[History of Medical Mycology in the former German Democratic Republic]. / Zur Geschichte der medizinischen Mykologie in der ehemaligen DDR.

After the Second World War the development of medical mycology in Germany had taken a very different course in the east and west parts depending on the political division. In this respect our contribution deals with the situation in the former German Democratic Republic. Efficient mycological centres were founded step by step almost in all medical universities on the basis of the mycological laboratories in dermatological hospitals competent for diagnostic work, but also for teaching and scientific research. In this context biologists were the main stay of mycology, they finally were integrated to the same degree in the universities like physicians. The effectiveness of the Gesellschaft für Medizinische Mykologie der DDR (GMM), its board of directors and its working groups as well as the topics of human and animal mycology during this period are described. Especially the merger of the GMM with the Deutschsprachige Mykologische Gesellschaft after the reunification of Germany without problems and the kind co-operation of Prof. Dr. Johannes Müller during this procedure are emphasized.

[IWhich are the conditions for Cryptococcus neoformans var. neoformans-strains from avian excrements as a cause for human infections?]. / Unter welchen Voraussetzungen sind Cryptococcus neoformans var. neoformans-Stämme aus Vogelexkrementen als Ursache menschlicher Infektionen anzusehen?

Detection of antigen factors of Cryptococcus with factor sera in slide agglutination confirms diagnosis of species and varieties of Cryptococcus neoformans (Cr. n). This method is important in investigations of sources of infections. Serotype D strains of Cr. neoformans were detected in pigeon breedings from Thuringia exclusively. Because of that an essential difference exists in comparison to human isolates in Germany and strains from breeding stocks of companion birds in Thuringia where serotype A strains are predominant in pet birds and in human infections. Using different primers in PCR fingerprinting Cr. neoformans isolates can be assigned to serotypes A, B, C and D and to varieties Cr. neoformans neoformans and Cr. neoformans gattii (primer FM 1). On the other hand, genetic heterogeneity of Cr. neoformans strains is detectable within the serotypes A and D (primer 60-26). This genetic heterogeneity can be demonstrated in investigations by Fourier Transform Infrared (FTIR) spectroscopy, too. Isolated Cr. neoformans strains from pigeons (serotype D) could be divided into 3 and from pet birds (serotype A) into 2 different clusters by FTIR spectroscopy. It is important to take into account heterogeneity of strains within serotypes for determination of infection chains of human disease.

Phenotypic and genetic characterization of NAD-dependent Pasteurellaceae from the respiratory tract of pigs and their possible pathogenetic importance.

Nicotinamide adenine dinucleotide (NAD)-dependent Pasteurellaceae other than Actinobacillus pleuropneumoniae and Haemophilus parasuis are frequently isolated from the respiratory tract of pigs. The taxonomic classification and relevance for pathogenicity of these bacteria deserves further attention. In the present study, 107 of these NAD-dependent isolates from the porcine respiratory tract, primarily from lungs with pathological changes, were investigated. On the basis of phenotypic criteria, such as haemolysis, urease, catalase, and indole formation as well as other fermentative activities, 50 of the isolates were assigned to Actinobacillus minor, 36 isolates to Actinobacillus porcinus and 21 isolates to Actinobacillus indolicus. However, many isolates among the three species showed fermentative activities differing from those of the respective type strain of the species. Serotyping on the basis of heat-stable polysaccharide antigens and 16 rDNA sequencing also revealed substantial heterogeneity within each of the three species although they clustered together in three distinct groups in the phylogenetic analysis. These three groups of NAD-dependent bacteria are different from, or in a borderline position, to the existing species or genera within the family Pasteurellaceae. A considerable number of isolates of these three groups were isolated in pure cultures from pneumonic lungs. Consequently, it will be necessary to critically review the opinion, that these NAD-dependent Pasteurellaceae are only "agents colonizing the mucosa". Further, taxonomic examinations of the strains within these three groups are indispensable to testing isolates for their virulence in gnotobiotic pigs.

