Abstract:
The present invention relates to the preparation of universal inactivated vaccines and their use in preparing compositions for the prophylaxis and therapy of dermatomycosis. Vaccines according to the present invention have the advantage of conferring immunity against all important causes of dermatomycosis in animals and are characterized by stable immunogenic properties, easy preparation, high content of microconidia and lack of side reactions in animals.

Description:
RELATED APPLICATIONS 
     The present application is a configuration of U.S. application Ser. No. 09/1256.915, filed Feb. 24, 1999, which is a continuation of U.S. application Ser. No. 08/1568,063, filed Dec. 6, 1995, now abandoned, which is a continuation of U.S. application Ser. No. 08/281,380, filed July 26, 1994, now abandoned, which is a continuation of U.S. application Ser. No. 08/1081,299 filed Aug. 11, 1993, now abandoned which is the National Stage of PCT/EP92/02391, filed Oct. 17, 1992, which claims priority under 35 USC § 119 to Russian Federation application Serial No. 50069611307308, filed Oct. 21, 1991; and claims priority benefit of all the above-listed applications. 
    
    
     BACKGROUND 
     This invention relates to the preperation of vaccines and their use in preparing compositions for specifically preventing and treating dermatomycosis. 
     Dermatomycoses in animals are anhropozoonotic diseases of the skin and related tissue. Clinical symptoms are characterized by loss of hair in the affected area, hyperemia, scaling and asbestos-like scabs. Inflammation is often accompanied by suppuration. Dermatomycoses are often also characterized by localized infection of the skin. 
     Dermatomycoses in animals carry a substantial socioeconomic impact. Diseased animals required prolonged treatment and can spread infection to both animals and humans. 
     Up till now, dermatomycoses have been treated using various types of medication applied locally to affected areas of the skin. These included the ointments YaM, Yuglon (I) and a number of other ointments, liniments, solutions and other substances containing fungicides and fungistatic agents. 
     The disadvantages of such treatments were:
         they were not very effective;   they required the adoption of quarantine measures and disinfection of areas where animals were kept (rearing pens, vivaria, farms, zoos, circuses, etc);   they required substantial funds to be spent on drug preparations and veterinary treatment;   they posed difficulties in immobilizing the animals (for wild animals held in captivity).       

     Later vaccines were developed to treat trichophytosis in cattle (see USSR Patent No. 268593, 1970), fur-bearing animals and rabbits (see USSR Patent No. 835446, 1980), camels (see USSR Patent No. 1190574, 1985) and others. 
     A vaccine had also been developed earlier for the prevention and treatment of trichophytosis in horses: S-P-I (see USSR Patent No. 548947, 1976). 
     The S-P-I vaccine contains the vaccinal strain  Trichophyton equinum  No. 2251/71, deposited with the USSR All-Union State Scientific Control Institute of Veterinary Preparations, which is cultivated in agar/wort for 20-25 days at a temperature of 26-28° C. The fungal mass is then lifted from the surface of the nutrient medium, mixed with sterile distilled water and homogenized, and the concentration of cells is brought to 600-900 million per ml. The homogenate is transferred to a separate flask and stabilized with a mixture containing 2-8% gelatine (gelatose) and 10-40% sucrose in the ratio 1:1 (±25%), then lyophilized. 
     For prophylactic and treatment purposes the vaccine is injected into the muscle tissue of the neck area of juvenile and mature horses in two doses of 1-2 cc, depending on the age of the horse, with an interval of 10-14 days. For therapeutic use the dosages were doubled. 
     Vaccines obtained using this method have the disadvantage that they do not provide immunity against microsporiae and trichophytiae caused by other agents. It has also been noted that the areas where a live vaccine is injected may become a specific focus in which cultures of vaccinal strains may at certain times be produced. Given that some species of domestic animals come into frequent contact with humans, the occurrence of such specific foci in these animals is unacceptable. 
    
    
     DESCRIPTION 
     This invention now provides universal inactivated vaccines for the specific treatment and prevention of dermatomnycosis in animals and corresponding immunogenic fungal strains. 
     This aim has been achieved by using the following fungal strains as vaccial strains:  Trichophyton verrucosum  (especially No. VKPGF-931/410),  Trichophyton mentagrophytes  (especially No. VKPGF-930/1032),  Trichophyton equinum  (especially No. VKPGF-929/381),  Trichophyton sarkisovii  (especially No. VKPGF-551/68),  Microsporum canis  (especially No. VKPGF-928/1393),  Microsporum canis  var.  obesum  (especially No. VKPGF-727/1311),  Microsporum canis  var.  distortum  (especially No. VKPGF-728/120),  Microsporum gypseum  (especially No. VKPGF-729/59). Vaccines can be produced by using various combinations of antigenic material from the above strains together with a suitable carrier. 
     A preferred combination consists of  Trichophyton verrucosum  No. VKPGF-931/410 , Trichophyton mentagrophytes  No. VKPGF-930/1032 , Trichophyton equinum  No. VKPGF-929/381 , Microsporum canis  No. VKPGF-928/1393 , Microsporum canis  var.  obesum  No. VKPGF-727/1311 , Microsporum canis  var.  distortum  No. VKPGF-728/120 , Microsporum gypseum  No. VKPGF-729/59, particularly for use in dogs, cats and horses. 
     Another preferred combination of vaccine strains consists of  Trichophyton verrucosum  No. VKPGF-931/410 , Trichophyton mentagrophytes  No. VKPGF-930/1032 , Trichophyton sarkisovii  No. VKPGF-551/68, particularly for use in cattle. 
     The antigenic material may comprise a single antigen of at least one, and more particularly of all of the above-mentioned dermatophytes or from a pluaity of antigens, provided that a sufficient immune response is stimulated to give resistance to a dermnatophyte infection. Antigenic material for such a purpose can be prepared using methods known from the prior art, e.g., homogenizing the above-mentioned dermnatophytes or parts thereof, fractionation of dermatophyte preparations, production of antigenic dermatophyte material by recombinant DNA technology, etc. It is preferable to use homogenized culture material having 40 to 12-million, preferably 90 million microconidia. 
     Suitable physiologically acceptable carriers for administering the vaccines are known from the prior art and may include buffers, gels, microparticles, implantable solids, solutions and other adjuvants. 
     To kill off the dermatophytes it is possible to use thiomersal (C 9 H 9 O 2 SNaHg), formaldehyde or 2-propiolactone. 
     In order to prepare a vaccine the following procedure may be used, for example: 
     Cultures of the strains are homogenized in an aqueous solution containing 0.2 ti 2.0% fermented, hydrolyzed muscle protein (FGM-s), 5 to 12% glucose and 0.1 to 1.2% yeast ha extract The concentration of the microconidia is adjusted to 40 to 120 million per milliliter and after 1 to 2 days the mixture is inactivated, e.g., with thiomersal (C 9 H 9 O 2 SNaHg) in the ration 1:10,000 to 1:25,000, or with another substance known from the prior art. The resulting suspension is packaged and is ready for use in animals. 
     The preparation of the vaccines, the dosage to be given and the method of administration for prevention and therapeutic treatment are explained in Examples 1 to 3. 
     The invention now makes it possible to prepare an inactivated vaccine that reduces the probability of reinfection and also implants a high degree of immunity. Unlike the known vaccines the vaccine according to the invention in practice gives immunity to all important causes of dernatomycosis in animals. 
     Briefly, the vaccine according to the invention offers the following advantages:
         in many species of disease-prone animals it establishes immunity after intramuscular injection,   it grants immunity against almost all causes of dermatomycosis in animals,   it has stable immunogenic properties,   it is easy to prepare,   it has a complete set of exo- and endo-antigens of dermatophyte cultures and shows no side reactions in animals.       

