Verrucosum Strain Research Articles

Drug-resistance genes and antifungal susceptibility of Trichophyton verrucosum variants isolated from bovine skin lesions and farm environments.

Trichophyton verrucosum causes a highly contagious disease in bovines and is occasionally transmitted to humans. Azoles are widely used as antifungal drugs in bovines, and act by targeting the ergosterol biosynthesis pathway. However, cases of treatment failure and recurrence of dermatophyte infections have been associated with mutations at the drug target site. Currently, information on T. verrucosum variants is lacking. This study aimed to classify T. verrucosum variants isolated from animals exhibiting clinical symptoms and cattle environments in the Republic of Korea based on their antifungal susceptibility and the presence or absence of ergosterol biosynthesis (ERG) and subtilisin genes. Of 139 clinical and 39 environmental samples, 86 and 21 were found to be infected, respectively. The positivity rate of calves aged 1-6 months was 73.1%, which was significantly higher than that of calves aged>6 months (55.2%). Twenty-seven T. verrucosum strains were identified as T. album (n=9), T. ochraceum (n=6), and T. discoides (n=12). Antifungal susceptibility testing showed that enilconazole had the lowest geometric mean antifungal activity of 1.08, 1, and 0.94µg/ml against T. album, T. ochraceum, and T. discoides, respectively. The detection ratios of ERG3 and ERG6 differed significantly among the three variants. In conclusion, our study suggests conducting sample cultures and antifungal susceptibility tests on isolates before administering antifungal drugs. Moreover, the expression profile of ERG appears to have a potential link to drug susceptibility. This information may contribute to the prevention and management of dermatophytosis in cattle.

Agreement between Clinical Assessment and Laboratory Diagnosis of Ringworm in Calves at Auction Markets

To limit the spread of bovine ringworm, control measures such as movement restrictions are highly recommended. In this context, calves at auction markets in Styria, Austria, displaying skin lesions characteristic for bovine ringworm, are excluded from the auctions. To investigate whether these clinical assessments correspond to laboratory diagnosis, a total of 166 samples taken from skin lesions assigned to the three clinical categories ‘ringworm very likely (v), likely (l) or unlikely (u)’ were mycologically examined using microscopy, culture, and nested PCR followed by amplicon sequencing. Further, the relationships of isolated dermatophytes were determined through multi-locus sequence typing (MLST). Overall, a high agreement between clinical assessment and laboratory results were observed with microscopy and nested PCR, providing more consistent results and molecular detection possessing an analytical sensitivity superior to that of cultural isolation (culture 21.7% vs. nested PCR 48.2%). Phylogenetic analyses revealed that most of the isolated dermatophytes belong to a unique Trichophyton verrucosum MLST genotype. In conclusion, clinical assessments were largely confirmed through laboratory diagnosis with nested PCR and sequencing, providing rapid, sensitive, and species-specific detection of dermatophytes in calves at auction markets displaying skin lesions typical for ringworm; this seems to be predominantly caused by a single Trichophyton verrucosum strain.

New Reference Genes for qRT-PCR Analysis as a Potential Target for Identification of Trichophyton verrucosum in Different Culture Conditions.

Dermatophytes are a group of filamentous fungi infecting skin, hair, and nails that raise great diagnostic difficulties. qRT-PCR is a reliable technique for quantifying gene expression with increasingly frequent use in mycological diagnostics. Knowledge of genes and molecular markers with potential to be used in the identification of dermatophytes is of great importance for the development of this branch of diagnostics. In this article, the suitability of six candidate reference genes (TUBB, ACTB, ADPRF, RPL2, SDHA, and EEF1A1) was investigated for gene expression analysis in the dermatophyte Trichophyton verrucosum, which was cultured in various mycological media that are commonly used in a diagnostic laboratory, i.e., Sabouraud, potato dextrose, and keratin-supplemented MM-Cove. The different culture conditions are extremely important factors for the growth and physiology of dermatophytes. Gene expression stability was evaluated using geNorm, NormFinder, BestKeeper, and RefFinder algorithms. Regarding the stability of expression, SDHA was the most stable housekeeping gene; hence, this gene is recommended for future qRT-PCR studies on T. verrucosum strains. These results allow us to conclude that the SDHA gene can be an additional good candidate as an identification target in the qRT-PCR technique.

