Tierärztliche Fakultät - Digitale Hochschulschriften der LMU - Teil 01/07 Archiv

This paper deals with the in vitro activity of the B. cereus enterotoxin-complexes Hemolysin BL (HBL) and non-hemolytic enterotoxin (Nhe) on cells. For that purpose the cytotoxic and hemolytic activity of crude B. cereus culture supernatants was analyzed in cell culture assays and on blood agar plates. Specific monoclonal antibodies were used for the detection of single components. By using neutralisation assays and subtractive immoaffinity chromatography the inactivation of the specific toxin complexes could be achieved. When testing different cell lines (Vero-, Neuro2A-, Jurkat-, hybridoma cells) for their sensibility against culture supernatants of toxic B. cereus isolates, Vero cells proved to be the most sensitive. The sensitivity pattern of hybridoma cells differed from the other cell lines, as HBL containing supernatants showed higher cytotoxicity than supernatants of single Nhe-producers. Analyses of 18 B. cereus isolates revealed that the 5 - 40 % of the cytotoxic activity measurable in culture supernatants could be attributed to HBL. A strong correlation (r2 = 0,938) could be found between remaining cytotoxic activity and the detectable L2 concentrations. The results of the neutralisation assays could be verified by subtractive immunoaffinity chromatography of Nhe B and testing the Nhe B negative, HBL positive B. cereus culture supernatants (Nhe B-/HBL+). Furthermore it could be demonstrated for the first time that Nhe B plays a major part in the appearance of the discontinuous hemolytic pattern typical for HBL producers. After sensibilisation with tunicamycin Vero cells showed a substantial increased cytotoxic activity (63 – 132 %). The same effect could be observed in Fumonisin B pretreated cells for crude supernatants, but not for the Nhe B-/HBL+ preparation. Neither treatment with swainsonine, castanospermine nor enzymatic treatment with sialidase and N-glycosidase F resulted in an alteration of toxicity.

In der vorliegenden Arbeit werden folgende- speziell für Mäuse entwickelte- Beatmungsgeräte, der MiniVent und der UNO-Mikroventilator hinsichtlich ihrer klinischen Tauglichkeit einerseits und eventueller schädlicher Auswirkungen auf das Versuchstier andererseits untersucht und prakisch erprobt. Anschließend werden die beiden Geräte anhand der erzielten Ergebnisse miteinander verglichen. Besondere Beachtung gilt neben der Erfassung der arteriellen Blutgase und des arteriellen Blutdruckes der Aufwachdauer, dem Verhalten kurz nach dem Erwachen und dem Verhalten nach 24 Stunden.

Relevant species of the canine parasite fauna in European Mediterranean countries and Portugal for dogs in Germany concerning epidemiology and travel veterinary medicine – a literature review The parasite fauna of the dog in European countries surrounding the Mediterranean Sea and in Portugal more or less corresponds with the spectrum of canine parasites in Germany, but is also characterised by species, which are not indigenous here or which occur only regionally or rarely such as Leishmania, piroplasms, Hepatozoon canis, Rhipicephalus sanguineus s.l. and filarial worms, which are of concern not only in regards to the increasing tourism with dogs but also from an epidemiological viewpoint. Accordingly, the geographical distribution of the respective parasites in the Mediterranean basin is illustrated in this overview with special emphasis on regional infection/infestation risks. Furthermore, for those parasites which are not present in Germany, the risk of establishing endemic foci is discussed, as for Ehrlichia canis and with special regard to the scientific literature concerning cases of imported parasites.

