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

The long-term toxicological effects of hepatic iron overload on the cancer initiating and promoting properties of fumonisin B1 (FB1) were investigated in male Fisher 344 rats. An initial pilot (dose response) study over 15 weeks was performed to determine a level of dietary iron that achieves a high hepatic iron concentration in the absence of significant side effects, to be used in the subsequent long-term carcinogenesis study with FB1. Doses of 1%, 1.5% and 2% dietary carbonyl iron (Fe) were used for 10 weeks, followed by a level of 0.5% Fe for another 5 weeks. After 10 weeks of feeding 1% Fe, the hepatic iron level was 30-fold that of controls. Following the reduction in dietary iron to 0.5%, the hepatic iron level was maintained and side effects were minimal. Irrespective the dose of iron, the iron deposition was initially confined to zone 1 (periportal region), but with increased iron loading, extended to zone 2 (mid-zonal region) and zone 3 (perivenular region). Iron overload was shown to increase hepatocellular proliferation as seen in labelling of cells with 5-bromo-2’-deoxy-uridine (BrdU). Lipid peroxidation, measured as MDA, was also significantly enhanced by excess iron and levels correlated the iron dosage. In the long-term study, half of the rats were submitted to an iron-loading regimen with 1% Fe for 10 weeks, while the other half received the powdered AIN-93M diet only. The iron was then reduced to 0.5%; the rats were divided into four treatment groups: FB1/Fe (n=15); Fe (n=14); FB1/AIN (n=15); and control group (n=15), respectively. FB1 was fed at 250 mg/kg AIN-93M diet for 5 weeks, followed by 100 mg/kg for 20 weeks. The effect of iron on FB1-induced initiation and promotion was assessed in 5 rats from each group at week 35. FB1 was removed from the diet of the remaining rats but iron supplementation continued for 25 weeks. The rats were terminated and the effect of dietary iron on the progression phase of FB1-induced carcinogenesis was assessed. The feed intake of rats from the Fe group (weeks 4-10), and FB1/Fe and FB1/AIN groups (weeks 10-60) was measured daily and the other groups were given averaged feeding accordingly; thus, the total body weight gain (tBWG) gain did not differ significantly between groups. Excess iron significantly increased the liver to body weight (LW/BW) ratio; however, iron could not counteract the mitoinhibitory effect of FB1 on hepatocyte proliferation and the LW/BW ratio of the FB1/Fe group was significantly lower than the Fe group. During the 25-week FB1-treatment period, more foci and nodules of dysplastic cells developed in the FB1/Fe group than in the FB1/AIN group. After removal of FB1, the number of nodules remained constant in the FB1/Fe group, while increasing in the FB1/AIN group. Initially, the pattern of iron deposition was the same as in the pilot study. Hepatocyte necrosis caused by FB1 then resulted in a shift of iron from hepatocytes to Kupffer cells and portal tract macrophages. FB1/Fe resulted in increased hepatocellular proliferation as demonstrated by increased BrdU labelling at 35 weeks. The withdrawal of FB1 reversed this effect and the index of BrdU labelling decreased to even lower levels than the Fe and FB1/AIN groups. Hepatic iron was almost 9-fold that of controls at 10 weeks; a significant increase in the hepatic iron levels in the FB1/Fe group was noticed as compared to all other groups. However, after removing FB1, the hepatic iron decreased to the same level as seen in the Fe group. During the FB1 treatment period, MDA increased in the FB1/Fe group to levels significantly higher than in the FB1/AIN and control groups. The present study suggests a dual role of iron overload in cancer development during FB1-induced carcinogenesis. During the cancer initiation/promotion phase, iron overload in combination with FB1 enhanced the susceptibility of the liver to the formation of foci and nodules; but after removal of FB1, continued supplementation of iron impaired the progression of pre-neoplastic hepatic lesions.

