An inactivated vaccine against ringworm.

A new vaccine against ringworm, containing the inactivated Trichophyton verrucosum strain, was assessed on guinea pigs and calves under experimental conditions and on three herds of cattle under natural conditions. The vaccine elicited a distinct immune response of the cellular type. This type of immunity assessed by the migration inhibition test of leukocytes corresponded to the immunity evaluated by the challenge. In herds in which there were from 30 to 67% of naturally infected animals with T. verrucosum, two doses of the vaccine resulted in after 4 weeks a decrease of the number of animals with clinical changes from 40.5 to 100% depending on the group of animals under study.

[Microbiological evaluation of the effectiveness of gynalgin in the treatment of vaginitis]. / Ocena mikrobiologiczna skutecznosci Gynalginu w zapaleniach pochwy u kobiet.

The microbiological effectiveness of the preparation Gynalgin produced by POLFA Pharmaceutical Works in Rzeszów was assessed in cases of vulvovaginitis in 55 patients with clinically diagnosed inflammatory conditions of the lower genital tract, who were given Gynalgin tablets in 10-day courses. Vaginal smears were examined three times for the presence of bacteria, fungi and trichomonas vaginalis (before and immediately after the treatment, and two weeks later). In the initial examination in five vaginal smears mixed bacterial flora was found, in 6 smears trichomonas was present, in 4--bacteria and fungi, and in one--trichomonas and fungi. After the treatment in control examinations I and II the number of the isolated bacterial strains was lower, trichomonas was no longer present, and the number of fungi was reduced evidently. In the light of these microbiological examinations Gynalgin was found to exert a strong fungicidal, bactericidal and antitrichomonal activity, and the results of laboratory investigations agreed with those of clinical trials of Gynalgin effectiveness.

Early immunization of calves with an inactivated vaccine against trichophytosis.

The investigations demonstrated that the inactivated vaccine against trichophytosis, elaborated by the authors, induced immune response in calves, aged 5-8 days. The state of immunity was assessed in vitro by the leukocyte migration inhibition test, and in vivo by the allergic test and the challenge test. In all vaccinated calves (10 animals) there occurred delayed hypersensitivity and in six cases also a significant leukocyte migration inhibition. Vaccinated animals exposed to challenge were to a large extent resistant to experimental infection with the virulent strain of T. verrucosum.

Immunological properties of selected strains of Trichophyton genus.

The investigations demonstrated that the strains under study, either virulent or in the form of inactivated vaccines, induced a delayed type allergy. The response depended upon the affiliation to a determined species and individual properties of a strain. The strain T. mentagrophytes 1 displayed the highest biological activity, since it was a good inducer of allergy and a manufacturer of active trichophytin in tests performed both in vivo and in vitro.

Intracellular keratinase of Trichophyton gallinae.

Trichophyton gallinae could grow on agar medium containing chicken feathers as sole sources of carbon and nitrogen, but it could not utilize human or guinea pig hair. The fungus grows rapidly when feather keratin solubilized in dimethyl sulphoxide was included in the growth medium. Keratinase activity could be detected by release of amino acids from solubilized keratin and by creation of zones of lysis in gel diffusion assay. However, the activity was observed only in homogenates of T. gallinae mycelia: it was not possible to detect it in culture filtrates.

[Micromorphology of Trichophyton verrucosum]. / Mikromorfologia Trichophyton verrucosum.

Using 6 samples of pathologically altered epidermis of guinea pigs and cattle, chosen strains Trichophyton verrucosum (6 strains) and the two layer method of inoculation on a classic Sabouraud's medium, arthropogenesis of the fungus was investigated. The authors described successive stages of the developmental cycle of T. verrucosum and they found that in all cases the process of arthropogenesis was very similar irrespective of the source of the material used for inoculation. Using various modifications of Sabouraud's medium and a rice medium microscopic examinations revealed in the examined strains of T. verrucosum the presence of chlamydospores (Sabouraud's medium without sugar) macroconidia (rice medium) and spiral forms (Sabouraud's medium with gluconic acid or natrium sulphite) analogical to the forms noted in Trichophyton mentagrophytes.

[Studies on using choline salt of N-glucosylpolyfungine as an antifungal preparation for cell culture]. / Badania nad zastosowaniem soli cholinowej N-glukozylopolifunginy jako preparatu przeciwgrzybowego do hodowli komórek.

Fungistatic and fungicidal activity as well as the toxicity of the polyfungine derivative of choline N-glucosylpolyfungine salt for chicken embryo fibroblasts were studied. This preparation was found to inhibit the growth of Candida albicans, Aspergillus niger and Penicillium sp. at 25-100 micrograms/ml concentrations of the medium. The highest sensitivity to polyfungine was shown by C. albicans strain, and the lowest by Aspergillus niger strain. The preparation studied did not lose its activity in the medium containing 20% of calf serum. Its toxicity for cells in vitro was considera bly lower than that of amphotericine B used for intravenous injections and reached over 156 micrograms/ml of the medium. In mixed cultures containing fibroblasts and cells of fungi, the polyfungine derivative inhibited the growth of microbes at concentrations lower than in the medium without cells.