Osteoporosis has a major impact on health care costs in the European Union. Not only for this reason effective therapy and early prophylaxis are necessary. Hormone replacement therapy was the treatment of choice until recently, but more and more risks have been proven. Thus research on alternatives is of current interest. Phytoestrogens, polyunsaturated fatty acids, as well as vitamins D and K were found to have a positive impact on bone in vitro and partially in vivo. In the present study effects of a combination of genistein, polyunsaturated fatty acids and vitamin D and K on bone metabolism and microarchitecture of bone in intact and overiohysterectomized dogs were investigated. In a feeding study twelve female beagle dogs were randomized into a study group and a control group. After five months all dogs were ovariohysterectomized and fed for another four months with control food or food with the additional substances. Blood and urine samples were taken every two or four weeks to analyse bone specific alkaline phosphatase in serum for bone formation and collagen-crosslinks pyridinoline and deoxypyridinoline in urine for bone resorption. Bone biopsies were obtained from the ilium at ovariohysterectomy and at the end of the study to do microcomputertomographic and biochemical analyses. The combination of substances was able to cause a prophylactic increase of bone mineral content. After ovariohysterectomy bone turnover was increased significantly (high turnover) and this rise could be normalized. Microstructure was altered only little by ovariohysterectomy, but a significant decrease in trabecular number could be prevented by the combined substances. The results of this study show, that a combination of genistein, polyunsaturated fatty acids and vitamin D and K can have a protective effect on bone in the intact as well as in the ovariohysterectomized beagle. This combination could be a possible alternative to hormone replacement therapy in postmenopausal osteoporosis.

Production and characterization of monoclonal antibodies specific for equine leucocytes This work was initiated to produce new monoclonal antibodies for the characterization of equine leucocyte-subpopulations. For immunisation, leucocytes isolated from the peripherial blood, lymph nodes and the intestinal epithelium of horses, have been used. Out of six cell-fusions 78 clons were isolated. To characterize the mAbs, the attention was focused on immunofluorescence analysis. Antibodies of the clones 4-13, 4-58, 1-19 and 6-39 have been purified by protein G-sepharoses chromatography and, except mAb 4-58, biotinylated. MAb 4-13, mAb 4-58 and mAb 5-50 are binding specifically to equine T-cells. Flow cytometer analyses demonstrated an increased frequency following the stimulation with Concanavalin A. The frequency of mAb 4-13- and mAb 4-58-positive cells matched the total frequency of CD4- and CD8-positive cells. The number of cells, marked by mAb 5-50, is slightly lower. Most likely these three mAbs recognize three different, as yet unknown antigens. MAb 4-58 stimulated the in vitro proliferation of leucocytes. Moreover mRNA for interleukin-4 and interferon gamma was detected in these cells after the stimulation. By contrast the antibodies 1-19, 2-52, 4-36, 4-55 and 6-39 are B-cell-specific, since they were present on cells that were Ig-positive. The monoclonal antibodies 6-5 and 6-17 stain all equine leucocytes. Possibly they are binding to an equine CD45-molecule. The antibodies of clone 4-18 recognize an antigen on granulocyctes. Preliminary data suggests that mab 4-39 is binding to a lektin, that can be found mainly on B-Lymphocytes and endothelial cells. No group of leucocytes and no antigen could be assigned to the other antibodies. The antibodies described here will be valuable tools to characterize different equine leucocyte-subpopulations by flow cytometry.