Ochratoxigenic fungi in post-fermented tea and inhibitory activities of Bacillus spp. from post-fermented tea on ochratoxigenic fungi

The contamination of ochratoxigenic fungi in the process of pile-fermentation of post-fermented tea is objective, but the fact that ochratoxigenic fungi have almost no risk to the safe drinking of post-fermented tea products has been widely concerned. This study aims to identify the diversity of ochratoxigenic fungi in post-fermented tea and to reveal the inhibitory effect of Bacillus strains isolated from post-fermented tea on ochratoxigenic fungi. Results showed: 8.33% of A. niger strains, 30% of A. tubingensis strains, 100% of A. carbonarius strains, A. nidulans strains, P. verrucosum strains and A. ochraceus strains isolated from post-fermented tea have the ability to produce ochratoxin A (OTA), and there are significant differences in the OTA production ability between different strains. The six different species of Bacillus isolated from post-fermented tea coexisted with ochratoxigenic fungi in liquid media can not only inhibit the growth of ochratoxigenic fungi, but also inhibit the production of OTA. Bacillus strains cocultured with ochratoxigenic fungi on the tea matrix also have significant effects on the growth of OTA-producing fungi and the effects of inhibiting fungi are influenced by the order of inoculation, culture time and the difference of fungal species. Bacillus crude antifungal extracts have inhibitory effects on the growth of OTA-producing fungi and anti-fungal substances can be roughly divided into two categories: peptides with molecular weight ≤10 kDa, and anti-fungal proteins with molecular weight >10 kDa according to their molecular weight. The research results have important guiding value for the correct understanding of the hazards of fungal contamination in post-fermented tea and searching for new technologies of producing post-fermented tea aimed at reducing the safety risks of OTA.

Molecular Characterization of Trichophyton Verrucosum Strains İsolated From Cattle by PCR-RFLP

Dermatofitler, insan ve hayvanlarda keratin içeren dokuları infekte ederek dermatofit infeksiyonuna neden olmaktadırlar. Trichophyton verrucosum sığır dermatofitozis olgularının en yaygın etkenidir. Trichophytosis, bütün dünyada hayvancılık sektöründe önemli ekonomik kayıplara neden olması yanında zoonoz olmasıyla da insan sağlığını tehdit etmektedir. Sığırlardan genellikle T. verrucosum izole edilmektedir. Sığırlar bu etkenin doğal rezervuarıdırlar. Bu çalışmanın amacı, sığırlarda hastalığa neden olan dermatofitlerinin izolasyonu ve izole edilen T. verrucosum suşlarının Internal Transcribed Spacer (ITS) bölgelerinin PCR-RFLP ile moleküler ayrımının yapılmasıdır. Bu amaçla dermatofitozisli sığırlardan 90 adet örnek alınarak kültürleri yapıldı. Bu örneklerin kültürü sonucunda 35 (%38,8) adet T. verrucosum izole ve identifiye edildi. Bu suşların DNA izolasyonu gerçekleştirilerek ITS bölgelerin amplifikasyonu gerçekleştirildi. T. verrucosum suşlarının MvaI ve HinfI enzimleri kullanılarak yapılan Restriction Fragment Lenght Polymorphism (RFLP) analizleri sonucunda bir adet RFLP profiline rastlandı. Sonuç olarak, izole edilen T. verrucosum suşlarının PCR-RFLP sonucunda tek bir profile sahip olduğu, farklı profil örneklerinin saptanması için farklı bölgelerden hatta farklı ülkelerden suşların PCR-RFLP’lerinin yapılması gerektiği kanısına varıldı.