The aim of this study was to evaluate and establish a large animal model of osteoporosis by a combination of ovariectomy and steroid treatment. A variety of densitometric, morphometric and biomechanical methods were used to quantify changes in ovine bone under estrogen-deficiency and glucocorticoid treatment. With DXA we monitored the progression of the loss of bone mineral density of integral bone at the central and the load-bearing skeleton. Peripheral QCT was employed to determine the different respons es of trabecular and integral bone. High-resolution µCT-images allowed the evaluation of precise morphometrical parameters. Histomorphometric analyses provided information about newly formed osteoid. Stiffness and strength of trabecular bone were assessed by uniaxial compression tests. For all these examinations we utilized integral or trabecular bone from various skeletal locations. The results of this study showed that a combination of ovariectomy and glucocorticoid treatment leads to osteopenia in sh e ep. Bone mineral density of integral bone was reduced during glucocorticoid treatment and returned to normal values thereafter. However, loss of bone mineral at trabecular sites was not reversible. Corticoid treatment also affected the microarchitecture of trabecular bone: bone volume, bone surface and trabecular thickness differed from the control group. However, trabecular number did not change under steroid treatment. Bone stiffness and strength of trabecular bone of corticoid treated animals were m arkedly reduced. These results showed the suitability of the ovariectomized and glucocorticoid treated sheep as an animal model in osteoporosis research. It could be used to develop new orthopaedic techniques or to test prosthetic designs or implants for the treatment of osteoporotic fractures.

Oncosuppressive properties and a low pathogenicity in adult animals make the two rodent parvoviruses MVMp and H1 promising vectors for cancer gene therapy. Recombinant vectors were developed from genomes of these viruses in which a part of the capsid-protein-coding sequence was replaced by a transgene. These recombinant viruses can replicate their genome and express their transgene, but they are unable to produce progeny viruses. Such vectors are produced by cotransfection of the recombinant virus genome and a helper plasmid which provides the capsid proteins. During this procedure, RCVs (Replication Competent Viruses) can be generated, probably by homologous recombination. As a consequence, RCVs can express their capsid proteins and produce progeny viruses. In the present work, second-generation H1- and MVMp- based recombinant virus genomes were developed in order to minimise RCV levels in the recombinant virus preparations. By constructing chimeras and pseudotypes between MVMp and H1 viruses, virus preparations with high titres were obtained which did not show any detectable RCVs in plaque-assays. This corresponds to a 100-17000fold decrease of RCVs for the chimeras and pseudotypes. In this work we also characterised the host-cell-tropism of the chimeras and the pseudotypes. The chimeric vectors and Pseudotypes allowed us to show that the restriction for viral replication of the rat H1 genome in murine A9 cells is not due to the H1 capsids, but can be ascribed to a 1800 bp long DNA region of the H1 genome, located in the NS coding sequence. To our knowledge this is the first report that convincingly shows that the tropism of a rodent parvovirus is determined by the viral genome. Since a recombinant MVMp genome packaged with either H1 or MVMp capsids led to viral DNA replication in A9 cells, virus entry is not responsible for the lack of recombinant H1 vector replication in these cells. After transfection of A9 cells with H1 viral DNA, replication and viral protein expression took place, whereas after H1 infection it did not. Thus we can conclude that the cause for the restricted H1 virus replication has to be found after virus entry but before the beginning of viral gene transcription. It is usually accepted that viral gene transcription needs the conversion of single-stranded Summary 134 DNA into double-stranded monomeric replicative form. Most probably this conversion reaction is disordered in A9 cells for the H1 genome. A defective double-stranded conversion might be due to an interaction of VP proteins with the viral single stranded DNA sequence. Another possibility is an interaction of a cellular protein with the viral single stranded DNA which then leads to an inhibition of the viral DNA conversion. It might be also possible that a cellular factor which is needed for the conversion reaction can not bind to the H1 genome in A9 cells. Interestingly, in most of the tested murine cell lines the tropism was determined by the MVMp genome, while for all human cell lines the tropism was determined by the virus capsid. The second part of this work focused on the effect that H1 virus infection on cellular gene expression. For this purpose, the cDNA array technique was used. It was previously shown that parvovirus H1 infection leads to the activation of caspase 3 and apoptotic cell death of the human monocytic U937 cell, in a way similar to the apoptotic cell death induced by TNFα. Moreover, the c-myc gene, overexpressed in these cells, is down-regulated during H1 infection. In agreement with these data, we were able to show -by comparing the gene expression profiles between buffer treated and H1 infected synchronised U937 cells- genes coding for caspases 2, 4 and 8 were upregulated after H1 infection. In addition, different genes associated with the TNFα complex were found to be up-regulated, as well as the mad4 gene, which is able to inhibit the transcriptional activity of c-myc by binding its main partner max. Moreover, 2 genes involved in oestrogen-synthesis were found to be up-regulated after infection. Oestrogen was shown to have some protective effect against TNFα induced apoptosis in U937 cells. Induction of these genes might reflect a defence mechanism of the cell. Also, a gene encoding for a subunit of the 26s proteosome and different genes involved in differentiation processes or in apoptosis were up-regulated in infected cells. Few genes were shown to be down-regulated after infection, such as the adenylate cyclase and cAMP dependent transcription factor 1. Altogether, our data give a better understanding of the molecular pathways which are used by the parvovirus and enlighten the diversity of cellular responses to a H1 virus infection in U937 cells.