Gene expression in the tooth development of mice (Mus musculus) The development of teeth and jaw bones are lead by mechanisms that determine cells to become, teeth, bones or cartilages. The ectomesenchymal cells of the lower jaw receives signals from overlying epithelium to differentiate into odontoblasts, osteoblasts and chondrocytes. Cells from the neural crest are influenced by ectodermal signals. An early signal (Fgf8) comes from the oral epithelium and induces the development of the lower jaw. Molar morphogenesis in the maxillary starts from the mesenchyme, influenced by genes like Barx1, Dlx2 and Dlx2. At the same time the absence of genes like Msx1 and genes from the Alx-family is necessary for a normal development (McCollum and Sharpe, 2001). The expression of Msx1 and Alx-genes causes the development of incisors. Mice with null mutation of Dlx1 and Dlx2 fail to develop maxillary molars. Also additional abnormalities appear in the upper jaw. All other teeth are regularly developed (Qiu et al., 1997; Thomas et al., 1997). Dlx5 and Dlx6 are believed to be involved in the development of the lower molar teeth. Ectopic expression of Barx1 in the distal mandibular mesenchym, accompanied by loss of Msx1, results in a transformation of incisors into molars (Tucker et al., 1998). Msx1 mutant mice have defects in the distal jaw skeletal tissue. In conclusion, genes that determine the expression of molar teeth also control the development of the proximal jaw and genes that regulate incisor morphogenesis also regulate distal jaw development (McCollum and Sharpe, 2001). Generation of canine and premolar teeth is caused by overlapping domains of homeobox genes like Msx1, Dlx1 and Dlx2. In mice, the development of teeth in the maxilla seems to be different compared to the mandibula. Activin ßA null mutant mice shows no incisor and molar teeth in the lower jaw. Growth of teeth stops at bud stage in the mandibula while tooth development in the upper jaw in unaffected (Matzuk et al., 1995b). Most of the basic genetic mechanisms during the development of the teeth in the upper and lower jaw are identical. Only some of them lead into a different developmental pattern. This could be due to the different origin of mesenchymal cells (McCollum and Sharpe, 2001). The lower jaw is formed by ectomesenchymal cells originating from the neural crest from the cranial rostral hindbrain and the caudal middlebrain while neural crest cells from the caudal forebrain and middlebrain contribute to the fronto-nasal region (Osumi-Yamashita et al., 1994).

2 SUMMARY Pathologically-anatomical and immunhistochemistric investigation of ischemic myocardium in the pig Background: Different degrees of histological alteration have been seen in hibernating myocardium. Hibernation is associated with structural myocardial changes, which involve both the cardiomyocytes and the cardiac interstitium. The purpose of this study is to investigate the effects of chronic myocardial ischemia on histological patterns to understand the underlying mechanism of hibernation. Methods: A model of ischemic injury was produced in 16 pigs (German land breed) by placement of a modified stent graft in the left anterior descending artery (LAD), which initially produced 75% stenosis, followed by a slow complete occlusion. Wall motion abnormalities were investigated by Magnetic Resonance Imaging (MRI) and ultrasound images at day 7 after implantation. Metabolism and perfusion were imaged by Positron Emission Tomography (PET) at day 7 (group 1) and at day 28 (group 2). After PET images the animals were sacrificed and tissue samples were taken for histology. Results: Viability in PET is defined by a relative decrease in perfusion in an area where there is a relative increase in FDG (fluorodeoxyglucose) concentration. This is often referred to as a “mismatch” pattern. In the study nearly all pigs showed this “mismatch” in the LAD area. The following significant results could be found in the mismatched samples: the accumulation of collagen (0,12 ± 0,12 %, p < 0,05) in LAD samples compared to those taken from remote area (0,02 ± 0,02 %) and glycogen rich perinuclear zones in LAD samples (0,06 ± 0,03 %, p < 0,05). Despite the difficulties of reproducing a long-term hibernating myocardium in an animal model, one pig (No.8) demonstrated physiological alterations that can be compared to those of human beings with hibernating myocardium. After 28 days the artery of the pig was completely occluded. A mismatch was determined by PET, and wall motion abnormalities were present. Furthermore in cells which were exposed to a repetitive ischemia, a small degree of fibrosis and glycogen richness were shown. Conclusion: In contrast to various short-term hibernating myocardium models, no equivalent model exists for long-term hibernating myocardium over a time period of weeks or months yet. For this reason, the complete mechanism of chronic hibernating myocardium is still unclear. The fluent transition of the different heart failure up to the infarct as seen in the clinic reflect the dynamic process, the interindividuell differences and last but not least the reduced compensation abilities of the heart. Due to this it is difficult to find a matching in-vivo model that shows the same mechanism as humans. Therefore every small and large animal model is helpful for clarification.