Production and migration of ochratoxin A and citrinin in Comté cheese by an isolate of Penicillium verrucosum selected among Penicillium spp. mycotoxin producers in YES medium

Moldy food products that are not subject to pathogenic bacterial contamination could be trimmed by consumers to remove fungal mycelium before consumption. However, prior to giving such recommendations to consumers, it is necessary to evaluate potential mycotoxin migration in these products. This study aimed at quantifying citrinin (CIT) and ochratoxin A (OTA) accumulation and migration in a French semi-hard Comté cheese after artificial inoculation with a CIT- and OTA-producing Penicillium verrucosum strain. At 8 °C, CIT and OTA production started after 14 days and 28 days incubation, respectively; while at 20 °C, both mycotoxins were produced from day 7. At 20 °C, maximum CIT concentration, about 50000 ng/g, was 20 fold that at 8 °C. Regardless of temperature, maximum OTA concentration was about 4000 ng/g cheese. Maximum concentrations were obtained in the upper part of the cheese, but depending on incubation time, mycotoxins were detected up to 1.6 cm in depth. As long as only white mycelium developed on the cheese surface, trimming can be acceptable, but a blue mold color (due to fungal sporulation) was associated with the accumulation of significant amounts of mycotoxins so the product should be discarded.

The host range of dermatophytes, it is at all possible? Phenotypic evaluation of the keratinolytic activity of Trichophyton verrucosum clinical isolates.

Dermatophytes are fungi that have an ability to invade keratinised structures. Enzymes secreted by dermatophytes can underlie fungal survival on the host and development of infection. It is possible that the range of activity of keratinases from various dermatophytes is limited to specific species of animals and groups of people. The aim of this study was to carry out phenotypic analysis of the degree of keratinolytic activity of Trichophyton verrucosum strains using hairs of humans and various animal species as substrates. Our results indicated that the activity of keratinases is substrate-induced. The host range of T. verrucosum can be defined as wide. The highest activity of keratinases was recorded in media containing keratin from cow (Bos taurus) and sheep (Ovis aries) hairs in comparison with that from other tested species. The production of keratin-degrading enzymes is a function of time, with the peak of their activity occurring on day 15 of incubation. The role of keratin-degrading enzymes in the pathogenesis of dermatophytosis is becoming increasingly clearer. Given the conceptual understanding that keratin breakdown may require more than just one enzyme, the use of phenotypic methods is an optimal approach to in vitro study of the decomposition of species-specific keratin.

Effect of cured meat product ingredients on the Penicillium verrucosum growth and ochratoxin A production

Ochratoxigenic penicillia can grow on the cured meat products surface during their ripening. Ingredients added to cured meat products throughout their processing, and specifically of dry-fermented sausages, consist of a potential strategy to prevent or minimise the hazard associated with ochratoxin A (OTA) in these products. The aim of this work was to evaluate the effect of different concentrations of NaCl, KCl and sucrose in a conducive medium on the growth and OTA production by two Penicillium verrucosum strains. In general, there were no statistical differences between their growth rates. Although OTA production was not completely avoided in the presence of KCl and sucrose, it was reduced by comparison with NaCl. The concentration of the ingredients also affected the OTA production by P. verrucosum. These results would mean that the replacement of NaCl with KCl would imply the production of potentially safer cured meat products in terms of the OTA presence, which is of great importance for the current trends to reduce the NaCl levels in this kind of products. Furthermore, the addition of sucrose should be considered one of the approaches of the hurdle technology for minimising OTA accumulation in cured meat products.

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English

MALDI-TOF MS analysis of bovine and zoonotic Trichophyton verrucosum isolates reveals a distinct peak and cluster formation of a subgroup with Trichophyton benhamiae.

The zoophilic dermatophyte Trichophyton verrucosum is the most important causative agent of bovine dermatophytosis. Additionally, it causes profound and poorly healing skin infections in humans indicating the high zoonotic potential. The objective of this study was to establish differentiation of T. verrucosum from other dermatophytes by mass spectrometry and to identify distinct features of the mass spectra. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was successful for identification of this pathogen only after extension of the database of the manufacturer with spectra from T. verrucosum strains, which were identified as such by sequencing of the internal transcribed spacer (ITS) region. MALDI-TOF MS analysis was conducted with 46 field isolates from cattle, two live vaccine strains, and 10 isolates from humans identified as T. verrucosum by sequence analysis of the ITS region. The results suggest a very good agreement of both methods. Comparison with the mass spectra of 68 strains of other keratinophilic fungi revealed that most T. verrucosum wild-type isolates showed a characteristic peak at 7950-7954m/z, which was missing in the spectra of other keratinophilic fungi and the live vaccine strains. The spectra of T. verrucosum were most similar to the spectra of T. benhamiae, an emerging zoophilic dermatophyte. In summary, MALDI-TOF MS is a powerful and reliable tool to identify T. verrucosum.

