HU225305B1 - Method for production of vaccine for preventing and treating of mycotic skin diseases of small animals and the vaccine - Google Patents

Abstract

A vaccine to prevent and treat fungal skin-infections on small animals, contains in solution antigens of single or combined fungal groups of the genus Dermatophyton, i.e. of Microsporum ccanis, Microsporum gypseum and Trichophyton mentagrophytes. A vaccine to prevent and treat fungal skin-infections on small animals, contains in solution antigens of single or combined fungal groups of the genus Dermatophyton, i.e. of Microsporum ccanis, Microsporum gypseum and Trichophyton mentagrophytes. Solutions contain phenol as preservative. Each fungal group is incubated separately having been inoculated in a liquid SABAURAUD-DEXTROSE nutrient medium, for 15-20 days at 24-30 degrees Centigrade and 12-18 days at 3-8 degrees Centigrade. After incubation, based on the volume of the culture media, 0.3-0.8% phenol preservative is added. The liquids are clarified and required antigen concentrations are set with PBS. Vaccines are packaged as a single or a combination of antigens.

Description

The present invention relates to a process for the production of a vaccine for the prevention and treatment of fungal skin disorders in small animals and to a vaccine. More particularly, the present invention relates to a process for the production of a vaccine comprising a soluble antigen of at least one of three fungal species of the genus Dermatophyton, Microsporum canis, Microspotum gypseum and Trichophyton mentagrophytes, and a vaccine produced by the process.

The vaccine produced by the method of the invention is particularly suitable for the prevention and treatment of fungal skin disorders in small animals such as dogs and cats by active immunization.

It is known that skin and fungus, dermatomophytes, dermatomycoses, infections that affect only the skin, hair, hair and nails are common in both humans and animals. The pathogens are morphologically filamentous fungi. Infection can spread from animal to human or from human to animal. It is a very common phenomenon that animals infect each other. The infected animal scratches, has hair loss on the body, which is prominent on the head, but can spread to the entire body, which can be accompanied by secondary bacterial infection, fever, loss of appetite, and eventually death.

Researchers have previously noted that dogs and cats do not have the disease caused by Microsporum canis, Microsporum gypseum, and Trichophyton mentagrophytes in an infected environment under 4 weeks of age. This fact suggests that there is a certain immunity. As a result, specific research on immunization based on immunization was started earlier in cattle, in the 1970s, in dogs and cats, and later in the early 1980s.

Researchers are trying to reduce the damage to Dermatophytons with vaccines in a specific way. Trichophytons in Germany (Polfakov, J., Ivanova, L., Farnow, D., Karle, J. 1998. Cattle Trichophytosis 10th Hungarian Buiatric Congress Central European Buiatric Meeting. Siofok; lecture; summary of lectures given). ), The Czech Republic and the United States reduce the damage caused by monospecific vaccines with strains of Microsporum canis. From the Czech Republic, the vaccine Biocan®, and from the United States Microsporum canis® was marketed in Hungary.

Both vaccines contain a fungal cell, called somatic antigen, adsorbed onto aluminum hydroxide gel, washed off the surface of the medium (Ribnikar, A., Vrzal, V., Chumela, J., Petrás, J.: "Protecting Cats against Microsporum canis", Acta Vet. Bmo, 1997). It should be noted that somatic antigen is not the same as the antigen, but is a fungal body. Both vaccines are monovalent, administered intramuscularly. Their disadvantage is that they often have abscesses at the injection site, which can be traced back to the fungal bodies and cell fragments in the vaccine and possibly to the carrier material.

Surgical treatment of abscesses causes additional problems besides existing dermatitis.

A further disadvantage is their monovalent property, ie one formulation protects against Trichophyton mentagrophytes infection only and the other vaccine only protects against Microsporum canis infection but clearly does not protect against mixed infection and also does not protect against the very common occurrence of Microsporum gypseum.

U.S. Patent No. 5,453,273 discloses a fungal disease vaccine comprising one or more, preferably all three, cultures of Microsporum canis, Microsporum gypsum (gypseum), Trichophyton mentagrophytes killed and preserved with formaldehyde. The cultures are prepared independently of each other in Sabouraud dextrose liquid medium. The cells are sacrificed and preserved by the addition of formaldehyde, the cultures are screened and loaded onto an aluminum hydroxide / methylcellulose gel carrier. The specification describes the preparation of a trivalent inoculum but does not teach the culture conditions of the fungal species and the antigen concentration of the final product, so that reproduction is difficult. A further disadvantage of the product obtained by the process is that the aluminum hydroxide / methylcellulose gel commonly used in vaccine production may cause local abscess.