Molecular phylogenetic differentiation of bovine Babesia In the present study, Babesia bovis, B. bigemina, B. divergens and B. ovata were differentiated with molecular biological techniques. These are four babesia species of cattle which are important in veterinary medicine. In addition to that, relationships within species were investigated. Therefore, two ribosomal DNA (rDNA) genes from babesia of different geographical origin were amplified and sequenced and then analyzed with regard of their phylogenetic correlation. Altogether 50 blood samples were examined, deriving from naturally infected animals or vaccine strains. 11 isolates originated from Europe, 22 from North- und South America, 5 from Africa, 4 from Asia and 8 from Australia. Previously, the Babesia contained in the samples had been assigned to the different species, using phenotypic characteristics. For the PCR amplifications of the 18S gene using the primers RIB-19 and RIB-20 and of the ITS region with the primers RIB-3 and RIB-13 PCR protocols developed for babesia of dogs were used. These were directly applied for B. divergens and, after optimization of MgCl2, DMSO, BSA concentrations and cycle number, with modifications also for B. bovis, B. bigemina and B. ovata. The length of the directly sequenced PCR products of the 18S rDNA was 1448 to 1552 base pairs for B. bovis, 1481 to 1485 bp for B. bigemina and 1516 to 1519 bp for B. divergens, exept one isolate which originated from a human infection and had a length of 1535 bp. For B. ovata, only a partial sequence of 889 bp was obtained. Compared with corresponding genebank sequences, the declared babesia species was confirmed with exception of one isolate. The PCR products of the ITS region were sequenced after cloning and their length resulted in 654 to 729 bp for B. bovis, 883 bp for B. ovata, 923 to 943 bp for B. bigemina and 995 to 997 bp for B. divergens. Despite the variability of the ITS sequence length of the B. bovis und B. bigemina isolates, this feature indicates characteristic values for each species. In the present study the ITS region of bovine Babesia was sequenced and analysed for the first time. With the phylogenetic analysis of the 18S sequences the four examined babesia species of the cattle could unequivocally be distinguished. Different phylogenetic trees (dendrograms) generated with the algorithms Distance Matrix, Maximum Parsimony and Maximum Likelihood showed all isolates in separated species groups. High bootstrap values (Distance Matrix and Maximum Parsimony) of 100 % for B. divergens, for B. bovis and B. bigemina, and 94 % resp. 70 % for B. ovata support the classification in four species. An additional phylogenetic analysis including also piroplasms of other hosts demonstrated a separation of bovine babesia species without overlap. Thereby, the present taxonomy and validity of the species B. divergens, B. bovis, B. bigemina and probably B. ovata were confirmed for the isolates examined in this study. It was not possible to differentiate between the B. divergens isolate collected from a human and the other B. divergens isolates which were very homogeneous. An additional intraspecific analysis for B. bovis and B. bigemina represented the heterogeneity of these two species in different dendrograms. The analysis of the ITS region was based on 95 clones, produced from 50 Babesia bovis, B. bigemina, B. divergens and B. ovata isolates of different geographic origin. Within the B. bovis and B. bigemina isolates an enormous variability was detected. This finding was indicated by intraisolate identities from 93,7 to 100 % for B. bovis and 95,8 to 100 % for B. bigemina. In contrast, the intraisolate identities of B. divergens clones (99,4 to 100 %) and the two B. ovata sequences (100 %) reached high values. On a species level, all clones within each species were compared. This procedure indicated the variability between isolates within each species. Thereby, the homogeneity of the B. divergens clones was confirmed by the identity of at least 99,1 % between two sequences. The corresponding values of B. bigemina (92,7 %) and B. bovis (82,7 %) turned out to be lower. The heterogeneity within the two species was confirmed by these identity scores and by the fact that clones from different isolates exhibited higher identities than clones from the same isolate. In the phylogenetic analysis of ITS sequences dendrograms for each species were generated with the algorithms Distance Matrix, Maximum Parsimony und Maximum Likelihood. The heterogeneity within B. bovis and B. bigemina was reflected by the fact that separated groups of isolates occurred in the phylogenetic trees. In addition, these groups correlated with their geographical origin. The phylogenetic relationship between isolates from Southern Africa and Australia was remarkably high. B. bigemina isolates were separated into two groups: one included the isolates from Australia und South Africa, the other isolates from Latin America. Within B. bovis four isolate groups of different geographic origin were detected. The first included isolates from Israel, Turkey and Morocco, the second isolates from Australia and South Africa. The Brazilian isolates appeared in the third group and the remaining isolates from the Americas in the fourth. There was no arrangement of isolates in phylogenetic trees of B. divergens.