Detection of ochratoxin A contamination in stored wheat using near-infrared hyperspectral imaging

Near-infrared (NIR) hyperspectral imaging system was used to detect five concentration levels of ochratoxin A (OTA) in contaminated wheat kernels. The wheat kernels artificially inoculated with two different OTA producing Penicillium verrucosum strains, two different non-toxigenic P. verrucosum strains, and sterile control wheat kernels were subjected to NIR hyperspectral imaging. The acquired three-dimensional data were reshaped into readable two-dimensional data. Principal Component Analysis (PCA) was applied to the two dimensional data to identify the key wavelengths which had greater significance in detecting OTA contamination in wheat. Statistical and histogram features extracted at the key wavelengths were used in the linear, quadratic and Mahalanobis statistical discriminant models to differentiate between sterile control, five concentration levels of OTA contamination in wheat kernels, and five infection levels of non-OTA producing P. verrucosum inoculated wheat kernels. The classification models differentiated sterile control samples from OTA contaminated wheat kernels and non-OTA producing P. verrucosum inoculated wheat kernels with a 100% accuracy. The classification models also differentiated between five concentration levels of OTA contaminated wheat kernels and between five infection levels of non-OTA producing P. verrucosum inoculated wheat kernels with a correct classification of more than 98%. The non-OTA producing P. verrucosum inoculated wheat kernels and OTA contaminated wheat kernels subjected to hyperspectral imaging provided different spectral patterns.

Molecular characterization of strains of theTrichophyton verrucosumcomplex from Tunisia

Trichophyton verrucosum is the most frequent etiologic agent of cattle dermatophytosis. Throughout the world, it was the second most common agent of zoophilic dermatophytes in human. The aim of our study was to evaluate the efficacy of the PCR- RFLP and PCR-sequencing methods for the identification and differentiation of T. verrucosum strains.Thirty-six clinical strains identified by morphological characteristics as T. verrucosum were isolated from patients referred to parasitology-mycology laboratory of Sfax University Hospital. Identification of our strains by conventional methods was confirmed by molecular methods in 94.4% of cases. Two strains were reclassified as T. violaceum PCR products digested with HinfI produced three profiles and two patterns with MvaI. Sequence analysis revealed a polymorphism in the ITS1and 5.8S regions. Analysis and alignment of consensus sequences has distinguished two types of genotypes among our T. verrucosum strains. The ITS type I was the dominant genotype (93.7%). Phylogenetic study showed that one cluster comprised T. verrucosum strains with ITS type I and species of T. mentagrophytes complex. It was related to Arthroderma vanbreuseghemii complex. The other cluster contained the two T. verrucosum strains with ITS type II, and was related to Arthroderma benhamiae complex. In this study, most of T. verrucosum isolates were type I, dissimilar to others rare studies where type II has been the most common. Specie and strain differentiation is relevant because it helps in prescribing the correct treatment and determining the source of the infection.