     The vaccine has been successfully tested on over 500 animals of different species, predominantly in affected regions. 
     The strains used to produce the vaccine have been deposited at the “All-Union Collection of Pathogenic Fungi within the USSR, Ministry of Health Centre for Deep Mycoses” in Leningrad and at the “DSM—Deutsche Sammlung von Mikroorganismen und Zellkulturen”, Mascheroder Weg 1B, W-3300 Braunschweig, Germany. 
     The following microorganisms were deposited with the Deutsche Samnmlung von Mikroorganismen und Zellkulturen (DSM) on Oct. 1, 1992 and received the following accession numbers:
           T. verrucosum  VKPGF-931/410 received accession No. DSM 7277     T. mentagrophytes  VKPGF-930/1032 received accession No. DSM 7279     T. equinum  VKPGF-929/381 received accession No. DSM 7276     T. sarkisovii  VKPGF-551/68 received accession No. DSM 7278     M. canis  VKPGF-928/1393 received accession No. DSM 7281     M. canis  var.  obesum  VKPGF-727/1311 received accession No. DSM 7280     M. canis  var.  distortum  VKPGF-728/120 received accession No. DSM 7275     M. gypseum  VKPGP-729/59 received accession No. DSM 7274.       

     DSM is located at Macheroder Weg 1B, W-3300 Braunschweig, Germany. “T” is an abbreviation for Trichophyton and “M” is an abbreviation for Microsporum. 
     Their characteristics are set out below: 
       Trichophyton verrucosum , No. VKPGF-931/410 
     The strain was deposited at the DSM—Deutschc Sammlung von Mikroorganismen und Zellkulturen”, Mascheroder Weg 1B, W-3300 Braunschweig, Germany. 
     The strain was obtained by directed selection based on spore production and attenuation of the epizootic Strain No. 410, which was identified on a deer in 1978. The strain was identified using the Rebell-Taplin key (Rebell, G., Taplin, D.: Dermatophytes, their recognition and identification, 1978) and according to Kashkin, P. N. et al. (Opredelitel patogennykn, toksigenykh vrednykh dlya cheloveka gribov, 1979). 
     The biological properties of the strain are described in Table 1. 
     Strain No. VKPGF-931/410 differs from the epizootic strain in its faster growth in nutrient medium, the enormous production of microconidia, lower virulence and the absence of any reaction with its antigens. 
       Trichophyton mentagrophytes  No. VKPGF-930/1032 
     The strain was deposited at the DSM—Deutsche Sammlung von Mikroorganismen und Zellkulturen”, Mascheroder Weg 1B, W-3300 Braunschweig, Germany. 
     The strain was obtained by directed selection based on spore production and attenuation of the epizootic Strain No. 1032, which was found on a horse in 1985. The strain was identified as described above Rebel, Taplin, loc. cit. and Kashkin, loc. cit.). The biological properties are described in Table 2. 
     Strain No. VKPGF-930/1032 differs from the epizootic strain by its faster growth in nutrient medium, the enormous production of microconidia, its lower virulence and the absence of any reaction with its antigens. 
       Trichopyton equinum  No.VKPGF-929/381 
     The strain was deposited at the DSM—Deutsche Sammlung von Mikroorganismen und Zellkulturen”, Mascheroder Weg 1B, W-3300 Braunschweig, Germany. 
     The strain was obtained by directed selection based on spore production and attenuation of the epizootic Strain No. 381 which was found on a horse in 1986. The strain was identified as describe above Rebel, Taplin, loc. cit. and Kashkin, loc. cit.). The biological properties are described in Table 3. 
     Strain No. VKPGF-929/381 differs from the epizootic strain by its faster growth in nutrient medium, lower virulence and the absence of any reaction with its antigens. 
       Microsporum canis  No. GF-928/1393 
     The strain was deposited at the DSM—Deutsche Sammlung von Mikroorganismen und Zellkulturen”, Mascheroder Weg 1B, W-3300 Braunschweig, Germany. 
     The strain was obtained by directed selection based on spore production and attenuation of the epizootic Strain No. 1393 which was found on a cat in 1988. The strain was identified as describe above Rebel, Taplin, loc. cit. and Kashkin, loc. cit.). The biological properties are described in Table 4. 
     Strain No. VKPGF-928/1393 differs from the epizootic strain by its faster growth in nutrient medium, its enormous capacity to carry spores, lower virulence and the absence of any reaction with its antigens. 
       Microsporum canis  var.  obesum  No. VKPGF-727/1311 
     The strain was deposited at the DSM—Deutsche Sammiung von Mikroorganismen und Zellkulturen”, Mascheroder Weg 1B, W-3300 Braunschweig, Germany. 
     The strain was obtained by directed selection based on spore production and attenuation of the epizootic Strain No. 1311 which was found on a tiger in 1986. The strain was identified as describe above Rebel, Taplin, loc. cit. and Kashkin, loc. cit.). The biological properties are described in Table 5. 
     Strain No. VKPGF-727/1311 differs from the epizootic strain by its faster growth in nutrient medium, its enormous capacity to carry spores, lower virulence and the absence of any reaction with its antigens. 
       Microsporum canis  var.  distortum  No. VKPGF-728/120 
     The strain was deposited at the DSM—Deutsche Sammlung von Mikroorganismen und Zellkulturen”, Mascheroder Weg 1B, W-3300 Braunschweig, Germany. 
     The strain was obtained by directed selection based on spore production and attenuation of the epizootic Strain No. 120 which was found on a black panther in 1987. The strain was identified as describe above Rebel, Taplin, loc. cit. and Kashkin, loc. cit.). The biological properties are described in Table 6. 
     Strain No. VKPGF-728/120 differs from the epizootic strain by its faster growth in nutrient medium, its enormous production of microconidia, its lower virulence and the absence of any reaction with its antigens. 
       Microsporum gypseum  No. VKPGF-729/59 
     The strain was deposited at the DSM—Deutsche Sammlung von Mikroorganismen und Zellkulturen”, Mascheroder Weg 1B, W-3300 Braunschweig, Germany. 
     The strain was obtained by directed selection based on spore production and attenuation of the epizootic Strain No. 59 which was found on a horse in 1985. The strain was identified as describe above Rebel, Taplin, loc. cit. and Kashkin, loc. cit.). The biological properties are described in Table 7. 
     Strain No. VKPGF-729/59 differs from the epizootic strain by its faster growth in nutrient medium, the enormous production of microconidia, the lower virulence and the absence of any reaction with its antigens. 
     