[Evidence of Cryptococcus neoformans in domestic and sports pigeons in Thyringia]. / Nachweis von Cryptococcus neoformans bei Haus- und Sporttauben in Thüringen.

19 strains of Cryptococcus neoformans var. neoformans were isolated from 17 (= 40.5%) of 42 investigated pigeon breeder flocks in Thuringia.

Occurrence of Cryptococcus spp. in excreta of pigeons and pet birds.

In pooled samples of faeces from 25 pet bird flocks in Thuringia, a high rate of contamination with Cryptococcus neoformans var. neoformans was found. The prevalence of Cr. neoformans in the bird-breeding establishments correlated with the numbers of the different pet bird species in these flocks. The differentiation between varieties of Cr. neoformans by means of proline assimilation and canavanine resistance detection as well as with the aid of Cr. neoformans factor sera, polymerase chain reaction (PCR) fingerprinting, sequencing of PCR products as well as Fourier transform infrared spectroscopy showed uniform results which also corresponded to the serological differentiation between serovars A and D. A predominance of serovar A could be observed among the pet bird breeding flocks. This corresponded to the frequency distribution of serovars A and D in cases of human diseases in Germany. In 50% of the samples of pigeon excreta examined (n = 30) in Innsbruck (Austria), Cryptococcus albidus could be isolated but not Cr. neoformans. However, this Cryptococcus species is of minor pathogenetic importance for man. Cryptococcus albidus may be clearly distinguished from Cr. neoformans by means of microbiological methods, PCR and Fourier transform infrared spectroscopy.

[Not Available]. / Beeinflussung der Zytokinsekretion von Maus-Thymom-Zellen (EL-4) durch Ochratoxin A.

The murine thymoma cell line EL-4 was used as an in vitro T-cell model to assess the immunomodulating effect of pure Ochratoxin A (OTA) and an Aspergillus ochraceus raw toxin preparation. Cytokine production (IL-2, IL-4, IL-5, IL-6) and cell viability of PMA-stimulated EL-4 cells were investigated in the presence of OTA. The cytotoxic effect of the raw toxin could be observed at lower concentrations than pure OTA. The IC50 values were 3 µg/ml and 11 µg/ml, respectively. Increasing concentrations of both OTA preparations caused an inhibition of cytokine production, but the inhibition effect of the raw toxin was stronger than of pure OTA. This is supposed to be the effect of further up to now not characterized substances in the raw toxin. Differences in the susceptibility of the mechanisms of production and regulation of each cytokine are indicated by the different concentrations for inhibition effects. Both toxin preparations showed also stimulating effects on some cytokines (IL-2 and IL-6) while others (IL-4 and IL-5) were depressed at these OTA concentrations. This indicates the immunomodulating properties of the toxin.

Studies on the influence of combined administration of ochratoxin A, fumonisin B1, deoxynivalenol and T2 toxin on immune and defence reactions in weaner pigs.

In weaners, combined administration of fumonisin, deoxynivalenol and T2 together with ochratoxin A in quantities expected to be present in feeds of central European origin resulted, as a rule, in changes identical to those observed after single administration of ochratoxin A. Such reactions were partly compensated. Synergistic amplification of immunosuppressive changes due to the simultaneous intake of these mycotoxins in low concentrations is not to be expected.

Studies of the influence of ochratoxin A on immune and defence reactions in weaners.

Even in subtoxic amounts, the mycotoxin, ochratoxin A, produced immunomodulation in weaner pigs in a dose-dependent mode. In addition to increased counts of total leukocytes and neutrophils in the blood, reduced lymphocyte levels were observed. There was a striking increase in the counts of eosinophils and of apoptotic phagocytes. Functionally, there was a predominance of the production of reactive oxygen radicals in whole blood, reduced phagocytosis performance and reduced expression of a swine-specific surface marker (SWC1) on lymphocytes. In a few single experiments, clinical manifestations could be demonstrated. Lung clearance and the degree of severity of experimental pneumonia as well as cutaneous hypersensitization may be influenced by ochratoxin A.