Quantification of Penicillium nalgiovense on Dry-Cured Sausage ‘Salchichón’ Using a SYBR Green-Based Real-Time PCR

To evaluate the effective implantation of a specific protective culture of Penicillium nalgiovense, a real-time quantitative PCR (qPCR) using SYBR Green methodology was developed. Two specific primers were designed on the basis of the published partial sequences of the Internal Transcribe Spacer (ITS)1–5.8S-ITS2 region of various strains of P. nalgiovense. Using the developed method, a PCR product of 51 bp with a Tm value 81.34 °C was detected. Tm values of the amplified product allowed specific differentiation between P. nalgiovense and the remaining mould species tested. The developed qPCR method was tested on inoculated slices of dry-cured sausage (‘salchichon’) showing an efficiency of 97.24 %, a R2 value of 0.99 and a detection limit of P. nalgiovense of 1 log colony-forming units (cfu)/cm2. The qPCR method demonstrated that the protective strain of P. nalgiovense grew and competed against an ochratoxin A (OTA)-producing Penicillium verrucosum strain on commercial dry-cured sausage. This qPCR method provides a specific, accurate and sensitive detection and quantification of P. nalgiovense on dry-cured sausage salchichon in order to estimate its colonization during their processing. This assay will improve strategies to prevent and control unwanted mould colonization and OTA risk in dry-cured meat commodities.

Relationship between ecophysiological factors, growth and ochratoxin A contamination of dry-cured sausage based matrices

Dry-cured sausages are colonised by moulds during the ripening process. The temperature and the salt content (which affects water activity, aw) predispose the surface to colonisation by Penicillium species, including Penicillium nordicum and Penicillium verrucosum which can lead to contamination of the sausages with ochratoxin A (OTA). The objective of this work was to obtain scientific data on the impact that interaction between ionic water stress (aw; 0.97, 0.94, 0.90, 0.87 and 0.84) and temperature (30, 25, 20, 15 and 10°C) may have on lag phases prior to growth, growth and OTA production by some P. verrucosum and P. nordicum strains isolated from dry-cured meat products on a dry-cured sausage-based medium over a period of 12days. Although P. nordicum had shorter lag phases than P. verrucosum, the latter grew faster than P. nordicum in most conditions tested. For both species, there was no growth and OTA production at 0.84 aw at all the temperatures tested. The fungi were more tolerant at moderate ionic aw conditions (0.94 and 0.90) and 20 and 25°C. In contrast, the patterns of production of OTA were very different from those for growth. Different OTA production profiles between the two OTA-producing species were found. While P. nordicum began producing OTA in most of the conditions tested by day 6, P. verrucosum only produced the toxin in these conditions when the temperature and aw were >10°C and >0.90, respectively. However, the P. verrucosum strain produced much higher concentrations of OTA than the P. nordicum strain in all conditions. We developed contour maps of the optimum and marginal aw×temperature conditions for growth/OTA production on dry-cured sausage-based medium for the first time. This suggests that these interacting conditions during the early phases of production must be effectively controlled as these favour growth of the toxigenic Penicillia. Knowledge on the ecophysiology of these two important Penicillium species on these matrices could help to make appropriate technological modifications during the sausage ripening process. Thus, our findings may help in informed decision-making in relation to temperature and salt additions at the beginning of processing/curing. Such changes may favour colonisation of starter cultures over OTA producing Penicillia and minimise OTA contamination risks in dry-cured sausages. This may be then effectively incorporated into the hygienic production system in the framework of HACCP.

Ochratoxin A and citrinin production by Penicillium verrucosum on cereal solid substrates

Cereals are among the most vulnerable substrates to mycotoxin contamination. One of the major fungal species colonising the ecosystem of stored grain is Penicillium verrucosum because of its ability to produce ochratoxin A (OTA). Knowledge of favourable environmental conditions promoting fungal growth and mycotoxin biosynthesis seems to be a crucial step towards preventing mycotoxin formation in stored grain. The aim of the study was to examine the effect of temperature and different cereal substrates (wheat, triticale, rye, barley, maize, rice) on fungal growth and OTA and citrinin (CIT) production by a mycotoxin-producing P. verrucosum strain at one moisture content. The correlation between fungal growth and mycotoxin biosynthesis was also investigated. For this purpose, cereal substrates moistened, autoclaved and inoculated with P. verrucosum spores were stored at 10°C, 20°C and 30°C for 40 days. After the storage period, the ergosterol (ERG) content as a fungal growth biomarker and OTA and CIT levels were determined using HPLC. Fungal growth was observed on all cereals at each temperature. Maximum ERG levels were found at 30°C and the values ranged from 401 to 3280 mg kg−1 of grain. Among tested cereals, the highest ergosterol content was found on rice at all applied temperatures. Accumulation of OTA and CIT was detected in samples stored at 20°C and 30°C, while optimal conditions for the production of both mycotoxins were observed at 20°C. The maximum concentration of OTA (31.9 ± 4.3 (SD) μg kg−1) and CIT (74.8 ± 5.7 μg kg−1) were found on rice. The ability to biosynthesise OTA and CIT at 10°C was not detected. Analysis of variance showed that single factors, temperature and cereal matrices and their interactions had significant effects on ERG, OTA and CIT levels. Optimal conditions for fungus growth did not coincide with the ones for mycotoxin formation.