Investigations on piroplasmosis in domestic cats in South Africa Domestic cats originating from an area in South Africa known to be endemic for babesiosis were included in an investigation on feline piroplasmosis. In order to receive some information about the range of species involved, molecular biological methods were used to characterize the causal agents genotypically. Two segments of the rDNA gene, the regions of the 18S and of the first and second internal transcribed spacers (ITS) were sequenced and phylogenetic analyses were performed. Based on these sequence data a modern and sensitive diagnostic test for a specific detection of B. felis, a real-time PCR, was developed and validated. Using this test, 206 blood samples of cats belonging to the patients of a small animal surgery in Port Elizabeth were screened for infections with B. felis and the test results were compared with results of examinations of stained blood smears and an indirect immunofluorescent antibody test (IFAT). Sequence analysis of the 18S rDNA and comparison with available sequence data from the Genbank NCBI demonstrated that at least 2, possibly 3 species are responsible for piroplasm infections in domestic cats in Port Elizabeth. The species B. felis was diagnosed in 11 of the 13 isolates. In one isolate, K 8, the parasites were identified as B. leo, a piroplasm species which had been documented to occur in lions before. This represents the first detection of B. leo in a naturally infected domestic cat. The sequence of the isolate K 68 could not be assigned to any known species, but it was closely related to the other feline piroplasms. The genetic relationship was slightly closer to B. leo with an identity of 98,3 % than to B. felis where an identity of 97,8 % was found. Compared to two other feline piroplasms isolated from a caracal, Babesia sp. Caracal Strain A and B, the isolate K 68 shared identities of 96,8 % and 96,7 % respectively. Phylogenetic trees clearly separated the isolate K 68 from the other feline piroplasms indicating a separate species status. Furthermore the results of the IFAT support a separation at least from B. felis. The isolate did not show any cross reactions with B. felis antigen. However, the parasites could not be differentiated from B. felis or B. leo in stained blood smears. In phylogenetic analyses including several Babesia and Theileria species all the feline piroplasm species of the present study were grouped together with B. rodhaini, B. microti and T. annae and seperated from the group of the `typical´ babesia and the classical theileria. When sequence analyses of the ITS rDNA of the 13 selected isolates were performed, the 11 isolates diagnosed as B. felis showed a polymorphism rate of 2,6 % and high pairwise identities between 98,6 % and 99,9 %. The sequences of the isolate K 8, already identified as B. leo, and of the isolate K 68 were less homologous with identities of 77,0 % and 76,0 % respectively compared with B. felis. In the phylogenetic tree the isolates K 8 and K 68 were clearly separated from each other and from the B. felis – isolates. This supports the existence of three distinct species. For one isolate, the isolate K 60, the intraisolate variation was determined in more detail. 7 different genotypes were found. 6 of them were very similar to B. felis and showed sequence identities between 97,2 % and 99,6 %. The sequence of the genotype 7, clone K 60 E.2, clearly differed from the other genotypes. With an identity of 76,9 % to B. felis the sequence could not be assigned to any hitherto described species. In phylogenetic analyses this clone K 60 E.2 was more closely related to B. leo and K 68 than to the B. felis isolates. Based on the sequence data of the ITS rDNA, a real-time PCR method was developed for the specific detection of B. felis. This test was proven to be sensitive and very specific in a blinded, externally controlled