       
         
               
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
               
                 Properties and characteristics of strain 
                 Strain No. VKPGF-931/410 
                 Epizootic Strain No. 410 
               
               
                   
               
             
             
               
                 Description of culture 
                 Mature 10-15 day single-spore colony 
                 Mature 25-30 day colony in agar/wort; 
               
               
                   
                 in agar/wort; white, velvety, convex, 
                 cream, leathery/velvety, folded, 
               
               
                   
                 narrow growing margin undersurface 
                 undersurface colorless, colony 
               
               
                   
                 colorless, colony diameter 10-15 mm 
                 diameter 9-13 mm 
               
               
                 Morphological characteristics 
                 Mature 10-15 day culture with septate 
                 Mature 25-30 day culture with septate 
               
               
                   
                 branching hyphae 1-3 μm wide, 
                 branching mycelium 1-3 μm wide, few 
               
               
                   
                 numerous oval, pyriform microconidia 
                 oval, pyriform, cylindrical microconidia 
               
               
                   
                 measuring 1.5 to 3 × 3 to 5 μm, 
                 measuring 1 to 3 × 3 to 7 μm, single 
               
               
                   
                 no macroconidia 
                 elongate irregular shape 
               
               
                   
                   
                 macroconidia with 
               
               
                   
                   
                 2-5 septates measuring 3 to 5 × 25 to 
               
               
                   
                   
                 30 μm, numerous arthrospores in 
               
               
                   
                   
                 chains 6-8 μm diameter, 
               
               
                   
                   
                 chlamydospores 10-12 μm diameter 
               
               
                 Pathogenic characteristics 
               
               
                 12 to 15 days after application of a dose of 500-600 
                 Thin necrotic scabs 
                 Dense asbestos-like scabs, possible 
               
               
                 thousand cells of fungal 
                   
                 suppuration 
               
               
                 matter per cm 2  to the scarified 
               
               
                 skin of a rabbit 
               
               
                 Spontaneous recovery after 
                 19-20 days 
                 25-30 days 
               
               
                 Reaction response 
               
               
                 Results of subcutaneous and intramuscular injection of 
                 No observed changes in clinical state 
                 Inflammation at point of injection, 
               
               
                 inactivated corpuscular antigens from cultures 
                   
                 edema 
               
               
                 Antigen response 
               
               
                 20 to 25 days after injecting rabbits with corpuscular 
               
               
                 antigens, antibody titers observed in blood serum 
               
               
                 By Passive Hemagglutination Reaction (PHR) 
                 1:320 to 1:640 
                 1:320 to 1:640 
               
               
                 By Enzyme-linked Immunosorbent Assay (ELISA) 
                 1:400 to 1:1600 
                 1:400 to 1:1600 
               
               
                 Immunogenic response 
               
               
                 Immunization of a group of rabbits with inactivated 
                 Establishes immunity 
                 Establishes immunity 
               
               
                 antigens from cultures (repeated at least 5 times)  
               
               
                   
               
             
          
         
       
     
     
       
         
               
               
               
             
           
               
                 TABLE 2 
               
               
                   
               
               
                 Properties and characteristics of strain 
                 Strain No. VKPGF-930/1032 
                 Epizootic Strain No. 1032  
               
               
                   
               
             
             
               
                 Description of culture 
                 Mature 10-15 day colony in agar/wort; 
                 Mature 25-30 day colony in agar/wort; 
               
               
                   
                 cream, velvety/powdered, flat with 
                 white, flat, narrow growing margin, 
               