Prevalence of Haemophilus parasuis serovars isolated in Spain from 1993 to 1997.

From 1993 to 1997, 327 strains of Haemophilus parasuis were isolated from spanish swine in our Diagnostic Laboratory and 174 strains (53.2%) were serotyped. Four serotypes, sv. 5 (18.4%), sv 4 (16%), sv. 2 (9.2%) and sv. 13 (8%) were the most frequently isolated and 29.3% of the studied strains were classified as non typable. The results obtained indicate that the distribution of the serotypes in Spain is very similar to that found by other researchers in Germany, Australia, Canada and alike to that found in the United States.

[Distribution, serovar affiliation and epidemiologic behavior of Cryptococcus neoformans isolates from ornamental bird breeds]. / Verbreitung, Serovarzugehörigkeit und epidemiologisches Verhalten von Cryptococcus-neoformans-Isolaten aus Ziervogelzuchten.

Investigations of faeces samples from breeding stocks of companion birds in the federal state of Thuringia revealed a high contamination rate of companion birds with Cryptococcus (Cr.) neoformans var. neoformans. The prevalence of Cr. neoformans var. neoformans correlated with the spectrum of bird species present in the respective breeding units. The causes for that are not clear at the moment. Sensitivity of Cr. neoformans var. neoformans towards alkaline agents was not confirmed and was ruled out as a reason for different tenacity of the yeast in various bird breedings. Differentiation of varieties within Cr. neoformans was possible on the basis of proline assimilation, determination of canavanine resistance, EDTA urease test, as well as Cr. neoformans var. neoformans factor sera and PCR fingerprinting. Serological differentiation of serovars and PCR fingerprinting resulted in subdivision of Cr. neoformans var. neoformans isolates into two groups, which corresponded to serovars A and D. A prevalence of serovar A isolates was found in investigated bird breeding stocks. This also corresponded to the distribution of Cr. neoformans var. neoformans described in literature in humans with cryptococcosis in Germany. Consequently, serovar A or D infections of patients may be connected with their contacts to Cr. neoformans-excreting companion birds.

[Variability of Trichophyton cerrucosum isolates from vaccinated herds with cattle ringworm]. / Zur Variabilität von Trichophyton verrucosum-Isolaten aus Impfbeständen mit Rindertrichophytie.

Twenty-seven strains of Trichophyton verrucosum from 14 cattle herds in the Federal States of Thuringia and Mecklenburg-Vorpommern were examined by culture morphological and molecular biological (PCR fingerprinting, AFLP analysis sequencing of ITS region) methods. Six reference strains of the same species, among them the so-called album and ochraceum varieties, were also included. Despite great variability in terms of culture morphology, which suggested their possible classification into 4 different colony types, all T. verrucosum isolates were genotypically almost identical. Even the 2 field isolates growing with yellow pigment, which could possibly be regarded as belonging to the ochraceum variety, could not be differentiated using molecular biological methods. The results do not provide indications of a separate taxonomic position of the 3 T. verrucosum varieties. Furthermore, there is no evidence confirming the suspected infection of cattle herds with ochraceum strains as the cause of the failure of immune prophylaxis using various T. verrucosum vaccines. The frequent occurrence of animals not responding to vaccination could not be explained either. It should be assumed that the main factors responsible for this situation include poor handling of the vaccine strains and errors in application, especially the absence of continuous and systematic immune prophylaxis in the herds.

Phenotypic and genotypic differentiation of several human and avian isolates of Cryptococcus neoformans.