evaluation. The specifity was shown to be 100 %. The sensitivity of the test was 75 %. The positive prediction value reached 100 % and the negative prediction value was 82,1 %. A detection limit of 7 to 77 parasites/µl blood was determined. With this real-time PCR a modern, sensitive and highly specific method for the detection of B. felis is now available. The performance of the real-time PCR were compared with those of stained blood smears and the IFAT by investigating 206 blood samples of domestic cats of a small animal surgery in Port Elizabeth.When the results of the blood smears were compared with the results of the real-time PCR an agreement was found in 94,6 % (192/203) of the results. Identical results of the blood smears and IFAT were demonstrated in 91,2 % (187/205) of the cases. The results of the PCR and the IFAT corresponded in 96,1 % (196/204) of the investigations. The isolates K 8 and K 68, which had been diagnosed as B. leo and a closely related species by 18S rDNA sequence analyses and which had revealed babesia in stained blood smears, showed a negative test result in the PCR as well as in the IFAT. 9 isolates that were found to be babesia negative in blood smears gave positive results in the PCR as well as in the IFAT. 8 isolates were positive in the IFAT but negative in the PCR. 7 out of these isolates were also proven negative in the blood smears whereas in the remainig one case the blood smear was not examined. Infections with piroplasms were diagnosed in a high portion of the cats examined. The prevalence of babesia determined by stained blood smear was 32,2 % (66/205). DNA of B. felis was detected in 35,3 % (72/204) of the cats by real-time PCR. B. felis specific antibodies were found in 39,3 % (81/206) of the examined cats in the IFAT. No specific breed predilection was evident. The results of blood smears and PCR did not correlate with the sex or age of the cats. However B. felis specific antibodies were detected more frequently in male than in female animals and in a higher portion of the cats older than 2 years. Concerning the housing conditions, significantly more “indoor/outdoor” cats were affected. DNA of B. felis and specific antibodies were found in a portion above average of the cats coming from the “Walmer” area. Typical clinical signs attributed to feline babesiosis were observed in 28,8 % (19/66) of the cats showing babesia in the blood smear, in 25 % (18/72) of the cats with DNA of B. felis detected by PCR and in 22,2 % (18/81) of the cats that showed a positive test result in the IFAT. The part of latently infected was thus very high. No specific breed or sex predilection for clinical affection was evident. However a correlation of the age of the cats with the manifestation of a clinical babesiosis is assumed. In the age group between 0,5 and 2 years the percentage of infected cats showing clinical signs was above average.

Some recently described receptor families on myeloid and lymphoid cells in man and mouse have both inhibitory and activating receptors with important functions in the regulation of the immune system. Due to the lack of information regarding these receptors in other vertebrates, in the present thesis seven new members of this receptor family were identified in the chicken. All receptors identified were located on one single clone of a chicken BAC library, which indicates that they are tightly clustered on the chromosome. This localisation and the high amino acid homology suggests, that they belong to a multigene-family. Analysis of the genomic organisation showed, that the signalpeptide was split into two exons, each Ig-domain and the transmembrane region were encoded by separate exons and the long cytoplasmic tail (in case of inhibitory receptors) was encoded by two exons, the last exon containing the ITIMs. The exon/intron boundaries conformed to the gt-ag rule and the exon phases were highly conserved. In