               
                   
                 slight flat elevation in center, narrow 
                 undersurface reddish-brown, colony 
               
               
                   
                 growing margin, fringed, undersurface 
                 diameter 15-20 mm 
               
               
                   
                 light brown, colony diameter 25-30 mm 
               
               
                 Morphological characteristics 
                 Septate, branching hyphae 1-3 μm 
                 Septate, branching straight and spiral 
               
               
                   
                 wide, numerous pyriform, oval 
                 hyphae 1-3 μm wide, round, flattened 
               
               
                   
                 microconidia measuring 1 to 3 × 2 to 6 
                 pyriform microconidia measuring 1 to 3 × 
               
               
                   
                 μm, no macroconidia 
                 2 to 6 μm, few elongate-oval 
               
               
                   
                   
                 macroconidia with 2-5 septates, 
               
               
                   
                   
                 measuring 2 to 6 × 15 to 25 μm 
               
               
                 Pathogenic characteristics 
               
               
                 9 to 10 days after application of a dose of 500-600 
                 Necrotic scabs 
                 Dense, asbestos-like scabs 
               
               
                 thousand cells of fungal matter per cm 2  to the 
               
               
                 scarified skin of a rabbit 
               
               
                 Spontaneous recovery after 
                 22-25 days 
                 30-35 days 
               
               
                 Reaction response 
               
               
                 Results of subcutaneous and intramuscular injection of 
                 No observed changes in clinical state 
                 Inflammation at point of injection, 
               
               
                 inactivated corpuscular antigens from cultures 
                   
                 edema 
               
               
                 Antigen response 
               
               
                 20 to 25 days after injecting rabbits with corpuscular 
               
               
                 antigens, antibody titers observed in blood serum 
               
               
                 By PHR 
                 1:320 to 1:640 
                 1:320 to 1:640 
               
               
                 By ELISA 
                 1:400 to 1:1600 
                 1:400 to 1:1600 
               
               
                 Immunogenic response 
               
               
                 Immunization of a group of 
                 Establishes immunity 
                 Establishes immunity 
               
               
                 rabbits with inactivated 
               
               
                 antigens from cultures 
               
               
                 (repeated at least 5 times)  
               
               
                   
               
             
          
         
       
     
     
       
         
               
               
               
             
           
               
                 TABLE 3 
               
               
                   
               
               
                 Properties and characteristics of strain 
                 Strain No. VKPGF-929/381 
                 Epizootic Strain No. 381  
               
               
                   
               
             
             
               
                 Description of culture 
                 Mature 10-15 day colony in agar/wort; 
                 Mature 15 day colony in agar/wort; 
               
               
                   
                 white, velvety/powdery, flat with 
                 white, velvety, slightly creased 
               
               
                   
                 slight elevation in center, narrow 
                 center, narrow growing margin, 
               
               
                   
                 growing margin, fringed, undersurface 
                 undersurface reddish-brown, 
               
               
                   
                 light brown, colony diameter 15-20 mm 
                 colony diameter 13-15 mm 
               
               
                 Morphological characteristics 
                 Septate, branching hyphae 1-3 μm 
                 Septate, branching hyphae with coil 
               
               
                   
                 wide, numerous oval pyriform 
                 end, 1-4 μm wide, few oval, pyriform 
               
               
                   
                 microconidia measuring 2 to 
                 microconidia measuring 2 to 3 × 
               
               
                   
                 3 × 3 to 6 μm, no macroconidia 
                 3 to 7 μm, lobar macroconidia 
               
               
                   
                   
                 measuring 4 to 7 × 15 to 25 μm 
               
               
                 Pathogenic characteristics 
               
               
                 10 to 12 days after application of a dose of 500-600 
                 Necrotic scabs 
                 Asbestos-like scabs 
               
               
                 thousand cells of fungal matter per cm 2  to the scarified 
               
               
                 skin of a rabbit 
               
               
                 Spontaneous recovery after 
                 20-22 days 
                 25-30 days 
               
               
                 Reaction response 
               
               
                 Results of subcutaneous and 
                 No observed changes in clinical 
                 Inflammation at point of injection, 
               
               
                 intramuscular injection of inactivated corpuscular 
                 state 
                 edema 
               
               
                 antigens from cultures 
               
               
                 Antigen response 
               
               
                 20 to 25 days after injecting rabbits with corpuscular 
               
               
                 antigens, antibody titers observed in blood serum 
               
               
                 By PHR 
                 1:320 to 1:640 
                 1:320 to 1:640 
               
               
                 By ELISA 
                 1:800 to 1:1600 
                 1:800 to 1:1600 
               
               
                 Immunogenic response 
               
               
                 Immunization of a group of rabbits with inactivated 
                 Establishes immunity 
                 Establishes immunity 
               
               
                 antigens from cultures (repeated at least 5 times)  
               
               
                   
               
             
          
         
       
     
     
       
         
               
               
               
             
           
               
                 TABLE 4 
               
               
                   
               
               
                 Proprieties and characteristics of strain 
                 Strain No. VKPGP-928/1393 
                 Epizootic Strain No. 1393  
               
               
                   
               
             
             
               
                 Description of culture 
                 Mature 10-15 day colony in agar/wort; 
                 Mature 15 day colony in agar/wort; 
               
               
                   
                 white, fluffy, convex, narrow growing 
                 greyish-beige, arachnoid, powdery in 
               
               
                   
                 margin, arachnoid, undersurface brown, 
                 center, growing margin fringed, 
               
               
                   
                 colony diameter 30-35 mm 
                 Undersurface yellowish, colony 
               
               
                   
                 diameter 20-25 mm 
               
               
                 Morphological characteristics 
                 Septate, branching hyphae 1-4 μm 
                 Septate, branching hyphae 2 to 6 μm 
               
               
                   
                 wide, numerous pyriform, cylindrical 
                 wide, few pyriform, cylindrical 
               
