterile, harmless, and immunogenic.

The vaccine was used to immunize commercial animals and poultry in order to prevent and treat forage toxicoinfections, wound complications, gas gangrene, post-labour complications, and enterotoxemia at animal and poultry farms affected with the diseases. The affection of the animals and poultry was characterized by signs of poisoning and quick lethal outcome.

Said vaccine was used to immunize 20G minks, 500 birds and 150 pigs. The preparation was given to minks in a single injection of 1-2 ml with subsequent re-vaccination, or in aerosol form (2-3 ml). Pigs were immunized twice with 5 ml doses. The parenteral dose to hens and ducks was 0.5-2 ml, which was given 1 or 2 times, depending on the epizootic situation, or in aerosol form (1-3 ml). The control group contained 100 minks, 250 birds and 20 pigs. The immunized minks delivered 150 cubs and the pigs delivered 1350 gilts without any signs of the disease in the mothers and young animals, and all of them survived. A mild form of the disease was observed with 10-15 per cent immunized birds. The disease was only transient (1-2 days), without lethal outcome.

The disease affected 95 per cent of the minks, poultry and pigs, including the mothers and young animals, in the control group, and 95 per cent of them perished.

Example 7 The vaccine contains the following components with the toxoids given as their antigenic activity, expressed in combining units per ml:

toxoid of Cl. perfringens type A 10 toxoid of Cl. perfringens type B 10 toxoid of Cl. perfringens type D 5 toxoid of C. oedematiens 4

toxoid of CL septicum 10 toxcoid of CL tetani 5 toxoid of CL botulinum type A 10 toxoid of Cl. botulinum type C 2 alumina gel (as Awl203) 5 mg mixture of phosphate buffer and physiological salt solution (1:1) balance The proposed vaccine is prepared by the method described in Example 1.

The finished preparation is sterile, harmless, and immunogenic. It is used for prophylaxis and treatment of commercial animals and poultry and is effective against the diseases indicated in Example 6.

The vaccine was used to immunize 150 minks and 300 birds. The minks were given the vaccine in a single injection of 1-2 ml with subsequent re-vaccination, or in aerosol form (2-3 ml). Poultry (hens and ducks) were given 0.5-2 ml of the vaccine, from 1 to 2 times, depending on the epizootic situation. The control group contained 50 minks and 100 birds. The immunized minks delivered 100 cubs without any signs of the disease. All of them survived. The posterity of the immunized poultry was not affected by the disease either, and all chicks survived. About 90-100 per cent of the non-immunized minks and poultry developed the diseases and perished.

Claims (7)

1. A vaccine for prophylaxis and treatment of clostridiosis of animals and poultry, comprising toxoids of Clostridium perfringins types A, B, and D, Cl.oedematiens, and CL septicum, separated from the microbial mass, a sorbent and a solvent, the antigenic activity of said components, expressed in combining units per ml of the vaccine, being as follows:toxoid of CL perfringens type A 2-100 toxoid of Cl. perfringens type B 2-100 toxoid of Cl. perfringens type D 1-80 toxoid of Cl. oedematiens 1-80 toxoid of cl. septicum 2-100

2.A vaccine as claimed in Claim 1, which also contains a toxoid of Cl. tetani, separated from the microbial mass, its antigenic activity, expressed in combining units per ml of the vaccine, being 1-80.

3. A vaccine as claimed in Claims 1 or 2, which also contains toxoids of Cl. botulinum of at least two of the types A, B, C, D, E, and F, their antigenic activity, expressed in combining units per ml of the vaccine, being as follows: toxoid of CL botulinum type A 2-100 toxoid of Cl. botulinum type B 0.2-80 toxoid of Cl. botulinum type C 0.2-80 toxoid of Cl. botulinum type D 0.2-80 toxoid of Cl. botulinum type E 0.2-80 toxoid of Cl. botulinum type F 0.2-80

4. A vaccine as claimed in Claims 1, 2, or 3, containing a mixture of a phosphate buffer and a physiological salt solution, taken in the weight ratio of 1:1, as a solvent.

5. A vaccine as claimed in any one of Claims 1 to 4which contains 1 to 10 mg of sorbent per ml.

6. A vaccine as claimed in any one preceding claim, containing alumina gel as the sorbent.

7. A vaccine substantially as described in any one of the Examples herein.