U. S. Patent No. 219,263 describes the preparation of tri- and polyvalent vaccines, which describes the preparation of antigen from seven strains of mycelium, including Microsporum canis, Microsporum gypseum and Trichophyton mentagrophytes. For the preparation of the vaccine, the strains are grown at 26 ° C for 15 days on agar / broth medium, then the fungal mass is harvested, homogenized and added to an aqueous solution containing 1% fermented muscle protein hydrolyzate, 10% glucose and 1% yeast extract. The microconidium concentration of the homogenate is 90 million milliliters. The vaccine is made by mixing the strains. Thiomersal is added to the cell suspension to inactivate the homogenate mixture.

The disadvantages of the process or the inoculum produced by the process are that: culturing at a single temperature of 26 ° C results in low microconidia, a proportionally low culture antigen content, and insufficient isolation of the antigen from the system. consequently, the vaccine contains fungal bodies or fragments thereof, the disadvantages of which have been described above, such as local reactions, dermatitis, abscess formation.

The object of the present invention is to develop a method which allows to obtain a higher antigen content than previously known methods, and to produce a polyvalent vaccine from the mycotic fungus culture which is suitable for the prevention and treatment of dermatomycosis in small animals without any side effects.

HU 225 305 Β1

The present invention is based on the discovery that when cultivated separately, the Dermatophyton strains of the fungal strains Microsporum canis, Microsporum gypseum and Trichophyton mentagrophytes are incubated at 24-30 ° C for 15-20 days and then cooled for 3-8 days. After incubation at 12 ° C and 12 to 18 days at this temperature, the amount of microconidia in the culture is greatly increased so that the dissolved antigen concentration in the system is also significantly higher. Thus, the higher microconidium content and, consequently, the higher antigen concentration is a consequence of the refrigeration period used in the cultivation of fungal strains.

The number of microconidia is determined microscopically, which is 50-150 million / milliliter according to the method of the invention, while the value is 40-120 million / milliliter according to the known method.

The cultures prepared by the method are removed by gentle filtration to remove fungal bodies and cell fragments causing side effects, and only the filtrate containing the dissolved antigen, free of fungal filaments and microconidia, can be used to prepare the vaccine.

The fungal strains used in the method produce antigens which, individually, are monovalent but, when combined, are highly suitable for the production of both di- or trivalent vaccines and for the prevention and treatment of dermatomycosis in animals, particularly small animals such as dogs and cats.

The fungal strains to be used in the process of the invention are known, available to the public, and described in the literature (Galgóczy, J., Fungal Diseases. Medicina Publishing House, Budapest, 1982, and Gy. Szemerédi et al. Application for Dr. Márkus Gy. Foundation, Budapest 1999, University of Veterinary Science).

The strains are characterized as follows.

Microsporum canis color culture

The strain was isolated from a cat's fur and leather scrap in Szolnok in 1997. Microsporum canis color colonies are fast growing, velvety, radial colonies of white color. A bright yellow pigmentation is observed along the edges of the colonies. After prolonged storage, the pigments turn red and then turn brown. They have a morphological structure in cellulose prints: macroconidia are sharpened, multi-compartmental, with thick and sinuous villages. The microconidia are slender, ovoid, slightly elongated.

Microsporum gypseum color culture

The strain was isolated from a cat's fur and leather scrap in 1997 in Leányvár. The fast-growing colonies of the Microsporum gypseum color culture initially develop in mostly yellowish to slightly bluish tones. Most morphologically, the colonies have a fine, gypsum-like dust deposit in the radial pattern.

After several days, their backside becomes beige. In cellulose prints, the macroconidia are enveloped in a grape-like arrangement, most of which are located at the end of the bracts.

Trichophyton mentagrophytes color culture

The strain was isolated from a dog's hair and skin scraper in 1996 in Budapest. Colonies of the Trichophyton mentagrophytes color culture develop rapidly, their surfaces are mostly velvety, with a white pigmentation at the center, pale yellow. The backside of the colonies is yellowish in color and becomes beige after several days. In cellulose prints, macroconidia are bud-shaped, grape-like or shaped like a Christmas tree.

The invention thus relates to a process for the preparation of a vaccine for the prevention and treatment of fungal skin disorders in small animals and to a vaccine produced by the process.

According to the invention, each of the three strains is cultured separately by inoculating Sabouraud dextrose in liquid medium. All three cultures were incubated at 24-30 ° C for 15-20 days. After this, the stock cultures were microscopically examined, cooled to 3-8 ° C, and incubated at this temperature for 12-18 days. At the end of the second incubation period, the culture is subjected to a microscopic examination again, followed by a sterility test. After the assay, 0.3-0.8% by volume of phenol is added to the liquid cultures, the filtrate is cleaned on a sintered glass filter, the antigen content of the filtrates is determined, the required antigen concentration is adjusted with PBS, and the resulting antigen solutions are separately after combining two solutions or combining all three solutions.