               
                   
                 microconidia, few fusiform 
                 microconidia measuring 1 to 3 × 3 to 7 
               
               
                   
                 macroconidia with 3-11 septates, 
                 μm, numerous fusiform macroconidia 
               
               
                   
                 measuring 10 to 20 × 40 to 75 μm 
                 with 3-11 septates, measuring 10 to 20 × 
               
               
                   
                 45 to 85 μm 
               
               
                 Pathogenic characteristics 
               
               
                 9 to 11 days after application of a dose of 500-600 
                 Necrotic scabs 
                 Dense, asbestos-like scabs 
               
               
                 thousand cells of fungal matter per cm 2  to the 
               
               
                 scarified skin of a rabbit 
               
               
                 Spontaneous recovery after 
                 20-24 days 
                 25-45 days 
               
               
                 Reaction response 
               
               
                 Results of subcutaneous and intramuscular injection of 
                 No observed changes in clinical state 
                 Edema and inflammation at point of 
               
               
                 inactivated corpuscular antigens from cultures 
                   
                 injection 
               
               
                 Antigen response 
               
               
                 20 to 25 days after injecting rabbits with corpuscular 
               
               
                 antigens, antibody titers observed in blood serum 
               
               
                 By PHR 
                 1:320 to 1:640 
                 1:320 to 1:640 
               
               
                 By ELISA 
                 1:400 to 1:1600 
                 1:400 to 1:1600 
               
               
                 Immunogenic response 
               
               
                 Immunization of a group of rabbits with inactivated 
                 Establishes immunity 
                 Establishes immunity 
               
               
                 antigens from cultures (repeated at least 5 times)  
               
               
                   
               
             
          
         
       
     
     
       
         
               
               
               
             
           
               
                 TABLE 5 
               
               
                   
               
               
                 Properties and characteristics of strain 
                 Strain No. VKPGF-727/1311 
                 Epizootic Strain No. 1311  
               
               
                   
               
             
             
               
                 Description of culture 
                 Mature 10-15 day colony in agar/wort; 
                 Mature 15 day colony in agar/wort; 
               
               
                   
                 white, fluffy, flat with a denser central 
                 greyish, fasciculate/arachnoid with 
               
               
                   
                 dome-like elevation, narrow growing 
                 pieces of cottony white mycelium, 
               
               
                   
                 margin, fringed, undersurface colorless 
                 growing margin fringed, undersurface 
               
               
                   
                 with brown center, colony diameter 30- 
                 brownish, colony diameter 23-28 mm 
               
               
                   
                 35 mm 
               
               
                 Morphological characteristics 
                 Septate, branching hyphae 1-3 μm 
                 Septate, branching hyphae 1-5 μm wide, 
               
               
                   
                 wide, numerous pyriform, oval and 
                 few oval, cylindrical microconidia 
               
               
                   
                 cylindrical microconidia measuring 1 to 
                 measuring 1 to 3 × 3 to 8 μm, numerous 
               
               
                   
                 3 × 3 to 7 μm, few short, elliptical, 
                 elliptical, fusiform, elongate-oval or 
               
               
                   
                 fusiform, elongate-oval macroconidia, 
                 irregularly-shaped macroconidia with 2- 
               
               
                   
                 some irregular shapes, less frequently 
                 5 septates, measuring 11 to 20 × 25 to 
               
               
                   
                 “beaked”, with 2-5 septates, measuring 
                 55 mm 
               
               
                   
                 11 to 20 × 25 to 50 μm 
               
               
                 Pathogenic characteristics 
               
               
                 12 to 15 days after application of a dose of 500-600 
                 Thin necrotic scabs 
                 Dense, asbestos-like scabs 
               
               
                 thousand cells of fungal matter per cm 2  to the 
               
               
                 scarified skin of a rabbit 
               
               
                 Spontaneous recovery after 
                 10-25 days 
                 25-30 days 
               
               
                 Reaction response 
               
               
                 Results of subcutaneous and intramuscular injection of 
                 No observed changes in clinical state 
                 Inflammation and edema at point of 
               
               
                 inactivated corpuscular antigens from cultures 
                   
                 injection 
               
               
                 Antigen response 
               
               
                 20 to 25 days after injecting rabbits with corpuscular 
               
               
                 antigens, antibody titers observed in blood serum 
               
               
                 By PHR 
                 1:320 to 1:640 
                 1:320 to 1:640 
               
               
                 By ELISA 
                 1:800 to 1:1600 
                 1:806 to 1:1600 
               
               
                 Immunogenic response 
               
               
                 Immunization of a group of rabbits with inactivated 
                 Establishes immunity 
                 Establishes immunity 
               
               
                 antigens from cultures (repeated at least 5 times)  
               
               
                   
               
             
          
         
       
     
     
       
         
               
               
               
             
           
               
                 TABLE 6 
               
               
                   
               
               
                 Properties and characteristics of strain 
                 Strain No. VKPGF-728/120 
                 Epizootic Strain No. 120  
               
               
                   
               
             
             
               
                 Description of culture 
                 Mature 10-15 day colony in agar/wort; 
                 Mature 15 day colony in agar/wort; 
               
               
                   
                 cream, velvety/powdery, button-like 
                 light-beige, powdery, umbonate, narrow 
               
               
                   
                 elevation in center, narrow growing 
                 growing margin, undersurface brown, 
               
               
                   
                 margin, finely-fringed, undersurface 
                 colony diameter 1 8-20 mm 
               
               
                   
                 light-brown with dark-brown center, 
               
               
                   
                 colony diameter 25-30 mm 
               
               
                 Morphological characteristics 
                 Septate, branching hyphae 1-3 μm 
                 Septate, branching hyphae 1-3 μm wide, 
               
               
                   
                 wide, numerous pyriform, oval, 
                 few pyriform, oval, cylindrical 
               
               
                   
                 cylindrical microconidia measuring 1 to 
                 microconidia measuring 1 to 3 × 3 to 8 
               