Lipid composition of the mycelial and spore forms of Trichophyton verrucosum.

Neutral lipid composition and that of phospholipids of mycelial and spore forms of Trichophyton verrucosum were examined. It was found that arthrospores had more than twice as high content of lipids (99.3 mg/g) than the corresponding mycelial form (44.7 mg/g). Differences were also found in the qualitative composition: almost two times more neutral lipids (58.5 mg/g) and three times more phospholipids (40.8 mg/g) occurred in the spores than in the mycelial form 30.6 and 14.1 mg/g, respectively). Analysis of the neutral lipid fraction composition of both examined forms of T. verrucosum showed that the basic component were triglycerides, constituting about 70% in the spores and 44% in the mycelium of all the lipids. In the case of phospholipids no significant differences were observed between the spore and mycelial forms, phosphatidylcholine and phosphatidylethanolamine were predominating in both forms. A much higher content of lipids in the infectious form of the fungus, the arthrospores, suggest a possibility of participation of this fraction in the pathogenicity of the fungus.

Screening the keratinolytic activity of dermatophytes in vitro.

Sixteen strains out of 12 species dermatophytes were examined in respect to their ability of utilizing keratin substrates as the only sources of C and N. The employed keratin substrates included a solubilized preparation of feather keratin (KS) and native keratin, guinea pig hair and chicken feathers. It has been shown that the preparation KS constitutes a convenient model for a preliminary estimation of fungal keratinolytic activity and it can be a source of information about the localization of these enzymes. It has been found that, among the 16 fungal strains, 13 strains synthesize mainly intracellular keratinases while 3 strains of T. verrucosum release enzymes mainly to the medium. Native keratin from hair and feathers was degraded only by some of the examined strains which, under the experimental conditions, developed characteristic spore forms. Keratin of guinea pigs hair was attacked only by the T. mentagrophytes strains, T. verrucosum and K. ajelloi, and only T. gallinae grew on native keratin from chicken feathers.

An evaluation of the resistance to Microsporum canis on the basis of the guinea pig.

The purpose of the work was to assess the immune response of guinea pigs after the experimental infection with Microsporum canis, and after immunization with a specific live vaccine. The guinea pigs after the recovery from infection showed a delayed type of hypersensitivity and in addition, 30 per cent of the animals were characterized by the presence of immediate hypersensitivity reactions. All the animals were resistant to reinfection with M. canis and to some extent to Trichophyton verrucosum, however, no cross-immunity against T. mentagrophytes was found. The vaccinated animals were characterized mainly by delayed type of hypersensitivity and the resistance to approximately 1000 infectious doses of M. canis.

Comparative characterization of proteolytic enzymes from Trichophyton gallinae and Trichophyton verrucosum.

The proteolytic activities of culture filtrates and cell homogenates of Trichophyton gallinae and Trichophyton verrucosum were compared when the fungi were grown in the presence of a readily assimilable source of C and N (Sabouraud's broth) and a poorly assimilable source (mineral medium containing soluble keratin (KS) protein prepared from either chicken feathers or guinea pig hair). The proteolytic activity of T. gallinae was found to be located predominantly in the cell homogenate although this depended somewhat on the nature of the C and N source in the medium. Enzyme activity in T. verrucosum on the other hand was located in the culture filtrate. The use of KS as a substrate revealed only one peak of enzymatic activity which occurred at pH 7.0 in both fungi. In the case of T. gallinae the total proteolytic activity was not related to the fungal biomass but did depend on the nature of the substrate; poorly assimilable substrates (native feathers and hair) stimulated a higher enzymatic activity per unit biomass than readily assimilable Sabouraud's broth. In T. verrucosum, proteolytic activity was related to the fungal biomass and was highest in Sabouraud's broth which proved most suitable for growth of the fungus. On the basis of the findings with six different inhibitors of protease activity, it is concluded that the proteolytic enzymes of T. gallinae differ from those of T. verrucosum in terms of their metal-dependence and contained serine as part of the active centre.

Feather keratin as a ligand in an affinity chromatographic technique for isolation of protease from Trichophyton verrucosum.

A technique of affinity chromatography was developed and optimized for proteases from the postculture fluid of Trichophyton verrucosum. The technique employs porous glass with adsorbed feather keratin or keratin covalently bound to the glass. Modifications of the amounts of proteases introduced into the columns and the manner of elution (pH gradient, buffer concentration, EDTA) made it possible to achieve yields of the isolated enzymes of the order of 80%. The degree of purification of four fractions isolated by the proposed technique was about 6-fold and allowed electrophoretically almost homogeneous enzymatic forms to be isolated. Substrate and inhibition tests on the four chromatographically purified proteolytic enzymes indicated a specifically keratinolytic nature of the enzymes studied.