RU2727766C1 - Method for producing an inactivated animal dermatophytosis vaccine - Google Patents

Abstract

FIELD: veterinary science; pharmaceutics.SUBSTANCE: invention relates to veterinary science and pharmaceutics, specifically to a method of producing an inactivated animal dermatophytosis vaccine, according to which one performs sowing and separate cultivation of fungi cultures Microsporum canis, Microsporum qypseum, Trichophyton mentagrophytes, suspensions are prepared from grown cultures, concentration of fungal elements in each suspension is subtracted to 20–50 million in 1 ml, obtaining suspensions with the same content of fungal elements, mixing suspensions Microsporum canis, Microsporum qypseum, Trichophyton mentagrophytes in volume ratio of 2:1:2, twice washed, vaccine is considered immunogenic with total content of 20–50 million elements of fungi in 1 ml.EFFECT: technical result consists in the immunogenicity and stability of the inactivated vaccine against animal dermatophytosis obtained using the disclosed method without using additional components, such as immunomodulating substances.1 cl, 13 ex

Description

The invention relates to veterinary immunology, in particular to methods of making vaccines against animal dermatophytosis.

The invention relates to veterinary mycology, in particular the prevention and treatment of animal dermatophytosis, although it indirectly helps to prevent their spread among humans, since dermatophytosis belongs to the group of zooanthroponous infections affecting both animals and humans. Dermatophytosis is widespread on all continents and is of great social importance, therefore, finding ways to obtain inexpensive, effective and safe vaccines for humans and animals against these infections is urgent.

At the moment, methods of making vaccines against animal dermatophytosis are known from the prior art.

A known method of manufacturing a vaccine against microsporia susceptible animals, including sowing and growing a culture of the fungus strain Microsporum canis VIEV 473 and preparing a homogenate with a concentration of 20-50 million microconidia in 1 ml, 4-6% saline solution sodium chlorine, followed by the addition of aluminum hydroxide from calculating 0.5 ml for every 100 ml of vaccine (RU 2084241 C1, 20.07.1997).

There is also known a method of making a vaccine for the prevention of dermatophytosis of domestic and laboratory animals, including the use as strains of virulent cultures of fungi species T. mentagrophytes, M. canis, M. qypseum, separate cultivation of cultures, obtaining homogenates and mixing them in equal volumes, additional inactivation of the resulting mixtures with a 0.5-0.6-n solution of formalin for 3-4 days at a temperature of 37-38 ° C and the addition of 0.8-0.9% solution of sodium nucleinate immunomodulator in an amount of 1: 1, packaging, freezing within 10-15 hours, drying in a freeze-drying unit for 32-36 hours.

For prophylactic purposes, a dermatophyte vaccine is used, resuspended in 1.9-2.1 ml of saline, which is administered to laboratory animals - dogs and cats, regardless of age in the area of the femoral muscle group twice with an interval of 10-14 days at doses of 0, 9-1.2 ml (200-250 million microconidia) (SU 1734762 A1, 23.05.1992).

A known method of manufacturing a vaccine for the prevention and treatment of dermatophytosis in domestic and laboratory animals (patent RU 2192885, 2001) is the closest analogue. The specified method includes sowing and separate cultivation of cultures of fungi Microsporum canis, Microsporum qypseum, Trichophyton mentagrophytes, preparing homogenates and mixing them in a ratio of 1: 1: 2, adding the immunomodulator Ribotan and an inactivator-formalin, and the results are taken into account by the number of fungal elements: mycelium, macroconidium, arthrospore, chlamydospore, microconidium and the vaccine are considered immunogenic with a total content of 25-50 million fungal elements in 1 ml. (RU 2192885 C2, 20.11.2002). The vaccine obtained in this way causes an overreaction in a number of vaccinated animals.

The aim of the claimed invention is to develop a method for the manufacture of an inactivated vaccine against animal dermatophytosis with high immunogenicity and low reactogenicity, reduced concentration of fungal cells in 1 ml of vaccine, stable physicochemical and biological properties, and not loaded with foreign components, in particular immunomodulatory substances.

The technical result of the claimed invention is to increase the immunogenicity and decrease the reactogenicity of the inactivated vaccine against animal dermatophytosis, obtained by the claimed method, having stable physicochemical and biological properties, a reduced concentration of fungal cells in 1 ml of the vaccine, unloaded with extraneous components, in particular immunomodulatory substances.