               
                   
                 3 × 3 to 8 μm, few irregular deformed 
                 μm, numerous irregular deformed or 
               
               
                   
                 macroconidia, distorted or fusiform 
                 fusiform macroconidia with 2-9 
               
               
                   
                 with 2-9 septates, measuring 8 to 20 × 
                 septates, measuring 8 to 20 × 25 to 80 
               
               
                   
                 25 to 70 μm 
                 μm 
               
               
                 Pathogenic characteristics 
               
               
                 12 to 15 days after application of a dose of 500-600 
                 Thin necrotic scabs 
                 Asbestos-like scabs 
               
               
                 thousand cells of fungal matter per cm 2  to the 
               
               
                 scarified skin of a rabbit 
               
               
                 Spontaneous recovery after 
                 20-25 days 
                 27-45 days 
               
               
                 Reaction response 
               
               
                 Results of subcutaneous and intramuscular injection of 
                 No observed changes in clinical state 
                 Inflammation and edema at point of 
               
               
                 inactivated corpuscular antigens from cultures 
                   
                 injection 
               
               
                 Antigen response 
               
               
                 20 to 25 days after injecting rabbits with corpuscular 
               
               
                 antigens, antibody tilers observed in blood serum 
               
               
                 By PHR 
                 1:320 to 1:640 
                 1:320 to 1:640 
               
               
                 By ELISA 
                 1:800 to 1:1600 
                 1:500 to 1:1600 
               
               
                 Immunogenic response 
               
               
                 Immunization of a group of rabbits with inactivated 
                 Establishes immunity 
                 Establishes immunity 
               
               
                 antigens from cultures (repeated at least 5 times)  
               
               
                   
               
             
          
         
       
     
     
       
         
               
               
               
             
           
               
                 TABLE 7 
               
               
                   
               
               
                 Properties and characteristics of strain 
                 Strain No. VKPGF-729/59 
                 Epizootic Strain No. 59  
               
               
                   
               
             
             
               
                 Description of culture 
                 Mature 10-l 5 day colony in agar/wort; 
                 Mature 15 day colony m agar/wort; 
               
               
                   
                 white, velvety/fluffy, flat with slight 
                 cream, velvety/powdery, flat with fluffy 
               
               
                   
                 elevation in center of colony, flat 
                 white mycelium in center, thin growing 
               
               
                   
                 growing margin, undersurface 
                 margin, undersurface brownish, colony 
               
               
                   
                 brownish, colony diameter 25-30 mm 
                 diameter 20-22 mm 
               
               
                 Morphological characteristics 
                 Septate, branching hyphae 2-3 μm 
                 Septate, branching hyphae 2-5 μm wide, 
               
               
                   
                 wide, numerous oval, pyriform, 
                 few oval, pyriform, cylindrical 
               
               
                   
                 cylindrical microconidia measuring 2 to 
                 microconidia measuring 2 to 4 × 3 to 7 
               
               
                   
                 4 × 3 to 6 μm, no or few macroconidia, 
                 μm, numerous elliptical, stretched-oval 
               
               
                   
                 elliptical, elongate-oval shape with 2-5 
                 macroconidia with 2-5 septates, 
               
               
                   
                 septates, measuring 7 to 15 × 25 to 40 
                 measuring 7 to 15 × 25 to 50 μm 
               
               
                   
                 μm 
               
               
                 Pathogenic characteristics 
               
               
                 12 to 15 days after application of a dose of 500-600 
                 Thin necrotic scabs 
                 Dense, asbestos-like scabs 
               
               
                 thousand cells of fungal matter per cm 2  to the 
               
               
                 scarified skin of a rabbit 
               
               
                 Spontaneous recovery after 
                 20-22 days 
                 25-28 days 
               
               
                 Reaction response 
               
               
                 Results of subcutaneous and intramuscular injection of  
                 No observed changes in clinical state 
                 Inflammation at point of injection 
               
               
                 inactivated corpuscular antigens from cultures 
               
               
                 Antigen response 
               
               
                 20 to 25 days after injecting rabbits with corpuscular 
               
               
                 antigens, antibody titers observed in blood serum 
               
               
                 By PHR 
                 1:320 to 1:640 
                 1:320 to 1:640 
               
               
                 By ELISA 
                 1:400 to 1:1600 
                 1:400 to 1:1600 
               
               
                 Immunogenic response 
               
               
                 Immunization of a group of rabbits with inactivated 
                 Establishes immunity 
                 Establishes immunity 
               
               
                 antigens from cultures (repeated at least 5 times)  
               
               
                   
               
             
          
         
       
     
     The vaccine may be prepared using the strain  Trichophyton sarkovii , No. 551/68. It is described for example in USSR Patent No. 1177972 dated Aug. 8, 1985, to which reference is made in its entirety. 
     This strain was also deposited at the DSM—Deutsche Sammlung von Mikroorganismen und Zellkulturen, Mascheroder Weg 1B, W-3300 Braunschweig, Germany. 
     In particular, the invention relates to the following: 
     a dermatomycosis vaccine, characterized in that it contains antigenic material from at least one of the following dematophytes: 
     
         
         
           
               Trichophyton verrucosum , particularly  Trichophyton verrucosum  Strain No. VKPGF-931/410 and/or 
               Trichophyton mentagrophytes , particularly  Trichophyton mentagrophytes  Strain No. VKPGF-930/1032 and/or 
               Trichophyton sarkisovii , particularly  Trichophyton sarkisovii  Strain No. VKPGF-551/68 and/or 
               Microsporum canis , particularly  Microsporum canis  Strain No. VKPGF-928/1393 and/or 
               Microsporum canis  var.  obesum , particularly  Microsporum canis  var.  obesum  Strain No. VKPGF-727/131 and/or 
               Microsporwn canis  var.  distortum , particularly  Microsporum canis  var.  distortum  Strain No. VKPGF-728/120 and/or 
               Microsporum gypseum , particularly  Microsporum gypseum  Strain No. VKPGF-729/59,
 
and a physiologically acceptable carrier.
 
