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 of the Invention

BACKGROUND

This invention relates to the preparation of vaccines and their use in preparing compositions for specifically preventing and treating dermatomycosis.

Dermatomycoses in animals are anthropozoonotic 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 26-25 days at a temperature of 26-28.degree. 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 no provide immunity against microsporiae and trichophytiae caused by other agents. 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. 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 dermatomycosis in animals and corresponding immunogenic fungal strains.

This aim has been achieved by using the following fungal strains as vaccinal 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 plurality of antigens, provided that a sufficient immune response is stimulated to give resistance to a dermatophyte infection. Antigenic material for such a purpose can be prepared using methods known from the prior art, e.g., homogenizing the above-mentioned dermatophytes 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 4 to 120 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.sub.9H.sub.9O.sub.2SNaHg), 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 extract. The concentration of the microconidia is adjusted to 4 to 120 million per milliliter and after 1 to 2 days the mixture is inactivated, e.g., with thiomersal (C.sub.9H.sub.9O.sub.2SNaHg) 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 dermatomycosis 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 Sammlung von und Zeflkulturen (DSM) on Oct. 1, 1992, under the provisions of the Budapest Treaty 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. 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 VKPGF-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--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. 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 patogennykh, toksigenykh vrednykh dlya cheloveka gribov, 1979).

The biological properties of the strain are described in Table 1 (see Original Patent).

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 (see Original Patent).

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.

Trichophyton 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 (see Original Patent).

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. VKPGF-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 (see Original Patent).

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 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. 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 (see Original Patent).

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 (see Original Patent).

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 described above Rebel, Taplin, loc. cit. and Kashkin, loc. cit.). The biological properties are described in Table 7 (see Original Patent).

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.

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 dermatophytes: 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-727/1311 and/or Microsporum 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-727/1311, 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, Trichophyton 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 4 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.

Claim 1 of 17 Claims

1. A method of inducing an immune response against dermatomycosis in an animal comprising the step of administering a vaccine to an animal in need thereof, said vaccine comprising antigenic material from at least one of the following inactivated dermatophyte strains: Trichophyton verrucosum Strain No. VKPGF-931/410 (accession No. DSM 7277), Trichophyton mentagrophytes Strain No. VKPGF-930/1032 (accession No. DSM 7279), Trichophyton sarkisovii Strain No. VKPGF-551/68 (accession No. DSM 7278), Trichophyton equinum Strain No. VKPGF-929/381 (accession No. DSM 7276), Microsporum canis Strain No. VKPGF-928/1393 (accession No. DSM 7281), Microsporum canis var. obesum Strain No. VKPGF-727/1311 (accession No. DSM 7280), Microsporum canis var. distortum Strain No. VKPGF-728/120 (accession No. DSM 7275), and Microsporum gypseum Strain No. VKPGF-729/59 (accession No. DSM 7274).

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