The proposed method for the manufacture of an inactivated vaccine against animal dermatophytosis is as follows.

Sowing and separate cultivation for 21-23 days of cultures of fungi Microsporum canis, Microsporum qypseum, Trichophyton mentagrophytes are carried out on a semi-liquid nutrient medium wort-agar with an agar content of 0.5 to 1.5%, at a temperature of 26-28 ° C. Then, suspensions are prepared from the grown cultures in a stable buffered saline solution with a pH of 7.0-7.6, 0.5% of an official 40% formalin solution is added to each resulting suspension of fungal cells, the amount of fungal elements is determined - microconidia, macroconidia, fragments mycelium and other cells of dermatophytes, titrate with a buffered saline solution the concentration of fungal elements in each suspension to 20-50 million in 1 ml, mix suspensions of Microsporum canis, Microsporum qypseum, Trichophyton mentagrophytes in a ratio of 2: 1: 2, respectively, with a concentration of 0.5 % of the official 40% formalin solution, count the number of fungal elements - microconidia, macroconidia, mycelium, arthrospores, chlamydospores in 1 ml of suspension. After that, they are washed twice in two volumes of buffered saline with a pH of 7.0-7.6 containing 0.2% of an official 40% formalin solution. The vaccine is considered immunogenic with a total content of 20-50 million fungal elements in 1 ml.

The method provides for the production of an immunogenic vaccine with stable physicochemical and biological properties during the entire shelf life of the drug, low reactogenicity and a reduced concentration of fungal cells in 1 ml of the vaccine. If used correctly, according to the instructions for use, the vaccine does not cause a negative reaction from the body and develops immunity for a period of at least 12 months.

The stated set of essential features ensures the achievement of an unexpected technical result. The retention period of the high immunogenic activity of the vaccine vakderm, obtained by the claimed method, unexpectedly increased in comparison with the closest analogue from 12 to 18 months, which makes it possible to use it for immunizing animals for 6 months longer. The shelf life of the vaccine obtained by the claimed method, packaged in vials under aseptic conditions and hermetically sealed is at least 18 months.

Example 1. Cultures of strains Microsporum canis, Microsporum qypseum, Trichophyton mentagrophytes are grown separately for 21-23 days on a semi-liquid medium - wort-wort-agar with an agar content of 0.5 to 1.5%, at a temperature of 26-28 ° C ... Then with a scraper remove the mushroom mass from the surface of the nutrient medium, grind separately in homogenizers and add 300-500 ml of sterile buffered saline solution (pH 7.0-7.6) at the rate of grinding 4-7 mattresses in 1 homogenizer with a working volume of a glass not less than 1.5-2.0 liters.

Then, 0.5% of an official 40% formalin solution is added to each obtained suspension of fungal cells, the amount of fungal elements (microconidia, macroconidia, fragments of mycelium and other dermatophyte cells) is determined. Next, the concentration of fungal elements in each suspension is titrated with a buffered saline solution to a concentration of fungal cells of 50 million in 1 ml, then mixed in the ratio: - 2 part M. canis - 1 part M. qypseum - 2 parts T. mentagrophytes. Then washed twice in two volumes of buffered saline with a pH of 7.0-7.6 containing 0.2% of the official 40% formalin solution. In the vaccine obtained in this way, the vakderm is checked for the appearance, the presence of foreign mechanical impurities and other foreign inclusions, sterility, the amount of fungal elements in 1 ml, harmlessness and reactogenicity on rabbits and / or dogs, immunogenic activity by intramuscular administration to animals 0.5-1, 0 ml of vaccine, which does not cause a negative reaction from the body and produces immunity for at least 12 months.

Example 2. For the manufacture of 1 liter of vaccine take 375 ml of suspension of culture of M. canis containing 50 million elements of the fungus in 1 ml and 0.2% of the official 40% formalin solution. Next, take 250 ml of M. qypseum culture suspension containing 50 million fungal elements in 1 ml and add 0.2% formalin. Then take 375 ml of a suspension of the culture of T. mentagrophytes containing 50 million elements of the fungus in 1 ml and add 0.2% of the official 40% formalin solution. Next, the vaccine is washed twice in two volumes of buffered saline with a pH of 7.0-7.6.