a dermatomycosis vaccine, particularly as an agent for treating dogs, cats and horses, characterized in that it contains antigenic material from the dermatophyte strains  Trichophyton verrucosum  No. VKPGF-931/410 , Trichophyton mentagrophytes  No. VKPGF-930/1032 , Trichophyton equinum  No. VKPGF-929/381 , Trichophyton sarkisovii  Strain No. VKPGF-551/68 , Microsporum canis  No. VKPGF-928/1393 , Microsporum canis  var.  obesum  No. VKPGF-727/311 , Microsporum canis  var.  distortum  No. VKPGF-728/120 , Microsporum gypseum  No. VKPGF-729/59, together with a physiologically acceptable carrier.
 
a dermatomycosis vaccine, more particularly as an agent for treating cattle, characterized in that it contains antigenic material from the dermatophyte strains  Trichophyton verrucosum  No. VKPGF-931/410 , Trichophyton mentagrophytes  No. VKPGF-930/1032 , Trichophylon equinum  No. VKPGF-929/381 , Trichophyton sarkisovii  Strain No. VKPGF-551/68, together with a physiologically acceptable carrier.
 
a dermatomycosis vaccine as described above, characterized in that it contains 40 to 120 million, preferably 90 million microconidia,
 
a dermatomycosis vaccine as described above, characterized in that it contains thiomersal or formaldehyde or 2-propiolactone as inactivator,
 
a dermatomycosis vaccine as described above, characterized in that the physiologically acceptable carrier used is an aqueous solution containing 0.2 to 2.0 percent weight of fermented, hydrolyzed muscle protein, 5 to 12 percent weight glucose and 0.1 to 1.2 percent weight yeast extract,
 
the dermatophyte strains:
 
               Trichophyton verrucosum  Strain No. VKPGF-931/410, 
               Trichophyton mentagrophytes  Strain No. VKPGF-930/1032, 
               Trichophyton equinum  Strain No. VKPGF-929/381, 
               Microsporum canis  Strain No. VKPGF-928/1393, 
               Microsporum canis  var.  obesum  Strain No. VKPGF-727/1311, 
               Microsporum canis  var.  distortum  Strain No. VKPGF-728/120, and 
               Microsporum gypseum  Strain No. VKPGF-729/59.
 
a process for preparing a vaccine, characterized in that:
 
             a. antigenic material is prepared from at least one of the following strains:
             Trichophyton verrucosum  Strain No. VKPGF-931/410,     Trichophyton mentagrophytes  Strain No. VKPGF-930/1032,     Trichophyton sarkovii  Strain No. VKPGF-551/68,     Microsporum canis  Strain No. VKPGF-928/1393,     Microsporum canis  var.  obesum  Strain No. VKPGF-727/1311,     Microsporum canis  var.  distortum  Strain No. VKPGF-728/120,     Microsporum gypseum  Strain No. VKPGF-729/59, and   
         
             b. the antigenic material is mixed with a physiologically acceptable carrier. 
             a process as described above, characterized in that an agent, particularly thiomersal, formaldehyde or 2-propiolactone is added to inactivate the dermatophytes. 
           
         
       
    
     The invention is illustrated by means of the Examples that follow. 
     EXAMPLES 
     Example 1 
     To produce 1 liter of vaccine, cultures are taken of the strains VKPGF-931/410, 930/1032, 929/381, 551/68, 928/1393, 727/1311, 728/120, and 729/59 and grown in agar/wort at 26° C. for 15 days. Each culture is grown in 8 mattress flasks. The fungal mass is then lifted off, homogenized, placed in 200 ml of solution and added to each mixer. The solution used is an aqueous solution containing 1% fermented hydrolyzed muscle protein, 10% glucose and 1% yeast extract The concentration of microconidia is brought to 90 million per ml of homogenate. After 2 days, 125 ml of each culture in suspension is taken and mixed in a single container. The vaccine may be prepared by mixing together various combinations of the given strains. 
     To inactivate the homogenate mixture, thiomersal is added directly to the cell suspension in the ration 1:20,000. 50 mg of thiomersal is added for every liter of homogenate. The cell mixture is allowed to stand at room temperature for 2 days. 
     The resulting vaccine is bottled, checked for sterility, safety and immunogenic properties in accordance with accepted methods, and kept refrigerated at 4° C. 
     Vaccine produced in this manner was used to immunize animals. 
     For prophylactic and treatment purposes the vaccine was used in the following doses (see Table 8): 
     
       
         
               
               
               
               
               
             
           
               
                 TABLE 8 
               
               
                   
               
               
                   
                   
                   
                 Dosage 
                 Dosage 
               
               
                   
                   
                   
                 (ml) 
                 (ml) 
               
               
                 Animal 
                   
                   
                 Pro- 
                 Treat- 
               
               
                 family 
                 Age 
                 Site of injection 
                 phylactic 
                 ment 
               
               
                   
               
             
             
               
                 Felidae medium/ 
                 1-6 months 
                 Gluteal muscles 
                 2-5 
                 3-6 
               
               
                 large cats 
                 6 months + 
                 Gluteal muscles 
                 3-7 
                  4-10 
               
               
                 Small cats 
                 1-5 months 
                 Gluteal muscles 
                   1-1.5 
                   1-1.5 
               
               
                   
                 5 months + 
                 Gluteal muscles 
                 1-2 
                 1-2 
               
               
                 Ursidae 
                 1-12 months 
                 Gluteal muscles 
                 1-3 
                 3-5 
               
               
                   
                 12 months + 
                 Guinea muscles 
                 3-5 
                 5-6 
               
               
                 Procyonidae 
                 1-10 months 
                 Gluteal muscles 
                 0.3-0.5 
                 0.5 
               
               
                   