conclusion, the genomic organisation showed conserved features of mammalian Ig-like receptor genes. The novel identified CHIR were grouped into three different types of receptors. The activating CHIR-A had two extracytoplasmic Ig-domains and a short cytoplasmic tail, but a positively charged amino acid in the transmembrane region, which could associate to an ITAM-containing adaptor molecule. CHIR-A2 had a relative molecular weight of ~ 42 kDa and was detected in the mRNA of bursa, spleen, PBL, NK-cells and different macrophage- and B-cell lines. The inhibitory CHIR-B consisted of two Ig-domains and a long cytoplasmic tail with two ITIMs in it. CHIR-B2 had a relative molecular weight of ~ 46 kDa and was only detectable on B-cells by a newly produced specific monoclonal antibody (mAb). Upon Pervanadate treatment the ITIMs in the cytoplasmic tail were phosphorylated and recruited the protein-tyrosine- phosphatases SHP-1 and -2. Additionally ligation of CHIR-B2 had an inhibitory effect on the cell proliferation of a B-cell line. The potentially inhibitory and activating CHIR-AB had one Ig-domain, a long cytoplasmic tail with one ITIM in it and a positively charged amino acid in the trans-membrane region, which could associate with an ITAM-containing adaptor molecule. The only CHIR-AB homologue in the literature is KIR2DL4, that represents a highly conserved Ig-like Receptor expressed on all primate NK-cells with both activating and inhibitory function. CHIR-AB1 had a molecular weight of ~ 40 kDa and its expression on B-cells, on the B-cell line DT-40, on primary macrophages, different macrophage cell lines and on NK-cells in the gut was detected by a specific mAb.

Das Ziel der vorliegenden Arbeit war den Krebspesterreger Aphanomyces astaci, in den drei nordamerikanischen Flusskrebsarten Signalkrebs (Pacifastacus leniusculus), Kamberkrebs (Orconectes limosus) und Roter Amerikanischer Sumpfkrebs (Procambarus clarkii) nachzuweisen. Diese Flusskrebsarten sind zwar gegen die Krebspest resistent, können aber als latent infizierte Tiere Ausscheider des Erregers sein und somit die für die Krebspest empfänglichen Edelkrebse (Astacus astacus) infizieren. Bei dieser in Europa heimischen Art geht die Erkrankung mit 100 % iger Mortalität einher. Daher ist die Bestimmung des Träger-Status der nordamerikanischen Krebse von großer Bedeutung um eine weitere Verbreitung dieser hochinfektiösen Erkrankung in europäischen Gewässern zu verhindern. Im Vergleich zu den Edelkrebsen konnten bereits bei der makroskopischen und post sectionem bei der mikroskopischen Betrachtung der nordamerikanischen Flusskrebse Unterschiede festgestellt werden. Die letztgenannten weisen im Bereich der weichen Abdominalkutikula sowie an den dünnen Gelenkhäuten der Schreitbeine häufig Melanisierungen auf. Bei der lichtmikroskopischen Untersuchung kann eine Wachstumshemmung der Pilzhyphen durch Melanineinlagerungen beobachtet werden. Unmittelbar nach der Invasion des Pilzes wird das Immunsystem aktiviert und verhindert normalerweise einen Krankheitsausbruch. Lediglich bei Einfluss von Stressoren kommt es zur Immunsuppression nordamerikanischer Krebse und daraufhin nach dem Auftreten der für die Krebspest typischen Symptome häufig zum Tod. Um mittels PCR Aphanomyces astaci in den nordamerikanischen Krebsen nachzuweisen musste zunächst die Pilz-DNA extrahiert werden. Dazu wurde das DNeasy Tissue-Kit (Qiagen) verwendet. Es wurden unterschiedliche Flusskrebsgewebe zur DNA-Extraktion eingesetzt um die am besten geeignete Stelle zu finden. Die bereits bei Edelkrebsen verwendete weiche Abdominalkutikula erwies sich auch bei den nordamerikanischen Krebsen als äußerst zweckmäßig. Des weiteren konnte bei DNA-Extraktionen aus den Beinansätzen, dem dorsalen Abdomen und dem Telson der Erreger in der nachfolgenden PCR nachgewiesen werden. Da die nordamerikanischen Flusskrebse aufgrund ihres angepassten Immunsystems häufig nur