Überprüfung der Wirksamkeit stallspezifischer Vakzinen zur Bekämpfung der Dermatitis digitalis bei Milchkühen Die Studie wurde als kontrollierter Doppelblindversuch durchgeführt. Die Untersuchungen fanden in einem Milchviehbetrieb in Norddeutschland statt (429 Tiere bei der ersten Untersuchung). Aus den Hautbioptaten typischer DD-Läsionen wurden folgende Erreger im anaeroben Bereich isoliert: Porphyromonas ssp., Porphyromonas levii, Porphyromonas endodonatales, Fusobacterium necrophorum, Bac. stercoris, Prevotella bivis, Peptostreptococcus indolicus, Treponema sp.. Daraus wurden zwei formalininaktivierte Adsorbatvakzinen hergestellt. Die eine enthielt alle Keimarten außer Treponema sp. (Impfgruppe A), die andere alle Keimarten und Treponema sp. (Keimanteil 20 %) aus einer Schüttelkulturvermehrung (Impfgruppe B). Als Placebo wurde die Impflösung ohne Antigen verwendet (Kontrollgruppe). Verabreicht wurden je 5 ml Impfstoff oder Placebo subkutan. Der Klauengesundheitsstatus wurde im Oktober 1998, März 1999, Oktober 1999 und im Januar 2000 im Rahmen der halbjährlich durchgeführten funktionellen Klauenpflege erfasst und die Befunde der Hintergliedmaßen dokumentiert. Die erste Impfung erfolgte im Dezember 1998, die zweite drei Wochen später, die weiteren Impfungen fanden im März 1999 und im November 1999 statt. Zwischen der ersten und vierten Untersuchung durchgeführte Klauen- und Antibiotikabehandlungen wurden vom Betriebsleiter protokolliert. Die Auswertung erbrachte folgende Ergebnisse: 1. Zu keinem der vier Untersuchungszeitpunkte bestanden signifikante Unterschiede bezüglich der Prävalenzen der DD in den zwei Impfgruppen und der Kontrollgruppe. Die Prävalenzen der DD bewegten sich zum Zeitpunkt der vier Untersuchungen um 36 %, 42 %, 51 % und 38 %. 2. An Hintergliedmaßen, die bei der Erstuntersuchung DD-Läsionen aufwiesen, konnten auch bei den Folgeuntersuchungen in den zwei Impfgruppen und der Kontrollgruppe jeweils etwa gleichhäufig derartige Veränderungen diagnostiziert werden. 3. An Hintergliedmaßen, die bei der Erstuntersuchung frei von DD waren, konnten bei den Folgeuntersuchungen in den zwei Impfgruppen und der Kontrollgruppe jeweils etwa gleichhäufig DD-Läsionen diagnostiziert werden. 4. Die Scorewerte (nach DÖPFER, 1994) DD positiver Tiere - als Maßzahl für den Schweregrad der DD-Veränderung - waren zu den vier Untersuchungszeitpunkten in den zwei Impfgruppen und der Kontrollgruppe statistisch nicht signifikant verschieden. 5. Der „maximale gewichtete Flächenindex (max. GFI)“ - als weitere Bewertungsgrundlage für den Schweregrad der DD - war bei den Untersuchungen U2 und U3 in den Impfgruppen und der Kontrollgruppe nicht statistisch signifikant verschieden. Bei U1 war in der Impfgruppe B ein statistisch signifikant höherer Mittelwert (x) festzustellen als in der Kontrollgruppe (x = 82; p = 0,006). Bei U4 war in der Kontrollgruppe ein statistisch signifikant niedrigerer Mittelwert (x) festzustellen als in den Impfgruppen (x = 22,5; p = 0,033). 6. Die Häufigkeit von Klauen- und Antibiotikabehandlungen sowie der Einfluss von DD-Läsionen auf die Abgangsursache waren in den zwei Impfgruppen und der Kontrollgruppe statistisch nicht signifikant verschieden. In der vorliegenden Impfstudie konnte weder eine prophylaktische noch eine therapeutische Wirkung durch die verwendeten stallspezifischen Vakzinen bei der Bekämpfung der Dermatitis digitalis festgestellt werden. Auch das Hinzufügen von Treponema sp. erbrachte keinen Effekt auf die Wirksamkeit des Impfstoffs.