                 10 months + 
                 Gluteal muscles 
                 0.3-0.5 
                 0.5-1.0 
               
               
                 Viverridae 
                 1-12 months 
                 Gluteal muscles 
                 0.3-0.5 
                 0.5 
               
               
                   
                 12 months + 
                 Gluteal muscles 
                 0.5-1.0 
                 0.5-1.0 
               
               
                 Hyaenidae 
                 1-12 months 
                 Gluteal muscles 
                 1-3 
                 1-3 
               
               
                   
                 12 months 
                 Gluteal muscles 
                 3-5 
                 5-6 
               
               
                 Canidae 
                 1-10 months 
                 Gluteal and 
                 0.3-0.5 
                 0.5-1.0 
               
               
                   
                 10 months + 
                 shoulder muscles 
                 0.3-1.0 
                 0.5-1.0 
               
               
                 Equidae 
                 3-12 months 
                 Neck area 
                 0.3-0.5 
                 0.5-1.0 
               
               
                   
                 12 months + 
                 Neck area 
                 0.5 
                 0.5-1.0 
               
               
                 Tyropodae 
                 1-6 months 
                 Shoulder and 
                 3-5 
                  5-10 
               
               
                   
                 6 months + 
                 neck area 
                 5-8 
                  7-10 
               
               
                 Bovidae 
                 1-12 months 
                 Neck area 
                 3-5 
                  5-10 
               
               
                   
                 12 months 
                 Neck area 
                 5-8 
                  7-10 
               
               
                   
               
             
          
         
       
     
     Example 2 
     The vaccine produced by the method described in Example 1 was tested on laboratory animals and various other animals for effectiveness in the prevention and treatment of disease. The results are given in Table 9. 
     Example 3 
     The vaccine produced by the method described in Example 1 was also used to treat animals suffering from dermatophytiae. The results are given in Table 10. 
     
       
         
               
               
               
               
             
           
               
                 TABLE 9 
               
               
                   
               
               
                 Type of 
                   
                 Dosage 
                   
               
               
                 animals 
                 Number 
                 (cm 3 ) 
                 Effectiveness 
               
               
                   
               
             
             
               
                 Rabbits 
                 10  
                 1.0 
                 No symptoms of disease after infection 
               
               
                 Dogs 
                 5 
                 0.3 
                 with virulent cultures of the fungi 
               
               
                 Domestic 
                 3 
                 1.0 
                   T. mentagrophytes ,  T. verrucosum , 
               
               
                 cats 
                   
                   
                   T. equinum ,  M. canis ,  M. gypseum . 
               
               
                 Horses 
                 5 
                 0.5 
                 No dermatophytiae linked to the fungi 
               
               
                 Ponies 
                 3 
                 0.3 
                   M. canis  and  T. mentagrophytes  after 
               
               
                 Camels 
                 2 
                 5.0 
                 being in direct contact with diseased 
               
               
                 Bears 
                 2 
                 3.0 
                 animals. 
               
               
                 Leopards 
                 2 
                 4.0 
               
               
                 Hyenas 
                 2 
                 2.0 
                 No dermatophytiae linked to the fungi 
               
               
                 Servals 
                 2 
                 3.0 
                   M. canis  and  T. mentagrophytes  after 
               
               
                 Ocelots 
                 2 
                 2.0 
                 being in direct contact with sources 
               
               
                 Lions 
                 2 
                 3.0 
                 of infection. 
               
               
                 Tigers 
                 3 
                 7.0 
               
               
                 Nasuas 
                 3 
                 0.5 
               
               
                 Civets 
                 2 
                 1.0 
               
               
                 Rabbits 
                 7 
                 1.5 
                 No symptoms of disease after infection 
               
               
                 Dogs 
                 3 
                 0.5 
                 with virulent cultures of the fungi 
               
               
                 Domestic 
                 3 
                 1.5 
                   T. sarkisovii  and  M. gypseum . 
               
               
                 cats 
               
               
                 Black 
                 2 
                 5.0 
                 No dermatophytiae linked to the fungi 
               
               
                 panthers 
                   
                   
                   M. canis ,  T. mentagrophytes  and 
               
               
                 Tigers 
                 5 
                 7.0 
                 
                   T. verrucosum after being in direct 
                 
               
               
                 Geese 
                 6 
                 3.0 
                 contact with sources of infection. 
               
               
                 Bears 
                 3 
                 1.0 
               
               
                 Dogs 
                 8 
                 0.5 
               
               
                 Llamas 
                 2 
                 3.0 
               
               
                   
               
             
          
         
       
     
                                 TABLE 10               Type of       Dosage           animals   Number   (cm 3 )   Effectiveness                   Black   5   7.0   Affected by microsporosis linked to the       panthers           fungi  M. canis.  Recovery took place       Black   3   4.0   12-25 days after immunization.       panthers       Horses   3   1.0       Ponies   2   0.5       Lions   3   10         Tigers   3   10         Dogs   4   0.5       Bear   1   5.0       Hyena   1   5.0       Domestic   15    1.5   Affected by microsporosis linked to the       cats           fungi  M canis . Recovery took place       Dogs   5   0.5   10-20 days after immunization.       Horses   5   0.7       Black   1   6.0   Affected by trichophytosis linked to the       panther           fungi  T. mentagrophytes.  Recovery took       Red foxes   4   1.0   place 12-15 days after       Bears   2   5.0   immunization.       Mountain   1   7.0       sheep       Horses   15    1.0   Affected by microsporosis linked to the                   fungi  M. equinum.  Recovery took place                   12-20 days after immunization.                    
Bibliograhy
 
     (1) Aisenberg, A. A., Noskow, A. I., Kolovatsky, P. P. “Primenenie Yuglona v Veterinarii” in Scientific and Technical Information Bulletin of the State and Scientific Control Committee under the Moldavian Council of Ministers (1958), p. 88. 
     (2) USSR Patent No. 548947 (1976).