The materials used in the process of the present invention are known and commercially available.

The invention is illustrated by the following examples, without limiting the scope thereof.

Example 1

Preparation of Microsporum canis antigen solution and monovalent vaccine; Strain Maintenance Microsporum canis strain required for inoculation is maintained on Sabouraud dextrose liquid medium (Sabouraud R., Les Teignes. Masson et Cie Edit. 1910), inoculated at 4-5 months intervals and stored in a refrigerator at 4-5 ° C. . For each production unit (batch), the fungal strain of freshly grafted material is used.

Into a 3-liter Erlenmayer flask, previously sterilized, was poured 1.5 liters of Sabouraud dextrose medium. A controlled color culture containing no foreign germ was inoculated with platinum on the surface of the medium, shaken and placed in a thermostat at 25-26 ° C. This temperature is maintained for 17 days. After 17 days, it is examined under a microscope. The culture should not contain any foreign germ. If it is found to be free of foreign germ, then te3

The culture was further incubated in a thermostat at 5-6 ° C for 14 days. Subsequently, it is subjected to a repeated microscopic examination, which must be free of any foreign germ. Thereafter, 0.5% phenol by volume of the culture was added, the contents of the flask shaken and stored at 20-25 ° C for 24 hours. After 24 hours, the culture was filtered through a sintered glass filter and its antigen content was determined as follows.

Six 2.5-3.5 kg rabbits without specific antibody were inoculated with 1 ml of filtrate and repeated 21 days later. Blood samples are taken immediately prior to the first, then the second vaccination, and on the 14th day after the second vaccination. Antibody titers of blood samples were determined using the Ochterlony two-dimensional, double-gel diffusion method. The filtrate is suitable if serum titres of 1:10 or greater are found in the serum of blood collected after the first vaccination. Blood samples collected after the second vaccination should have a serum titre of at least 1:20 or greater in at least 4 rabbits. At the appropriate antigen content, the filtrate was diluted 1: 5 with PBS and subjected to a safety study (0.5 ml of the diluted filtrate was inoculated with 10 x 20-25 g mice subcutaneously. During the 8-day observation period, the animals were healthy). must remain). If the safety test is negative and the aim is to produce a monovalent vaccine, the product is formulated. If a di- or trivalent vaccine is to be prepared, the diluted filtrate should be stored at 5 ± 2 ° C until further processing.

Example 2

Preparation of Microsporum gypseum antigen solution and monovalent vaccine

All the procedures described in Example 1 were followed except that the strain Microsporum gypseum was used to prepare the antigen solution and the vaccine.

Example 3

Preparation of Trichophyton mentagrophytes Antigen Solution and Monovalent Vaccine The procedure described in Example 1 was repeated except that the Trichophyton mentagrophytes strain was used to prepare the antigen solution and the vaccine.

Example 4

Production of divalent vaccine

1-3. Two of the purified, controlled and adjusted antigen concentrations described in Examples 1 to 8 are mixed and formulated at a 1: 1 volume ratio.

Example 5

Production of a trivalent vaccine

1-3. 3 of the pure filtered, controlled and adjusted antigen concentrations described in Examples 1 to 2 are mixed and formulated in a 1: 1: 1 volume ratio.

Example 6

Technology Testing of the Vaccines Produced by the Method of the Invention Six dogs of different breeds and ages were treated twice with 2-3 week intervals with the trivalent vaccine prepared according to Example 5. In addition, one treatment and six preventive treatments were performed on seven cats. One animal also had dermatitis in addition to mycrosporosis, but no additional treatment was performed. Overall, treatment with the vaccine revealed that the infected animal had healed and that animals treated for preventive purposes did not develop skin disease or abscess.

Claims (3)

CLAIMS 1. A method of producing a phenol-preserved vaccine for the prevention and treatment of fungal skin disorders in small animals comprising the steps of separately injecting Microsporum canis strain, Microsporum gypseum strain, Trichophyton mentagrophytes strain into Sabouraud dextrose liquid medium, incubate at 15 ° C for 15-20 days, then cool the cultures again and incubate at 3-8 ° C for 12-18 days, then add 0.3-0.8% phenol to the cultures by volume, strain clear of fungal and adjusting the antigen content of the filtrate, free of microconidia, with PBS to the desired concentration, and finally formulating the solutions individually, or by combining two or three. Vaccine according to claim 1, characterized in that it contains a solubilized antigen of at least one of the fungal species or species of the genus Dermatophyton, free of fungal filaments and microconidia. Vaccine according to Claim 2, characterized in that it contains a soluble antigen of at least one of the fungi species Microsporum canis and / or Microsporum gypseum and / or Trichophyton mentagrophytes as species antigen of the genus Dermatophyton.