Morphological fundamentals for the sonographic examination of the feline thyroid gland The thyroid glands of 101 deceased cats were removed for morphological, sonographical, morphometrical, volumetrical and pathohistological assessment to investigate the morphological fundamentals for sonographic examination. The determined reference range extends over 0,08 – 0,27 ml in volume, 13,8 – 25,2 mm in length, 2,3 – 4,1 mm in width and 3,9 – 7,1 mm in height of the feline thyroid gland. These parameters are independent of body weight, age, sex and castration status of the cat. A correction factor of 0,45 has been established from the volumetrically and morphometrically measured values by sonographic survey. This makes it possible to apply the ellipsoid formula for the specific determination of the feline thyroid gland volume. However, comparative studies have shown a remarkably broad standard deviation for this method of calculating the thyroid gland volume in the cat. Its assessment by surveying the thyroid gland lobes` width and height is more expressive: the ratio should not be below 1:1,5. The number of detected thyroid alterations in the cat was surprisingly high. Mainly - in 48 out of 101 animals - nodular hyperplasia and adenomas have been found. Sonographic examination revealed characteristic features of nodular hyperplasia and adenoma cases. However, these examinations could not provide reliable distinctive marks. In addition, two adenocarcinomas were found. Their sonographic examination resulted in a list of characteristics of thyroid malignancies (e.g. outstanding size, knobby surface, inhomogeneous echostructure with poor echogenity) as reported in literature. Analogous to the proven etiology of human nodular hyperplasia and adenoma hypothetically the high incidence of those alterations in cats could be caused by chronic iodine deficiency. This hypothesis is in line with the recently detected deficitary iodine content of an average of commercial cat food. Nodular hyperplasia and adenoma are one of the major causes for feline hyperthyroidism. In the recent years their incidence has increased. The studies on hand emphasize the importance of the sonographic examination for detection of such morphologic thyroid gland alterations as a supplementary measure to laboratory diagnostics of hyperthyroidism.

The fetlock joint (articulatio metacarpophalangea) plays an exceptional role within the limb, as it is the only joint which is in hyperextension in the standing horse. It is therefore exposed to special loading situations compared to other joints. The objective of this study is to describe in a detailed way the morphological parameters of loading in the equine fetlock joint (metacarpophalangeal articulation). 45 specimens of 26 horses of different breeds and sizes, male and female were examined. Their age ranged from foal to 30 years. Using Computertomographic Osteoabsorptiometry (CT-OAM) and Chondrocrassometry (CT-CCM) the distribution of the subchondral bone density and the cartilage thickness were determined topographically in the 3D reconstruction of the joint. Generally the subchondral bone density increases in all bones forming the fetlock joint with increasing age of the horses. Up to the age of 2 years no significant increase in the subchondral bone density can be stated. From the age of 3 years onward a distinct increase can be measured until it comes to stagnation in horses older than 13 years of age. In most cases the areas with homogeneous subchondral bone density extend within the articular surface. In the dorso-distal area of the trochlea a broad transversal band of high bone density can be observed, which at the angle of stance corresponds to a respectively dense part of the phalangeal articular surface. The dorsal and palmar area at the edge of the trochlea, as well as the palmar edge of the fovea articularis show low bone density. Altough the lateral edges of the trochlea and the fovea articularis show a higher density. The method of split lines showed no definite preferential orientation of the collagen fibres in the subchondral bone of the fetlock joint. Therefore it is not possible to draw any conclusions concerning physiological incongruence of this articulation. In animals from 3 months to 2 years of age a change in the trochlear profile can be observed from a cylindrical condyle to a concave - convex shape in abaxial direction. This could be related to the results described above. Surfaces of the trochlea (palmar area) and the sesamoid bones articulating in the standing horse show a higher subchondral density. In contrast to the subchondral bone density the cartilage thickness generally decreases in older horses. After the period of maximum growth in horses the decrease of cartilage thickness stagnates at the age of 3 to 4 years. Articular surfaces in the trochlea, the proximal phalanx and the sesamoid bones which are in contact during stance show low cartilage thickness. It seems that areas which are loaded more statically way are covered by a thinner cartilage layer while areas loaded more dynamically are covered by a thicker cartilage layer. Generally speaking the tendency towards a negative correlation between subchondral bone density and cartilage thickness can be observed in the fetlock joint of the horse. An exception to this rule is the vertex area in the sagittal groove, where both a high density and a high cartilage thickness can be found.