Intraspecific variability of HOG1 phosphorylation in Penicillium verrucosum reflects different adaptation levels to salt rich habitats

Mycotoxin biosynthesis in Penicillium verrucosum is modulated by different molecular regulation mechanisms. One important mechanism is the HOG1 (high osmolarity glycerol) MAP kinase signaling pathway. In a comparative analysis three different P. verrucosum strains were selected from six strains with different ability to produce the mycotoxins ochratoxin and citrinin. The fungal strains were grown on laboratory medium supplemented with different concentrations of NaCl. It could be shown that there exists an interrelationship between the growth rate, the level of HOG phosphorylation and the mycotoxin biosynthesis under the respective growth condition. The weak to non ochratoxin producing P. verrucosum strain, BFE875, showed only a poor growth rate but the strongest HOG1 phosphorylation; the strong ochratoxin and citrinin producing strain BFE575 showed a reasonable HOG1 phosphorylation with an average growth rate; and the strong ochratoxin and weak citrinin producing strain BFE495 showed only a poor phosphorylation but the highest growth rate in 7days of incubation at 25°C. The magnitude of phosphorylation of the HOG1 protein seems to be inversely correlated with the degree of adaption of the fungus to hyperosmotic growth conditions.

EVALUATION OF THE IMMUNE RESPONSE TO <i>T. VERRUCOSUM</i> VACCINES

Dermatophytosis is considered as one of the most important fungal skin disease affecting both humans a nd animals otherwise its importance as zoonotic diseas e. Treatment of ringworm in cattle is expensive and time consuming especially on herd level. Vaccination can be a giant step forward to prevent the occurrence of mycozoonosis between the affected animals with ringworm and susceptible man. Fifty skins scraping samples were collected from animals (cattle, sheep and calves) suffering from skin lesions suspected t o be ringworm. Then these collected samples were subjected to mycological examination and identification. T. verrucosum is the main causative agent of collected skin scra pings. Two types of vaccines were prepared from this strain (Culture filtrate and formalin ina ctivated vaccines). Rabbit model was used for chall enge testing of the vaccines. The rabbits were separated into four groups as follow: Each group contained 3 rabbits each one was injected subcutaneously with 1 ml of culture filtrate with adjuvant twice for seve n days intervals. Group No. 2 rabbits each one was injecte d intramuscularly with 1 mL formalin-inactivated wi th adjuvant. The 3rd group is control positive and 4th one is control negative. Vaccination programme is done via injection twice with one week interval and the challenge by scarification of the skin with 0.2 mL of 5×10 7 fungal elements/mL. For 3 days. Humeral immune response was assessed by measuring anti-T. verrucosum antibodies using ELISA technique (direct method). By using statistical analysis of ANOVA, the mean optical density of anti- T. verrucosum specific IgG level in rabbits artificially immuniz ed with culture filtrate with adjuvant vaccine significantly increa se from (1.97) post first vaccination to reach (2.4 3) post second vaccination and still high after challenge w ith the virulent T. verrucosum strain (2.32) when compared with control positive (1.57). The results revealed that both type of vaccines induce good hum eral immune response in vaccinated animals. Culture filt rate vaccine used as a treatment in infected farm animals by two doses within 2 weeks and noted that healing occurred.

Elastolytical activity in vitro and in vivo of the mycelial and spore forms of the Trichophyton Verrucosum strains.