In the vaccine obtained in this way, the appearance, the presence of foreign mechanical impurities and other foreign inclusions, sterility, the amount of fungal elements in 1 cm3, harmlessness and reactogenicity on rabbits or dogs, immunogenic activity by intramuscular administration of 0.5-1.0 ml of the vaccine to animals are checked , which does not cause a negative reaction from the body and produces immunity for at least 12 months.

Example 3. For 1 liter of vaccine, 250 ml of M. canis culture suspension is taken with the addition of 0.3% official 40% formalin solution with a cell concentration of 50 million in 1 ml, 250 ml of M. qypseum culture suspension with 0.3% official 40 % formalin solution with a concentration of 50 million fungal elements in 1 ml, and then take 500 ml of a suspension of T. mentagrophytes culture with the addition of 0.3% official 40% formalin solution. After mixing the suspensions with each other and washing it in a buffered saline solution, the resulting vaccine, similarly to the above Example 1, checked the appearance, the presence of foreign mechanical impurities and other foreign inclusions, sterility, the amount of fungal elements in 1 cm3, harmlessness and reactogenicity in rabbits or dogs , immunogenic activity by intramuscular injection of 0.5-1.0 ml of vaccine to animals, which does not cause a negative reaction from the body and develops immunity for at least 12 months.

Example 4. For 1 liter of vaccine was taken 250 ml. suspensions of cultures of M. canis and M. qypseum with a concentration in each of them of 20 million elements of the fungus in 1 ml with 0.2% official 40% formalin solution and 500 ml was added to them. suspensions of T. mentagrophytes culture with a concentration of 20 million / ml of fungal elements, with preliminary addition of 0.2% official 40% formalin solution to it and washing it in buffered saline.

Example 5. For 1 liter of vaccine, suspensions of cultures of Microsporum canis 400 ml, Microsporum qypseum, Trichophyton mentagrophytes were taken in a ratio of 2: 1: 2 (200 ml and 400 ml) with a concentration of 20 million fungal elements in 1 ml and 0 was added, 3% official 40% formalin solution.

Example 6. For 1 liter of vaccine suspensions of cultures of Microsporum canis, Microsporum qypseum, Trichophyton mentagrophytes were taken in a ratio of 2: 1: 2 with a concentration of 35 million fungal elements in 1 ml and with the addition of 0.25% official 40% formalin solution.

In all examples, in the samples of the obtained vaccines, the appearance, the presence of foreign mechanical impurities and other foreign inclusions, sterility, the amount of fungal elements in 1 ml, harmlessness and reactogenicity in rabbits and / or dogs, immunogenic activity by intramuscular administration to animals 0.5-1 , 0 ml vaccine as described in Example 1.

An increase in the concentration of fungal elements in 1 ml over 50 million leads to the appearance of reactogenicity of the vaccine, and a decrease in the concentration of less than 20 million fungal elements in 1 ml reduces its immunogenicity, therefore, the optimal immunogenic doses of the vaccine are the concentration of fungal elements 20-50 million in 1 ml.

For prophylactic purposes, the vakderm vaccine is administered intramuscularly, twice with an interval of 10-14 days at doses of 0.5-0.6 (10-25 million fungal elements) to dogs weighing up to 5 kg, kittens from 1 to 3 months of age, puppies of fur animals and rabbits from 1 to 2 months of age and 1.0-1.2 ml (20.0 million - 50.0 million of fungal elements) dogs weighing more than 5 kg, cats over 3 months of age, fur animals and rabbits over 2 months of age.

In case of dermatophytosis (microsporia or trichophytosis), vaccines are used intramuscularly three times with an interval of 10-14 days between injections in the same doses as for prophylaxis - 0.5-0.6 -1.0-1.2 ml.

Example 7.5 dogs weighing up to 5 kg, 3 kittens 1-3 months of age, 12 puppies of fur animals (5 puppies of a fox and 7 puppies of a fox) and 7 rabbits of 30 days of age were inoculated twice, intramuscularly with an interval of 10 days, at a dose of 0 , 5 ml, with the content of fungal elements in 0.5 ml of the vaccine 10 million. Immunological restructuring of the body was determined by examining blood serum in PMA 21 days after the 2nd vaccination. The obtained antibody titers (AT) and AT JgG, expressed in logarithms, were 6.47 and 5.32, respectively, while in the same animals before immunization these indicators were AT - 2.54 and AT JgG - 0.92.