Zur Klärung der Frage, ob das BCS-System nach EDMONSON et al. (1989) in der Version von METZNER et al. (1993) unter Praxisbedingungen auch beim Fleckvieh zur Überwachung des Ernährungszustandes geeignet ist, wurden die Komponenten des Systems und einige andere Beurteilungsmethoden mit der ultrasonographisch bestimmten Rückenfettdicke verglichen. Dazu wurden 421 Fleckviehkühe in 30 Betrieben während des Kalbezeitraums und in der Frühlaktation insgesamt mindestens fünfmal im monatlichen Abstand untersucht. Zusätzlich wurden Untersuchungen zur Reproduzierbarkeit der angewandten Methoden angestellt. Die Ergebnisse der Milchleistungsprüfung wurden für den Untersuchungszeitraum dokumentiert. In dem untersuchten Kollektiv von 421 Fleckviehkühen spiegelte die Rückenfettdicke die für die Frühlaktation typische Entwicklung des Ernährungszustandes von Milchkühen wider. Selbst im Bereich des Tiefpunktes dieser Entwicklung (36-95 Tage p.p.) lag die mittlere Rückenfettdicke mit zirka 51 mm deutlich über dem in der Literatur angegebenen Wert für verfettete HF-Kühe (35 mm). Die maximale fett-eiweißkorrigierte Milchleistung übte keinen nachweisbaren Einfluß auf die Veränderungen der Rückenfettdicke aus. Als Faktoren mit einer nachweisbaren Wirkung auf die Körperfettmobilisation konnten der Betrieb und die Rückenfettdicke bei der Kalbung identifiziert werden. Bei stärkerem Körperfettabbau traten signifikant höhere maximale Fett-Eiweiß-Quotienten auf. Das leicht modifizierte BCS-System ist prinzipiell auch beim Fleckvieh geeignet, die aktuelle Rückenfettdicke der Kuh so zu beschreiben, wie es auch von der Anwendung des BCS bei anderen Rassen angegeben wird.Die Bewertung der Dornfortsätze (BCS1) und der Lendenwirbelquerfortsätze (BCS3) bringt bei dieser Rasse - zumindest unter den in der Untersuchung vorliegenden Haltungs und Fütterungsbedingungen - keinen Vorteil. Kleine Änderungen der Körperkondition sind mit dem System insbesondere deshalb schlecht darstellbar, weil es nicht genügend Differenzierungsmöglichkeiten für die bei Fütterung von Maissilage ausgeprägten Fettdepots im Beckenbereich bietet.Das System beschreibt unter Vernachlässigung von BCS1 und BCS3 beim Fleckvieh die Rückenfettdicke mit r=0,77 und ihre Änderungen mit maximal r=0,56. Seine Komponenten sind innerhalb eines Untersuchers sehr gut reproduzierbar (r=0,88 bis r=0,95). Die getrennte palpatorische Beurteilung der Fettauflage von Hüft- und Sitzbeinhöcker erscheint aufgrund der meist erheblichen Unterschiede notwendig. Dabei sollte zur Dokumentation von starken Fettauflagerungen am Sitzbeinhöcker, die mit keiner Stelle der eigenen Hand mehr vergleichbar sind, eine zusätzliche Beurteilungsstufe eingeführt werden. Das angewandte BCS-System ist also auch bei Zweinutzungsrassen brauchbar, aber verbesserungswürdig. Mit den untersuchten Methoden zur Ergänzung oder weiteren Modifikation des Systems konnten aber keine deutlichen Fortschritte in dieser Richtung erzielt werden.