sehr geringe Mengen an Pilz-DNA aufweisen, stellt der Nachweis hier ein Problem dar. Bei nur 50 mg eingesetzter Kutikula, laut Insekten-Protokoll des Tissue Kits, ist unsicher ob gerade in diesem kleinen Gewebestück Pilz-DNA vorhanden ist. Daher wurden auch Zusammenfassung ______________________________________________________________________________________________________________________________________________________ 132 Extraktionsversuche vorgenommen bei denen ein kompletter Krebspanzer eines Kamberkrebses (3000 mg) zur DNA-Isolierung eingesetzt wurde. Die benötigte Lysis-Puffermenge wurde dementsprechend erhöht und die Inkubationszeit wurde verlängert. Das Ergebnis der PCR war bei diesem Krebs positiv. Trotzdem wurde beim Großteil der Kamberkrebse nur die weiche Kutikula in die DNA-Extraktion eingesetzt. Hierfür wurden Kutikula-Segmente ausgewählt, die bereits im Lichtmikroskop Melanisierungen oder sogar Pilzhyphen aufwiesen. Bei zahlreichen Krebsen war das Ergebnis der nachfolgenden PCR ebenfalls positiv. Zusammenfassend wird somit deutlich, dass der molekularbiologische Nachweis des Krebspesterreger Aphanomyces astaci auch bei nordamerikanischen Flusskrebsen möglich ist. Sowohl die DNA-Extraktion als auch die PCR, mit teilweise neu entwickelten Primern, können zur Feststellung des „Carrier-Status“ eines nordamerikanischen Krebses dienen.

Summary In the here presented study, the zinc serum response was investigated after the application of 4 different zinc compounds with varying dosages. To be able to consider species differences, dogs and cats were also supplemented with the same compounds and the zinc serum values measured. A total of 4 ponies, 12 dogs and 11 cats were alternately fed with zinc oxide, zinc sulfate, zinc lactate and B-Traxim using a single dose of 10 mg zinc/kg KM and the zinc serum levels measured over the next 24 hours. For the ponies, the effect of a single dose of 20 mg zinc/kg KM (except zinc lactate) was also measured. In the third approach, the ponies were supplemented 2,5 mg zinc/kg KM per day for a total of 14 days. The results were as follows: • The zinc serum level increased significantly after the application of zinc sulfate and B-traxim in relation to the dosage (Tab. 1). Zinc lactate also showed signs of a potential increase but due to the reduced tolerance was only tested in the lower dosages. In contrast, zinc oxide did not show a significant zinc serum response regardless of the applied amount. Tab. 1: Mean values of the maxima with standard deviation (SD) and minimum / maximum zinc serum values for the ponies (Mean values, not indicated with the same letter differ significantly) 10 mg Zn / kg KM 20 mg Zn / kg KM Compound Mean Maxima SD Range Mean Maxima SD Range Control 860  200b 660-1102 854  209b 649-1146 Zinc oxide 1100  340ab 776-1564 1136  463b 842-1785 Zinc sulfate 2080  800a 1030-2805 4134  1323a 2880-5888 B-Traxim 2120  670a 1397-2885 4895  1497a 3136-6206 Zinc lactate 1810  630ab 1171-2638 • No significant increase of the zinc serum levels could be found after the daily application of 2,5 mg zinc/kg KM over the 2 weeks. All levels were within the range of the ones presented in current literature. • For the dogs and cats, no significant zinc serum response could be determined after the supplementation of 10 mg zinc/kg KM, regardless of the zinc compound. For the cats, it could be speculated that the zinc serum response showed a certain tendency to higher serum levels after the application of zinc sulfate, B-traxim and zinc lactate. For these investigations, it can be concluded that zinc sulfate and B-traxim are available in the horse and are thus suited for the use in studies on zinc supplementation in regards to hoof quality. But one needs to consider, that from the results presented in this study, it can not be excluded that zinc oxide or zinc lactate are also available in the horse under certain circumstances.