Summary It was found in vitro that 31 laboratory strains of Trichophyton verrucosum decomposed elastin to a various degree, whereas none of 20 newly isolated strains had this property. In further experiments, 4 T. verrucosum strains were used each in the spore and mycelial forms. Their pathogenicity for quinea pigs was studied, as well as their capability to decompose elastin in vitro and in vivo. It was shown that the transition from the mycelial form (passage through a sensitive animal) into the spore one is followed by a loss of the capability to decompose elastin in vitro. The spore form caused more marked and longer lasting inflammatory changes in the experimentally infected guinea pigs than the corresponding mycelial form did. The mycelial form in contrast to the spore form was characterized by the distinc elastolytical activity. This indicate the elastase enzyme does not play any essential role that in the pathogenesis of skin changes caused by the T. verrucosum strains in guinea pigs. Zusammenfassung Es wurde in vitro festgestellt, das 31 Laborstamme von Trichophyton verrucosum in unterschiedlich hohem Grade Elastin spalten, wahrend keiner von 20 frisch isolierten Stammen diese Eigenschaft aufweist. Zu weiteren Untersuchungen gelangten 4 T. verrucosum-Stamme jeweils in Sporen-und Myzelform. Es wurde die Pathogenitat dieser Stamme fur Meerschweinchen und die Fahigkeit uberpriift, Elastin in vitro und in vivo zu spalten. Es wurde nachgewiesen, das der Ubergang der Myzelform in die Sporenform (Passage durch ein empfindliches Tier) vom Verlust der Fahigkeit, Elastin in vitro zu spalten, begleitet wird. Die Sporenform der untersuchten Stamme verursachte deutlichere und langer andeuernde entzundliche Veraderungen bei den experimentell infizierten Meerschweinchen, als das bei der Myzelform der Fall war. Dagegen war fur die Myzelform im Gegensatz zur Sporenform eine deutliche elastolytische Aktivitat kennzeichnend. Das koonnte darauf hinweisen, das das Elastase-Ferment bei der Pathogenese der durch T. verrucosum-Stamme hervorgerufenen Hautveranderungen bei Meerschweinchen keine wesentliche Rolle spielt.

Verrucine F, a Quinazoline from Penicillium verrucosum

Verrucine F (3), a quinazoline similar to verrucines A (1) and B (2), contains one anthranilic acid residue, one L-glutamine residue, and one alpha,beta-unsaturated phenylalanine residue, as determined by NMR, MS, and chemical methods. Compounds 1 and 3, but not 2, were produced by Penicillium verrucosum J255 and eight additional P. verrucosum strains. Verrucines were typically more concentrated in the inner (older) parts of the colony, and 3 peaked 4-8 days after 1, but at 10-fold lower concentrations (approximately 200 microg/g). Verrucine F (3) formed spontaneously from 1 in buffered water solutions, probably by oxidation at C-1 followed by water elimination.

Clinical assessment of postinfection, postcontact and postvaccination immunity manifestation after experimental inoculation of calves with Trichophyton verrucosum culture

Immunity against ringworm has been well examined, but the knowledge of its development after repeated infection is limited. Therefore, the immunity developed after a single or double contact with various doses of Trichophyton verrucosum strain was investigated in calves which had or had not shown any signs of ringworm after contact with dermatophyte. In total six individual experiments were performed on 117 calves. Experimental infection with the virulent T. verrucosum strain was used to investigate the animals' immunity. Calves showing severe trichophytosis after application of high doses of T. verrucosum culture were immune to reinfection by equally high doses of this dermatophyte. A similar protection level was reached after intramuscular revaccination or after recovery from trichophytosis with moderate course induced by two inoculations of low doses. If moderate course of illness had been induced by same single dose, the immunity was lower. Animals displaying no signs of trichophytosis after inoculation with the low doses developed insufficient immunity. There was no immunity against reinfection in animals having previous contact with subinfectious doses through undamaged skin. The results obtained suggest that the level of postinfection immunity depends on the disease extent and the frequency of administration of the infectious agent. The development of postcontact immunity cannot be excluded in calves.