The strength of immunity was determined by the method of direct infection of immune (experimental group) and intact (control group) animals with virulent strains of M. canis, M. qypseum and T. mentagrophytes fungi. Infection was carried out 30 days after the second immunization. After infection, the animals of the experimental and control groups were monitored daily. When observing experimental animals (vaccinated), clinical signs of the disease were not found, which was confirmed by mycological studies of material samples from the site of infection.

3 dogs, 2 kittens and 6 puppies of fur-bearing animals (3 foxes and 3 polar foxes) and 3 rabbits, not vaccinated against dermatophytosis (control group), infected with virulent cultures of M. canis, M. qypseum, T. mentagrophytes at identical infectious doses, fell ill as microsporia and trichophytosis with the manifestation of pronounced clinical signs of dermatophytosis.

Example 8. 6 dogs weighing more than 5 kg, 4 cats over 3 months of age, 9 heads of fur-bearing animals and 7 rabbits over 3 months of age were vaccinated twice with the vakderm vaccine obtained by the claimed method, with a prophylactic purpose at a dose of 1.0 ml with the content of fungal elements is 50 million in 1 ml. A study of blood serum 21 days after the second vaccination in the PMA showed the presence of a titer AT - 7.62 and AT JgG - 5.55. Cutaneous infection of immunized animals (experimental group) with virulent cultures of M. canis, M. gypseum, T. mentagrophytes showed a high degree of protection of the body of immune animals. There were no clinical signs of dermatophytosis in these animals.

Animals of the control group (not vaccinated against dermatophytosis) and infected with virulent cultures of M. canis, M. qypseum, and T. mentagrophytes developed dermatophytosis with characteristic signs of classical experimental microsporia and trichophytosis.

Example 9. The therapeutic effect of the vaccine vakderm, obtained by the claimed method, was tested on 5 fox puppies, 7 polar fox puppies, 5 rabbits and 4 dogs weighing up to 5 kg, 7 kittens 3-8 months of age with dermatophytosis. For this purpose, the animals were vaccinated with the vaccine three times, intramuscularly, in doses of 0.6-0.8 ml containing 10-12.5 million fungal elements. When examining all animals on days 10-12 after the last vaccination, the animals were healthy. At the site of the formation of mycotic areas, characterizing the specificity of the lesions, where crusts and scales had previously formed, a smooth, even surface without signs of mycotic inflammation and active growth of young hair were observed. Mycological analyzes of material samples from the sites of infection were negative. In the control group of animals, 3 fox puppies, 4 arctic fox puppies, 3 rabbits, 4 kittens and 3 dogs, sick with dermatophytosis and not exposed to vaccine therapy, continued to get sick. Mycological analyzes of material samples from the sites of infection were positive in all cases.

Example 10. Carrying out treatment of 5 foxes, 2 polar foxes, 3 rabbits over 4 months of age, cats of 1-3 years of age and 5 dogs weighing more than 5 kg against dermatophytosis, the vaccine was administered three times intramuscularly with an interval of 10-14 days at a dose of 1 , 0 ml containing 50 million elements of the fungus. 7-10 days after the third vaccination, all animals recovered. By this time, all the treated animals had no clinical signs of the disease, and active growth of young hair was observed.

Animals of the control group (3 foxes, 2 polar foxes, 3 rabbits, 4 dogs, 2 cats), sick with dermatophytosis and not exposed to vaccine therapy, by this time continued to get sick.

Example 11.3 dogs weighing up to 5 kg, 2 kittens 1-3 months of age, 4 puppies of fur animals (2 puppies of a fox and 2 puppies of a polar fox) and 3 rabbits of 30 days of age were vaccinated with the Vakderm vaccine twice, intramuscularly with an interval of 12 days, in dose of 0.5 ml, containing 10 million fungal elements in 0.5 ml of the vaccine. For immunization, the vaccine was used, stored in a refrigerator for 18 months. The strength of immunity was determined by the method of cutaneous infection of immune (experimental group) and intact (control group) animals with virulent strains of M. canis, M. qypseum and T. mentagrophytes fungi. After the second immunization, 30 days later, the animals of the experimental and control groups were infected. After infection, all animals in the experiment were monitored daily. There were no clinical signs of experimental dermatophytosis in vaccinated animals, which was confirmed by mycological studies of material samples from the site of infection.