The Cryptococcus neoformans strains isolated from two human cases could be diagnosed as Cr. neoformans var. neoformans by differentiation on the basis of their characteristics determined by proline, canavanine and EDTA urease tests. The results of the serovar assignment were: for the isolate from the meningoencephalitis patient with lethal outcome, serovar A; for the strain isolated from the osteomyelitis patient with benign course, serovar D. Also, the PCR fingerprinting using primers (GACA)4, (CAC)5 and FM 1 resulted in a clear and reproducible assignment of the Cr. neoformans strains to the varieties neoformans and gattii, respectively, and, in addition, it confirmed the serovar assignment. No statistically confirmed differences in virulence between the osteomyelitis and the meningoencephalitis strain could be established by i.v. testing in mice, nor did the PCR with several primers provide any clues to a genetically determined higher virulence of the meningoencephalitis strain. The different classification as serovars A and D does not allow any conclusions concerning different virulence. It was not possible to retrospectively establish the sources of infection of the two Cr. neoformans infections, but pigeon faeces may well have played a role as a reservoir for one of the illnesses.

[Examination of primary SPF swine after experimental infection with Haemophilus parasuis. Clinical symptoms, changes in hematological parameters and in the parameters of the cerebrospinal fluid]. / Untersuchungen an primären SPF-Ferkeln nach experimenteller Infektion mit Haemophilus parasuis. Klinische Symptomatik, Veränderungen von hämatologischen Parametern und von Parametern des Liquor cerebrospinalis.

The objectives of this work were to cause the Glässer's disease (GD) in primary specific pathogen free piglets after experimental infection, to observe the clinical symptoms and to examine the influence of the infection on the haematological parameters. GD was caused by experimental infection of Haemophilus parasuis in seven to eight weeks old specific pathogen free piglets. In relation to the infection route the morbidity was high (83-100%) and 20% of the infected piglets died. Based on the physical examination fever, respiratory distress, cramps and paralysis were observed which are typical for GD. Arthritis and nerval symptoms are also typical but less common in Glässer's disease. PCV was significantly decreased and WBC significant increased before the piglets were euthanatized.

[Studies on the ecological behavior of Cryptococcus neoformans]. / Untersuchungen zum ökologischen Verhalten von Cryptococcus neoformans.

Tenacity studies of Cryptococcus neoformans in bird droppings originated from different ornamental birds and chickens showed that there is less chance for this fungus species to survive in non-sterile or bacteria-free droppings of large parakeets and chickens in comparison with droppings of small parakeets. Survival rates of Cr. neoformans in buffer solutions with pH-values ranging from 8.5-9.5 allow to conclude that this species is not alkali-sensitive. Therefore, the increase of pH is not regarded responsible for the survival of Cr. neoformans in bird droppings. Possibly fungistatic substances present in droppings are involved.

Studies of the influence of ochratoxin A on immune and defense reactions in the mouse model.

In the mouse model, the mycotoxin ochratoxin A has a non-selective suppressive effect on various immune and defence reactions. Apart from weight depression, lymphopenia, neutrophilia and eosinophilia, antibody-producing cells, antibody titres in blood serum and phagocytosis of Escherichia coli by blood phagocytes become suppressed. Moreover, immunized animals show a lower survival rate after experimental infection with Pasteurella multocida as well as an increase in oxygen radicals in blood cells.

Designation of 15 serovars of Haemophilus parasuis on the basis of immunodiffusion using heat-stable antigen extracts.

Previous independent investigations of the serotyping of Haemophilus parasuis strains have led to the designation of serovars A to D, 1 to 7, Jena 6 to Jena 12, and ND1 to ND5. Heat-stable antigen preparations from the reference strains for these serovars were tested by immunodiffusion with rabbit hyperimmune antisera. The existence of 15 distinct serologic groups was apparent, for which we propose the designations serovars 1 to 15. Examination of 290 field isolates from swine in the former German Democratic Republic indicated a prevalence of serovars 4 and 5, which together accounted for 41% of the isolates examined. However, 26.2% of the isolates were nontypeable with this test procedure and available antisera. Intraperitoneal inoculation of specific-pathogen-free pigs with cells representing the 15 serovars indicated differences in virulence which may be serovar related. Cells of strains representing serovars 1, 5, 10, 12, 13, and 14 were the most virulent, causing death or moribundity in inoculated pigs. Cells of serovars 2, 4, 8, and 15 caused polyserositis, but not death, in inoculated pigs. However, inoculation of cells of strains representing serovars 3, 6, 7, 9, and 11 resulted in no clinical symptoms or lesions indicative of H. parasuis infection.