2 dogs, 3 kittens and 5 puppies of fur-bearing animals (2 foxes and 3 polar foxes) and 2 rabbits, previously not vaccinated against dermatophytosis (control group of animals), infected with virulent cultures of M. canis, M. qypseum, T. mentagrophytes, in the same doses, as the animals of the experimental group, got sick with both microsporia and trichophytosis with the manifestation of characteristic clinical signs of dermatophytosis. The diagnosis was confirmed by mycological research methods. Retrocultures of infecting strains were isolated from samples of pathological material samples.

Example 12. 3 dogs weighing more than 5 kg, 2 cats over 3 months of age, 5 heads of fur animals (2 foxes, 3 polar foxes) and 3 rabbits over 3 months of age were vaccinated twice with the vaccine obtained by the claimed method and stored for 18 months in refrigerator conditions, for prophylactic purposes in a dose of 1.0 ml with a content of fungal elements of 20 million in 1 ml. Cutaneous infection of the immunized animals of the experimental group with virulent cultures of the fungi M. canis, M. gypseum, T. mentagrophytes showed a sufficient degree of protection of the body of immune animals. After the control infection of vaccinated animals, no clinical signs of microsporia and trichophytosis were found in them. Animals of the control group (not vaccinated with the vaccine) and infected with virulent cultures of M. canis, M. qypseum, and T. mentagrophytes developed dermatophytosis with obvious signs of microsporia and trichophytosis. The diagnosis was confirmed by methods of mycological examination of samples taken from mycotic foci.

Example 13. The therapeutic efficacy of a vaccine stored for 18 months in a refrigerator was tested on fur animals (3 foxes, 3 polar foxes), 3 rabbits, 4 cats and 3 dogs. For this purpose, the animals of the experimental group, patients with microsporia and trichophytosis, were vaccinated 3 times for therapeutic purposes intramuscularly with the vaccine with an interval between injections of 13 days, in doses of 0.6-0.8 and 1.0-1.2 ml with a content of fungal elements 10-12.5 and 20-50 million, respectively. After 10-25 days after the last therapeutic immunization, a clinical examination of all the experimental animals was carried out, as a result of which it was established that all the animals recovered. Mycological findings were negative.

Obtained by the proposed method inactivated vaccine vakderm against dermatophytosis of animals, when introduced into the body, forms a tense long-term immunity against dermatophytosis. The shelf life of the vaccine has been increased to 18 months. The vaccine obtained by the proposed method has been tested with a positive result in wide industrial practice and in a number of farms; it will find application in fur farming, dog breeding and the private sector to combat dermatophytosis in cats, dogs and other species of susceptible animals.

Claims (1)

A method for the manufacture of an inactivated vaccine against animal dermatophytosis, characterized by the fact that sowing and separate cultivation for 21-23 days of cultures of fungi Microsporum canis, Microsporum qypseum, Trichophyton mentagrophytes on a semi-liquid nutrient medium wort-agar with an agar content of 0.5 to 1, 5%, at a temperature of 26-28 ° C, suspensions are prepared from the grown cultures in a stable buffered saline solution with a pH of 7.0-7.6, 0.5% of the official 40% formalin solution is added to each resulting suspension of fungal cells, determine the amount of fungal elements - microconidia, macroconidia, fragments of mycelium and other dermatophyte cells, titrate with a buffered saline solution the concentration of fungal elements in each suspension to 20-50 million in 1 ml, obtaining suspensions with the same content of fungal elements, mix suspensions of Microsporum canis, Microsporum qypseum , Trichophyton mentagrophytes in a volume ratio of 2: 1: 2, respectively, n Count the number of fungal elements - microconidia, macroconidia, mycelium, arthrospores, chlamydospores in 1 ml of suspension, washed twice in two volumes of buffered saline with a pH of 7.0-7.6 with a 0.2% official 40% formalin solution, the vaccine is considered immunogenic with a total content of 20-50 million fungal elements in 1 ml.