[Relationship between serotype, virulence and SDS-PAGE protein patterns of Haemophilus parasuis]. / Beziehungen zwischen Serotyp, Virulenz und SDS-PAGE-Proteinmustern von Haemophilus parasuis.

141 Haemophilus (H.) parasuis and 8 H. parasuis-like strains from different farms were serotyped according to Morozumi and Nicolet (1986 b) as well as to Bakos et al. (1952). It was possible to classify 72.8% of the investigated strains. 7 out of 12 serotypes have been described for the first time. The high specificity in the agar gel precipitation test was not reproducible in the more sensitive dot-blot procedure. The dot-blot results point to a participation of non-immunogenic polysaccharides in the detection reaction. The serotypes SV 1, SV 5, SV Jena 6 and SV Jena 10 proved to be highly virulent in SPF pigs, SV 2 and SV 4 were of medium virulence. The other serotypes were found to be nonvirulent. Unencapsulated strains and isolates of serotype SV 5 prevailed in animals with Glasser's disease. 23 H. parasuis and 3 H. parasuis-like strains were examined in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). On the basis of protein profiles of whole-cell lysates, 23 of them could be assigned to 5 groups. Apart from the highly virulent strains of serovar 1, which belonged to PAGE type III, all other highly virulent strains of the serovars SV 5, SV Jena 6 and SV Jena 10 were grouped into PAGE type I. No correlation could be found between PAGE type on the one hand and virulence or origin of isolates on the other hand.

Typing of heat-stable soluble Haemophilus parasuis antigen by means of agargel precipitation and the dot-blot procedure.

According to Morozumi's and Nicolet's (1986) investigations, a serological classification procedure for H. parasuis and to a certain extent, for H. parasuis-like strains was proposed on the basis of heat-stable cell antigens in the immunodiffusion test. It was possible to classify 72.8% of the investigated strains serologically using this procedure. 7 of 12 serotypes were described for the first time. 60.1% of the classified strains belonged to the already known serotypes SV 1 to SV 5, whereas the new serotypes SV Jena 6 to SV Jena 12 amounted to only 12.7% of the field isolates. The serotypes SV Jena 7 to 9 are represented by H. parasuis-like strains. Unencapsulated strains and isolates of serotype SV 5 dominate in animals with Glasser's disease. The serotypes SV 1, SV 5, Jena 6 and SV Jena 10 proved to be highly virulent in SPF pigs, SV 2 and SV 4 were of medium virulence. The other serotypes were non-virulent. The high specificity in AGPT was not reproducible in the more sensitive dot-blot procedure. This must be taken into account, if the dot-blot is to be used for the classification of serotypes of H. parasuis. The results point to a participation of nonimmunogenic polysaccharides in the detection reaction.

Systematic control of dermatophytosis profunda of cattle in the former GDR.

Human occupational diseases orginated from Trichophyton verrucosum infection of cattle belonged to the most frequent zooanthroponoses since 1960. Morbidity peaks of this human dermatophytosis could be observed in 1970 and 1971 with about 740 cases of occupational diseases per year. The ecological properties of Tr. verrucosum (compulsory monoxenic parasite), the pathogenetic development of bovine dermatophytosis (rising immunity with subsequent elimination of the agent), systematic medical therapy and prophylaxis with griseofulvin and other drugs as well as the application of Tr. verrucosum live vaccines were the preconditions for a successful control of this zooanthroponosis. Since then the number of human and animal diseases could be essentially reduced.